JAC Advance Access originally published online on September 14, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):942-949; doi:10.1093/jac/dkl377
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Influence of tannins from Stryphnodendron adstringens on growth and virulence factors of Candida albicans
1 Mestranda do Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá Avenida Colombo, 5790, 87020-900, Maringá, Paraná, Brazil 2 Departamento de Farmácia e Farmacologia, Universidade Estadual de Maringá Avenida Colombo, 5790, 87020-900, Maringá, Paraná, Brazil 3 Departamento de Análises Clínicas, Universidade Estadual de Maringá Avenida Colombo, 5790, 87020-900, Maringá, Paraná, Brazil
*Corresponding author. Tel: +55-44-3261-4863; Fax: +55-44-3261-4860; E-mail: cvnakamura{at}uem.br
Received 29 May 2006; returned 23 June 2006; revised 10 August 2006; accepted 22 August 2006
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Objectives: The main objective of this work was to investigate the antifungal activity of a crude extract, fractions and subfractions from Stryphnodendron adstringens (Mart.) Coville, known as ‘barbatimão’.
Methods: The growth inhibition by ‘barbatimão’ of 103 isolates of yeasts from vaginal fluid was determined using the broth microdilution method. In addition, the effect of the most active subfraction on cell surface hydrophobicity (CSH), germ-tube formation, budding, ultrastructure and phagocytosis of Candida albicans was analysed. Fluconazole and nystatin were used as reference drugs. The cytotoxicity of ‘barbatimão’ to Vero cells, macrophages and red blood cells was assessed. The most active subfraction was characterized by mass and 13C NMR spectroscopy.
Results: Subfraction F2.4 had the best antifungal action at concentrations above 7.80 mg/L. Its action was similar to nystatin, and only slightly less effective than fluconazole. CSH and the capacity for adhering to Vero cells and a glass surface were lower in treated yeasts. In addition, the inhibition of formation of the germ tube, the increase in the number of buds and changes in the cell wall ultrastructure of C. albicans were also demonstrated. ‘Barbatimão’ extracts showed low cytotoxicity to Vero cells, macrophages and red blood cells. Subfraction F2.4 is composed of proanthocyanidin polymers of prodelphinidin and prorobinetinidin units and gallic acid of molecular weight 2114 Da.
Conclusions: The antifungal action of subfraction F2.4 on C. albicans can be attributed to condensed tannins. It is considered moderate antifungal activity. These properties of ‘barbatimão’ on the growth of C. albicans, putative virulence factors and its low cytotoxicity justify further studies to investigate the mechanisms of action and the possible development of a new antifungal agent.
Keywords: antifungal , yeasts , proanthocyanidin , electron microscopy , hydrophobicity
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Introduction |
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There are between 250 000 and 500 000 plant species on Earth, a small percentage of which are utilized for treatment of diseases.1 In the past decade interest in natural products has increased, and medicinal plants have been assessed for possible bioactive compounds. These compounds have been isolated and subjected to detailed structural analysis, and their potential modes of action and target sites elucidated. Several plants used in folk medicine have been studied for antimicrobial activity, as a source of new antifungal compounds with fewer side effects, a wider spectrum of action and lower cost.2
Stryphnodendron adstringens (Mart.) Coville, Leguminosae, known as ‘barbatimão’, occurs in the central savannah region of Brazil. Stem bark from this plant is used popularly as an anti-inflammatory and astringent, and in the treatment of wounds and vaginal infections.3,4 Anti-ulcerogenic, anti-inflammatory and antibacterial activities of fractions of the crude extract from the bark of S. adstringens were reported by Lima et al.5 and Audi et al.6,7 Other studies have shown antiprotozoan activity against Leishmania amazonensis, Trypanosoma cruzi and Herpetomonas samuelpessoai.8–10 The bark of S. adstringens is rich in condensed tannins composed of several flavan-3-ols, such as prodelphinidins and prorobinetinidins.11–13
Candida spp. are commensal yeasts found on the mucosal surfaces and epithelial tissues of vertebrates. However, these yeasts often cause opportunistic fungal infections in human patients who have become immunocompromised by anticancer therapy, HIV infection, organ transplantation or therapy with broad-spectrum antibiotics, leading to a severe fungal infection. The physiological and immune condition of the host and the yeasts' versatility in surviving in many anatomical sites are important factors in the transition from commensal to disease-causing yeasts. Many putative virulence factors can contribute to the yeasts' invasiveness and pathogenicity, such as their ability to adhere to tissues, cell surface hydrophobicity (CSH), conversion of unicellular yeasts into filamentous forms and expression of extracellular enzymes such as aspartyl proteinases and phospholipases.14 Several antifungal drugs, such as fluconazole, ketoconazole, nystatin, amphotericin B and 5-fluorocytosine, can interfere with certain virulence factors.15,16 Innate immunity by phagocytic cells is involved in the response to invasion by Candida, and many drugs may be associated with immunomodulatory mechanisms, aiding in antifungal activity.16
Vulvovaginal candidiasis (VVC) is one of the most common vaginal infections. Women in the range of 50–75% suffer symptomatic disease, and 
5% develop recurrent VVC. About 90% of isolates from vaginal fluid are identified as Candida albicans; a small minority of cases are caused by non-albicans species. Many drugs available for the treatment of fungal infections have problems, such as a narrow spectrum of action and toxicity. Polyene agents are highly nephrotoxic and hepatotoxic. However, with the emergence of azole agents, decreasing the toxic effects of polyene agents, many fungal infections can now be treated. Although few strains resistant to antifungal agents have been isolated, VVC continues to be a common problem in immunocompetent or healthy women.17
The discovery of new antifungal agents thus remains an important challenge for the scientific community, and plants may supply promising material for studies on the development of antifungal drugs. The aim of the present study was to evaluate the effects of a crude extract, fractions and subfractions from the bark of S. adstringens on growth, putative virulence factors and ultrastructure of C. albicans isolated from vaginal fluid. In addition, we analysed the influence of ‘barbatimão’ subfraction F2.4 on the phagocytosis of yeast by macrophages.
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Preparation of crude extract, fractions and subfractions from S. adstringens
Stem bark from the plants was collected in São Jerônimo da Serra, Paraná, Brazil, in November 2004. A voucher herbarium specimen was deposited under number HUM 11386 at the Universidade Estadual de Maringá, Paraná, Brazil.
The bark was dried at room temperature and then pulverized. The crude extract was obtained by turbo-extraction (Skymsen) of 100 g of the bark with 70% acetone in water for 20 min. The organic solvent was eliminated by reduced pressure and lyophilized to yield a crude extract (F1). Next, the F1 (36 g) was suspended in water (360 mL) and partitioned with ethyl acetate (360 mL) to obtain a water fraction (F2) and an ethyl acetate fraction (F3). The F2 fraction (2 g) was chromatographed on a Sephadex® LH-20 column (h = 170 mm; 
= 21 mm, Pharmacia), using one sequence of eluent system of volumetric proportions with water (50% ethanol, 70% ethanol, 90% ethanol and 70% acetone), obtaining four subfractions: F2.1 (0.35 g), F2.2 (0.54 g), F2.3 (0.03 g) and F2.4 (1.08 g; 54%).
Chemical characterization of subfraction F2.4 from S. adstringens
Subfraction F2.4 was analysed by ES-MS mass spectrometry, using Quattro equipment (Micromass, Manchester, UK). Solid-state 13C NMR spectroscopy was carried out using an NMR Varian spectrometer, model Mercury plus BB 300 MHz, with a frequency of 75 MHz. The sample was packed into a 7 mm diameter silicon nitrite rotor, retained with Kel-F end caps and spun at 5 kHz. Each proton preparation pulse was 4.9 µs at 33.9°, followed by 50 ms of data acquisition and recovery delay of 10 s. Transients from 5120 such sequences were averaged.
Microorganisms
The yeasts studied included 103 isolates from vaginal fluid (96 Candida albicans, 1 Candida parapsilosis, 1 Candida tropicalis, 4 Candida sp. and 1 unidentified genus) obtained from women with no previous history of immunodeficiency;18 and two standard yeasts (C. albicans ATCC 10231 and C. parapsilosis ATCC 22019). The standards were kindly provided by the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. These isolates were stored as water suspensions at room temperature. For the tests, each strain stored in distilled water was cultured in Sabouraud dextrose agar to obtain isolated colonies, which were then subcultured in Sabouraud dextrose broth.
Cell culture
Murine macrophage J774G8 cells and green monkey kidney cells (Vero) were maintained in RPMI 1640 medium and Dulbecco's modified Eagle's medium (DMEM) (Gibco Invitrogen Corporation, New York, USA), respectively, and supplemented with 2 mM L-glutamine, heat-inactivated 10% fetal bovine serum (FBS) and 50 mg/L gentamicin and buffered with sodium bicarbonate. The cultures were maintained at 37°C in a 5% CO2/air mixture.
Antifungal susceptibility tests
MIC determination.. The MICs of the extracts were determined using the broth microdilution technique, as described by the Clinical and Laboratory Standards Institute in document M27–A2.19
Stock solutions of extracts of S. adstringens were prepared in RPMI 1640 medium without sodium bicarbonate (Sigma Chemical Co., Missouri, USA) buffered with MOPS, pH 7.0. Serial 2-fold dilutions of extracts were made in 96-well microtitre trays to obtain concentrations of 1000 to 0.98 mg/L. The yeast inoculum was adjusted to concentrations of 1–5 x 106 cfu/mL, comparable to the turbidity of a 0.5 McFarland standard, and an aliquot was dispensed into each well. The microtitre trays were incubated at 37°C for 48 h, in a humid dark chamber. Antifungal activity was interpreted by analysing the endpoint concentration of the extracts, which was considered the lowest concentration that resulted in a visually observable inhibition of yeast growth. Fluconazole (Pfizer, São Paulo, Brazil) and nystatin (Sigma Chemical Co., Missouri, USA) were utilized as control drugs.
Minimum fungicidal concentration (MFC) determination.. The contents of each well containing drug concentrations that inhibited growth after 48 h of incubation were transferred onto drug-free Sabouraud dextrose agar plates. The plates were incubated at 37°C for 48 h. The MFC was defined as the lowest concentration yielding negative subcultures.
Effect of subfraction F2.4 from S. adstringens on putative virulence factors
Effect on CSH..
Eight organisms were selected for this test (C. albicans ATCC 10231, C. parapsilosis ATCC 22019 and six isolates: 3 C. albicans, 1 C. parapsilosis, 1 C. tropicalis and 1 Candida sp.). Yeasts treated with 7.80 and 125 mg/L of subfraction F2.4 were washed with 50 mM PBS buffer, pH 7.4, and resuspended in the same buffer. Turbidity was determined in a spectrophotometer (Spectronic 21D—Milton Roy) at 660 nm. Next, the suspended cells were mixed with xylene 2.5:1 (v/v) (Merck) and shaken for 2 min, and the tube was left for 20 min at room temperature in order to obtain separation of the phases. The turbidity of the aqueous phase was read at 660 nm. The hydrophobicity index (HI) was calculated as described by Teixeira et al.20
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where Acontrol = optical density of the strains before xylene treatment and Atest = optical density of the strains after xylene treatment.
Inhibition of adherence capacity of yeast on Vero cells and glass surface.. In order to obtain a confluent monolayer of Vero cells, a volume of 500 µL containing 2.50 x 105 cells was dispensed onto 24-well plates in DMEM with 10% FBS, 2 mM glutamine, 50 IU/mL penicillin and 50 mg/L streptomycin, and incubated at 37°C and 5% CO2 for 24 h. Eight samples of previously selected yeasts were treated with 7.80 and 125 mg/L of subfraction F2.4 for 24 h at 37°C, washed with 10 mM PBS buffer at pH 7.2 and counted in a Neubauer chamber to obtain 1 x 106 cfu/mL. A 500 µL aliquot of the yeast suspension was added to each well with a Vero cell monolayer or on coverslips alone. The interaction of the yeast with the Vero cells or the abiotic surface was carried out for 1 h at 37°C and 5% CO2. The coverslips were washed in 10 mM PBS, pH 7.2, fixed in Bouin's solution overnight, stained with Giemsa (Gibco, Invitrogen Co., New York, USA) for 30 min and mounted on slides with Entellan (Merck). In counting the adhered cells, yeasts with buds were considered to be one cell. In the analysis of adherence of yeasts on the Vero cells, at least 200 cells were counted; and for the abiotic surface, 20–100 fields were counted with a 100x objective.21
Effect on germ-tube formation and budding of C. albicans
Different concentrations (7.80, 31.25 and 125 mg/L) of subfraction F2.4 from S. adstringens, diluted in RPMI 1640 medium, were tested to assess the effect on budding of C. albicans. Cells (0.50–2.50 x 103 cfu/mL) were incubated at 37°C for 48 h and observed by a negative-staining technique using 7% aqueous nigrosin. Three hundred cells were counted in each smear by light microscopy, and the percentage of budding cells was calculated.22
The effect of subfraction F2.4 (1.90–125 mg/L) on germ-tube formation of C. albicans was also investigated. A yeast suspension of 1 x 106 cfu/mL was treated with the subfraction diluted in RPMI 1640 medium and observed by phase-contrast microscopy (Zeiss Axiovert 25) after 2–3 h of incubation at 37°C.23 Nystatin was used as the standard antifungal drug.
Effect on phagocytosis by macrophages
A 500 µL aliquot of the yeast suspension (1 x 106 cfu/mL) previously treated with 7.80 mg/L of subfraction F2.4 for 2 h at 37°C, washed with 10 mM PBS, pH 7.20, was added to each well with a macrophage J774G8 monolayer grown on coverslips with RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 50 IU/mL penicillin and 50 mg/L streptomycin. The yeast–macrophage interaction was carried out for 1 h at 37°C. The coverslips were washed in 10 mM PBS at pH 7.2, fixed with Bouin's solution overnight, stained by Giemsa for 30 min and mounted on slides with Entellan. The yeasts internalized by the macrophages were counted in at least 200 macrophages. The internalization index was calculated by multiplying the percentage of macrophages with internalized yeasts by the average number of yeast cells per macrophage.
Ultrastructural analysis
Transmission electron microscopy.. C. albicans treated with subfraction F2.4 in RPMI 1640 medium was fixed for 2 h at room temperature with 2.50% glutaraldehyde in 0.10 M cacodylate buffer, pH 7.2. Post-fixation was carried out in 1% osmium tetroxide in cacodylate buffer containing 0.80% potassium ferrocyanide and 5 mM CaCl2. Next, the cells were dehydrated in acetone and embedded in epon. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed in a Zeiss CEM-900 electron microscope.
Scanning electron microscopy.. C. albicans treated with subfraction F2.4 were fixed with 2.50% glutaraldehyde for 2 h at room temperature. After fixation, small drops of the sample were placed on a specimen support with poly-L-lysine. Post-fixation was carried out with 1% osmium tetroxide in cacodylate buffer containing 0.80% potassium ferrocyanide and 5 mM CaCl2 for 30 min. Subsequently, the samples were dehydrated in graded ethanol, critical-point dried in CO2, coated with gold and observed in a SHIMADZU SS-550 scanning electron microscope.
Cytotoxicity assay
Viability test.. Aliquots (100 µL) of Vero and macrophage J774G8 cell suspensions at 5 x 104 cells/mL were dispensed onto a 96-well microtitre tray and incubated for 24 h. Next, the monolayers were treated with several concentrations of S. adstringens extracts (1–1000 mg/L) for 48 h at 37°C and 5% CO2. Cytotoxicity was analysed by the sulforhodamine B method.24 The 50% cytotoxic concentration (CC50) and the selectivity index (SI = CC50/MIC) were calculated.
Red blood cells (RBCs) lysis.. Sheep RBCs were washed in 0.85% saline solution, and 5% glucose was added to obtain a 4% RBC suspension. This suspension was treated with several concentrations of S. adstringens extracts (0.10–1000 mg/L) and Triton X114 (as an indicator of complete haemolysis), and incubated at 37°C for 2 h. The RBCs were centrifuged at 2000 g for 10 min, and the supernatant was removed for estimation of haemolysis using a spectrophotometer at 540 nm.9 The results were expressed as the percentage of haemolysis, and the SI was calculated.
Statistical analysis
Statistical analysis was performed by Graph Pad Prism 3.0 (Graph Pad Software Inc., USA), using one-way ANOVA. A Tukey test was used to compare antifungal activity of extracts, and a Dunnet test to compare strains with and without antifungal treatment. A 5% significance level was adopted.
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Chemical characterization of subfraction F2.4 of S. adstringens
Subfraction F2.4 is composed of flavan-3-ol units with an average molecular weight of 2000 Da, suggesting that proanthocyanidin polymers are arranged as 6 U with one or more gallic acid units, confirmed by analysis of ES-MS and 13C NMR spectrum. The 13C NMR spectrum exhibited resonances typical of a polymer containing subunits with 2,3-cis (76 ppm) and 2,3-trans (82 ppm) configuration, the presence of 3',4',5'-trihydroxylated B-rings (102 and 107 ppm) and with or without a hydroxyl group at C-5 (158 ppm), which are characteristic of prodelphinidin and prorobinetinidin units. Also a methoxyl group can occur at C-4' of B-ring (56 ppm) and a gallic acid unit (122, 140, 170 and 177 ppm). Linkages 4
8 and 46 between units cannot be determined by 13C NMR. Therefore, we propose in Figure 1 the chemical structure of a proanthocyanidin polymer of molecular weight 2114 Da, which may compose the subfraction F2.4 from S. adstringens.
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Antifungal susceptibility tests
In vitro susceptibility tests of the extract, fractions and subfractions from S. adstringens were performed with 103 isolates of Candida from vaginal fluid (Table 1). In the partition of the crude extract of ‘barbatimão’, the fractions F2 (aqueous fraction) and F3 (ethyl acetate fraction) were obtained. Fraction F2 was more active than F1, with an inhibitory effect on yeast growth with MICs in the range of 0.97–7.80 mg/L. Fraction F3 showed less antifungal activity than F1 (P < 0.001). Consequently, fraction F2 was selected for the purification process, and four subfractions were obtained (F2.1, F2.2, F2.3 and F2.4). These subfractions were tested on 38 isolates. Subfraction F2.4 showed better antifungal effect than F2.2 and F2.3 (P < 0.001), inhibiting yeast growth from 0.97 to 7.80 mg/L, whereas the subfractions F2.2 and F2.3 inhibited growth from 1.9 to 62.50 and 0.97 to 31.25 mg/L, respectively. Subfraction F2.1 showed no antifungal activity at the concentrations tested. The crude extract, fractions and subfraction F2.4 showed low fungicidal activity against most of the isolates. MFCs for isolates were from 31.25 to 1000 mg/L, with yeast death observed at concentrations from 16 to 128 times higher than the MICs for isolates.
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Susceptibility assays also were done with fluconazole and nystatin on all clinical isolates (Table 1). It was observed that 92.24% of samples were susceptible to fluconazole and only 7.76% were resistant and dose dependent. For nystatin, all samples were susceptible.
Effect on CSH
Exposure of Candida spp. to 7.80 and 125 mg/L of subfraction F2.4 from S. adstringens for 48 h interfered with CSH. In C. albicans ATCC 10231 and LC 352 treated with 125 mg/L of subfraction 2.4, the HI decreased from 68.36% to 14.31% and from 78.53% to 29.96%, respectively (Table 2). In C. parapsilosis ATCC 22019 and LC 144, the HI decreased from 54.13% to 30.91% and from 85.47% to 46.60%. On the other hand, in samples treated with 7.80 mg/L, the observed increase in HI was not significantly different (P > 0.05).
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Effect on adherence of yeast on Vero cells and abiotic surface
The previous treatment of Candida spp. with 7.80 and 125 mg/L of subfraction F2.4 for 48 h inhibited the process of adherence of yeasts on the Vero cell monolayer and the glass surface. The adherence inhibition by subfraction F2.4 was dose dependent (Table 2). The adherent capacity on the Vero cell and the glass surface decreased in samples treated with 7.80 and 125 mg/L. In C. albicans ATCC 10231 treated with 125 mg/L, the yeasts adhered on Vero cell and glass surface decreased from 99.77 to 41.89 yeasts/100 cells and from 94.10 to 14.57 yeasts/field, respectively. The adherent capacity on the glass surface was also observed in C. albicans LC 111 and LC 235 and C. tropicalis LC 299, treated with 125 mg/L of subfraction F2.4.
Effect on germ-tube formation and budding of C. albicans
Concentrations higher than 15.75 mg/L were able to inhibit the formation of the germ tube, whereas only 6.25 mg/L nystatin was necessary to inhibit it. However, in C. albicans treated with subfraction F2.4 for 48 h, budding increased. The budding percentage in 7.80 mg/mL (29.22%) was similar to the control cell (22.02%). However, the difference was statistically significant for 125 mg/L (59.75%) (P < 0.001), with an increase of 188.34% in treated cells.
Effect on phagocytic process
C. albicans treated previously with 7.8 mg/L of subfraction F2.4 increased the phagocytic process, with an internalization index of 55.38 ± 11.05, indicating an increase of 28% in phagocytosis compared with the control group (43.33 ± 1.89).
Ultrastructural alterations
In order to investigate the effect of subfraction F2.4 on the morphology and ultrastructure of C. albicans, scanning and transmission electron microscopy were utilized. In cells treated with subfraction F2.4, both yeast agglutination (Figure 2c and d) and material deposited on the cell walls of the fungi (Figure 2d) were observed. Yeasts treated with subfraction F2.4 underwent pronounced morphological alterations (Figures 2 and 3), low electrodensity and loss of integrity of the cell wall (Figures 2b and 3b, c and d). Figure 3c and d shows the deformed cells. In addition, the cytoplasm did not appear to be homogeneous (Figure 3b and d) and the effect of ‘barbatimão’ on the cell wall changed the cytoplasm contents. Untreated cells had a normal cell wall and cytoplasmic membrane, separated by a low-density space (Figure 3a).
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Cytotoxic effect of S. adstringens
To evaluate the cytotoxic effect of S. adstringens, two experimental models were used. As shown in Table 3, the CC50s of all extracts for Vero cells and macrophage J774G8 cells were above 100 mg/L, except for fraction F1 with a CC50 of 70 mg/L for macrophages. In addition, no haemolytic effect of extracts on sheep RBCs was observed for concentrations below 1000 mg/L. However, a haemagglutinant action could be seen with 500 and 1000 mg/L of ‘barbatimão’ extracts (data not shown). Fraction F2 was more selective for the experimental model used, in comparison with fractions F1 and F3 and subfraction F2.4.
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Many antifungal agents are available to treat superficial and systemic mycosis. The emergence of drug-resistant strains and dose-limiting toxic effects has impeded antifungal treatment. Moreover, immunocompromised and hospitalized patients are more susceptible to severe fungal infections.25 Many investigators have searched for new compounds with some antifungal action in natural products. Some plant extracts have been shown to possess activity against several pathogens and may be a good source of new active agents. Many plants have been screened for activity against a wide array of diseases on the basis of ethnopharmacological data.2,26
Extracts from the bark of S. adstringens have been assessed for various kinds of biological activity and are now known to act against many microorganisms.7–10 In the present study, we investigated the antifungal potential of an extract, fractions and subfractions obtained from S. adstringens on Candida spp. Purification of the crude extract was monitored by determination of MIC, which showed that the antifungal activity of the aqueous fraction was greater than that of the ethyl acetate fraction (P < 0.001). Subfraction F2.4 was obtained from this fraction by Sephadex LH-20 column chromatography and contained compounds of high molecular weight, with even better antifungal activity (MICs from 0.90 to 7.8 mg/L). These data were confirmed by mass spectrometry, which showed the presence of polymers with a molecular weight of 2114 Da. Subfraction F2.4 may consist of 6 U of flavan-3-ols and a galoil group. Solid-state 13C NMR spectra demonstrated that subfraction F2.4 consists of a polymer of prodelphinidin and prorobinetinidin units, with 2,3-cis and 2,3-trans configuration. A methoxyl group may also occur in the polymer. The interpretation of the 13C NMR spectrum was based on Czochanska et al.27 and Newman and Porter.28 The chemical structure of the polymer proposed in Figure 1 is based on Mello et al.11–13
There are many reports that condensed tannins and gallic acid are responsible for different antimicrobial activities.10,29 Santos et al.30 reported that the degree of polymerization is an important factor in biological activity. This has been discussed by Field and Lettinga,31 who demonstrated that an increase in the degree of polymerization progressively increases the degree of reaction to tannins. Previous studies showed that the number of hydroxyl groups in the B-ring affects the level of growth inhibition of several microorganisms, suggesting that proanthocyanidin trihydroxylated B-rings may have better antimicrobial action.32 The high degree of polymerization and hydroxylation of the condensed tannins from subfraction F2.4 appears to be an important factor in its antifungal activity against Candida spp. Scalbert proposed different mechanisms to explain tannin antimicrobial activity. These include (i) inhibition of extracellular microbial enzymes; (ii) deprivation of substrates and metal ions required for microbial growth and (iii) direct action on microbial metabolism through inhibition of oxidative phosphorylation.32 In addition, Haslam proposed that tannins are able to complex with other molecules, including macromolecules such as proteins and polysaccharides.33
In comparison with fluconazole and nystatin, subfraction F2.4 showed good antifungal activity (Table 1). About 92% of the Candida spp. isolates were more susceptible to fluconazole than to subfraction F2.4. However, samples resistant (>64 mg/L) or dose-dependent susceptible (16–32 mg/L) to fluconazole were susceptible to subfraction F2.4, with MICs of 7.80–15.75 and 1.90–7.80 mg/L, respectively. In addition, nystatin was no more active on the clinical isolates than was subfraction F2.4. We also evaluated the fungicidal effect of ‘barbatimão’ extracts by observing yeast death at concentrations 16–128 times higher than MICs for isolates. Despite their fungistatic action, the ‘barbatimão’ extracts showed satisfactory antifungal activity in relation to the standard drugs tested.
The hydrophobicity assay revealed that S. adstringens subfraction F2.4 at 125 mg/L significantly decreased the CSH for some of the isolated yeasts (C. albicans ATCC 10231, C. parapsilosis ATCC 22019, LC 144 and LC 352). In addition, yeasts previously treated with subfraction F2.4 were less able to adhere to Vero cells and to a glass surface (Table 2). Positive correlations were detected in the adhesion to glass surfaces, with the adhesion to Vero cells and CSH. Several investigators have suggested that CSH is involved in adherence to epithelial cells and is associated with the pathogenic potential.34 In contrast, other studies have shown that CSH has little effect on adherence.35 Many antifungal agents such as amphotericin B, nystatin, ketoconazole, fluconazole and 5-fluorocytosine decrease adherence to buccal epithelial cells, but do not interfere with the CSH of the yeasts.15
Morphogenesis, the transition of unicellular yeast cells to the filamentous form, is an attribute of Candida species such as C. albicans and C. dubliniensis. The presence of the filamentous form and budding is associated with virulence and pathogenicity, but both forms may be involved in the development and progress of disease.14 Several antifungal drugs and plant extracts can inhibit germ-tube formation and budding of yeast cells.16,22,23 The ‘barbatimão’ subfraction F2.4 inhibited morphogenesis at concentrations above 15.62 mg/L. However, at a concentration of 125 mg/L there was a significant increase in budding yeast cells compared with the control, although growth was inhibited by at least 1.5 log. Based on these results, we can suggest that the condensed tannins of subfraction F2.4 act on the separation process of buds from yeast cells (Figure 3c and d).
Many antifungal drugs and plant extracts can affect the morphology and ultrastructure of yeasts.22 Recent studies have demonstrated the effects of tannins from S. adstringens on the ultrastructure of protozoans. Maza et al.8 observed significant morphological changes in L. amazonensis, such as the appearance of two flagella and two nuclei, which suggests interference in cell division. Moreover, Holetz et al.10 observed in H. samuelpessoai, evident vesiculation of the Golgi complex, marked mitochondrial swelling and a reduction in activity of the enzyme succinate cytochrome c reductase, interfering with the energy metabolism of the cell. In C. albicans treated with ‘barbatimão’ subfraction F2.4, the cell wall showed loss of integrity and low electrodensity (Figure 3). In consequence, it could induce modifications of plasmalemma permeability and changes in cytoplasm contents, as shown by the difference in electrodensity of treated cells compared with the controls (Figure 3c and d).
In this study, C. albicans exposed at the MIC (7.80 mg/L) of subfraction F2.4 from S. adstringens for 2 h increased the internalization of yeasts by macrophages by 28%. Similar results were obtained when C. albicans was pre-treated with fluconazole at 0.75x MIC for 26 h before interaction, with an increase in phagocytosis. The exposure of yeast to subinhibitory concentrations of fluconazole changed the CSH, which was correlated with structural changes in the yeast cell wall.16
Previous studies have shown that CSH, which is determined by the cell wall, correlates with germ-tube formation, adhesion to epithelial cells and extracellular matrix proteins, cell surface fibril organization and phagocytosis.36 Drugs such as fluconazole can interfere in germ-tube formation, adherence to buccal cells and interactions with phagocytic cells.15,16 These studies suggested that stimulus of phagocytosis and killing of C. albicans by fluconazole is associated with alterations in the yeast cell wall and plasmalemma. The modifications in the cell wall of C. albicans by subfraction F2.4 may aid in the process of phagocytosis, stimulating internalization of yeasts.
In terms of the cytotoxic effect of the ‘barbatimão’ extract, fractions and subfraction F2.4 on Vero cells and macrophages, CC50s > 100 mg/L were observed, except for F1 on macrophages, with a CC50 of 70 mg/L. Neither the crude extract, fractions nor subfraction F2.4 affected RBC integrity at concentrations 
1000 mg/L. Fraction F2 had a higher SI than did the other extracts (Table 3). In contrast, polyene agents were revealed as largely haemolytic in comparison with the crude extract of S. adstringens.9 Probably this selective action of subfraction F2.4 occurs because of the action of the tannins on the cell wall of Candida spp.
Our results suggest that ‘barbatimão’ subfraction F2.4 inhibits C. albicans growth by affecting the integrity of the yeast cell wall, which is related to the change in CSH, decreased capacity to adhere to eukaryote cells and glass surfaces, inhibition of germ-tube formation, effect on the budding process and stimulus of phagocytosis by macrophages. These potent properties of condensed tannins from ‘barbatimão’ on the growth of C. albicans, putative virulence factors and their low cytotoxicity justify further studies to investigate the mechanisms of action and the possible development of a new antifungal agent.
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None to declare.
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We are grateful to Dr Heinrich Luftmann of the University of Münster, Germany, for critical analysis of the mass spectroscopy. This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP) and Programa de Pós-Graduação em Ciências Farmacêuticas da Universidade Estadual de Maringá. K. I. is supported by a fellowship from CAPES, Brazil.
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