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Antifungal activity of extracts and isolated compounds from Buchenavia tomentosa on Candida albicans and non-albicans Guilherme R Teodoro1, Fernanda L Brighenti3, Alberto C Botazzo Delbem4, Ádina Cléia B Delbem4, Sonia Khouri5, Aline Vidal L Gontijo2,6, Aislan CRF Pascoal6, Marcos J Salvador6 & Cristiane Y Koga-Ito*,1,2

Abstract Aim: This study aimed to evaluate the antifungal activity of Buchenavia tomentosa extract and bioactive compounds on six Candida species. Materials & methods: The antimicrobial activity of extract was evaluated using standard strains and clinical isolates. Cytotoxicity was tested in order to evaluate cell damage caused by the extract. Extract was chemically characterized and the antifungal activity of its compounds was evaluated. Results: Extract showed antifungal activity on Candida species. Candida non-albicans were more susceptible than Candida albicans. Low cytotoxicity for extract was observed. The isolated compounds presented antifungal activity at least against one Candida spp. and all compounds presented antifungal effect on Candida glabrata. Conclusion: Extracts from Buchenavia tomentosa showed promising antifungal activity on Candida species with low cytotoxicity. Gallic acid, corilagin and ellagic acid showed promising inhibitory activity on Candida glabrata.

Candida spp. are considered opportunistic pathogens and affect mainly immunocompromised individuals, such as HIV-positive patients, cancer patients undergoing chemotherapy, individuals submitted to transplantation using immunosuppressive drugs and diabetic patients [1,2] . Fungal infections have substantially increased in number and severity in recent decades and Candida spp. are cited as the main fungal pathogens causing nosocomial infections [3,4] . Epidemiological data showed that Candida spp. are the 4th agent responsible for nosocomial systemic infections and the mortality rate is 40% among patients with Candida infections [5] . The statistic has shown a gradual increase of deaths caused by opportunistic fungi (from 1980 to 1997 there was a fourfold increase), and Candida spp. are considered the majority of these agents (over 30%) [5] . The Candida genus presents approximately 200 species of fungi with distinguished morphological, biochemical and genetic characteristics. Among these 200 species, only approximately 20 are related to infections; the most common being Candida albicans [6] . However, the number of patients infected by Candida non-albicans, such as Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida dubliniensis, Candida krusei and Candida dubliniensis, is currently increasing [7–9] .

Keywords 

• antifungal agents • Candida albicans • Candida non-albicans • natural

compounds

1 Oral Biopathology Graduate Program, São José dos Campos Institute of Science & Technology, Universidade Estadual Paulista/UNESP, São Paulo, Brazil 2 Department of Environmental Engineering, São José dos Campos Institute of Science & Technology, Universidade Estadual Paulista/UNESP, São Paulo, Brazil 3 Department of Orthodontics & Pediatric Dentistry, Araraquara Dental School, Universidade Estadual Paulista (UNESP), São Paulo, Brazil 4 Department of Pediatric Dentistry, Araçatuba Dental School, Universidade Estadual Paulista (UNESP), São Paulo, Brazil 5 Faculdade de Ciências da Saúde, Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, São Paulo, Brazil 6 Department of Plant Biology, PPGBTPB, Institute of Biology, Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil *Author for correspondence: Tel.: +55 123 947 9000; [email protected]

10.2217/FMB.15.20 © 2015 Future Medicine Ltd

Future Microbiol. (2015) 10(6), 917–927

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ISSN 1746-0913

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Research Article  Teodoro, Brighenti, Delbem et al. The number of available drugs for the treatment of infections by Candida spp. is limited and the antifungal resistance of Candida species has increased in recent decades [10,11] . There is an urgent need for new therapeutic alternatives for fungal infections. Therefore, the discovery of natural compounds with antifungal potential could be promising. Many plant species of the Combretaceae family have been used as medicinal plants around the world, especially in Africa and Asia [12] . Plants of the Combretaceae family occur in tropical and subtropical regions and include 20 genera [13] . The genus Buchenavia, belonging to the Combretaceae family, presents promising biological activities  [14] . A recent study reported that Buchenavia tomentosa presents antimicrobial activity [15] , nonetheless data on antifungal activity of genus Buchenavia, including Buchenavia tomentosa, are scarce. The widespread popular use of plants of this family raises the interest on systematic scientific studies. Thus, the first aim of this study was to evaluate the antifungal activity of extracts from Buchenavia tomentosa against Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei and Candida dubliniensis. The chemical characterization of the most effective extract was then performed to obtain a qualitative chemical profile of Buchenavia tomentosa extract and to select compounds previously identified in plants of the same family or genus and related with antifungal activity. Finally, the antifungal effect of these selected standard compounds was evaluated on these six species of Candida spp. Materials & methods ●●Plant material

The leaves of Buchenavia tomentosa were collected in the city of Corumbá, state of Mato Grosso do Sul (19°0′42.95″ south; 57°40′22.99″ west). The plant was identified by Antônio Arantes B Sobrinho and voucher specimen is deposited at the herbarium of the Campinas State University. ●●Preparation of extracts

The leaves were dried in an oven at 38°C and powdered in a knife mill [16] . The proportion of 20 g of powder to 400 ml (concentration of 50 mg ml-1) of solvent (water or ethanol) was used for all extracts. Extracts were prepared using five different obtaining protocols. The aqueous extracts were obtained by decoction in deionized water for 5 min at 100°C (extract B),

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for 1 h at 55°C (extract C) or for 72 h at 25°C (extract D) [17] . Ethanolic extracts were macerated for 72 h at 25°C, filtered in qualitative paper (Whatman Filter Paper, Grade 1, from Sigma-Aldrich, MO, USA) and concentrated in a rotary evaporator (Heidolph 03600250 Hei-Vap Advantage Rotary Evaporator, USA) under reduced pressure and temperature of 60 to 70°C. Aqueous and ethanolic extracts were sterilized by filtering in 0.22 μm cellulose ester membranes and lyophilized. The yield of extract was calculated for all used protocols. ●●Agar diffusion test

The screening of extracts was performed by agar diffusion test reference strains (Candida albicans ATCC 18804, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019, Candida glabrata ATCC 90030, Candida krusei ATCC 6258 and Candida dubliniensis NCPF 3108). Double agar layer-well technique was adopted for the tests [18] . Aliquots of 10 ml of Roswell Park Memorial Institute (RPMI) agar (RPMI 1640, without sodium bicarbonate, with L-alanine; 2% glucose; buffered at pH 6.5 with MOPS, and 3% agar) formed the base layer. Standardized suspensions containing 106 cells ml-1 were obtained by spectrophotometry. The parameters of optical density (OD) and wavelengths were previous established: Candida albicans: OD = 0.380, λ = 550 nm; Candida parapsilosis: OD = 0.380, λ = 530 nm and OD = 0.280, λ = 530 nm for the other species. The ‘seed’ layer was obtained by adding 100 μl of inoculum in 10 ml of RPMI agar and, after homogenization, the medium was poured on the layer base. The final volume (base layer + seed layer) in the dish was 20 ml. After complete solidification of the medium, standardized orifices with approximate diameter of 5 mm were fabricated in the agar. Then the wells received 50 μl of the extracts resuspended in the culture medium RPMI 1640 (pH 6.5) in a concentration of 50 mg ml-1. Sterile distilled water was used as negative control and amphotericin B at a concentration of 2 μg ml-1 was used as positive control. After 1 h, the dishes were placed in an oven at 37°C for 24 h to allow diffusion of extracts before incubation. After the incubation period, the inhibition halos were measured. The extracts that produced halos equal to or greater than 7 mm were selected for the MIC tests, as previously proposed [19] . The tests were performed in duplicate.

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Anticandidal activity of Buchenavia tomentosa extract  ●●Antifungal activity against standard

strains & clinical isolates: determination of the MIC & minimum fungicidal concentration of the extract

The MIC and the minimum fungicidal concentration (MFC) values were obtained for the same reference strains described above. In addition to the standard samples, the study also analyzed 35 nosocomial samples of Candida albicans, 19 of Candida tropicalis, 20 of Candida parapsilosis, eight of Candida glabrata and Candida krusei, supplied by the Institute of Biomedical Sciences of the University of São Paulo after approval by the Institutional Ethical Review Board under number 702/CEP. The study also included five strains of Candida dubliniensis isolated from oropharyngeal candidiasis of hospitalized patients, supplied by Prof José Pontón, of the University of the Basque Country (Spain). The study also used 30 isolates of Candida albicans, five of Candida glabrata and three of Candida tropicalis obtained from denture stomatitis lesions, isolated in a previous study (Institutional Ethical Review Board approval number 070/2006-PH/CEP). All samples were stored in brain heart infusion (BHI) and glycerol at 20%, frozen at -20°C. Prior the experiments, microorganisms were plated in Sabouraud dextrose agar. After 24 h of incubation at 37°C, standardized suspensions were prepared in 0.9% sterile saline solution at a concentration of 106 cells ml-1 with the aid of a spectrophotometer following the same OD parameters cited above. The broth microdilution technique was used following the M27-A3 document by Clinical and Laboratory Standards Institute (CLSI) (formerly National Committee on Clinical Laboratory Standards, NCCLS) [20] with slight adaptations, in which 100 μl of Buchenavia tomentosa extract resuspended in the culture medium RPMI 1640 (pH 6.5) in a concentration of 50 mg ml-1 were placed in column 1 in a 96-well plate. Serial dilutions in ratio 2 of the extract were obtained in mg ml-1 ranging from 25 to 0.2. Then, 50 μl of the inoculum were pipetted in the wells. The MIC was analyzed after 24 h of incubation. This evaluation was performed by plating aliquots of the dilutions and of the control in BHI agar (50 μl of RPMI and 50 μl of the inoculum were pipetted in the last column). After incubation for 24 h at 37°C, the minimum concentration of the extract able to inhibit fungal growth (MIC) or eliminate the cells (MFC) were determined by visual

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comparison with control (without antifungal agents). Amphotericin B at 2 μg ml-1 was used as positive control. The minimal and maximal obtained values of MICs (intervals of MICs) were presented for all clinical isolates. The concentration required to inhibit the growth in the 50 and 90% of samples (MIC50 and MIC90, respectively) were showed only for clinical species presenting more than ten samples, as previous described [21] . All experiments were performed at least in triplicate. ●●Cytotoxicity assay

The 3T3 fibroblast cells were grown in RPMI 1640 medium supplemented with 100 U ml-1 of penicillin, 100 μg ml-1 of streptomycin and 5% of inactivated fetal calf serum, and maintained at 37°C in 5% CO2. A cell suspension was seeded at a concentration of 106 cells ml-1 in a 96-well microplate containing RPMI 1640 medium. Thereafter, the cells were treated with the extract solubilized in RPMI 1640 at different concentrations (25.0 to 1.56 mg ml-1). The plates were incubated at 37°C for 24 h, and the bioactivity was evaluated using the MTT colorimetric method [22] , in a microplate reader at 570 nm. Nontreated cells and RPMI 1640 medium were used as negative control. Doxorubicin at 50 μg/ml was used as the positive control. All the experiments were performed in triplicate. The percentage of mitochondrial activity was calculated considering the negative control as 100%. ●●Chemical characterization of bioactive

extract by electrospray ionization-mass spectrometry

The fingerprinting electrospray ionization-mass spectrometry (ESI-MS) analyses were performed using UPLC-MS equipment, model ACQUITY TQD (Waters Corporation, MA, USA). The general conditions were: source temperature of 100oC, capillary voltage of 3.0 kV and cone voltage of 30 V. ESI-MS was performed by direct infusion using a syringe pump, with a flow rate of 10 μl min/ml analysis was performed in positive and negative ion modes. For that purpose, 1 mg of the samples was solubilized in 1 ml of methanol (S1). Then, 100 μl of S1 were diluted in 900 μl of a methanol/water solution (1:1) added to 0.1% of ammonium hydroxide (NH4OH), yielding the first dilution (D1). Following, 100 μl of D1 were diluted in 900 μl of a methanol/water solution (1:1) added to 0.1%

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Research Article  Teodoro, Brighenti, Delbem et al. of NH4OH. Structural analysis of single ions in the mass spectra from the extract was performed by ESI-MS/MS. The ion with the m/z of interest was selected and submitted to 15–45 electron volts collisions with argon. The collision gas pressure was optimized to produce extensive fragmentation of the ion under investigation [23] . The constituents were identified by comparing their ESI-MS/MS fragmentation spectra to those in the literature [24–33] . ●●Antifungal activity of standard

compounds present in the extract of Buchenavia tomentosa

After the chemical characterization of the extracts by ESI-MS, the antifungal activity on the same Candida reference strains of compounds present in extract B was assessed. The MIC and MFC values were obtained for the standard compounds such as gallic acid, kaempferol, (-)epicatequin, ellagic acid, vitexin and corilagin using broth microdilution test as described above. All standard compounds were supplied by Santa Cruz Biotechnologies,

Inc. (TX, USA), apart from (-)epicatechin and ellagic acid, which were provided by Cayman Chemical (MI, USA). Serial dilutions in DMSO 5% and RPMI were obtained ranging from 10 to 0.004 mg ml-1. Due to alterations in the RPMI color into a dark solution observed in the pilot tests with gallic acid, the culture medium for the tests with this compound was substituted by Müeller Hinton. The amount of growth in the wells containing the antimicrobial agent was compared visually with the growth in the controls (without antimicrobial agent). Amphotericin B at 2 μg ml-1 was used as positive control. Results ●●Yield of extracts

Five extracts from leaves of Buchenavia tomentosa were obtained. The yield for extracts A, B, C, D and E was 22, 63, 52, 39 and 14%, respectively. The highest yield was observed for extract B (63%), and the lowest for the extract E (14%). The time for preparation of the extract B was the shortest (5 min). Candida albicans ATCC 18804† Candida tropicalis ATCC 13803† Candida parapsilosis ATCC 22019† Candida glabrata ATCC 90030† Candida krusei ATCC 6258‡

25

Candida dubliniensis NCFP 3108†

Diameter of halo (mm)

20

15

10

5

0 A

B

C Extracts

D

E

Figure 1. Result of the inhibition test of the extract from Buchenavia tomentosa. Value of the inhibition zone corresponding to the halo (diameter) of inhibition of microbial growth measured in millimeters. † Presence of microcolonies in the halos. ‡ Halo without microcolonies.

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Anticandidal activity of Buchenavia tomentosa extract 

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Table 1. Values of MIC and minimum fungicidal concentration (mg ml-1) of extract B from Buchenavia tomentosa observed for standard strains of Candida spp. Microorganism  

Origin  

Extract B MIC (mg ml ) 

MFC (mg ml-1) 

Candida albicans Candida tropicalis Candida parapsilosis Candida glabrata Candida krusei Candida dubliniensis

ATCC 18804 ATCC 13803 ATCC 22019 ATCC 90030 ATCC 6258 NCPF 3108

12.5 12.5 6.25 0.20 0.78 0.20

–† – –

-1

6.25 –

Tests were performed at least in triplicate.  † Indicates no effect detected. MFC: Minimum fungicidal concentration. ●●Screening of extract using agar

diffusion test

aqueous extracts B of Buchenavia tomentosa leaves at concentration the 25.0 mg ml-1 was observed (cell viability >60 ± 5%).

The results of this screening (Figure 1) demonstrated that using a concentration of 50 mg ml-1 the ethanolic (A and E) and aqueous extracts (B, C and D) formed inhibition halos whose diameter was greater than 7 mm for all standard strains investigated, except for extracts A and E, which did not present any effect against Candida tropicalis, and for that reason were excluded from the further investigations. Yet since the results were similar between the three extracts (Figure 1) , and considering the highest yield and the shortest time of preparation, only the extract B was selected for further analysis.

The ESI-MS/MS analyses (Figure 2 & Table 3) showed the following constituents in the extracts of Buchenavia tomentosa: gallic acid, quinic acid, kaempferol, (-)epicatechin, ellagic acid, buchenavianine, eschweilenol b, eschweilenol c, vitexin, corilagin, 1α,23β-dihydroxy-12-oleanen-29-oicacid-23β-o-α-l-4-acetylramnopiranoside and punicalin. All the analyses were performed in negative-ion mode, apart from buchenavianine, which was performed in positive-ion mode.

●●Antifungal activity on reference strains

●●Antifungal activity of the compounds of

●●Chemical characterization

& clinical isolates

extract

Fungistatic effect (MIC) was observed for most of the standard strains (Table 1) , with MIC values ranging from 0.2 to 12.5 mg ml-1. Fungicidal activity (MFC) was observed only for Candida krusei, with MFC value equal to 6.25 mg ml-1. The lowest MIC values were showed for Candida glabrata and Candida dubliniensis strains (0.2 mg ml-1 for both). MIC values for Candida albicans and Candida tropicalis were the highest (12.5 mg ml-1 for both). Table 2 shows the values of MIC of extract B from Buchenavia tomentosa evaluated for clinical isolates of Candida spp. The highest value of MIC was observed for Candida albicans (25 mg ml-1); while one of the lowest values of MIC was observed for Candida glabrata (0.2 mg ml-1).

Six compounds detected in the extracts (gallic acid, kaempferol, (-)epicatechin, ellagic acid, vitexin and corilagin) were used in their isolated form to evaluate antifungal activity (Table 4) . Ellagic acid showed antifungal effect on all the studied species, with MIC values ranging between 0.004 and 1 mg ml-1. Candida glabrata was susceptible to all six compounds. Candida krusei and Candida tropicalis were susceptible to almost all standard compounds, except for vitexin. Fungicide effect was observed for ellagic acid and vitexin on Candida glabrata (with MFC at concentrations of 0.25 and 1 mg ml-1, respectively).

●●Evaluation of the cytotoxicity of the

bioactive extracts

In order to evaluate the cytotoxicity of the bioactive extracts B, MTT assays on 3T3 fibroblast cells were performed. Low cytotoxicity of the

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Discussion This study demonstrated the antifungal effect of the extract from Buchenavia tomentosa against all Candida strains analyzed. The isolation of the bioactive compounds is also an interesting possibility and could be performed in future work. Nonetheless, in this present study we have

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Research Article  Teodoro, Brighenti, Delbem et al. Table 2. Minimal and maximal values of MICs (mg ml-1), MIC50 and MIC90 (mg ml-1) of extract B from Buchenavia tomentosa observed for clinical isolates of Candida spp. Microorganism 

Samples (n)  Origin 

Extract B Intervals of MICs (mg ml ) 

MIC50 (mg ml-1)

MIC90 (mg ml-1)

1.26–25 0.78–12.5 1.56–12.5 3.12 0.78–6.25 0.20–1.56 0.20–12.5 1.56 0.39–6.25

12.5 6.25 3.12 NA NA NA 3.12 NA NA

25 12.5 6.25 NA NA NA 6.25 NA NA

-1

Candida albicans Candida albicans Candida tropicalis Candida tropicalis Candida glabrata Candida glabrata Candida parapsilosis Candida krusei Candida dublininesis

34 30 19 3 8 5 20 1 5

Nosocomial Eryth. Cand.† Nosocomial Eryth. Cand.† Nosocomial Eryth. Cand.† Nosocomial Nosocomial Oropharyngeal candidiasis

The concentration required to inhibit the growth in the 50 and 90% of samples (MIC50 and MIC90, respectively) was showed only for clinical species presenting more than ten samples accordingly to Schwarz et al. [21]. Tests were performed at least in triplicate. † Eryth. Cand.: Erythematous oral candidiasis. NA: Not available.

precisely decided to use the extract of plants, since it is used by population in regions of tropical and subtropical climates as an alternative therapy. For this reason, it is also important to investigate the pharmaceutical properties of the extract and its toxicity. For instance, the antimicrobial activity of several extracts from different plants have been investigated in previous studies  [34–37] , attesting thus the scientific interest of these natural products for the treatment of infections. Our study is important because it confirmed an antifungal property of Buchenavia tomentosa, which is a plant used in traditional medicine by populations from tropical and subtropical regions for the treatment of candidosis. Additionally, we also worked with some commercially available compounds of the extract that are related in the literature with antifungal activity. Interestingly, some clinical isolates of Candida used in our study showed resistance to conventional drugs [38] . For instance, among 19 nosocomial isolates of Candida tropicalis, two presented resistance to fluconazole and another two showed resistance to amphotericin B. Furthermore, two and three among 20 nosocomial isolates of Candida parapsilosis presented resistance to amphoterecin B and fluconazole, respectively. Finally, 50% of eight nosocomial isolates of Candida tropicalis showed resistance to fluconazole and itraconazole. One should remind that the extract B showed a significant antifungal activity against all these clinical isolates (Figure 1) . Further studies should be performed to attest the enhanced effect of extract B compared with conventional antifungal drugs.

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Moreover, low cytotoxicity (>60 ± 5% of cell viability) was observed for extract B at 25.0 mg ml-1, the higher concentration of MIC found in this study. By comparison, an in vitro cytotoxicity test [39] showed a cell viability of only 20% for amphotericin B at 20.0 μg ml-1. The ESI-MS analysis showed important bioactive molecules in Buchenavia tomentosa aqueous extract, some of which were previously shown to present activity against Candida species. Examples include gallic acid [40–42] , kaempferol  [43,44] , (-)epicatechin [45] , ellagic acid [41,42,46–47] , vitexin [44] and corilagin [41,48] . The antifungal activity on Candida species of these compounds was investigated. The most noticeable result was that all tested compounds showed antifungal effect against Candida glabrata, with significantly low MIC values (between 0.004 and 0.25 mg ml-1). Moreover, a promising fungicide effect was observed for ellagic acid and vitexin on Candida glabrata, and this finding should be further investigated in future studies. This fungus is the second most frequently yeast isolated from normal flora of humans [49] . It has recently become an emerging agent of nosocomial infections and yet little is known about its epidemiology [50] . This yeast is particularly important because it has high intrinsic resistance to certain antifungal agents, especially azoles [51] . Studies have shown resistance cases for azoles (fluconazole, voriconazole, itraconazole and pozaconazol) as well as for echinocandins anidulafungin, caspofungin and micofungina [4,52–54] . Gallic acid is considered a promising antifungal agent in the literature [40] . Its mechanism of action is not fully understood, but it has

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Anticandidal activity of Buchenavia tomentosa extract  been proposed that it may act by a disruption of fungi membrane preventing the development of cells [40] . The antifungal effect of gallic acid on Candida albicans and non-albicans was found in previous studies [40–42] . MIC values for gallic acid reported in the literature varied according to the species. They ranged from 0.0025 to 16 mg ml-1 to Candida albicans and from 0.1 to 4 mg ml-1 to Candida parapsilosis and Candida krusei [40–42,55] . The MIC value obtained in this

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study was 10 mg ml-1, similar to that observed by Govindarajan et al. [42] . The presence of the kaempferol in this extract may be related to its Candida non-albicans activity. In this study, kaempferol showed activity against Candida krusei and Candida glabrata, and these findings are in accordance with Salvador et al. [44] . Although this previous study did not report activity on Candida tropicalis  [44] , in our study the concentration of 1 mg ml-1 showed inhibitory

A 207

100

165

Relative intensity (%)

207

(2) 191 165 153

541 542

117

(4) (5)

(1)

120

0 100

171

150

B 100

200

223

541 541

269 301 268 325

208

169

(3)

228 267 205 236 237

250

542 542 577 (7) (8) 431 447 577 327 577 367 423 583 305 447 484 499 (10) (11) 538 543 343 361 675 701 712 610 619 416 466 636 655

300

(9)

350

400

481

450

500

455

503

(12) 781 782 784

840 866 883 901 915

976

550 600 m/z

650

700

750

800

850

900

950

1000

550 600 m/z

650

700

750

800

850

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950

1000

Relative intensity (%)

140

165 130 104 110

0 100

(6) 368

147

366

167

150

181 205

247

200

250

425

291 307

300

350

400

427

450

500

Figure 2. ESI-MS spectra of aqueous extract from Buchenavia tomentosa. ESI-MS fingerprints of aqueous extract B from Buchenavia tomentosa in negative (A) and in positive (B) ion mode. (1) gallic acid; (2) quinic acid; (3) kaempferol; (4) (-)epicatechin; (5) ellagic acid; (6) buchenavianine; (7) eschweilenol B; (8) vitexin; (9) eschweilenol C; (10) corilagin (11) 1α,23β-dihydroxy-12-oleanen-29-oic-acid-23β-O-αL-4-acetylramnopiranoside and (12) punicalin.

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Research Article  Teodoro, Brighenti, Delbem et al. Table 3. Compounds identified in water extracts B and C of Buchenavia tomentosa by ESI-MS analysis. MW (u) 

[M+H]+ (m/z)

[M-H]- (m/z)

Compound

MF

170 192 286 290 302 365 424 432 448 636 676

          366          

169 191 285 289 301   423 431 447 635 675

C7H6O5 C7H12O6 C15H10O6 C15H14O6 C14H6O8 C22H23NO11 C20H8O11 C21H20O10 C20H16O12 C27H24O18 C38H60O10

782

 

781

Gallic acid Quinic acid Kaempferol (-)Epicatechin Ellagic acid Buchenavianine Eschweilenol B Vitexin Eschweilenol C Corilagin 1α,23β-dihydroxy-12-oleanen-29-oic-acid-23βO-α-L-4-acetylramnopiranoside Punicalin

C34H22O22

[M-H]-: Negative ion mode analysis; [M+H]+: Positive ion mode analysis; MF: Molecular formula; MW: Molecular weight.

effect against this species, corroborating with Araújo et al. [43] , which also observed effectiveness of kaempferol (1 mg ml-1) on Candida tropicalis. The effect of the (-)epicatechin against Candida albicans was not observed in this study as well as previously described [56] . Nonetheless, Hirasawa and Takada [45] observed effect against Candida albicans. Although previous studies evaluated the effect of its epimer, the cathequin, on non-albicans species [40,55,57] , as far as we know, our study reported for the first time the antifungal effect of (-)epicatequin on Candida krusei, Candida glabrata and Candida tropicalis. The antifungal activity against Candida yeasts of ellagic acid has been reported in the literature  [41,42,46,47] . In this study, ellagic acid showed inhibitory effect on all the studied species with promising results. The f lavonoid vitexin had good activity against Candida glabrata, with fungicidal effect at a concentration of 1 mg ml-1. No effect was

observed for Candida albicans, Candida tropicalis and Candida krusei, and these results are in accordance to Salvador et al.  [44] . However, the same study [44] showed effect on Candida parapsilosis, which was not detected in this study. The corilagin had antifungal action demonstrated against all non-albicans (with MIC ranging between 0.004 and 0.5 mg ml-1), which was comparable with a previous study [41] that has shown activity against Candida krusei and Candida parapsilosis, both at MIC of 1 mg ml-1. No activity was observed for Candida albicans, despite a previous study [41] showing effect against this species with a MIC of 1 mg ml-1. To the best of our knowledge, no previous reports were found considering the antifungal activity of crude extracts from any genus of the family Combretaceae, including the genus Buchenavia, against non-albicans Candida species. Non-albicans species are frequently related to antifungal resistance occurrence [4] .

Table 4. Values of MIC and minimum fungicidal concentration (mg ml-1) of the compounds identified in extracts B by ESI-MS. Compounds   

Species (MIC and MFC [mg ml-1])  Ca

Gallic acid Kaempferol (-)Epicatequin Ellagic acid Vitexin Corilagin

Ck

Cg

Cp

Cd

Ct

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

10 – – 1 – –

–† – – – – –

10 1 0.125 0.125 – 0.08

– – – – – –

0.008 0.125 0.031 0.004 0.25 0.004

– – – 0.25 1 –

10 – – 1 – 0.5

– – – – – –

10 – – 1 – 0.5

– – – – – –

>10 1 0.5 1 0.125

– – – – – –

No effect detected. Ca: Candida albicans ATCC 18804; Cd: Candida dubliniensis NCPF 3108; Cg: Candida glabrata ATCC 90030; Ck: Candida krusei ATCC 6258; Cp: Candida parapsilosis ATCC 22019; Ct: Candida tropicalis ATCC 13803; MFC: Minimum fungicidal concentration. †

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Future Microbiol. (2015) 10(6)

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Anticandidal activity of Buchenavia tomentosa extract  Buchenavia tomentosa aqueous extracts showed antifungal activity against all reference strains of Candida species. Candida glabrata, Candida krusei, Candida dubliniensis and Candida parapsilosis were the most susceptible to the tested extracts, while the most resistant strains were Candida albicans and Candida tropicalis. Conclusion The aqueous extract from Buchenavia tomentosa showed a promising antifungal activity with MIC values ranging from 0.2 to 25 mg ml-1. Moreover, it presented low cytotoxicity, with more than 60% of cell viability after an exposure of 25 mg ml-1 to the extract. Its chemical characterization showed some compounds which had previously presented effect on Candida species; such as gallic acid, kaempferol, (-)epicatechin, ellagic acid, vitexin and corilagin. These isolated molecules showed an antifungal effect against at least one species of Candida. All isolated compounds showed an effect on Candida glabrata, with significant results for gallic acid, ellagic acid and corilagin, which presented fungistatic activity in a low concentration. We conclude that bioactive natural products and compounds could be a good alternative to conventional antifungal drugs for the treatment of infections caused by Candida spp., especially by non-albicans species. Future perspective The treatment of infections caused by Candida spp. is a health problem, due to the emergence

Research Article

of resistance and the limited number of available antifungal drugs. Alternatives to the conventional therapy are currently requested, as for example the use of natural products, which have shown antifungal effect. We have demonstrated a promising antifungal effect of extract and isolated compounds from plants of tropical and subtropical regions, therefore our study could contribute in the next years to overcome the problem of fungal resistance. In vivo studies should be performed to confirm the beneficial effect of these natural products and compounds against Candida species. Financial & competing interests disclosure The authors are grateful to CAPES for GR Teodoro fellowship and to FAPESP for financial support. AVL Gontijo is supported by FAPESP. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary ●●

This work evaluated the antifungal activity of extract and compounds from Buchenavia tomentosa against six Candida species.

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Standard strains and clinical isolates were tested.

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MIC and cytotoxicity of the extract were evaluated.

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Extract showed antifungal activity for all species and low cytotoxicity was observed.

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The chemical analysis of extract was performed and its compounds showed antifungal effect on several Candida spp., mainly for Candida glabrata.

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Gallic acid, corilagin and ellagic acid showed a promising inhibitory activity on Candida glabrata.

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