HOSTED BY Contents lists available at ScienceDirect Asian Pac J Trop Biomed 2016; 6(10): 841–845 841 Asian Pacific Journal of Tropical Biomedicine journal homepage: www.elsevier.com/locate/apjtb Original article http://dx.doi.org/10.1016/j.apjtb.2016.08.003 *Corresponding author: Luis Octavio Regasini, Laboratory of Green and Me- dicinal Chemistry, Department of Chemistry and Environmental Sciences, Institute of Biosciences, Letters and Exact Sciences, São Paulo State University (UNESP), São Jos�e do Rio Preto, Sao Paulo, Brazil. Tel: +55 17 3221 2362 E-mail: regasini@ibilce.unesp.br Foundation Project: Supported by Support Foundation of São Paulo Research (FAPESP, Grant No. 2014/05445-3), National Council of Technological and Scien- tific Development (CNPq), and Office of Research of the São Paulo State University. Peer review under responsibility of Hainan Medical University. The journal implements double-blind peer review practiced by specially invited international editorial board members. 2221-1691/Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Hainan Medical University NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Anti-Candida and anti-Cryptococcus evaluation of 15 non-alkaloidal compounds from Pterogyne nitens Caroline Sprengel Lima1, Carlos Roberto Polaquini1, Mariana Bastos dos Santos1, Fernanda Patrícia Gullo2, 2 2 3 2 Fernanda Sangalli Leite , Liliane Scorzoni , Vanderlan da Silva Bolzani , Maria Jos�e Soares Mendes-Giannini , Ana Marisa Fusco-Almeida2, Andr�eia Alves Rezende4, Luis Octavio Regasini1* 1Laboratory of Green and Medicinal Chemistry, Department of Chemistry and Environmental Sciences, Institute of Biosciences, Letters and Exact Sciences, São Paulo State University (UNESP), São Jos�e do Rio Preto, Sao Paulo, Brazil 2Department of Clinical Analysis, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, São Paulo, Brazil 3Department of Organic Chemistry, Institute of Chemistry, São Paulo State University (UNESP), Araraquara, São Paulo, Brazil 4Department of Biology and Animal Sciences, Faculty of Engineering, São Paulo State University (UNESP), Ilha Solteira, São Paulo, Brazil ARTICLE INFO Article history: Received 19 Nov 2015 Received in revised form 2 Dec, 2nd revised form 7 Dec 2015 Accepted 5 Jun 2016 Available online 26 Aug 2016 Keywords: Candida Cryptococcus Antifungal Pterogyne nitens Flavonoid Opportunistic fungi ABSTRACT Objective: To evaluate anti-Candida and anti-Cryptococcus activities of 15 non- alkaloidal compounds from Pterogyne nitens Tulasne (Leguminosae), a South Amer- ican medicinal plant. Methods: Compounds were submitted to antifungal assays, using microdilution method described by Clinical and Laboratory Standards Institute document, with minor modifi- cations. Five species of Candida and two species of Cryptococcus, including clinical isolates were screened. Antifungal activity was expressed by minimum inhibitory con- centration (MIC). Amphotericin B and fluconazole were used as standard antifungal drugs. Results: Among tested compounds, six substances presented fungal growth inhibition (MIC< 31.2 mg/mL) [three flavone derivatives (1–3), a glycosylated flavonol derivative (5) and two phenolic acids (10 and 12)]. Sorbifolin (1), exhibited potent antifungal activity, demonstrating MIC value of 3.90 mg/mL against Candida glabrata ATCC 90030, Cryp- tococcus gattii 118 and fluconazole-resistant clinical isolate of Cryptococcus neoformans var. grubii. Pedalin (2) and nitensoside B (3), two glycosylated flavone derivatives, were active against Cryptococcus neoformans ATCC 90012 (MIC = 7.80 mg/mL). Conclusions: Flavone derivatives from Pterogyne nitens can serve as prototypes for the design and development of innovative anti-Candida and anti-Cryptococcus hits. 1. Introduction In the last decades, there has been a significant increase in the incidence and prevalence of opportunistic fungi infections, including candidiasis and cryptococcosis. This increase is related to the growing number of immunocompromised patients, including those with AIDS, cancer, transplant recipients and premature neo- nates [1,2]. Seven Candida species are classified as having major clinical relevance, namely, Candida albicans (C. albicans), Candida tropicalis (C. tropicalis), Candida glabrata (C. glabrata), Candida parapsilosis (C. parapsilosis), Candida krusei (C. krusei), Candida stellatoidea and Candida kyfer [3–6]. Candidiasis, the most common opportunistic yeast infection in the world has been in majority with C. albicans. This yeast is a causative agent of mucocutaneous and vulvovaginal infections, among other more invasive infections, such as septicemia, endocarditis, meningitis and peritonitis [3,4,7]. Cryptococcosis is an important globally systemic mycosis and the third most . This is an open access article under the CC BY- http://dx.doi.org/10.1016/j.apjtb.2016.08.003 mailto:regasini@ibilce.unesp.br http://crossmark.crossref.org/dialog/?doi=10.1016/j.apjtb.2016.08.003&domain=pdf www.sciencedirect.com/science/journal/22211691 http://www.elsevier.com/locate/apjtb http://dx.doi.org/10.1016/j.apjtb.2016.08.003 http://creativecommons.org/licenses/by-nc-nd/4.0/ Caroline Sprengel Lima et al./Asian Pac J Trop Biomed 2016; 6(10): 841–845842 prevalent disease in AIDS patients [8]. The most common clinical manifestation is cryptococcal meningitis, which has been mainly caused by Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii). However, there are reports of human infections caused by C. albidus and Cryptococcus laurentii [9]. On the other hand, the inefficacy of conventional antifungal drugs against resistant strains, as well as their severe side effects, limited spectrum of action and drug–drug interactions justify the urgent search for novel antifungal compounds [10]. In this way, natural products have long been used as prototypes for design of innovative drugs, which may be useful against infectious diseases, such as artemisinin, quinine, b-lactams, aminoglycosides, tetra- cyclines, echinocandins, griseofulvin, etc. [11]. Several metabolites of diverse structural patterns have proven to be active against fungi, as well as the screening of plant extracts is a valid strategy being exploited to discover novel antifungal agents [12,13]. Pterogyne nitens Tulasne (Leguminosae) (P. nitens), popularly named as “bálsamo”, “cocal”, “amendoim-bravo”, “amendoin- zeiro” and “yvi-raró” is the sole member of the genus. It is found in non-protected South America areas, belonging to the list of species recommended for conservation genetics in Brazil. Also,P. nitens is admired for the beauty and odor of its flowers, leaves and fruits [14]. Ethnopharmacological studies in Guarani communities revealed cold aqueous preparations from P. nitens stem barks have been used for the treatment of helminthic infestations, mainly against Ascaris lumbricoides [15]. Chemically, P. nitens presented a variety of compounds, including guanidine alkaloids, flavonoids (flavones, flavonols, flavan-3-ols and catechins), phenolic acids, triterpenes and sterols [16–19]. Guanidine alkaloids from P. nitens have demonstrated a broad spectrum of biological activities, including cytotoxic, pro-apoptotic, antibacterial and trypanocidal activity [20–25]. Flavones and flavonols from P. nitens exhibited myeloperoxidase inhibitory and antioxidant activities [26–29]. In our previous study, we identified antimicrobial activity of P. nitens extracts and their four guanidine alkaloids against C. albicans, C. krusei, C. parapsilosis and C. neoformans [30]. Our goal with present work was to evaluate anti-Candida and anti-Cryptococcus activities of 15 non-alkaloidal compounds against five Candida species and two Cryptococcus species. 2. Materials and methods 2.1. Non-alkaloidal compounds from P. nitens Flavonoids (flavone, flavonol and catechin derivatives) (1–8) and phenolic acids (9–13) were isolated and identified, using chemical procedures reported previously (Figure 1). Flavone de- rivatives, sorbifolin (1), pedalin (2) and nitensoside B (3), were isolated from leaves [26]. Flavonol derivatives, quercetin (4), isoquercitrin (5), quercetin 3-O-sophoroside (6) and rutin (7) were obtained from fruits and flowers [27,31]. Ourateacatechin (8) and the phenolic acids (9–13), such as caffeic acid (9), ferulic acid (10), sinapic acid (11), chlorogenic acid (12) and gallic acid (13) were isolated from flowers [18]. Triterpene acids (14) and (15) were purified from P. nitens leaves for the first time. Leaves of P. nitens were collected from Institute of Biosciences, Letters and Exact Sciences, São Paulo State University, São Jos�e do Rio Preto, Sao Paulo, Brazil (20�47002.400 S, 49�21036.000 W) in July 2014 and a voucher specimen (10291) was deposited in the Herbarium of Ilha Sol- teira (HISA) of Faculty of Engineering, Ilha Solteira, São Paulo, Brazil. Shade-dried leaves (600 g) were ground and extracted with hexane (1.8 L × 3, at room temperature). Dry hexane extract (10 g) was subjected to purification by successive chromatography columns over silica gel, eluted with mixtures of hexane and ethyl acetate, as well as furnishing betulinic acid (14 and 20 mg) and oleanonic acid (15 and 14 mg) (Figure 1). Structures of compound 14 and 15 were identified according to literature data, including 13C nuclear magnetic resonance spec- trum analysis [32]. 2.2. Microorganisms Six ATCC biological standards were used in our preliminary experiments, including C. albicans ATCC 90028, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, C. glabrata ATCC 90030, C. tropicalis ATCC 750 and Cryptococcus neoformans var. grubii (C. neoformans var. grubii) ATCC 90012. Two clin- ical isolates of C. neoformans var. grubii, fluconazole-resistant [C. neoformans clinical resistant (CnR)] and fluconazole- susceptible [C. neoformans clinical susceptible (CnS)], were obtained from AIDS patient with recurrent cryptococcosis [33]. Fluconazole-resistant isolate of C. gattii (118) was obtained from psittacine birds [34]. All yeasts were obtained from the collection of Laboratory of Clinical Mycology, Department of Clinical Analyses, School of Pharmaceutical Sciences, Universidade Estadual Paulista (UNESP), Araraquara, São Paulo, Brazil. 2.3. Minimum inhibitory concentration (MIC) Dissolution of compounds was performed with dime- thylsulfoxide on 96-well plates and their concentration ranged from 250.00 to 0.48 mg/mL. Anti-Candida and anti-Crypto- coccus activity experiments were carried out using reference broth microdilution method, as outlined in M27-A3 document produced by Clinical and Laboratory Standards Institute [35], with minor modifications [36]. Amphotericin B and fluconazole (FCZ) were used as standard antifungal drugs. MIC values were determined as the lowest concentration of test samples which showed complete fungal growth inhibition. Some 96- well plates were analyzed visually and spectrophotometrically. All tests were performed in triplicate and in the three indepen- dent experiments. 3. Results MIC values for all yeasts were given in Table 1. Out of 15 non-alkaloidal compounds (1–15), six substances presented fungal growth inhibition (MIC � 31.20 mg/mL) including three flavone derivatives (1–3), a glycosylated flavonol derivative (5) and two phenolic acids (10 and 12). Compound 1 demonstrated potent antifungal activity against both human opportunistic fungi, with MIC values ranging from 3.90 to 31.20 mg/mL. In anti-Candida assays, the most potent effect of compound 1 was against C. glabrata (MIC = 3.90 mg/ mL), followed by C. krusei and C. parapsilosis (MIC = 7.80 mg/ mL). The lowest potency of compound 1 was against C. albicans and C. tropicalis (MIC = 31.20 mg/mL). In the anti- Cryptococcus assays, compound 1 was active against three strains of C. neoformans var. grubii (MIC values of 3.90 and 7.80 mg/mL), including fluconazole-resistant clinical isolate (CnR). For CnR strain, compound 1 exhibited a MIC value of 1 O OH O OH HO O O OOH O OH O OH O OH HO HO HO 2 O OOH O OH O OH O OH O HO HO 3 O HO HO OH O OOH HO OH OH 4 OH O OOH HO OH OH O O OH OH OHOH 5 O OOH HO OH OH O O OH OHO O OH OH HO HOHO 6 O OOH HO OH OH O O O OHOH 7 HO O OH OH OH O OH HO O OH OH OH 8 R OH O HO R1 R R1 9 OH H 10 OCH3 H 11 OCH3 OCH3 OH HO 12 O O O HO HO OH OH HO OH OH O OH 13 HO 14 O OH O 15 O OH Figure 1. Structure of non-alkaloidal compounds from P. nitens. 1: Sorbifolin; 2: Pedalin; 3: Nitensoside B; 4: Quercetin; 5: Isoquercitrin; 6: Quercetin 3-O-sophoroside; 7: Rutin; 8: Ourateacatechin; 9: Caffeic acid; 10: Ferulic acid; 11: Sinapic acid; 12: Chlorogenic acid; 13: Gallic acid; 14: Betulinic acid; 15: Oleanonic acid. Caroline Sprengel Lima et al./Asian Pac J Trop Biomed 2016; 6(10): 841–845 843 3.90 mg/mL, four times less potent than amphotericin B, which has been administered as gold standard for cryptococcosis treatment [37]. Compound 1 was active against C. gattii (118), displaying a MIC value of 3.90 mg/mL, four times less potent than amphotericin B. For C. neoformans var. grubii ATCC 90012 and CnS strains, compound 1 (MIC = 7.80 mg/mL) was two times less potent than FCZ (MIC = 4.00 mg/mL). Compounds 2 and 3, two glycosylated flavone derivatives, were active against C. neoformans var. grubii (ATCC 90012), with MIC values of 7.80 mg/mL, two times less potent than FCZ (MIC = 4.00 mg/mL). On the other hand, compounds 2 and 3 exhibited weak fungitoxicity against Candida species (MIC > Table 1 MIC values of non-alkaloidal compounds (1–15) from P. nitens. mg/mL. Compounds C. albicans ATCC 90028 C. krusei ATCC 6258 C. parapsilosis ATCC 22019 C. gl ATCC 1 31.20 7.80 7.80 2 250.00 31.20 125.00 3 250.00 62.50 125.00 4 � 250.00 � 250.00 � 250.00 � 2 5 250.00 62.50 250.00 6 250.00 250.00 250.00 7 � 250.00 � 250.00 � 250.00 � 2 8 � 250.00 � 250.00 � 250.00 � 2 9 � 250.00 � 250.00 � 250.00 � 2 10 � 250.00 125.00 � 250.00 � 2 11 � 250.00 � 250.00 � 250.00 � 2 12 � 250.00 � 250.00 � 250.00 � 2 13 � 250.00 � 250.00 � 250.00 � 2 14 � 250.00 � 250.00 � 250.00 � 2 15 � 250.00 � 250.00 � 250.00 � 2 FCZa 8.00 – 16.00 Amphotericin Ba – 2.00 – a: Standard antifungal drugs. 62.50 mg/mL), except compound 2 which was moderately active against C. krusei (MIC = 31.20 mg/mL). Interestingly, flavonols, compound 4 and its glycosylated derivatives (5–7) were significantly less fungitoxic than flavone derivatives (1–3). Among flavonol derivatives, compound 5 demonstrated potent anti-Cryptococcus activity against ATCC strain, displaying a MIC value of 15.60 mg/mL, four times less potent than FCZ (MIC = 4.00 mg/mL). For this strain, com- pounds 4, 6 and 7 were weakly active, exhibiting MIC values of 125.00, 62.50 and 125.00 mg/mL, respectively. The comparison of MIC values for compounds 4–7 indicated number of sugar units influenced anti-Cryptococcus effect. Thus, order of abrata 90030 C. tropicalis ATCC 750 C. neoformans ATCC 90012 CnS CnR C. gattii (118) 3.90 31.20 7.80 7.80 3.90 3.90 – – 7.80 – – – – – 7.80 – – – 50.00 � 250.00 125.00 – – 125.00 – – 15.60 – – – – – 62.50 – – – 50.00 � 250.00 125.00 – – 125.00 50.00 � 250.00 125.00 – – 125.00 50.00 � 250.00 125.00 – – 125.00 50.00 � 250.00 31.20 – – 31.20 50.00 � 250.00 125.00 – – 125.00 50.00 � 250.00 31.20 – – 31.20 50.00 � 250.00 125.00 – – 125.00 50.00 � 250.00 125.00 – – 125.00 50.00 � 250.00 125.00 – – 125.00 – 8.00 4.00 4.00 – – 0.50 – – – 1.00 1.00 Caroline Sprengel Lima et al./Asian Pac J Trop Biomed 2016; 6(10): 841–845844 antifungal potency was monoglycosylated (5) > diglycosylated (6 and 7) > free aglycone (4). Among phenolic acids (9–13), compounds 10 and 12 exhibited moderate anti-Cryptococcus activity (MIC = 31.20 mg/ mL) against C. neoformans (ATCC 90012) and C. gattii (118). Compounds 8, 14 and 15 were not active against both yeasts species (MIC � 125 mg/mL). 4. Discussion MIC values of compounds 1–3 corroborate antifungal po- tential of flavone derivatives, which have shown fungitoxicity against a broad spectrum of fungi species, including yeasts (Saccharomyces cerevisiae), halohyphomycetes (Aspergillus) and dermatophytes (Trichophyton and Epidermophyton) [38–40]. Nevertheless, our results to compound 1 were significantly opposite to those described by Taleb-Contini et al., who re- ported absent growth inhibition until 500 mg/mL against C. albicans ATCC 1023 and C. tropicalis (clinical isolate from oral cavity), by using well diffusion assay [41]. This difference may be related to strain types, susceptibility tests and/or purity grade of compounds. Reviewof literature data on anti-Candida activity of compounds 4, 10 and 13 is conflicting. A commercial sample of compound 4 presented higher anti-C. albicans activity (MIC = 8 mg/mL) than isolate samples from Buddleja salviifolia (MIC = 125 mg/mL) and Halimodendron halodendron (MIC = 250 mg/mL) [42–44]. Similar behavior was observed for commercial gallic acid (MIC = 8 mg/ mL) in comparison to the one obtained from Lythrum salicaria (MIC = 2500 mg/mL), Paeonia rockii (MIC = 30 mg/mL) and Pelargonium reniforme subsp. reniforme (MIC = 500 mg/mL) [42,45–47]. Also, commercial sample of compound 10 (MIC = 20 mg/mL) was quite different to sample obtained from Halimodendron halodendron (MIC = 200 mg/mL) [44,48]. Our anti-C. albicans MIC value data were more similar to plant isolates than commercial samples. Commercial samples of compounds 9 and 12 displayed MIC values of 8 and 16 mg/mL againstC. albicans andC. parapsilosis, respectively [42]. In contrast, our MIC value data for compounds 9 and 12 against both yeasts were equal or superior to 250 mg/mL. Compound 7 from P. nitens was not able to exhibit anti- C. albicans activity (MIC � 250 mg/mL), on the other hand, rutin commercial sample displayed the potent effect (MIC = 40 mg/mL) [48]. Martins et al. suggested that these differences could be probably assigned to purity grade of tested compounds [49]. Additionally, we inferred this difference may be correlated to strain types. In summary, 15 non-alkaloidal compounds from P. nitens were evaluated against Candida and Cryptococcus species. Of these, compound 1 may be considered suitable template for design of innovative hits for the treatment of opportunistic yeast infections, including candidiasis and cryptococcosis. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgments This work was supported by the Support Foundation of São Paulo Research (FAPESP), National Council of Technological and Scientific Development (CNPq), and Office of Research of the São Paulo State University. The authors thank the Support Foundation of São Paulo Research (FAPESP), for fellowship to Sprengel Lima (Proc. 2014/05445-3). References [1] de Oliveira RB, Atobe JH, Souza SA, de Castro Lima Santos DW. Epidemiology of invasive fungal infections in patients with ac- quired immunodeficiency syndrome at a reference hospital for in- fectious diseases in Brazil. Mycopathologia 2014; 178(1–2): 71-8. [2] Vandeputte P, Ferrari S, Coste AT. Antifungal resistance and new strategies to control fungal infections. Int J Microbiol 2012; 2012: 713687. [3] Greenberg MS, Glick M. Burket's oral medicine: diagnosis and treatment. 10th ed. Hamilton: BC Decker Inc.; 2003. [4] Sardi JC, Scorzoni L, Bernardi T, Fusco-Almeida AM, Mendes Giannini MJ. Candida species: current epidemiology, pathoge- nicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol 2013; 62(Pt 1): 10-24. [5] Silva F, Ferreira S, Duarte A, Mendonça DI, Domingues FC. Antifungal activity of Coriandrum sativum essential oil, its mode of action against Candida species and potential synergism with amphotericin B. Phytomedicine 2011; 19(1): 42-7. [6] Bailly S, Maubon D, Fournier P, Pelloux H, Schwebel C, Chapuis C, et al. Impact of antifungal prescription on relative distribution and susceptibility of Candida spp. – trends over 10 years. J Infect 2016; 72(1): 103-11. [7] Brunke S, Hube B. Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiol 2013; 15(5): 701-8. [8] Gullo FP, Rossi SA, Sardi Jde C, Teodoro VL, Mendes- Giannini MJ, Fusco-Almeida AM. Cryptococcosis: epidemiology, fungal resistance, and new alternatives for treatment. Eur J Clin Microbiol Infect Dis 2013; 32(11): 1377-91. [9] Khawcharoenporn T, Apisarnthanarak A, Mundy LM. Non-neo- formans cryptococcal infections: a systematic review. Infection 2007; 35(2): 51-8. [10] Perlin DS, Shor E, Zhao Y. Update on antifungal drug resistance. Curr Clin Microbiol Rep 2015; 2(2): 84-95. [11] Butler MS, Robertson AA, Cooper MA. Natural product and nat- ural product derived drugs in clinical trials. Nat Prod Rep 2014; 31(11): 1612-61. [12] Abiodun OO, Sood S, Osiyemi OA, Agnihotri VK, Gulati A, Ajaiyeoba EO, et al. In vitro antimicrobial activity of crude ethanol extracts and fractions of Terminalia catappa and Vitex doniana. Afr J Med Med Sci 2015; 44(1): 21-6. [13] Abubacker MN, Devi PK. In vitro antifungal potentials of bioactive compound oleic acid, 3-(octadecyloxy) propyl ester isolated from Lepidagathis cristata Willd. (Acanthaceae) inflorescence. Asian Pac J Trop Med 2014; 7: S190-3. [14] Lorenzi H. Brazilian trees: manual of identification and cultivation of arboreal plants. 2nd ed. Nova Odessa: Plantarum; 1998. [15] Crivos M, Martínez MR, Pochettino ML, Remorini C, Sy A, Teves L. Pathways as “signatures in landscape”: towards an ethnography of mobility among the Mbya-Guaraní (Northeastern Argentina). J Ethnobiol Ethnomed 2007; 3: 2. [16] Ferreira FG, Regasini LO, de Oliveira AM, Campos JAD, Silva DHS, Cavalheiro AJ, et al. Evaluation of mutagenicity and antimutagenicity of different fractions of Pterogyne nitens (Legu- minosae), using Tradescantia pallida micronuclei assay. Rev Bras Farmacogn 2009; 19: 61-7. [17] Regasini LO, de Oliveira CM, Vellosa JCR, de Faria Oliveira OM, Siqueira Silva DH, da Silva Bolzani V. Free radical scavenging activity of Pterogyne nitens Tul. (Fabaceae). Afr J Biotechnol 2008; 7(24): 4609-13. [18] Regasini LO, Fernandes DC, Castro-Gamboa I, Siqueira Silva DH, Furlan M, da Silva Bolzani V, et al. Chemical constituents of the flowers of Pterogyne nitens (Caesalpinioideae). Quim Nova 2008; 31(4): 802-6. http://refhub.elsevier.com/S2221-1691(15)30965-5/sref1 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref1 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref1 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref1 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref2 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref2 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref2 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref3 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref3 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref4 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref4 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref4 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref4 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref5 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref5 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref5 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref5 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref6 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref6 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref6 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref6 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref7 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref7 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref8 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref8 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref8 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref8 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref9 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref9 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref9 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref10 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref10 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref11 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref11 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref11 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref12 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref12 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref12 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref12 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref13 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref13 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref13 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref13 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref14 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref14 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref15 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref15 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref15 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref15 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref16 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref16 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref16 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref16 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref16 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref17 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref17 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref17 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref17 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref18 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref18 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref18 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref18 Caroline Sprengel Lima et al./Asian Pac J Trop Biomed 2016; 6(10): 841–845 845 [19] Regasini LO, Vieira-Júnior GM, Fernandes DC, da Silva Bolzani VS, Cavalheiro AJ, Siqueira Silva DH. Identification of triterpenes and sterols from Pterogyne nitens (Fabaceae-Caesalpi- nioideae) using high-resolution gas chromatography. J Chil Chem Soc 2009; 54(3): 218-21. [20] Regasini LO, Castro-Gamboa I, Siqueira Silva DH, Furlan M, Barreiro EJ, Ferreira PMP, et al. Cytotoxic guanidine alkaloids from Pterogyne nitens. J Nat Prod 2009; 72(3): 473-6. [21] Tajima Y, Nakagawa H, Tamura A, Kadioglu O, Satake K, Mitani Y, et al. Nitensidine A, a guanidine alkaloid from Pterogyne nitens, is a novel substrate for human ABC transporter ABCB1. Phytomedicine 2014; 21(3): 323-32. [22] Tajima Y, Murase H, Satake K, Mitani Y, Regasini LO, da Silva Bolzani V, et al. Nitensidine A, a guanidine alkaloid from Pter- ogyne nitens, induces osteoclastic cell death. Cytotechnology 2015; 67: 585-92. [23] Duarte RA, Mello ER, Araki C, Bolzani Vda S, Siqueira e Silva DH, Regasini LO, et al. Alkaloids extracted from Pterogyne nitens induce apoptosis in malignant breast cell line. Tumour Biol 2010; 31(5): 513-22. [24] Coqueiro A, Regasini LO, Stapleton P, da Silva Bolzani V, Gibbons S. In vitro antibacterial activity of prenylated guanidine alkaloids from Pterogyne nitens and synthetic analogues. J Nat Prod 2014; 77(8): 1972-5. [25] Siqueira MC, Silva MTA, Regasini LO, Silva DHS, Cicarelli RMB. Trypanocidal activity of pterogynidine and nitensidine E using two distinct Trypanosoma cruzi strains. Planta Med 2009; 75: 945. [26] Fernandes DC, Regasini LO, Vellosa JC, Pauletti PM, Castro- Gamboa I, Bolzani VS, et al. Myeloperoxidase inhibitory and radical scavenging activities of flavones from Pterogyne nitens. Chem Pharm Bull (Tokyo) 2008; 56(5): 723-6. [27] Regasini LO, Vellosa JC, Silva DH, Furlan M, de Oliveira OM, Khalil NM, et al. Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors. Phytochemistry 2008; 69(8): 1739-44. [28] Okumura LL, Regasini LO, Fernandes DC, da Silva DH, Zanoni MV, Bolzani Vda S. Fast screening for antioxidant prop- erties of flavonoids from Pterogyne nitens using electrochemical methods. J AOAC Int 2012; 95(3): 773-7. [29] Vellosa JC, Regasini LO, Belló C, Schemberger JA, Khalil NM, de Araújo Morandim-Giannetti A, et al. Preliminary in vitro and ex vivo evaluation of afzelin, kaempferitrin and pterogynoside ac- tion over free radicals and reactive oxygen species. Arch Pharm Res 2015; 38: 1168-77. [30] Regasini LO, Pivatto M, Scorzoni L, Benaducci T, Fusco- Almeida AM, Giannini MJS, et al. Antimicrobial activity of Pterogyne nitens Tul., Fabaceae, against opportunistic fungi. Rev Bra Farmacogn 2010; 20(5): 706-11. [31] Regasini LO, Lopes AA, Silva DHS, Furlan M, Young MCM, Maria DA, et al. Antiproliferative effect of Pterogyne nitens on melanoma cells. Rev Ciênc Farm Básica Apl 2007; 28: 335-40. [32] Mahato SB, Kundu AP. 13C NMR spectra of pentacyclic triterpenoids-a compilation and some salient features. Phytochem- istry 1994; 37(6): 1517-75. [33] Matsumoto MT, Fusco-Almeida AM, Baeza LC, Melhem Mde S, Medes-Giannini MJS. Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil. Rev Inst Med Trop São Paulo 2007; 49(1): 41-7. [34] Raso TF, Werther K, Miranda ET, Mendes-Giannini MJ. Crypto- coccosis outbreak in psittacine birds in Brazil. Med Mycol 2004; 42(4): 355-62. [35] Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. 2nd ed.Wayne, PA:Clinical andLaboratoryStandards Institute; 2008. [36] de Paula E Silva AC, Costa-Orlandi CB, Gullo FP, Sangalli- Leite F, de Oliveira HC, da Silva Jde F, et al. Antifungal activity of decyl gallate against several species of pathogenic fungi. Evid Based Complement Altern Med 2014; 2014: 506273. [37] de Aguiar Cordeiro R, Mourão CI, Rocha MF, de Farias Marques FJ, Teixeira CE, de Oliveira Miranda DF, et al. Anti- folates inhibit Cryptococcus biofilms and enhance susceptibility of planktonic cells to amphotericin B. Eur J Clin Microbiol Infect Dis 2013; 32(4): 557-64. [38] Hamada Y, Takano S, Ayano Y, Tokunaga M, Koashi T, Okamoto S, et al. Structure-activity relationship of oligomeric flavan-3-ols: importance of the upper-unit b-ring hydroxyl groups in the dimeric structure for strong activities. Molecules 2015; 20(10): 18870-85. [39] Cao H, Chen X, Jassbi AR, Xiao J. Microbial biotransformation of bioactive flavonoids. Biotechnol Adv 2015; 33(1): 214-23. [40] Singh G, Kumar P, Joshi SC. Treatment of dermatophytosis by a new antifungal agent ‘apigenin’. Mycoses 2014; 57(8): 497-506. [41] Taleb-Contini SH, Schorr K, Da Costa FB, de Oliveira DCR. Detection of flavonoids in glandular trichomes of Chromolaena species (Eupatorieae, Asteraceae) by reversed-phase high-perfor- mance liquid chromatography. Rev Bras Ciênc Farm 2007; 43(2): 315-21. [42] Ozçelik B, Kartal M, Orhan I. Cytotoxicity, antiviral and antimi- crobial activities of alkaloids, flavonoids, and phenolic acids. Pharm Biol 2011; 49(4): 396-402. [43] Pendota SC, Aderogba MA, Ndhlala AR, Van Staden J. Antimi- crobial and acetylcholinesterase inhibitory activities of Buddleja salviifolia (L.) Lam. leaf extracts and isolated compounds. J Ehnopharmacol 2013; 148(2): 515-20. [44] Wang J, Lou J, Luo C, Zhou L, Wang M, Wang L. Phenolic compounds from Halimodendron halodendron (Pall.) voss and their antimicrobial and antioxidant activities. Int J Mol Sci 2012; 13(9): 11349-64. [45] Manayi A, Saeidnia S, Faramarzi MA, Samadi N, Jafari S, Vazirian M, et al. A comparative study of anti-Candida activity and phenolic contents of the calluses from Lythrum salicaria L. in different treatments. Appl Biochem Biotechnol 2013; 170(1): 176-84. [46] PicernoP,Mencherini T, SansoneF,DelGaudioP,Granata I, PortaA, et al. Screening of a polar extract of Paeonia rockii: composition and antioxidant and antifungal activities. J Ethnopharmacol 2011; 138(3): 705-12. [47] Latt�e KP, Kolodziej H. Antifungal effects of hydrolysable tannins and related compounds on dermatophytes, mould fungi and yeasts. Z Naturforsch C 2000; 55(5–6): 467-72. [48] Siler B, Zivkovi�c S, Banjanac T, Cvetkovi�c J, Nestorovi�c �Zivkovi�c J, Ciri�c A, et al. Centauries as underestimated food ad- ditives: antioxidant and antimicrobial potential. Food Chem 2014; 147: 367-76. [49] Martins N, Barros L, Henriques M, Silva S, Ferreira ICFR. Activity of phenolic compounds from plant origin against Candida species. Ind Crop Prod 2015; 74: 648-70. http://refhub.elsevier.com/S2221-1691(15)30965-5/sref19 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref19 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref19 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref19 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref19 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref20 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref20 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref20 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref21 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref21 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref21 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref21 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref22 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref22 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref22 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref22 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref23 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref23 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref23 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref23 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref24 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref24 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref24 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref24 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref25 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref25 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref25 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref26 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref26 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref26 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref26 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref27 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref27 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref27 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref27 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref28 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref28 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref28 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref28 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref29 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref29 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref29 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref29 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref29 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref29 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref30 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref30 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref30 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref30 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref31 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref31 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref31 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref31 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref31 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref32 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref32 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref32 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref32 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref33 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref33 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref33 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref33 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref33 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref34 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref34 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref34 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref35 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref35 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref35 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref36 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref36 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref36 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref36 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref37 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref37 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref37 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref37 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref37 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref38 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref38 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref38 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref38 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref38 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref39 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref39 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref40 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref40 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref41 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref41 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref41 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref41 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref41 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref41 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref42 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref42 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref42 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref43 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref43 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref43 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref43 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref44 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref44 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref44 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref44 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref45 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref45 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref45 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref45 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref46 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref46 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref46 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref46 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref47 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref47 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref47 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref47 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref48 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref48 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref48 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref48 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref48 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref48 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref48 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref48 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref49 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref49 http://refhub.elsevier.com/S2221-1691(15)30965-5/sref49 Anti-Candida and anti-Cryptococcus evaluation of 15 non-alkaloidal compounds from Pterogyne nitens 1. Introduction 2. Materials and methods 2.1. Non-alkaloidal compounds from P. nitens 2.2. Microorganisms 2.3. Minimum inhibitory concentration (MIC) 3. Results 4. Discussion Conflict of interest statement Acknowledgments References