Anti-Candida and anti-Cryptococcus evaluation of 15 non-alkaloidal compounds from Pterogyne nitens

Caroline Sprengel Lima，Carlos Roberto Polaquini，Mariana Bastos dos Santos，Fernanda Patrícia Gullo，Fernanda Sangalli Leite，Liliane Scorzoni，Vanderlan da Silva Bolzani，Maria Josˊe Soares Mendes-Giannini，Ana Marisa Fusco-Almeida，Andrˊeia Alves Rezende，Luis Octavio Regasini*

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 Josˊe 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



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，Fernanda Sangalli Leite2，Liliane Scorzoni2，Vanderlan da Silva Bolzani3，Maria Josˊe Soares Mendes-Giannini2，Ana Marisa Fusco-Almeida2，Andrˊeia 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 Josˊe 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：

in revised form 2 Dec，2nd revised form 7 Dec 2015

Accepted 5 Jun 2016

Available online 26 Aug 2016

Candida

Cryptococcus

Antifungal

Pterogyne nitens

Flavonoid

Opportunistic fungi

Objective：To evaluate anti-Candida and anti-Cryptococcus activities of 15 nonalkaloidal compounds from Pterogyne nitens Tulasne（Leguminosae），a South American medicinal plant.

Methods：Compounds were submitted to antifungal assays，using microdilution method described by Clinical and Laboratory Standards Institute document，with minor modifications.Five species of Candida and two species of Cryptococcus，including clinical isolates were screened.Antifungal activity was expressed by minimum inhibitory concentration（MIC）.Amphotericin B and fluconazole were used as standard antifungal drugs. Results：Among tested compounds，six substances presented fungal growth inhibition（MIC＜31.2μg/mL）［threeflavonederivatives（1-3），aglycosylatedflavonolderivative（5）and two phenolic acids（10 and 12）］.Sorbifolin（1），exhibited potent antifungal activity，demonstrating MIC value of 3.90μg/mL against Candida glabrata ATCC 90030，Cryptococcus 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=7.80μg/mL）.

Conclusions：Flavone derivatives from Pterogyne nitens can serve as prototypes for the design and development of innovative anti-Candida and anti-Cryptococcus hits.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2016.08.003

研究生师生关系主要包括教育关系、心理关系、伦理关系。教育关系是师生关系的核心，表现为导师与研究生的教学指导关系。心理关系是师生为完成共同的教学任务而产生的心理交往和情感交流。伦理关系处于师生关系体系中的最高层次，表现为导师与研究生构成一个特殊的道德共同体，各自承担和履行一定的伦理责任和伦理义务。当前研究生师生关系在上述各个层面均出现了不同程度的异化和失衡。

1.Introduction

In the last decades，there has been a significant increase in the incidence and prevalence of opportunistic fungi infections， includingcandidiasisandcryptococcosis.Thisincreaseisrelatedto the growing number of immunocompromised patients，including those with AIDS，cancer，transplant recipients and premature neonates［1，2］.Seven Candida species are classified as having major clinical relevance，namely，Candida albicans（C.albicans），Candidatropicalis（C.tropicalis），Candidaglabrata（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 mostprevalent 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，β-lactams，aminoglycosides，tetracyclines，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“b´alsamo”，“cocal”，“amendoim-bravo”，“amendoinzeiro”and“yvi-rar´o”isthesolememberofthegenus.Itisfoundin non-protected SouthAmerica areas，belonging tothelistofspecies recommendedforconservationgeneticsinBrazil.Also，P.nitensis admiredforthebeautyandodorofitsflowers，leavesandfruits［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 derivatives，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 Josˊe do Rio Preto，Sao Paulo，Brazil（20°47′02.4′S，49°21′36.0′W）in July 2014 and a voucher specimen（10291）was deposited in the Herbarium of Ilha Solteira（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，including13C nuclear magnetic resonance spectrum analysis［32］.

2.2.Microorganisms

2.3.Minimum inhibitory concentration（MIC）

Dissolution of compounds was performed with dimethylsulfoxide on 96-well plates and their concentration ranged from 250.00 to 0.48μg/mL.Anti-Candida and anti-Cryptococcus 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 independent 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≤31.20μg/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 31.20μg/mL.In anti-Candida assays，the most potent effect of compound 1 was against C.glabrata（MIC=3.90μg/ mL），followed by C.krusei and C.parapsilosis（MIC=7.80μg/ mL）.Thelowestpotencyofcompound1wasagainst C.albicans and C.tropicalis（MIC=31.20μg/mL）.In the anti-Cryptococcus assays，compound 1 was active against three strains of C.neoformans var.grubii（MIC values of 3.90 and 7.80μg/mL），including fluconazole-resistant clinical isolate（CnR）.For CnR strain，compound 1 exhibited a MIC value of3.90μg/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 3.90μg/mL，four times less potent than amphotericin B.For C.neoformans var.grubii ATCC 90012 and CnS strains，compound 1（MIC=7.80μg/mL）was two times less potent than FCZ（MIC=4.00μg/mL）.

Compounds 2 and 3，two glycosylated flavone derivatives，were active against C.neoformans var.grubii（ATCC 90012），with MIC values of 7.80μg/mL，two times less potent than FCZ（MIC=4.00μg/mL）.On the other hand，compounds 2 and 3 exhibited weak fungitoxicity against Candida species（MIC＞ 62.50μg/mL），except compound 2 which was moderately active against C.krusei（MIC=31.20μg/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 15.60μg/mL，four times less potent than FCZ（MIC=4.00μg/mL）.For this strain，compounds 4，6 and 7 were weakly active，exhibiting MIC values of 125.00，62.50 and 125.00μg/mL，respectively.The comparison of MIC values for compounds 4-7 indicated number of sugar units influenced anti-Cryptococcuseffect.Thus，orderofantifungal potency was monoglycosylated（5）＞diglycosylated（6 and 7）＞free aglycone（4）.

Figure 1.Structure of non-alkaloidal compounds from P.nitens.

Table 1 MIC values of non-alkaloidal compounds（1-15）from P.nitens.μg/mL.

Among phenolic acids（9-13），compounds 10 and 12 exhibited moderate anti-Cryptococcus activity（MIC=31.20μg/ mL）against C.neoformans（ATCC 90012）and C.gattii（118）. Compounds 8，14 and 15 were not active against both yeasts species（MIC≥125μg/mL）.

4.Discussion

MIC values of compounds 1-3 corroborate antifungal potential 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 reported 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.

Reviewofliteraturedataonanti-Candidaactivityofcompounds 4，10 and 13 is conflicting.A commercial sample of compound 4 presented higher anti-C.albicans activity（MIC=8μg/mL）than isolate samples from Buddleja salviifolia（MIC=125μg/mL）and Halimodendron halodendron（MIC=250μg/mL）［42-44］.Similar behavior was observed for commercial gallic acid（MIC=8μg/ mL）in comparison to the one obtained from Lythrum salicaria（MIC=2500μg/mL），Paeonia rockii（MIC=30μg/mL）and Pelargonium reniforme subsp.reniforme（MIC=500μg/mL）［42，45-47］.Also，commercialsampleofcompound10（MIC=20μg/mL）was quite different to sample obtained from Halimodendron halodendron（MIC=200μg/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 valuesof8and16μg/mL againstC.albicansandC.parapsilosis，respectively［42］.Incontrast，ourMICvaluedataforcompounds9 and 12 against both yeasts were equal or superior to 250μg/mL. Compound 7 from P.nitens was not able to exhibit anti-C.albicans activity（MIC≥250μg/mL），on the other hand，rutincommercialsampledisplayedthepotenteffect（MIC=40μg/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）.

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19 Nov 2015

Luis Octavio Regasini，Laboratory of Green and Medicinal Chemistry，Department of Chemistry and Environmental Sciences，Institute of Biosciences，Letters and Exact Sciences，São Paulo State University（UNESP），São Josˊe 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 Scientific 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.

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