Antifungal Constituents from the Roots of Piper dilatatum Rich .

1 Departamento de Estudos Básicos e Instrumentais, Universidade Estadual do Sudoeste da Bahia, BR 415, Km 03, s/n, 45700-000 Itapetinga, BA, Brazil 2 Departamento de Ciências Moleculares, Universidade Federal Rural de Pernambuco, Rua DomManoel de Medeiros, s/n, 52171-030 Recife, PE, Brazil 3 Seção de Fisiologia e Bioquı́mica de Plantas, Instituto de Botânica, CP 3005, 01061-970 São Paulo, SP, Brazil 4Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Rodovia Ilhéus-Itabuna, Km 16, 45662-900 Ilhéus, BA, Brazil 5 Instituto de Quı́mica, Universidade de São Paulo, CP 26077, 05513-970 São Paulo, SP, Brazil 6 Instituto de Quı́mica, Universidade Federal da Bahia, Rua Barão de Geremoabo, s/n, Ondina, 40170-290 Salvador, BA, Brazil


Introduction
An alarming and remarkable increase in the incidence of deep-seated disseminated mycoses has been observed in the last decades, and it may be credited to the advent of aggressive cancer chemotherapy, highly effective immunosuppressants for organ transplantation, long-term use of corticoids, widespread use of powerful broad spectrum antibacterial agents, and the explosion in the number of cases of human immunodeficiency virus (HIV) infection [1].Taking into account the increasing emergence of resistance to current antimycotic agents, new efforts have been devoted to the discovery of new antifungal lead compounds with different mechanisms of action [2].Within this context, natural products have been proven to be an excellent source of novel chemical entities in drug discovery [3,4].
Piper is one of the most diverse genera among the basal lineages of angiosperms in tropical wet forest around the world.The diversification centers of Piper species include Southeast Asia, Southeast Mexico, Andes, Colombian (Chocó department) and Brazilian Amazon, and Atlantic forest in Brazil [5].The Brazilian forests harbor 283 Piper species [6], and approximately 10% of them have already been chemically investigated [7].
Piper dilatatum Rich. is a shrub, 1.5-2 m tall, usually found in gaps and clearings with white spicate inflorescences consisting of several thousand flowers [8].The chemical investigation on its leaves revealed the presence of six prenylated benzoic acid derivatives and three chalcones [9].The essential oil from the leaves has been found to contain phellandrene, Δ-3-carene, and bicyclogermacrene as major constituents [10].Despite these investigations, no previous phytochemical studies have been conducted on P. dilatatum growing in Brazil.

Materials and Methods
2.1.General Procedures.IR spectra were measured in KBr pellets on a Perkin-Elmer infrared spectrometer model 1750.Circular dichroism (CD) spectrum was measured in CH 3 OH with a JASCOORD/UV-6 spectropolarimeter and optical rotation on a Perkin-Elmer 241 polarimeter.HREIMS spectra were obtained on a Bruker Daltonics MicrOTOF mass spectrometer.LREIMS spectra were measured at 70 eV on a VG Platform II spectrometer. 1H and 13 C NMR spectra were recorded at 200 and 50 MHz, respectively, on a Bruker AC200 apparatus.CDCl 3 (Aldrich) was used as solvent and TMS as internal standard.Chemical shifts were reported in  units (ppm) and coupling constants () in Hz.Silica gel (Merck, 230-400 mesh) was used for column chromatographic separations, while silica gel 60 PF254 (Merck) was used for analytical (0.25 mm) TLC chromatography.HPLC analyses of extracts and pure compounds were performed on a Shimadzu instrument using a C18 column (250 × 4.6 mm, 5 m) from Supelco eluted in a gradient mode starting with CH 3 CN : H 2 O (3 : 7) for 8 min, raising to 100% of CH 3 CN in 37 min, with detection at 254 nm.GC-FID analyses were carried out on a Shimadzu 17A instrument equipped with an HP DB-5 capillary column (30 m × 0.25 mm i.d., 0.25 m film thickness, and cross-linked 5% phenylmethyl silicone).Temperature gradient: from 100 ∘ to 280 ∘ at 3 ∘ min −1 and then held at 300 ∘ during 15 min.The flow rate of carrier gas (He) was 1.6 mL min −1 .

Plant Material.
Roots and leaves of P. dilatatum were collected in Ilhéus, Bahia, Brazil, in March 2008.The species was identified by Dr. André Márcio Amorim (Universidade Estadual de Santa Cruz, Brazil), and a voucher specimen (Piper 001) was housed at the Herbarium of CEPEC/CEPLAC in Ilhéus, Bahia, Brazil.

Extraction and Isolation of Constituents.
The dried and powdered roots of P. dilatatum (203 g) were extracted with CH 2 Cl 2 (3 × 500 mL) by maceration for two days.The resulting solution was filtered and concentrated under vacuum to afford the crude dichloromethanic extract of the roots (DER, 2.99 g), which was partially suspended (2.90 g) in hexanes to yield a yellow precipitate, which was filtered and dried (1.32 g).The hexanic solution was then concentrated under vacuum to give the hexanic fraction residue (1.68 g).

Antifungal Bioassay.
The microorganisms used in the antifungal assays C. cladosporioides (Fresen.)de Vries SPG 140 and C. sphaerospermum (Penzig) SPC 491 have been maintained at the Instituto de Botânica, São Paulo, SP, Brazil.Assays were performed in triplicate using the direct bioautography method in agreement with the literature procedure [17][18][19][20].Ten microliters of the solutions was prepared, in different concentrations, corresponding to 20, 10, 5, and 1 g for pure compounds and 400, 200, and 100 g for the crude extracts.The samples were applied to TLC plates, with these being eluted with hexanes-EtOAc (4 : 1) followed by complete removal of the solvent at room temperature.The chromatograms were then sprayed with a spore suspension of fungi in glucose and salt solution and incubated for 72 h in the darkness in a moistened chamber at 25 ∘ C. Clear inhibition zones appeared against a dark background, indicating the minimal amount of compound required (Table 1).Nystatin was used as the positive control (detection limit 1 g), whereas ampicillin and chloramphenicol were used as negative controls.

Results and Discussion
Extracts DER and DEL were assessed for their antifungal activity against Cladosporium fungi, and the results are shown in Table 1.As can be seen, only the root extract was active, inhibiting growth of both fungal strains at the 200 g treatment.
The crude extracts from roots and leaves were fractionated using a silica gel chromatography and afforded compounds 1-4 (Figure 1).Their structures were determined on the basis of their IR, MS, 1 H, and 13 C NMR data as well as confirmed by comparison with the literature data.
The crude CH 2 Cl 2 extract from the roots of P. dilatatum afforded (+)-(7S,8R)-epoxy-5,6-didehydrokavain (1) and flavokavain B (2). Compound 1 was found to compose approximately 40% of the extract or 0.6% of the dry weight.In addition, HPLC analysis (Figure 2) revealed 1 as the major component, representing 87% of 1 and 5% of 2. 1 H and 13 C NMR spectral data as well as CD curve for 1 were identical to those reported for (+)-(7S,8R)-epoxy-5,6-didehydrokavain, which was previously isolated from Piper rusbyi leaves [11].The literature search reveals that this is the first report on the occurrence of the pyrone 1 from P. dilatatum and represents the first report of the occurrence of this class of compounds in a plant species native to Brazilian and perhaps also to American forests.
The structure of compound 2, a yellow crystalline solid of molecular formula C 17 H 16 O, was identified as the chalcone flavokavain B, and its spectroscopic data were identical to those reported in the literature [12][13][14].Flavokavain B has been isolated from several Piper species [21], including P. dilatatum leaves [9], but it is the first report from the roots of P. dilatatum.
Given the interesting in vitro and in vivo biological activities already described for 1 and 2 [11,22], and bearing in mind that these compounds were found as the main chemical constituents of the roots of P. dilatatum, it was assumed that both substances 1 and 2 would be responsible for the crude extracts antifungal activity (Table 1).Thus, these secondary metabolites were assayed against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum, and the results are shown in Table 1.Both pyrone 1 (1 g) and chalcone 2 (100 g) exhibited antifungal activity, with pyrone 1 as the most potent substance (1 g).Considering that pyrone 1 is the most abundant constituent on the roots of P. dilatatum, the present study identifies this plant species as a candidate for the development of novel antifungal phytotherapic products.Furthermore, the potent antifungal activity of pyrone 1 provides a new and promising hit for the pursuit of more active and selective antifungal agents.

Conclusions
This study describes the first report of the occurrence of (+)-(7S,8R)-epoxy-5,6-didehydrokavain (1) in Piper dilatatum.The potent antifungal activity observed for 1 identifies this plant species as a promising candidate for the development of novel antifungal phytotherapic products.Moreover, the antifungal activity of pyrone 1 provides a new hit for the development of new antifungal derivatives.

Table 1 :a
Antifungal activity of extracts and compounds from P. dilatatum against Cladosporium cladosporioides and C. sphaerospermum.Minimum amount required for the inhibition of fungal growth on a thinlayer chromatographic plate (TLC).