Antifungal Activity of the Extracts and Neolignans from Piper regnellii ( Miq . ) C . DC . var . pallescens ( C . DC . ) Yunck

Extratos de folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck (Piperaceae), uma planta medicinal utilizada no Brasil para tratar doenças infecciosas, foram testados para a atividade antifúngica sobre as leveduras Candida albicans, C. krusei, C. parapsilosis e C. tropicalis. O extrato em acetato de etila apresentou boa atividade contra C. albicans com uma CIM de 125 μg mL, moderada atividade contra C. krusei e C. parapsilosis (CIM de 500 μg mL) e foi inativo contra C. tropicalis (CMI> 1000 μg mL). Com base nestes resultados, o extrato em acetato de etila foi fracionado em nove frações em cromatografia de coluna de sílica gel. As frações hexano e clorofórmio mostraram variados níveis de atividade antifúngica contra as leveduras testadas. Posterior separação da fração hexânica em cromatografia de coluna resultou nas substâncias puras eupomatenóide-6, eupomatenóide-5, eupomatenóide-3 e conocarpano. A elucidação estrutural das substâncias foi baseada em dados espectrais (RMN de H e de C, HSQC, HMBC, gNOE, IV e EM). O conocarpano foi a única substância isolada, com atividade contra as leveduras. As propriedades antifúngicas do extrato de P. regnellii demonstraram preliminar validação científica do uso da planta na medicina popular.


Introduction
Piper regnellii (Miq) C. DC. var.pallescens (C.3][4] In a screening of Brazilian medicinal plants, we have reported the antimicrobial activity of the hydroethanolic extract of the leaves of P. regnellii (Miq) C. DC against the bacteria Staphylococcus aureus and Bacillus subtilis and against the yeasts Candida krusei and Candida tropicalis. 5he search for active constituents from different Piper species has been intensified in recent years, with the finding that several species have been shown to have a number of biological activities. 6Phytochemical study of P. regnellii roots has shown the accumulation of several phenylpropanoids and dihydrobenzofuran neolignans including (+)-conocarpan as major compound.Conocarpan, a dihydrobenzofuran neolignan, has been isolated from several Piperaceae species. 7,8This compound displays a variety of biological activities including anti-PAF, 9 antifungal 10,11 and insecticidal activity. 7,12n the present study we describe the in vitro antifungal activity of ethanolic extract and fractions from leaves of Piper regnellii as well as of the bioactivity-directed isolation of eupomatenoid-6, eupomatenoid-5, eupomatenoid-3 and conocarpan.

Plant material
The leaves of Piper regnellii (Miq.) C. CD. var.pallescens (C.DC.) Yunck were collected in August 2001 in Horto of Medicinal Plants "Prof.Irenice Silva" in the Campus of Universidade Estadual de Maringá.The plant material was identified by Marilia Borgo from the Botanical Department of Universidade Federal do Paraná, and a voucher specimen (n.HUM 8392) is deposited at the Herbarium of Universidade Estadual de Maringá, Paraná, Brazil.

Isolation of the constituents
Dried leaves (200 g) of P. regnellii were ground and extracted with EtOH:H 2 O (9:1) at room temperature.The solvent was removed under vacuum at 40 °C to give an aqueous extract and a dark green residue.The aqueous extract from the crude hydroalcoholic extracts was lyophilized (13.9 g) and the residue from crude extract was washed with EtOAc.The organic solvent was removed to give the EtOAc extract (15.3 g).The aqueous and EtOAc extracts were assayed against S. aureus by bioautography and broth microdilution assay to determine the MICs as described below.

Microorganisms used and growth conditions
A single clinical isolated of each species (Candida albicans, C. krusei, C. parapsilosis, and C. tropicalis), obtained from vaginal mucosa, was selected to testing.The yeasts were maintained in agar Sabouraud-dextrose (Merck).

Antifungal susceptibility testing
The minimal inhibitory concentrations (MICs) of all the extracts, compounds and reference antifungal compounds were determined by microdilution techniques in Sabouraud broth (Merck) for yeasts. 15Inoculates were prepared in the same medium at a density adjusted to a 0.5 McFarland turbidity standard (10 4 colony-forming units [CFU] mL -1 ) and diluted to 1:10 for the broth microdilution procedure.Microtiter trays were incubated at 37 o C and the MICs were recorded after 24 h of incubation.Two susceptibility endpoints were recorded for each isolate.The MIC was defined as the lowest concentration of compounds at which the microorganism tested does not demonstrate visible growth.MBC was defined as the lowest concentration yielding negative subcultures or only one colony.Nystatin (Sigma Chemical Co., St.Louis, MO, USA) was included in the test as control.

Results and Discussion
The results of the antifungal activities of extracts, fractions and compounds appear in results were classified as follow: when the MIC is equal or smaller than 100 μg mL -1 , the antifungal activity was considered significant.If the extracts or compounds displayed a MIC from 100 to 500 μg mL -1 the antifungal activity was considered moderate; from 500 to 1000 μg mL -1 the antifungal activity was considered weak; over 1000 μg mL -1 the extracts were considered inactive.The EtOAc extract obtained from leaves of P. regnellii presented a moderate activity against C. albicans with MIC of 125 μg mL -1 , a weak activity on both C. krusei and C. parapsilosis with MIC of 500 μg mL -1 , and was inactive against C. tropicalis at a concentration >1000 μg mL -1 .On the other hand, the aqueous extract was inactive against all yeasts tested (MIC > 1000 μg mL -1 ) (Table 1).
On the basis of these findings, the EtOAc extract was fractionated on silica gel into nine fractions.The hexane and CHCl 3 fractions showed varied levels of antifungal activity against all yeasts.The hexane fraction showed significant anticandidal activity with a MIC from 62.5 to 125 μg mL -1 .CHCl 3 fraction was less active than hexane fraction with a MIC from 31.2 to 500 μg mL -1 .The CHCl 3 /EtOAc, EtOAc, acetone, MeOH, and MeOH/H 2 O fractions showed no activity against the organisms tested.The minimal fungicidal concentrations were within twofold dilution of the MIC for these organisms.
Further separation of the hexane fraction by column chromatography afforded the pure compounds eupomatenoid-6 (1), eupomatenoid-5 (2), eupomatenoid-3 (3) and conocarpan (4).The pure compound conocarpan (4) was the only active compound on the yeasts at concentrations of 6.3 to 12.5 μg mL -1 (Table 1).Conocarpan was methylated to afford O-methylconocarpan (4a), which was inactive against all the yeasts (data not shown).It is suggested that the antifungal activity of conocarpan can be related with the presence of the phenolic hydroxyl.
Under the conditions employed in the present study, 1-3 compounds were also inactive against all yeasts tested.The higher activity of conocarpan could also possibly be attributed to the absence of a double bond at C-2, since the derivatives 1-3 were inactive.
Studies on the antimicrobial properties of conocarpan have shown that it was active against C. albicans, Cryptococcus neoformans and Saccharomyces cerevisiae, as well as against the dermatophytes Microsporum gypseum and Tricophyton mentagophytes. 16n recent years, a number of plants of Piper species have been found to possess antifungal activity ( a Samples with MIC>1000 μg mL -1 were considered inactive.n.d.not determined. mentagrophytes) which has allowed the isolation of the active principles followed by their characterization as benzofuran neolignans in leaves of P. fulvescens 16 , benzoic acid derivatives in leaves of P. dilatatum, 17 and pyrrolidine and pyperidine amides in leaves and stems of P. hispidum, 18,19 seeds and leaves of P. tuberculatum 19,20 and of the leaves of P. arboreum. 20he findings revealed that the antifungal properties of P. regnellii extract provide preliminary scientific validation for the traditional medicinal use of this plant.However, the extracts and active compound isolated from P. regnellii should be further studied in animal models in order to evaluate the in vitro efficacy and toxicity.

Table 1 .
The in vitro

Table 1 .
Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of extracts, fractions and compounds obtained from leaves of P. regnellii