Antifungal piperamides from Piper mollicomum Kunth ( Piperaceae )

Herlle Aparecido da Silva, Lydia Fumiko Yamaguchi , Maria Cláudia Marx Young , Clécio Souza Ramos , André Márcio Araújo Amorim, Massuo Jorge Kato , Ronan Batista 1 State University of Southeast of Bahia, BR 415 Hwy, Km 03, Itapetinga, Bahia, Brazil 2 University of São Paulo (USP), Institute of Chemistry, 748 Prof. Lineu Prestes Av, São Paulo, São Paulo, Brazil 3 Institute of Botany, 3687 Miguel Stéfano Av, São Paulo, São Paulo, Brazil 4 Federal Rural University of Pernambuco (UFRPE), Dom Manoel de Medeiros St, Recife, Pernambuco, Brazil 5 State University of Santa Cruz (UESC), Ilhéus-Itabuna Hwy, Km 16, Ilhéus, Bahia, Brazil 6 Federal University of Bahia (UFBA), Barão de Jeremoabo St, Salvador, Bahia, Brazil


Introduction
Piperaceae family belongs to the Piperales order and it is one of the most primitive families among Angiosperms.It is predominantly a tropical family, comprising the genera Piper, Peperomia, Sarchorhachis and Ottonia, from which Piper and Peperomia are the most representative ones with approximately 2000 and 1700 species, respectively 1,2 .Piperaceae species have long been used as food additive and folk medicine agents mainly due to their antimicrobial properties, including potent fungicidal action 3 .
Piper species usually possess strong and pleasant aroma and spicy flavor, which explain their use as condiments, flavorings and medicinal materials.Piper compounds are often grouped into seven general classes, including amides, lignans, neolignans, flavonoids, kavalactones, butenolides, and volatile oils 4,5 .Amides are one of the most characteristic constituents of Piper species 6 .The biological activities presented by Piper's amides (also known as "piperamides") have inspired
The present study aimed to further investigate the chemical composition of

General procedures
Optical rotation was measured on a Perkin-Elmer 241 polarimeter. 1 H and 13 C NMR spectra were recorded at 500 and 125 MHz, respectively, on a Bruker DRX.CDCl3 (Aldrich) was used as solvent and TMS as internal standard.Chemical shifts were reported in δ units (ppm) and coupling constants (J) in Hz.MS spectra were obtained on a GGEM SHIMADZU mass spectrometer (70 eV) apparatus GCMS-QP5050A, equipped with BPX5 capillary column (30m x 0.25mm ID).Silica gel (Macherey-Nagel, 70-230 mesh) was used for column chromatographic separations, while silica gel 60G (Acros Organic) was used for analytical TLC chromatography.

Plant material
The plant was collected in Firmino Alves, southern Bahia, Brazil, in April 2009, and a voucher specimen was housed in the Herbarium of the Executive Committee of the Cacao Plan (CEPLAC) in Ilhéus, Bahia, under the code "PIPER-007", and identified as Piper mollicomum Kunth by Dr. André Márcio Araújo Amorim (Bahia State University of Santa Cruz -UESC).The collected material was dried in an oven (50 °C) for 48 hours, and their separated parts (stem, leaf and root) were pulverized in a knife mill.

Obtaining extracts
The dried and pulverized leaves (28 g), stem (128 g) and roots (200 g) were thoroughly extracted by maceration with dichloromethane, and then concentrated under reduced pressure (38 °C) until complete removal of the solvent, affording the dichlorometane extracts of the roots (1.68 g), stem (1.31 g) and leaves (1.19 g).

Antifungal assay by direct bioautography
The microorganisms used in the antifungal assays Cladosporium cladosporioides (Fresen.)de Vries SPG 140 and C. sphaerospermum (Penzig) SPC 491 were grown in Sabouraud dextrose agar, and maintained at the Instituto de Botânica, São Paulo, SP, Brazil.Both fungi were cultured in Sabouraud medium before the assay.Direct bioautography assays were performed in triplicate in agreement with the literature procedure 9,13 .Ten microliters of dichloromethane solutions prepared for each pure compound, corresponding to 100, 50, 25, 10, 5, and 1 µg, were applied to TLC plates and eluted with hexanes-EtOAc (7:3) followed by complete removal of the solvent at room temperature.The chromatograms were then sprayed with a spore suspension (3 x 10 6 cells/mL) in glucose and salt solution and incubated for 48 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 to inhibit the growth of each fungus (Table 1).Nystatin was used as the positive control (detection limit 1 µg).

Results and discussion
The phytochemical study on dichlorometane extracts of the leaves, stem and roots of Piper mollicomum Kunth led to the isolation of the known compounds tembamide (1), (R)-(-)tembamide acetate (2) and riparin I (3) (Figure 1), from which only 1 and 2 were found to exhibit remarkable antifungal properties against Cladosporium cladosporioides (5 μg) and C. sphaerospermum (1 μg) through direct bioautography analyses.When tested against C. sphaerospermum, compounds 1 and 2 were shown to be as potent as nystatin, the positive control (Table 1).The acetate 2 could be easily hydrolysed by fungal enzymes and this could be one likely explanation for the same antifungal activity observed for both compounds 1 and 2. In spite of being already known chemical entities, the compounds 1-3 are reported here, for the first time, as chemical constituents of P. mollicomum Kunth.In addition, bearing in mind that only 1 and 2 were active against the assayed fungi, but not 3, one can hypothesize that such antifungal activity may be related to the oxygenation at the benzylic carbon, explaining why 3 was inactive against the both fungi assayed.
Tembamide is widely described as a chemical constituent of plants belonging to the Rutaceae family, such as Clausena brevistyla Oliver 14 , Zanthoxylum ekmanii (URB.)ALAIN 15 , Aegle marmelos 16 , Clausena lansium 17 , and Feroniella lucida 18 .This amide was also obtained by hydrolysis from tembamide acetate isolated from the dichloromethane fraction of Piper guayranum (Piperaceae) 19 , a plant species traditionally used in Indian medicine due to its hypoglycemic activity 20 .In addition, tembamide displayed one of the most potent activities against HIV virus among 67 substances isolated from the bark and root of Zanthoxylum ailanthoides (Rutaceae) 21 .Isolated from Zanthoxylum capense (Rutaceae), tembamide was also evaluated along with (R)-(+)-tembamide acetate in tests of antibiotic modulators 22 .Its synthesis has been previously described by Aguirre  et al. (2001) and Kamal et al. (2004)  23,24 .According to literature data, riparine I, isolated from the fruits of Aniba riparia (Nees) Mez (Lauraceae), presented anxiolytic effects 25 and it was shown to be a potent muscle relaxant 26 .
Our findings call attention to the importance of P. mollicomum Kunth as a source of bioactive natural compounds for further studies on promising antimicrobial and pharmacological agents.

Conclusions
The present study reports on the chemical constituents of Piper mollicomum Kunth, in which against Cladosporium strains by means of direct bioautographic analyses.

a
Minimum amount required for inhibition of fungal growth on thin layer chromatography (TLC) plate.

Table 1 .
Antifungal activity of compounds from P. mollicomum Kunth against Cladosporium species.