Journal of Ethnopharmacology 138 (2011) 633636

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Ethnopharmacological communication

Detection of antifungal compounds in Polygonum ferrugineum Wedd. extracts by bioassay-guided fractionation. Some evidences of their mode of action
S.N. Lpez, R.L.E. Furlan, S.A. Zacchino
Farmacognosia, Facultad de Ciencias Bioqumicas y Farmacuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: Polygonum ferrugineum Wedd. (Polygonaceae) is used to heal infected wounds and as antiseptic, antibiotic or antifungal in the traditional Argentinean medicine. The present investigation was carried out to evaluate the antifungal properties of different extracts of aerial parts of Polygonum ferrugineum, in order to give support to its ethnopharmacological use and to isolate the compounds responsible for the antifungal properties. The most active compounds were tested for their capacity of producing hyphae malformations, similar to those previously observed for crude extracts. Materials and methods: Agar Dilution Method (ADM) and Agar Overlay Bioautography (AOB) were used for bioassay-guided fractionation of the aerial part extracts against a panel of human opportunistic pathogenic fungi. The Neurospora crassa assay, followed by Optical Microscopy and Scanning Electron Microscopy observation, was used for studies of mechanisms of action. Results: MeOH extract and DCM and Hex sub-extracts, but not Aq, EtOAc or BuOH ones possess antifungal activity. Of the seven isolated compounds, cardamonin 2 showed a selective inhibition of Epidermophyton occosum with a very low MIC (=6.2 g/mL) and pashanone 1 possessed moderate antifungal activity (MICs = 2550 g/mL) but a broader spectrum of action. Chalcone 2, but not 1, induced swelling and shortening of the Neurospora crassa hyphae, similar as those caused by the crude DCM extract. Conclusions: The bioassay-guided fractionation of Polygonum ferrugineum DCM extract allowed the isolation of ve active compounds. Among them, cardamonin 2 showed the highest antifungal activity and selectivity towards Epidermophyton occosum; in addition, it induced Neurospora crassa malformations that are similar than those produced by the crude DCM extract. These results give additional support to the ethnopharmacological use of Polygonum ferrugineum as antifungal agent. 2011 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 19 June 2011 Received in revised form 8 September 2011 Accepted 20 September 2011 Available online 28 September 2011 Keywords: Polygonum ferrugineum Pashanone Cardamonin Antifungal activity Hyphal malformations

1. Introduction Among the twenty species of Polygonum genus growing in Argentina, Polygonum ferrugineum Wedd. (Polygonaceae) (common name caatay guaz), is used by the Argentinean people in traditional medicine to heal infected wounds, as antiseptic, antibiotic or antifungal agent (Del Vitto et al., 1997). A previous study showed that a crude DCM extract displayed high antifungal activities on dermatophytes and showed the ability to induce hyphae malformations in Neurospora crassa (Zacchino et al., 1998) suggesting that the mechanism of action would be the inhibition of the fungal cell-wall. Since fungal but not mammalian cells possess a wall (Farka s, 2003), this is an ideal target for antifungal agents (Espinel-Ingroff, 2009) and therefore, this extract gained importance for further research.

Subsequently, a phytochemical study of this plant revealed the presence of an unknown homoisoavanone and the related dihydrochalcone along with known avanones and chalcones (Lpez et al., 2006). In order to establish the connections between the antifungal properties of Polygonum ferrugineum extracts and some of its chemical components, here we studied the antifungal activity-guided fractionation of the active extracts, the antifungal properties of the isolated compounds, and their capacity to induce hyphae malformations similar to those previously observed for crude DCM extract. 2. Materials and methods 2.1. Plant material Aerial parts of Polygonum ferrugineum were collected at Puerto Gaboto, Santa Fe, Argentina on December 2002 by Dr. S. Gattuso (voucher specimen UNR 99, Biologa Vegetal Herbarium UNR, Suipacha 531, Rosario, Argentina).

Corresponding author. Tel.: +54 341 4375315; fax: +54 341 4375315. E-mail address: szaabgil@citynet.net.ar (S.A. Zacchino). 0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.09.038

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Table 1 MIC values (in g/mL) of the crude MeOH extract of aerial parts of Polygonum ferrugineum (tested up to 1000 g/mL), its Hex and DCM sub-extracts and the isolated compounds 15 (tested up to 250 g/mL) against fungi. The inactive sub-extracts EtOAc, BuOH and Aq or the inactive pinostrobin and avokawin B are not shown.

R2

R3

HO

O
3

R1 OH O

O OH O

OH

1 R1 = -OCH3; R2 = -OCH3; R3 = -OH 2 R1 = -H ; R2 = -OH ; R3 = -OCH3

HO OCH3 HO O O OH

3
OH OH

OH

OCH3

5
Ca Sc i i 750 50 i i i i 0.62

4
Cn i i i 50 i i i i 0.40 0.01 0.04 0.04 0.01 0.004 Mc 125 125 50 50 i 200 250 i Mg 125 125 125 50 I 250 i 250 Tm 125 62.5 62.5 25 i 125 200 250 Tr 50 125 50 50 250 125 250 250 Ef 50 125 50 25 6.2 200 Nt 250

Extract MeOH Sub-extracts Hex DCM Compounds 1 2 3 4 5 Antifungals Amph Terb

i i i 50 i i i i 0.75

Ca, Candida albicans ATCC 10231; Sc, Saccharomyces cerevisiae ATCC 9763; Cn, Cryptococcus neoformans ATCC 32264; Mg, Microsporum gypseum CCC 115; Tm, Trichophyton mentagrophytes ATCC 9972; Tr, Trichophyton rubrum CCC 113; Ef, Epidermophyton occosum CCC 114; Amph, amphotericin B; Terb, terbinane. Candida tropicalis and Aspergillus spp. were not sensitive. i: MIC > 1000 g/mL and >250 g/mL for extracts and pure compounds, respectively. Nt, not tested.

2.2. Preparation of extracts and antifungal-guided fractionation Air-dried powdered aerial parts of Polygonum ferrugineum (1.00 kg) were macerated with MeOH (3 1.5 L, 24 h each), resuspended in H2 O (70:30, v/v) and partitioned successively with hexane (HE ), dichloromethane (DCME ), ethyl acetate (AE ) and nbutanol (BE ) as reported previously (Lpez et al., 2006). Extract and sub-extracts, including the resting aqueous (AqE ) one, were submitted to antifungal evaluation. DCME and HE were fractionated by vacuum liquid chromatography (VLC) on silica gel (CHCl3 EtOAc gradient/Me2 CO/MeOH) to give 13 fractions (fr) each (113 for HE and IXIII for DCME ). Further chromatographic fractionation of active fractions led to the isolation of compounds 15 from fr IV of DCME and pinostrobin and avokawin B from fr 1 of HE which were evaluated for antifungal properties. 2.3. Antifungal evaluation 2.3.1. Microorganisms and media All the microorganisms were either from ATCC [American Type Culture Collection (Rockville, MD)], or from CCC [CEREMIC (Centro de Referencia en Micologa, UNR, Rosario, Argentina)] species belonging to: Candida genus, Candida albicans ATCC 10231 and Candida tropicalis CCC 131; Aspergillus genus, Aspergillus avus ATCC 9170, Aspergillus fumigatus ATCC 26934 and Aspergillus niger ATCC 9029; Trichophyton genus, Trichophyton mentagrophytes ATCC 9972 and Trichophyton rubrum CCC 113; Microsporum genus, Microsporum canis CCC 112 and Microsporum gypseum CCC 115;

Saccharomyces cerevisiae ATCC 9763; Cryptococcus neoformans ATCC 32264 and Epidermophyton occosum CCC 114 were used. Inocula were prepared and quantied as follows (Wright et al., 1983): 5.0 L of conidia or yeasts stock suspensions were added to each test tube containing SDA previously mixed with the solution test, to reach a nal concentration of 2.5 104 elements/mL. 2.3.2. Agar Dilution Method (ADM) Stock solutions dissolved in DMSO were two-fold diluted to give dilutions ranging from 1000 to 10 g/mL for extracts or from 250 to 0.1 g/mL for pure compounds according to reported procedures (Svetaz et al., 2004). Concentrations ranging from 1000 to 10 g/mL for extracts and from 250 to 0.1 g/mL for pure compounds, according to reported procedures (Svetaz et al., 2004). Amphotericin B (Sigma) and terbinane (Pzer) were used as positive controls: drug-free medium and drug-free inoculated medium were the negative and growth controls, respectively. Minimum Inhibitory Concentration (MIC) was dened as the lowest concentration of a compound or an extract that produced no visible growth after the incubation time (48 h for yeasts, 72 h for Aspergillus spp. and 7 d for dermatophytes). MICs 1000 or 250 g/mL for extracts or pure compounds, respectively, were considered active. 2.3.3. Agar Overlay Bioautography (AOB) AOB method was carried out according to reported procedures (Lpez et al., 2007) with Microsporum gypseum CCC 115. Extracts were tested at 100 g/spot.

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Fig. 1. Optical microscopic appearance (A and C) and Scanning Electronic Microscopy (B and D) of Neurospora crassa hyphae, untreated (A, 100 and B, 1350) and treated (C, 400 and D, 1320) with 40 g of 2 ,4 -dihydroxy-6 -methoxychalcone (cardamonin 2). Arrows in C and D show hyphal malformations. (SEM, bar = 10 m.)

3. Results and discussion 3.1. Bioassay-guided fractionation with non-targeted assays The MeOH extract of aerial parts of Polygonum ferrugineum in ADM showed MIC values = 50125 g/mL only in dermatophytes. Regarding the sub-extracts, DCME (MIC = 50125 g/mL) and HE (62.5125 g/mL) but not AE , BE or AqE showed signicant activity against dermatophytes (Table 1). DCME also showed marginal activity (MIC = 750 g/mL) against Saccharomyces cerevisiae. HE was fractionated by VLC to give 13 fractions (fr 113 for HE ), of which fr 1 showed activity in AOB performed with Microsporum gypseum. This fraction was submitted to column chromatography rendering the known compounds 5-hydroxy7-methoxyavanone (pinostrobin) (Burke and Nair, 1986) and 2 -hydroxy-4 ,6 -dimethoxychalcone (avokawin B) (Ahmed et al., 1981). However, none of them showed signicant antifungal properties by ADM (MICs > 250 g/mL). DCME was also fractionated by VLC to give 13 fractions (fr IXIII for DCME ), of which fr IV showed the best antifungal activity in ADM (MIC = 12.5250 g/mL). Fr IV was submitted to column chromatography rendering ve known compounds: 2 ,6 dihydroxy-3 ,4 -dimethoxychalcone (common name pashanone, 1) (Ramakrishnan et al., 1974), 2 ,4 -dihydroxy-6 -methoxychalcone (cardamonin or alpinetin chalcone, 2) (Krishna and Chaganty, 1973), 5,7-dihydroxy-6-methoxy-3-(9-hydroxy-phenylmethyl)chroman-4-one (homoferrugenone, 3) (Lpez et al., 2006), 2 ,4 ,6 -trihydroxy-3 -methoxy--hydroxymethyl--hydroxydihydrochalcone (homoferrugen-dihydrochalcone 4) (Lpez et al., 2006) and 5,8-dimethoxy-7-hydroxyavanone (5) (Bratoeff and Perez-Amador, 1994). The evaluation by ADM showed that compounds 15 have diverse antifungal properties. Chalcone 2 showed selective high activity against Epidermophyton occosum (MIC = 6.2 g/mL) and marginal activity (MIC = 250 g/mL) against Trichophyton rubrum.

Chalcone 1 possessed moderate activity (MICs 2550 g/mL) but broader spectrum of action, inhibiting the whole fungal panel. Compounds 35 showed low activity (125250 g/mL) only against dermatophytes. The most striking result was the high selectivity of 2 towards Epidermophyton occosum, which is one of the most common causes of dermatophytosis in healthy as well as immunocompromised people all over the world. It infects different skin areas producing tinea corporis, tinea cruris and tinea pedis and also nails (tinea unguium or onychomycosis) which are very difcult to eradicate (Weitzmann and Summerbell, 1995). Since there is a considerable overlap of therapies for all supercial infections (caused by Trichophyton and Epidermophyton spp.) leading to a high resistance to the available antifungal agents (Ghannoum and Rice, 1999); the treatment of fungal infections with an appropriate narrow spectrum agent (Di Domenico, 1999) is highly welcome. Regarding the activity displayed by both, the homoisoavanone 3 and its related derivative 4, it is interesting to highlight that, although the activity is very weak, this is the rst report on their antifungal activity. 3.2. Studies of mechanism of action Polygonum ferrugineum DCM extract showed to induce Neurospora crassa hyphae malformations (Zacchino et al., 1998), suggesting that it could act by inhibiting the fungal cell wall. In order to reveal whether the most active compounds 1 and 2 act by a similar mechanism of action than the original extract, they were tested at rst with the agar diffusion Neurospora crassa assay. When this fungus grows in the presence of inhibitors of its wall, a blotchy halo is observed around the paper disk, which, under microscopic observation, shows malformed hyphae (Escalante et al., 2008). In contrast, a clear halo is observed when antifungal compounds act by another mechanism of action. Compound 2, but not 1, produced a hazy halo around the paper disk which, under Optical Microscopy

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S.N. Lpez et al. / Journal of Ethnopharmacology 138 (2011) 633636 Burke, B., Nair, M., 1986. Phenylpropene, benzoic acid and avonoid derivatives from fruits of Jamaican Piper species. Phytochemistry 25, 14271430. Del Vitto, L.A., Petenatti, E.M., Petenatti, M.E., 1997. Recursos herbolarios de San Luis (Repblica Argentina). Primera parte: plantas nativas. Multequina 6, 4966. Di Domenico, B., 1999. Novel antifungal drugs. Current Opinion in Microbiology 2, 509515. Escalante, A.M., Gattuso, M.A., Prez, P., Zacchino, S.A., 2008. Evidence for the mechanism of action of the antifungal phytolaccoside B isolated from Phytolacca tetramera Hauman. Journal of Natural Products 71, 17201725. Espinel-Ingroff, A., 2009. Novel antifungal agents, targets or therapeutic strategies from the treatment of invasive fungal diseases: a review of the literature (20052009). Revista Iberoamericana de Microbiologa 26, 1522. Farka s, V., 2003. Structure and biosynthesis of fungal cell walls: methodological approaches. Folia Microbiologica 48, 469478. Ghannoum, M.A., Rice, L.B., 1999. Antifungal agents: mode of action, mechanisms of resistance and correlation of these mechanisms with bacterial resistance. Clinical Microbiology Reviews 12, 501517. Krishna, B.M., Chaganty, R.B., 1973. Cardamonin and alpinetin from seeds of Alpinia speciosa. Phytochemistry 12, 238242. Lpez, S.N., Gonzlez Sierra, M., Gattuso, S.J., Furlan, R.L.E., Zacchino, S.A., 2006. An unusual homoisoavanone and a structurally-related dihydrochalcone from Polygonum ferrugineum (Polygonaceae). Phytochemistry 67, 21522158. Lpez, S.N., Ramallo, I.A., Gonzlez Sierra, M., Zacchino, S.A., Furlan, R.L.E., 2007. Chemically engineered extracts as an alternative source of bioactive natural product-like compounds. Proceedings of the National Academy of Sciences of the United States of America 104, 441444. Maerz, S., Funakoshi, Y., Negishi, Y., Suzuki, T., Seiler, S., 2010. The Neurospora peptide:N-glycanase ortholog PNG1 is essential for cell polarity despite its lack of enzymatic activity. Journal of Biological Chemistry 285, 23262332. Ramakrishnan, G., Banerji, A., Chadha, M., 1974. Chalcones from Onychium auratum. Phytochemistry 13, 23172318. Seiler, S., Plamann, M., 2003. The genetic basis of cellular morphogenesis in the lamentous fungus Neurospora crassa. Molecular Biology of the Cell 14, 43524364. Svetaz, L.A., Tapia, A., Lpez, S.N., Furlan, R.L.E., Petenatti, E., Pioli, R., SchmedaHirschmann, G., Zacchino, S., 2004. Antifungal chalcones and new caffeic acid esters from Zuccagnia punctata acting against soybean infecting fungi. Journal of Agricultural and Food Chemistry 52, 32973300. Weitzmann, I., Summerbell, R., 1995. The dermatophytes. Clinical Microbiology Reviews 8, 240259. Wright, L., Scott, E., Gorman, S., 1983. The sensitivity of mycelium, arthrospores and microconidia of Trichophyton mentagrophytes to imidazoles determined by in-vitro tests. Journal of Antimicrobial Chemotherapy 12, 317327. Zacchino, S., Santecchia, C., Lpez, S., Gattuso, S., Munoz, J., Cruanes, A., Vivot, E., M., Salinas, A., Ruiz, R., Ruiz, S., 1998. In vitro antifungal evaluation and Cruanes, studies on mode of action of eight selected species from the Argentine Flora. Phytomedicine 5, 389395.

(Fig. 1A and C) and Scanning Electron Microscopy (Fig. 1B and D) showed round tips and swelling of the hyphae. Interestingly, the observed malformations matched to png-1 polarity defecting mutants of Neurospora crassa (Seiler and Plamann, 2003) which showed to be involved in cell wall integrity (Maerz et al., 2010). 4. Conclusions The bioassay-guided fractionation of DCME of Polygonum ferrugineum allowed the detection of cardamonin 2 as the compound with the highest antifungal activity, selective towards Epidermophyton occosum. Chalcone 1 possessed moderate activity against the whole panel; 3 and 4 showed weak activities only against dermatophytes and 5 showed marginal activities against dermatophytes. The two compounds isolated from HE , pinostrobin and avokawin, were devoid of antifungal activities. Among active compounds, only 2 showed to be inducer of similar Neurospora crassa malformations than the crude DCM extract. These results give additional support to the ethnopharmacological use of Polygonum ferrugineum as antifungal agent in traditional medicine. Acknowledgements Authors thank to International Foundation for Science (IFS, Stockholm); Organisation for the Prohibition of Chemical Weapons (OPCW, The Hague) (F/3114 2F); and Agencia de Promocin Cientca y Tecnolgica de Argentina, PICT328 to SNL, PICT995 to RLEF and PICT608 to SAZ. SNL and RLEF are members of the Research Career of Consejo Nacional de Investigaciones Cientcas y Tcnicas (CONICET). References
Ahmed, M., Khaleduzzaman, M., Rashid, M., 1981. Chalcone derivatives from Polygonum lapathifolium. Phytochemistry 20, 25032506. Bratoeff, E.A., Perez-Amador, M.C., 1994. Phytochemical study of Typha dominguensis Pers. (Typhaceae). Phyton 55, 7175.