Phytomedicine 17 (2010) 926–929Contents lists available at ScienceDirect Phytomedicine journal homepage: www.elsevier.de/phymed Antimicrobial, antiviral and cytotoxic activity of extracts and constituents from Polygonum spectabile Mart.夽 Geraldo Célio Brandão a , Erna Gessien Kroon b , Maria Gorette R. Duarte a , Fernão Castro Braga a , José Dias de Souza Filho c , Alaíde Braga de Oliveira a,∗ a b c Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, UFMG, Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil Departamento de Microbiologia, Instituto de Ciências Biológicas, UFMG, Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil Departamento de Química, Instituto de Ciências Exatas, UFMG, Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil a r t i c l e Keywords: Polygonaceae Polygonum spectabile Antimicrobial Antiviral Cytotoxicity i n f o a b s t r a c t Polygonum spectabile is used in Brazil for treatment of several infection diseases. Extracts and constituents isolated from this species were evaluated for cytotoxicity and effects on 15 bacterias and yeasts as well on 4 viruses strains (HHV-1, VACV-WR, EMCV, DEN-2). Less polar extracts were effective against Staphylococcus aureus, Bacillus subtillis, Micrococcus luteus, M. canis and Tricophyton mentagrophytes and T. rubrum. Two known chalcones and 3-O--d-glucosyl--sitosterol were isolated. The ethanol extract was the only one to show antiviral activity (CE50 < 30 g/ml). One chalcone has inhibited the growth of several bacteria and was significantly active against dermathophytes. The 3 compounds isolated have shown moderate cytotoxicity against Vero and LLCMK2 cells (CC50 <50 g/ml). These results support the use of P. spectabile as antimicrobial agent. © 2010 Elsevier GmbH. All rights reserved. Introduction Emergent viruses, bacterial strains resistant to antibiotics clinically available and the incidence of opportunistic fungal infections, especially involving inmunocompromised patients, have increased in recent decades (Mishra et al. 2007). In particular, some forms of dermatomycoses are cause of great morbidity in patients receiving antineoplastic chemotherapy, undergoing organ transplants, or suffering from AIDS (De Lencastre et al. 2007). In the last years, reemergence of vaccinia virus (Trindade et al. 2007) and outbreaks of dengue virus have been registered in Brazil. The quest of new antimicrobial and antiviral drugs by evaluation of in vitro antimicrobial and antiviral activity of plants of traditional and/or popular use is of great interest (Bhattacharjee et al. 2006; Koduru et al. 2006). Polygonum spectabile Mart. (Polygonaceae) is a plant native to swampy areas of South America, with wide occurrence in Brazil, Uruguay and Argentina (Pio Corrêa 1978). In Brazil, it is popularly named erva-de-bicho, and is used for the treatment of diarrhea, ulcers, gingivitis, rheumatism, and skin affections, among others (Pio Corrêa 1978). The present paper reports on 夽 Part of G. C. Brandão’s PhD thesis at the Curso de Pós-Graduac¸ão em Ciências Farmacêuticas – CPGCF, Faculdade de Farmácia, UFMG, Belo Horizonte, MG, Brazil. ∗ Corresponding author. E-mail address:
[email protected] (A.B. de Oliveira). 0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.03.004 the investigation of the antimicrobial and antiviral activity of different extracts and compounds isolated from the aerial parts of P. spectabile. Material and methods Plant material Aerial parts of P. spectabile were collected in the municipality of Belo Horizonte, Minas Gerais state, Brazil. The species were identified by Dr. J. F. Macedo, Empresa Agropecuária de Minas Gerais (EPAMIG), Belo Horizonte, Brazil, where voucher specimens are deposited under the code PAMG-55256. Extraction and isolation of compounds 1–3 After drying in a ventilated oven, at 40 ◦ C for 72 h, aerial parts of P. spectabile (2.320 kg) were powdered and extracted by exhaustive sequential percolation affording the hexane extract (HE, 25.1 g), the dichloromethane extract (DE, 18.7 g), the ethyl acetate extract (AE 10.1 g) and the ethanol extract (EE, 253.8 g). HE and AE were shown to be active in the antimicrobial assays and EE was significantly active against HHV-1, VACV and DEN-2. HE (8.5 g) and AE (10.0 g) were submitted to a series of separations by column chromatography and HPLC affording compounds 1 (40 mg), 2 (53 mg) and 3 (53 mg). 7 0. Figueiredo (USP. amphotericin B (32.0 mg/disc) and compounds 1. Dengue virus 2 (DEN-2).6 ± 1. Microdilution method The Minimal Inhibitory Concentrations (MIC) were determined for the microorganisms which were sensitive to compound 2 in the disc-diffusion assay (Eloff 1998).1 20. Tricophyton mentagrophytes.6 16. in 5% CO2 atmosphere. HHV-1 was a clinical isolate of Human herpes virus 1 (HHV-1) obtained in the Laboratory of Virus. 2 and 3 (10. L.5 12. HMBC) and comparison with literature data allowed the identification of compounds 1–3.7 ± 0.8 10. London. 1 H NMR.9 Gen ± ± ± ± ± Amp B 0.5 ± 0.8 14. aureus M. MS. Gen = gentamicin.7 0. 1. luteus B.2 9.5 ± 0. aeruginosa P.7 ± 0.5 ± 0. rubrum. Dr.8 ± 0. Brazil.9 NA NA NA NA NA NA NA NA NA 12. Brazil) and the following yeasts clinically isolated from humans: Mycrosporum canis.7 0. The dried plant extracts (100 mg) and isolated compounds (10 mg) were dissolved in the same solvent of the extract from their origin (n-hexane.44 m Millipore membranes. UFMG. Encephalomyocarditis murine virus (EMCV) and Vaccinia virus strain Western Reserve (VACV-WR) were kindly donated by Dr.4 -dihydroxy-3 . 50 g/ml gentamicin.9 0. tropicalis S.95 g/ml.1 12.1 12.1 14.9 ± 0.6 14. niger T. Identification of compounds 1–3 Spectral analysis (IR. subtilis S. UFMG (Belo Horizonte.0 ± 0.1 9. NA: no activity.G. DE: dichloromethane extract.5 0. gentamicin (10.5 12.5 ± 0. Amp B = amphotericin B. ethyl acetate and methanol) and the solutions were sterilized by filtration through 0.0 mg/disc).8 12.0 mg/disc) were used as positive reference standard antimicrobial discs.4 13.8 11. vulgares S.7a 11. epidermides E.8 12. Chemical structures of 2 -hydroxy-4 .7 0.3 ± 0.125 g/ml) to Vero and LLCMK2 cells was determined by the MTT assay (Merck solution 2 mg/ml in PBS) (Twentyman and Luscombe 1987). dichloromethane. HMQC.9 ± ± ± ± ± ± 0.5 ± 0.8 10.5 12. Ribeirão Preto. cerevisiae A. C.2 ± 0. Chloramphenicol (Sigma) and gentamicin (Sigma) were used as positive controls. Microorganisms S. 2 .8 0.6 -dimethoxychalcone (2). Belo Horizonte.8 0. canis Inhibition zone diameter around test discs (mm) HE DE AE EE 1 2 3 Chlo NA NA NA NA NA NA NA NA NA NA NA NA 18. Stock solution of compound 2 in DMSO was diluted to give serial twofold dilutions that were added to each medium resulting in concentrations ranging from 250 to 1.8a NA 7. HE: n-hexane extract.8 14. UK) and Dr. MG.3 ± 0.9 0.C. I. and 3-O--d-glucosyl--sitosterol (3).0 ± 0. Jungwirth (University of Würzburg.7 ± 1. NOESY.1 14.8 NA NA NA NA 24.9 8.3 ± 0. Brazil). gentamicin (10 g/disc) and amphotericin B (32 g/disc) were used as positive reference standards. Cytotoxicity assay Cytotoxicity of extracts and compounds 1–3 (500–0. Bacteria and fungi The microbial panel included laboratory control strains from the American Type Culture Collection (Rockville.5 15.5 ± 0. Cell culture and virus Vero cells (ATCC CCL-81) and LLCMK2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) at 37 ◦ C. USA).6 ± 0. UV. AE: ethyl acetate extract.0 mg/disc) and. ICB. Chlo = chloramphenicol. Each Fig.9 9.6 -dimethoxychalcone (1). Antimicrobial activity Disc-diffusion method.3 ± 0. Brandão et al.3 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 15. 13 C NMR. chloramphenicol (3.2 24.3 24.5 ± 1. marcescens C.4 NA NA NA NA NA NA NA NA NA NA NA NA 11. albicans C. Brazil) from the Laboratório de Biologia Geral. MD. EE: ethanol extract. supplemented with 5% fetal bovine serum. SP. Antimicrobial tests were carried out by the disc-diffusion method (Gavin 1957). Drug free solution was used as a blank control.8 ± 0. 100 U/ml penicillin and 5 g/ml fungizone. Kerr (London Research Institute. Chloramphenicol (3 g/disc).5 ± 1.9 ± 0.2 11. Each sample was tested in six replicas (Table 1). .5 ± 0.6 – 7.8 ± 0. TOCSY.5 9. respectively.5 8.0 g/disc) by the disc-diffusion method. / Phytomedicine 17 (2010) 926–929 927 Table 1 Antimicrobial activity of Polygonum spectabile extracts (2.5 ± 0. Fundac¸ão André Tosello (Campinas.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 15. mentagrophytes Tricophyton rubrum M.8 10. T.8 a Inhibition zone diameter around test discs with n = 6. coli P. Germany). 8 VACV-WRb EC50 g/ml NA NA NA 34. aureus.4 ± 2.3 <0. respectively. spectabile were shown to be effective against S.5 <0.5 ± 1. The effective concentrations (EC50 ) were 24. rubrum M.6 ± 4.2 ± 6.3 <0.1 1. DEN-2.2 ± 3.0 125.0 <0.5 <0.0 12. for HHV-1. Other Polygonum species have shown activity against several viral strains such as HHV-1.0 250.5 117.8 ± 1. subtillis. e 2. aeruginosa P.6. Human respiratory syncytial virus.2 and 21. 1. EMCV.8 7.5 <0. 2001). 1.0 ± ± ± ± ± ± ± ± ± ± ± 2. a Viral titer TCID100 /ml 2. Compound 2 S.3 ± 3.0 to >250. subtilis E.6 1. The determined titers were 2.0 125.5 1.5 × 103 d . VACV-WR and DEN-2 virus. senegalense (Maradufu and Ouma 1978). MIC and IC50 values for chalcone 2 ranged from 12.4 20. 1) by the usual spectrometric techniques (UV.3 Gentamicin MIC (g/ml) IC50 (g/ml) 8.9 ± 1.5 × 106 in 48 h.9 62.6 EMCVb EC50 g/ml DENV-2c EC50 g/ml NA NA NA NA NA NA NA NA NA NA 24.0 g/ml and from 1.3 Amphotericin B MIC (g/ml) IC50 (g/ml) 1. marcescens T.0 × 104 in 72 h.5 g/ml.0 mg per disk. rubrum. VACV-WR and HHV-1.6 2. in addition to literature data (López et al. Acyclovir (Calbiochem. b Viral titer TCID100 /ml 1.0 1.0 >250.928 G. 2001). Brazil) were used as positive controls.5 × 102 d . 34.0 × 106 and 1. respectively. vulgares S. 2005).8 2.6 -dimethoxychalcone (1). respectively (Table 2) and it is clear that it has a good antifungal activity but a low antibacterial activity. The antiviral activity (EC50 ) was evaluated by the MTT colorimetric assay (BetancurGalvis et al.4 -dihydroxyTable 3 In vitro cytotoxic and antiviral activity of P.4 1.1 to 67. Experiments were carried out with four different concentrations within the inhibitory range of the samples.5 × 102 d . M. spectabile have shown no activity against the assayed viral strains but have been moderately cytotoxic to Vero and LLCMK2 cells exhibiting CC50 between 7.9 >500 >500 11.6 ± 3. 3 . NA. Compounds 1–3 were isolated from HE and AE and were identified as 2 -hydroxy-4 . It has inhibited the growth of Gram-positive and Gram-negative bacteria and was remarkably active against the dermatophytes T. mentagrophytes and T.2 g/ml. 2 .0 1.2 31. chloramphenicol and gentamicin were used as positive reference standard antibiotics. 1990) and P. These results.7 NA NA NA 1.6 ± 1.9 16.0 1. mentagrophytes with an inhibition zone diameter of approximately twofold that one of amphotericin B and close to the one of this reference compound for DE and AE (Table 1).2 >500 >500 16. 1999.6 -dimethoxychalcone (2) and 3-O--d-glucosyl--sitosterol (3) (Fig. aureus M. Hepatitis B virus (HBV) (Zheng 1988.0 12.7 71. Further investigation is required to identify the antiviral compounds of the ethanol extract which was sample was assayed in four replicates with at least four concentrations. spectabile extracts and compounds 1–3. IR.8 and 31. respectively. e Concentration in UI/ml. mentagrophytes T. 1990). VACV-WR and HHV-1 (Tables 1 and 3). MIC minimal inhibition concentration.9 g/ml for DEN-2.0 8.0 × 106 .2 ± 2. spectabile extracts and compounds 1–3 against four virus strains are depicted in Table 3.9 ± 7. selective index.0 1.3 .7 ± 1.3 27.1 0. coli P.0 × 106 in 48 h. T.0 8. Chalcone 2 was the only compound to show antimicrobial activity (Table 1) with a broad spectrum of action. occurrence of chalcone 2 is rarely reported.0 67.0 8.0 250. The therapeutic index (i.e. Human adenovirus.2 25.0 <0.6 32.3 <0. No inhibition of Gram-negative bacteria and yeasts has been observed with extracts at the concentration of 2. luteus.C. Cytotoxicity (CC50 ) was evaluated by the MTT colorimetric method. 1 H and 13 C NMR and MS) and comparison with literature data (Maradufu and Ouma 1978).2 0. Inhibition of viral replication was observed only with the EE extract which has shown low cytotoxicity for Vero and LLCMK2 cells (CC50 > 500 g/ml). T.3 0. B. Results and discussion Extracts from aerial parts of P. Kott et al. canis. SI) is defined as CC50 /EC50 . USA) and ␣-2a interferon (Bergamo. Results of the antiviral assays of P. Brandão et al.5 × 106 .1 29. luteus S.0 × 104 TCID100/ml .6 128.4 36.5 NA NA NA 21. c Viral titer TCID100 /ml 1. Extracts/compounds Vero cells CC50 g/ml LLCMK2 cells CC50 g/ml HHV-1a EC50 g/ml HE DE AE EE 1 2 3 Acyclovir ␣-2a Interferon 83.4 NA NA NA 2. epidermides B. e SI >20. lapathifolium (apud Ahmed et al.0b 250.5 18. d 80–100% inhibition of cytopathic effect. / Phytomedicine 17 (2010) 926–929 Table 2 MIC and IC50 (g/ml) for chalcone 2 against the microorganisms tested by the microdilution assay. selective index. e SI >14.1 0.9 1.4 3. rubrum (Table 1).0 >50 Literature data (López et al. Compounds 1–3 isolated from P.0 125. IC50 median inhibitory concentration. no activity in the assayed concentrations. reinforces that chalcones are promising as new drugs to treat dermathophyte infections. SI >22. M.2 35. Chang et al. canis a b Chloramphenicol MIC (g/ml) IC50 (g/ml) >250. mentagrophytes.0 18.8 NA NA NA 40d SI.5 MICa (g/ml) 6. Antiviral assay The viral samples were titrated by the TCID microculture assay after 48 h incubation for HHV-1 and EMCV and 72 h for VACV-WR and DEN-2 (Rodriguez et al. Chalcones 1 and 2 have been firstly isolated from P. 1999). HE was significantly active against T. . Pharm. M. S. Cavallaro. Martino. A... H. 1999. Chin. Microbiol.C. Oliveira. De Lencastre.. 1999. Inst. F.. Chem. Macedo/EPAMIG for collection and taxonomic identification of P. J.. R. 2009–2011.. Polar compounds are expected to be responsible for the antiviral activity of the EE extract which has disclosed significant effect against HHV-1. Saez.. IBDF.. 1998. Gupta..... Chen. 1978. An experimental study of antiviral action of 472 herbs on herpes simplex virus. Betancur-Galvis. Br. Salazar... D. Koduru. A. M. Cancer 56.S.M. V. J. Mariotte. Emerg. Tomasz. E. L. 29–34. L.G.. 9. Med. Damon.. 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