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Antimicrobial, antiviral and cytotoxic activity of extracts and constituents from Polygonum spectabile Mart.

ABSTRACT

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-[beta]-D-glucosyl-[beta]-sitosterol were isolated. The ethanol extract was the only one to show antiviral activity (CE50 < 30 [mu]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 [LLCMK.sub.2] cells ([CC.sub.50] <50 [mu]g/ml). These results support the use of P. spectabile as antimicrobial agent.

[C] 2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Polygonaceae Polygonum spectabile Antimicrobial Antiviral Cytotoxicity

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 at. 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 Correa 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 Correa 1978). The present paper reports on 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 Agropecuaria 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 [degrees]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. 18.7g), 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).

Identification of compounds 1-3

Spectral analysis (IR, UV, MS, (1) H NMR, (13) C NMR, NOESY, TOCSY, HMQC, HMBC) and comparison with literature data allowed the identification of compounds 1-3.

Bacteria and fungi

The microbial panel included laboratory control strains from the American Type Culture Collection (Rockville, MD, USA), Fundacao Andre Tosello (Campinas, SP, Brazil) from the Laboratorio de Biologia Geral, ICB, UFMG (Belo Horizonte, MG, Brazil) and the following yeasts clinically isolated from humans: Mycrosporum canis, Tricophyton mentagrophytes, T. rubrum.

Cell culture and virus

Vero cells (ATCC CCL-81) and [LLCMK.sub.2] cells were cultured in Dulbecco's modified Eagle's medium (DMEM) at 37 [degrees]C, in 5% [CO.sub.2] atmosphere, supplemented with 5% fetal bovine serum, 50[mu]g/ml gentamicin, 100U/ml penicillin and 5 [mu]g/ml fungizone. HHV-1 was a clinical isolate of Human herpes virus 1 (HHV-1) obtained in the Laboratory of Virus, UFMG, Belo Horizonte, Brazil. Dengue virus 2 (DEN-2), Encephalomyocarditis murine virus (EMCV) and Vaccinia virus strain Western Reserve (VACV-WR) were kindly donated by Dr. L. Figueiredo (USP, Ribeirao Preto, Brazil), Dr. I. Kerr (London Research Institute, London, UK) and Dr. C. Jungwirth (University of Wurzburg, Germany), respectively.

Antimicrobial activity

Disc-diffusion method. 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, dichloromethane, ethyl acetate and methanol) and the solutions were sterilized by filtration through 0.44 [mu]m Millipore membranes. Antimicrobial tests were carried out by the disc-diffusion method (Gavin 1957). Chloramphenicol (3 [mu]g/disc), gentamicin (10[mu]g/disc) and amphotericin B (32 [mu]g/disc) were used as positive reference standards. Each sample was tested in six replicas (Table 1).
Table 1

Antimicrobial activity of Polygonum spectabile extracts (2.0mg/disc)
and compounds 1, 2 and 3 (10.0 [mu]g/disc) by the disc-diffusion
method.

Microorganisms Inhibition zone diameter around test discs (mm)

 HE DE AE

S. aureus NA NA 8.8 [+ or -] 0.8
 (a)

M. luteus NA NA NA

B. subtilis NA NA 7.9 [+ or -] 0.9

S. epidermides NA NA NA

E. coli NA NA NA

P. aeruginosa NA NA NA

P. vulgares NA NA NA

S. marcescens NA NA NA

C. albicans NA NA NA

C. tropicalis NA NA NA

S. cerevisiae NA NA NA

A. niger NA NA NA

T. 18.5 [+ or -] 0.8 11.5 [+ or -] 0.8 12.0 [+ or -] 0.9
mentagrophytes

Tricophyton 10.5 [+ or -] 1.1 10.5 [+ or -] 0.5 9.5 [+ or -] 0.6
rubrum

M. canis 9.5 [+ or -] 1.4 8.5 [+ or -] 0.9 -

Microorganisms Inhibition zone diameter around test discs (mm)

 EE 1 2 3

S. aureus 7.5 [+ or -] 0.5 NA 15.2 [+ or -] 0.7 (a) NA

M. luteus 9.5 [+ or -] 0.5 NA 11.3 [+ or -] 0.5 NA

B. subtilis NA NA 12.7 [+ or -] 0.8 NA

S. epidermides NA NA 11.3 [+ or -] 0.5 NA

E. coli NA NA 12.7 [+ or -] 0.4 NA

P. aeruginosa NA NA 13.8 [+ or -] 0.1 NA

P. vulgares NA NA 14.3 [+ or -] 0.6 NA

S. marcescens NA NA 14.6 [+ or -] 0.8 NA

C. albicans NA NA NA NA

C. tropicalis NA NA NA NA

S. cerevisiae NA NA NA NA

A. niger NA NA NA NA

T. NA NA 24.7 [+ or -] 1.2 NA
mentagrophytes

Tricophyton NA NA 24.3 [+ or -] 0.8 NA
rubrum

M. canis NA NA 14.6 [+ or -] 1.3 NA

Microorganisms Inhibition zone diameter around test discs (mm)

 Chlo Gen Amp B

S. aureus 15.1 [+ or -] 0.9

M. luteus 20.1 [+ or -] 0.7

B. subtilis 14.3 [+ or -] 0.9

S. epidermides 24.1 [+ or -] 0.7

E. coli 12.9 [+ or -] 0.8

P. aeruginosa 12.8 [+ or -] 0.6

P. vulgares 16.5 [+ or -] 0.8

S. marcescens 10.0 [+ or -] 0.8

C. albicans 12.9 [+ or -] 0.8

C. tropicalis 14.5 [+ or -] 0.5

S. cerevisiae 15.1 [+ or -] 0.8

A. niger 12.2 [+ or -] 0.7

T. 9.5 [+ or -] 0.7
mentagrophytes

Tricophyton 12.2 [+ or -] 0.8
rubrum

M. canis 11.9 [+ or -] 0.8

(a) Inhibition zone diameter around test discs with n = 6, NA: no
activity, HE: n-hexane extract, DE: dichloromethane extract, AE: ethyl
acetate extract, EE: ethanol extract, chloramphenicol (3.0 mg/disc),
gentamicin (10.0 mg/disc) and, amphotericin B (32.0 mg/disc) were used
as positive reference standard antimicrobial discs, Chlo =
chloramphenicol, Gen = gentamicin, Amp B = amphotericin B.


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). 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 1.95 [mu]g/ml. Chloramphenicol (Sigma) and gentamicin (Sigma) were used as positive controls. Drug free solution was used as a blank control.

Cytotoxicity assay

Cytotoxicity of extracts and compounds 1-3 (500-0.125 [mu]g/ml) to Vero and [LLCMK.sub.2] cells was determined by the MTT assay (Merck solution 2 mg/ml in PBS) (Twentyman and Luscombe 1987). Each sample was assayed in four replicates with at least four concentrations.
Table 2

MIC and [IC.sub.50] ([mu]g/ml) for chalcone 2 against the
microorganisms tested by the microdilution assay.

 Compound 2 Chloramphenicol

 MIC ([mu]g/ml) [IC.sub.50] MIC ([mu]g/ml)
 ([mu]g/ml)

S. aureus >250.0 (b) 67.2 [+ or -] 2.4 8.0
M. luteus 250.0 35.9 [+ or -] 1.6 8.0
S. epidermides >250.0 62.4 [+ or -] 2.8 8.0
B. subtilis 250.0 36.6 [+ or -] 2.5 8.0
E. coli 250.0 32.5 [+ or -] 1.0
P. aeruginosa 125.0 18.9 [+ or -] 1.3
P. vulgares 125.0 16.4 [+ or -] 0.9
S. marcescens 125.0 20.6 [+ or -] 1.1
T. mentagrophytes 12.0 1.1 [+ or -] 0.2
T. rubrum 12.0 1.4 [+ or -] 0.1
M. canis 18.0 3.0 [+ or -] 0.3

 Chloramphenicol Gentamicin

 [IC.sub.50 MIC [IC.sub.50]
 ([mu]g/ml) ([mu]g/ml) ([mu]g/ml)

S. aureus <0.3
M. luteus <0.3
S. epidermides <0.3
B. subtilis <0.3
E. coli 1.0 <0.5
P. aeruginosa 1.0 <0.5
P. vulgares 1.0 <0.5
S. marcescens 1.0 <0.5
T. mentagrophytes
T. rubrum
M. canis

 Amphotericin (B)

 MIC (a) ([mu]g/ml)
S. aureus
M. luteus
S. epidermides
B. subtilis
E. coli
P. aeruginosa
P. vulgares
S. marcescens
T. mentagrophytes 6.2
T. rubrum 25.0
M. canis >50

(a) Literature data (Lopez et al. 2001).
(b) MIC minimal inhibition concentration, [IC.sub.50] median inhibitory
concentration, chloramphenicol and gentamicin were used as positive
reference standard antibiotics.


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 at. 1990). The determined titers were 2.5 x [10.sup.6], 1.0 x [10.sup.6], 1.0 x [10.sup.6] and 1.0 x [10.sup.4] [TCID.sub.100/ml], respectively, for HHV-1, EMCV, VACV-WR and DEN-2 virus. The antiviral activity ([EC.sub.50]) was evaluated by the MTT colorimetric assay (Betancur-Galvis et al. 1999). Acyclovir (Calbiochem, USA) and [alpha]-2a interferon (Bergamo, Brazil) were used as positive controls. Cytotoxicity ([CC.sub.50]) was evaluated by the MTT colorimetric method. Experiments were carried out with four different concentrations within the inhibitory range of the samples. The therapeutic index (i.e. selective index. SI) is defined as [CC.sub.50]/[EC.sub.50].

Results and discussion

Extracts from aerial parts of P. spectabile were shown to be effective against S. aureus, B. subtillis, M. luteus, M. canis, T. mentagrophytes, T. rubrum, DEN-2, VACV-WR and HHV-1 (Tables 1 and 3). HE was significantly active against T. 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). No inhibition of Gram-negative bacteria and yeasts has been observed with extracts at the concentration of 2.0 mg per disk.

Compounds 1-3 were isolated from HE and AE and were identified as 2'-hydroxy-4',6'-dimethoxychalcone (1), 2',4'-dihydroxy-3',6'-dimethoxychalcone (2) and 3-O-[beta]-D-glucosyl-[beta]-sitosterol (3) (Fig. 1) by the usual spectrometric techniques (UV, IR, [.sup.1.H] and [.sup.13.C] NMR and MS) and comparison with literature data (Maradufu and Ouma 1978). Chalcones 1 and 2 have been firstly isolated from P. lapathifolium (apud Ahmed et al. 1990) and P. senegalense (Maradufu and Ouma 1978), respectively; occurrence of chalcone 2 is rarely reported. Chalcone 2 was the only compound to show antimicrobial activity (Table 1) with a broad spectrum of action. It has inhibited the growth of Gram-positive and Gram-negative bacteria and was remarkably active against the dermatophytes T. mentagrophytes and T. rubrum (Table 1). MIC and [IC.sub.50] values for chalcone 2 ranged from 12.0 to >250.0 [mu]g/ml and from 1.1 to 67.2 [mu]g/ml, respectively (Table 2) and it is clear that it has a good antifungal activity but a low antibacterial activity. These results, in addition to literature data (Lopez et al. 2001), reinforces that chalcones are promising as new drugs to treat dermathophyte infections.

[FIGURE 1 OMITTED]

Results of the antiviral assays of P. spectabile extracts and compounds 1-3 against four virus strains are depicted in Table 3. Inhibition of viral replication was observed only with the EE extract which has shown low cytotoxicity for Vero and [LLCMK.sub.2] cells ([CC.sub.50] > 500 [mu]g/ml). The effective concentrations ([EC.sub.50]) were 24.6, 34.2 and 21.9 [mu]g/ml for DEN-2, VACV-WR and HHV-1, respectively. Other Polygonum species have shown activity against several viral strains such as HHV-1, Human respiratory syncytial virus, Human adenovirus, Hepatitis B virus (HBV) (Zheng 1988; Kott et al. 1999; Chang et al. 2005). Compounds 1-3 isolated from P. spectabile have shown no activity against the assayed viral strains but have been moderately cytotoxic to Vero and [LLCMK.sub.2] cells exhibiting [CC.sub.50] between 7.8 and 31.5 [mu]g/ml. Further investigation is required to identify the antiviral compounds of the ethanol extract which was shown by RP-HPLC-DAD to contain stilbene glycosides as major constituents (data not shown).
Table 3

In vitro cytotoxic and antiviral activity of P. spectabile extracts and
compounds 1-3.

Extracts/compounds Vero cells [CC.sub.50] [LLCMK.sub.2] cells
 [mu]g/ml [CC.sub.50] [mu]g/ml

HE 83.6 [+ or -] 4.6 71.3 [+ or -] 3.5

DE 128.2 [+ or -] 6.9 117.9 [+ or -] 7.2

AE >500 >500

EE >500 >500

1 11.7 [+ or -] 1.2 16.4 [+ or -] 2.1

2 31.5 [+ or -] 1.3 29.6 [+ or -] 1.8

3 27.2 [+ or -] 3.7 7.8 [+ or -] 1.5

Acyclovir

[alpha]-2a
Interferon

Extracts/compounds HHV-1 (a) SI VACV-WR (b) SI
 [EC.sub.50] [EC.sub.50]
 [mu]g/ml [mu]g/ml

HE NA NA

DE NA NA

AE NA NA

EE 21.9 [+ or -] 1.8 >22.8 34.2 [+ or -] 2.4 >14.6

1 NA NA

2 NA NA

3 NA NA

Acyclovir 40 (d)

[alpha]-2a 2.5 x [10.sup.2]
Interferon (d), (e)

Extracts/compounds EMCV (b) [EC.sub.50] DENV-2 (c) SI
 [mu]g/ml [EC.sub.50] [mu]g/ml

HE NA NA

DE NA NA

AE NA NA

EE NA 24.6 [+ or -] 3.7 >20.3

1 NA NA

2 NA NA

3 NA NA

Acyclovir

[alpha]-2a 1.5 x [10.sup.2] (d), 2.5 x [10.sup.3] (d),
Interferon (e) (e)

SI, selective index. NA, no activity in the assayed concentrations.
(a) Viral titer [TCID.sub.100]/ml 2.5 x [10.sup.6] in 48 h.
(b) Viral titer [TCID.sub.100]/ml 1.0 x [10.sup.6] in 48 h.
(c) Viral titer [TCID.sub.100]/ml 1.0 x [10.sup.4] in 72 h.
(d) 80-100% inhibition of cytopathic effect.
(e) Concentration in UI/ml.


Conclusion

The present results support the ethnopharmacological use of this species as anti-infectious agent. Chalcone 2 was shown to present good in vitro activity against dermatophytes. Polar compounds are expected to be responsible for the antiviral activity of the EE extract which has disclosed significant effect against HHV-1, VACV and DEN 2. This is the first report on the chemistry, antimicrobial, antiviral and cytotoxic activities of P. spectabile.

Acknowledgements

To CNPq and FAPEMIG for financial support (ABO, EGK) and PhD fellowship (GCB); to Dr. E. A. Nunan, Faculdade de Farmacia/UFMG for helpful assistance in the antimicrobial assays, and to Dr. J. F. Macedo/EPAMIG for collection and taxonomic identification of P. spectabile.

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Antimicrobial, antiviral and cytotoxic activity of extracts and constituents from Polygonum spectabile Mart. *

Geraldo Celio Brandao (a), Erna Gessien Kroon (b), Maria Gorette R. Duarte (a), Fernao Castro Braga (a), Jose Dias de Souza Filho (c), Alaide Braga de Oliveira (a), *

(d) Departamento de Produtos Farmaceuticos, Faculdade de Farmacia, UFMG, Av. Antonio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil

(b) Departamento de Microbiologia, Instituto de Ciencias Biologicas, UFMG, Av. Antonio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil

(c) Departamento de Quimica, Instituto de Ciencias Exatas, UFMG, Av. Antonio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil

* Part of G. C. Brandao's PhD thesis at the Curso de Pos-Graduacao em Ciencias Farmaceuticas - CPGCF, Faculdade de Farmacia, UFMG, Belo Horizonte, MG, Brazil.

* Corresponding author.

E-mail address: alaidebraga@terra.com.br (A.B. de Oliveira).

0944-7113/$ - see front matter [C] 2010 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2010.03.004
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Author:Brandao, Geraldo Celio; Kroon, Erna Gessien; Duarte, Maria Gorette R.; Braga, Fernao Castro; de Souz
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Oct 1, 2010
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