Phytomedicine 17 (2010) 926929

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Phytomedicine
journal homepage: www.elsevier.de/phymed

Antimicrobial, antiviral and cytotoxic activity of extracts and constituents from

Polygonum spectabile Mart.
Geraldo Clio Brando a , Erna Gessien Kroon b , Maria Gorette R. Duarte a ,
Ferno Castro Braga a , Jos Dias de Souza Filho c , Alade Braga de Oliveira a,
a
b
c

Departamento de Produtos Farmacuticos, Faculdade de Farmcia, UFMG, Av. Antnio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
Departamento de Microbiologia, Instituto de Cincias Biolgicas, UFMG, Av. Antnio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
Departamento de Qumica, Instituto de Cincias Exatas, UFMG, Av. Antnio 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 signicantly 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 Corra 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 Corra 1978). The present paper reports on

Part of G. C. Brandos PhD thesis at the Curso de Ps-Graduaco em Cincias

Farmacuticas CPGCF, Faculdade de Farmcia, UFMG, Belo Horizonte, MG, Brazil.
Corresponding author.
E-mail address: alaidebraga@terra.com.br (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 identied by Dr. J. F. Macedo, Empresa Agropecuria de Minas Gerais
(EPAMIG), Belo Horizonte, Brazil, where voucher specimens are
deposited under the code PAMG-55256.

Extraction and isolation of compounds 13

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 signicantly 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).

G.C. Brando et al. / Phytomedicine 17 (2010) 926929

927

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

S. aureus
M. luteus
B. subtilis
S. epidermides
E. coli
P. aeruginosa
P. vulgares
S. marcescens
C. albicans
C. tropicalis
S. cerevisiae
A. niger
T. mentagrophytes
Tricophyton rubrum
M. canis

Inhibition zone diameter around test discs (mm)

HE

DE

AE

EE

Chlo

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
18.5 0.8
10.5 1.1
9.5 1.4

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
11.5 0.8
10.5 0.5
8.5 0.9

8.8 0.8a
NA
7.9 0.9
NA
NA
NA
NA
NA
NA
NA
NA
NA
12.0 0.9
9.5 0.6

7.5 0.5
9.5 0.5
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

15.2 0.7a
11.3 0.5
12.7 0.8
11.3 0.5
12.7 0.4
13.8 0.1
14.3 0.6
14.6 0.8
NA
NA
NA
NA
24.7 1.2
24.3 0.8
14.6 1.3

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

15.1
20.1
14.3
24.1
12.9

Gen

Amp B

0.9
0.7
0.9
0.7
0.8
12.8 0.6

16.5 0.8
10.0 0.8
12.9 0.8
14.5
15.1
12.2
9.5
12.2
11.9

0.5
0.8
0.7
0.7
0.8
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.

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

Bacteria and fungi

The microbial panel included laboratory control strains from the
American Type Culture Collection (Rockville, MD, USA), Fundaco
Andr Tosello (Campinas, SP, Brazil) from the Laboratrio 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 LLCMK2 cells were cultured in
Dulbeccos modied Eagles medium (DMEM) at 37 C, in 5% CO2
atmosphere, supplemented with 5% fetal bovine serum, 50 g/ml
gentamicin, 100 U/ml penicillin and 5 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, Ribeiro Preto, Brazil), Dr. I. Kerr (London
Research Institute, London, UK) and Dr. C. Jungwirth (University of
Wrzburg, 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 ltration
through 0.44 m Millipore membranes. Antimicrobial tests were
carried out by the disc-diffusion method (Gavin 1957). Chloramphenicol (3 g/disc), gentamicin (10 g/disc) and amphotericin B
(32 g/disc) were used as positive reference standards. Each sample was tested in six replicas (Table 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). 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 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 13 (5000.125 g/ml)
to Vero and LLCMK2 cells was determined by the MTT assay (Merck
solution 2 mg/ml in PBS) (Twentyman and Luscombe 1987). Each

Fig. 1. Chemical structures of 2 -hydroxy-4 ,6 -dimethoxychalcone (1), 2 ,4 -dihydroxy-3 ,6 -dimethoxychalcone (2), and 3-O--d-glucosyl--sitosterol (3).

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G.C. Brando et al. / Phytomedicine 17 (2010) 926929

Table 2
MIC and IC50 (g/ml) for chalcone 2 against the microorganisms tested by the microdilution assay.
Compound 2

S. aureus
M. luteus
S. epidermides
B. subtilis
E. coli
P. aeruginosa
P. vulgares
S. marcescens
T. mentagrophytes
T. rubrum
M. canis
a
b

Chloramphenicol

MIC (g/ml)

IC50 (g/ml)

>250.0b
250.0
>250.0
250.0
250.0
125.0
125.0
125.0
12.0
12.0
18.0

67.2
35.9
62.4
36.6
32.5
18.9
16.4
20.6
1.1
1.4
3.0

2.4
1.6
2.8
2.5
1.0
1.3
0.9
1.1
0.2
0.1
0.3

Gentamicin

MIC (g/ml)

IC50 (g/ml)

8.0
8.0
8.0
8.0

<0.3
<0.3
<0.3
<0.3

Amphotericin B

MIC (g/ml)

IC50 (g/ml)

1.0
1.0
1.0
1.0

<0.5
<0.5
<0.5
<0.5

MICa (g/ml)

6.2
25.0
>50

Literature data (Lpez et al. 2001).

MIC minimal inhibition concentration, IC50 median inhibitory concentration, chloramphenicol and gentamicin were used as positive reference standard antibiotics.

3 ,6 -dimethoxychalcone (2) and 3-O--d-glucosyl--sitosterol (3)

(Fig. 1) by the usual spectrometric techniques (UV, IR, 1 H and
13 C NMR and MS) and comparison with literature data (Maradufu
and Ouma 1978). Chalcones 1 and 2 have been rstly 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 IC50 values
for chalcone 2 ranged from 12.0 to >250.0 g/ml and from 1.1 to
67.2 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 (Lpez et al. 2001), reinforces that
chalcones are promising as new drugs to treat dermathophyte
infections.
Results of the antiviral assays of P. spectabile extracts and compounds 13 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 LLCMK2 cells
(CC50 > 500 g/ml). The effective concentrations (EC50 ) were 24.6,
34.2 and 21.9 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 13 isolated from P. spectabile have
shown no activity against the assayed viral strains but have been
moderately cytotoxic to Vero and LLCMK2 cells exhibiting CC50
between 7.8 and 31.5 g/ml. 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.

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. 1990). The determined titers were
2.5 106 , 1.0 106 , 1.0 106 and 1.0 104 TCID100/ml , respectively,
for HHV-1, EMCV, VACV-WR and DEN-2 virus. The antiviral activity
(EC50 ) was evaluated by the MTT colorimetric assay (BetancurGalvis et al. 1999). Acyclovir (Calbiochem, USA) and -2a interferon
(Bergamo, Brazil) were used as positive controls. Cytotoxicity
(CC50 ) 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 dened as CC50 /EC50 .
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 signicantly 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 13 were isolated from HE and AE and were identied as 2 -hydroxy-4 ,6 -dimethoxychalcone (1), 2 ,4 -dihydroxyTable 3
In vitro cytotoxic and antiviral activity of P. spectabile extracts and compounds 13.
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

83.6 4.6
128.2 6.9
>500
>500
11.7 1.2
31.5 1.3
27.2 3.7

71.3 3.5
117.9 7.2
>500
>500
16.4 2.1
29.6 1.8
7.8 1.5

NA
NA
NA
21.9 1.8
NA
NA
NA
40d

SI, selective index. NA, no activity in the assayed concentrations.

a
Viral titer TCID100 /ml 2.5 106 in 48 h.
b
Viral titer TCID100 /ml 1.0 106 in 48 h.
c
Viral titer TCID100 /ml 1.0 104 in 72 h.
d
80100% inhibition of cytopathic effect.
e
Concentration in UI/ml.

SI

>22.8

VACV-WRb
EC50 g/ml
NA
NA
NA
34.2 2.4
NA
NA
NA
2.5 102 d , e

SI

>14.6

EMCVb
EC50 g/ml

DENV-2c
EC50 g/ml

NA
NA
NA
NA
NA
NA
NA

NA
NA
NA
24.6 3.7
NA
NA
NA

1.5 102 d , e

2.5 103 d , e

SI

>20.3

G.C. Brando et al. / Phytomedicine 17 (2010) 926929

shown by RP-HPLC-DAD to contain stilbene glycosides as major

constituents (data not shown).
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 signicant effect against HHV-1,
VACV and DEN 2. This is the rst report on the chemistry, antimicrobial, antiviral and cytotoxic activities of P. spectabile.
Acknowledgements
To CNPq and FAPEMIG for nancial support (ABO, EGK) and PhD
fellowship (GCB); to Dr. E. A. Nunan, Faculdade de Farmcia/UFMG
for helpful assistance in the antimicrobial assays, and to Dr. J. F.
Macedo/EPAMIG for collection and taxonomic identication of P.
spectabile.
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