SUMMARY. Aqueous EtOH (70%) crude extract of Alternanthera brasiliana leaves and dichloromethane, ethyl acetate and butanolic fractions of the crude extract were tested against a panel of microrganisms by the broth microdilution method to determine the Minimal Inhibitory Concentration (MIC). The results demonstrate that the crude extract and some fractions showed moderate activities against Staphylococcus aureus, Pseudomonas aeruginosa, Saccharomyces cerevisae and Prototheca zopffi. All samples did not present activity against the bacteria Escherichia coli, Candida albicans and Candida glabrata. The strongest effect occurred with dichloromethane fraction against the algae P. zopfii, (MIC= 312.5 μg.mL -1 ). These results demonstrate that A. brasiliana have a weak antimicrobial activity that not support the ethno-pharmacological indication of the plant for the treatment of infection diseases nor it&apos;s popular names such as &quot;penicillin&quot; and &quot;terramycin&quot; in Brazil . RESUMEN. &quot;Actividad Antimicrobiana del Extracto Bruto y Fracciones de las Hojas de Alternanthera brasiliana (L.) O. Kuntze&quot;. El extracto etanólico crudo (70%) y las fracciones diclorometánica, de acetato de etilo y butanólica de las hojas de A. brasiliana fueron analizados en relación a su actividad antimicrobiana. Las concentraciones inhibitorias mínimas (MIC) fueron determinadas usando el método de microdilución en caldo. Los experimentos demostraron que el extracto y algunas fracciones exhibieron moderada acción antimicrobiana, particularmente contra Staphylococcus aureus, Pseudomonas aeruginosa, Saccharomyces cerevisae y Prototheca zopffi. El efecto más fuerte ocurrió con la fracción diclorometánica frente a P. zopfii (MIC 312,5 μg.mL -1 ). La bacteria Escherichia coli y las leveduras Candida albicans y C. glabrata no fueron inhibidas por ninguno de los extractos probados. La baja actividad antimicrobiana de la planta no respalda las indicaciones planteadas por la comunidad para su uso en el tratamiento de procesos infecciosos, ni tampoco sus nombres populares en Brasil, tales como &quot;penicilina&quot; y &quot;terramicina&quot;

Danielle F Pereira

Margareth L Athayde

Mariane Dos Santos

Marli M A De Campos

Patrícia Pozzatti

Sydney H Alves

CiteSeerX

893ISSN 0326-2383
KEY WORDS: Alternanthera brasiliana, Amaranthaceae, Antimicrobial activity.
PALABRAS CLAVE: Actividad antimicrobiana, Alternanthera brasiliana, Amaranthaceae.
* Author to whom correspondence should be addressed. E-mail : marga@ccs.ufsm.br
Latin American Journal of Pharmacy
(formerly Acta Farmacéutica Bonaerense)
Lat. Am. J. Pharm. 26 (6): 893-6 (2007)
Comunicaciones breves
Recibido el 31 de julio de 2007
Aceptado el 12 de agosto de 2007
Antimicrobial Activity of a Crude Extract and Fractions
from Alternanthera brasiliana (L.) O. Kuntze Leaves
Danielle F. PEREIRA 1, Mariane dos SANTOS 2, Patrícia POZZATTI 1,
Sydney H. ALVES 3, Marli M. A. de CAMPOS 4 & Margareth L. ATHAYDE 1,5 *
1 Programa de Pós-Graduação em Ciências Farmacêuticas, 2 Curso de Graduação em Farmácia,
3 Departamento de Microbiologia e Parasitologia, 4 Departamento de Analises Clínicas e Toxicológicas,
5 Departamento de Farmácia Industrial, Centro de Ciências da Saúde,
Universidade Federal de Santa Maria, Campus Universitário Camobi,
Prédio 26, sala 1115. Santa Maria, CEP 97105-900, Brasil.
SUMMARY. Aqueous EtOH (70%) crude extract of Alternanthera brasiliana leaves and dichloromethane,
ethyl acetate and butanolic fractions of the crude extract were tested against a panel of microrganisms by
the broth microdilution method to determine the Minimal Inhibitory Concentration (MIC). The results
demonstrate that the crude extract and some fractions showed moderate activities against Staphylococcus
aureus, Pseudomonas aeruginosa, Saccharomyces cerevisae and Prototheca zopffi. All samples did not pre-
sent activity against the bacteria Escherichia coli, Candida albicans and Candida glabrata. The strongest ef-
fect occurred with dichloromethane fraction against the algae P. zopfii, (MIC= 312.5 μg.mL–1). These re-
sults demonstrate that A. brasiliana have a weak antimicrobial activity that not support the ethno-phar-
macological indication of the plant for the treatment of infection diseases nor it’s popular names such as
“penicillin” and “terramycin” in Brazil .
RESUMEN. “Actividad Antimicrobiana del Extracto Bruto y Fracciones de las Hojas de Alternanthera brasilia-
na (L.) O. Kuntze”. El extracto etanólico crudo (70%) y las fracciones diclorometánica, de acetato de etilo y bu-
tanólica de las hojas de A. brasiliana fueron analizados en relación a su actividad antimicrobiana. Las concentra-
ciones inhibitorias mínimas (MIC) fueron determinadas usando el método de microdilución en caldo. Los experi-
mentos demostraron que el extracto y algunas fracciones exhibieron moderada acción antimicrobiana, particular-
mente contra Staphylococcus aureus, Pseudomonas aeruginosa, Saccharomyces cerevisae y Prototheca zopffi.
El efecto más fuerte ocurrió con la fracción diclorometánica frente a P. zopfii (MIC 312,5 μg.mL–1). La bacteria
Escherichia coli y las leveduras Candida albicans y C. glabrata no fueron inhibidas por ninguno de los extractos
probados. La baja actividad antimicrobiana de la planta no respalda las indicaciones planteadas por la comunidad
para su uso en el tratamiento de procesos infecciosos, ni tampoco sus nombres populares en Brasil, tales como
“penicilina” y “terramicina”.
INTRODUCTION
Alternanthera brasiliana (L.) O. Kuntze,
commonly known as “Penicilina”, “Terramicina”
or “Doril” belongs to the Amaranthaceae family.
It is a perennial herbaceous plant widely dis-
tributed in Brazil’s eastern beaches and certain
Amazonian’s regions 1.The plant is popularly
used in Brazilian folk medicine as an analgesic
and anti-inflammatory remedy for the treatment
of infectious processes 2,3 . Previous phytochem-
ical studies on this plant furnished the presence
of triterpenoids and betacianin and triterpenes
and steroids such as β-sitosterol, besides pheno-
lic compounds 2,4. Various bioactivities, includ-
ing antinociceptive 2, lymphocyte proliferation
inhibition 4 and antiviral 5 properties of crude
extracts, fractions or isolated compounds from
this plant were reported. Regarding antimicro-
bial activity some controversial results were re-
ported, such as the activity against Staphylococ-
cus aureus reported by Caetano et al. 6. and the
ineptness of a methanolic extract of the aerial
parts of the plant against various microrganisms
reported by Souza et al. 7 both using the agar-
diffusion method. These previous results en-
couraged us to deepen the studies on antimicro-
bial properties of A. brasiliana by assessing the
minimal inhibitory concentration (MIC) using
the micro dilution method for the evaluation of
the crude extract and fractions against gram-
894
PEREIRA D.F., dos SANTOS M., POZZATTI P., ALVES S.H., CAMPOS M.M.A. de & ATHAYDE M.L.
positive and gram-negative bacteria, yeasts like
fungi such as Candida albicans and Candida
glabrata and the algae Prototheca zopfii, the last
species also with clinical importance in im-
munologically suppressed patients 8. At the
same time, the study was conducted with the
objective to validate or not the ethnopharmaco-
logical claim of this plant concerning the popu-
lar names associated to antibiotics (Penicillin
and Terramycin) used by the population for this
species. 
MATERIALS AND METHODS
Plant material
Leaves of A. brasiliana were harvested in
Santa Maria (State of Rio Grande do Sul, Brazil)
on January of 2005. Samples of the collected
material were identified by Botanist Dr. Thais
Scott do Canto Dorow and archived as voucher
specimens in the herbarium of Department of
Biology at Federal University of Santa Maria by
register number SMD 10038.
Extraction and partition of the leaves 
Air dried, powdered leaves of A. brasiliana
(472.33 g) were extracted with ethanol (70%) at
room temperature for seven days with daily agi-
tation. After filtration, the extract was evaporat-
ed under reduced pressure to remove the
ethanol in order to obtain an aqueous suspen-
sion. The aqueous suspension was partitioned
successively with dichloromethane, ethyl acetate
and n-butanol (3 x 100 mL for each solvent),
yielding dichloromethane, ethyl acetate, and n-
butanol extracts, respectively. The extracts were
evaporated to dryness under vacuum, to yield
dichloromethane (11.80 g, 2.5%), ethyl acetate
(2.83 g, 0.6%) and n-butanol-soluble (9.44 g,
2.0%) fractions respectively. At the same time
100 g of the dried and powdered leaves was ex-
tracted with ethanol (70%) at room temperature
for seven days and after filtration the extract
was evaporated under reduced pressure to ob-
tain the crude extract.
Antimicrobial Screening
The crude extract and the fractions were in-
dividually tested against a panel of microorgan-
isms including C. albicans ATCC 28367, C.
glabrata (clinical isolate), Saccharomyces cere-
visiae ATCC 28952, S. aureus ATCC 25293,
Bacillus subtilis ATCC 6633, Escherichia coli
ATCC2792, Pseudomonas aeruginosa ATCC
27853, and P. zopfii (clinical isolate). Bacterial
strains were cultured overnight at 37 °C in
Mueller-Hinton Agar (MHA). Yeasts and the al-
gae were cultured overnight at 30 °C in
Sabouraud dextrose agar. 
Microdilution Method for
MIC Determination 
The MICs of the crude extract and fractions
against the test microorganisms were deter-
mined by the broth microdilution method ac-
cording to National Committee for Clinical Labo-
ratory Standards 9,10. Seven different dilutions of
each fraction (dichloromethane, ethyl acetate
and butanolic) and the crude extract (1.250, 625,
312.5, 156.25, 78.125, and 39.06 µg.mL–1) in DM-
SO were prepared. Bacteria were inoculated in-
to Mueller-Hinton agar and, after overnight
growth four or five colonies were directly sus-
pended in saline solution so that the turbidity
matched the turbidity of the McFarland standard
(≈108cfu/mL). By further progressive dilutions
with the test medium, the required concentra-
tions were obtained. The suspension was dilut-
ed by 1:100 in saline followed by a new dilution
of 1:20 in Mueller-Hinton broth, resulting in a fi-
nal inoculum concentration of 5 x 104 CFU per
well. Yeasts like fungi were inoculated into
potato dextrose agar. Tests were performed in
sterile 96-well plates. The first column of the
plate was reserved for negative control wells
(without inoculants) and the last column, for the
positive growth control wells (without antimi-
crobial agents). The plates were sealed and in-
cubated at 35 °C for 24 h for bacteria and Can-
dida and 72 h for S. cerevisae and growth or a
lack thereof in the antimicrobial agent contain-
ing wells was determined by comparing with
the growth control, indicated by turbidity. The
experiments were repeated twice and the results
were determined as an average value. The result
readings were made visually. The MIC endpoint
was considered as the lowest concentration of
the extract or fraction inhibiting the total growth
of microrganisms. MIC was detected by lack of
visual turbidity (matching the negative growth
control). Subcultures were made from the clear
wells which did not show any growth after in-
cubation during the MIC assays on Mueller-Hin-
ton agar for bacteria and Sabouraud agar for
fungi and algae in order to achieve the Minimal
Bactericidal Concentration (MBC) the Minimal
Fungicidal Concentration (MFC) and the Mini-
mal Algaecidal Concentration (MAC). The lowest
concentration that yielded no growth after this
sub-culturing was taken as the MMC, MFC and
895
Latin American Journal of Pharmacy - 26 (6) - 2007
MAC. Standard antibiotics (ampicillin, imipen-
em, and cefoperazone) were used to control the
sensitivity of the tested bacteria, whereas flu-
conazole and amphotericin B were used as con-
trol against the tested fungi and the algae. 
Crude Crude CH2Cl2 CH2Cl2 AcOet AcOet BuOH BuOH Control
extract extract µg.mL–1
MBC MBC MBC MBC
MIC MFC MIC MFC MIC MFC MIC MFC
or MAC or MAC or MAC or MAC
E. coli >2500 * >2500 * >2500 * >2500 * Amp 8.0
P. aeruginosa >2500 * 1250 1250 >2500 * >2500 * Cpz. 16.0
S. aureus 1250 1250 1250 >1250 1250 1250 >2500 * Amp 2.0
B. subtilis 2500 2500 1250 1250 2500 2500 2500 2500 Imi 0.06
C. albicans >1250 * >1250 * >1250 * >1250 * Flu 16
C. glabrata >1250 * >1250 * >1250 * >1250 * Flu 32
S. cerevisae 625 625 625 >1250 625 1250 >1250 * Flu 2.0
P. zopfii >1250 * 312.5 312.5 >1250 * >1250 * Amph 0.5
Table 1. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), minimal fungicidal
concentration (MFC) and minimal algaecidal concentration (MAC) for crude extract and fractions of Alternan-
thera brasiliana leaves. Amp = ampicillin, Cpz = cefoperazone, Imi = Imipenem, Flu = fluconazole, Amph =
Amphotericin B. * not tested because the MIC was not determined. Tests were carried out twice. Values are ex-
pressed in µg ml–1.
Determination of Percent activity
The percent activity was determinate accord-
ing Ellof 11 and Rangasamy et al. 12, and demon-
strates the total antimicrobial potency of a par-
ticular extract or fraction [Ec. 1].
100 x nº of susceptible strains to a specific extract
Activity (%) = [Ec. 1]
Total nº of tested strains
Determination of total activity
Total activity was determinate according
Ellof 11 and Rangasamy et al. 12. The value ob-
tained would indicate the largest volume to
which biologically active compounds in 1 g of
plant material can be diluted and still inhibits
the growth of microrganism [Ec. 2].
quantity of material extracted from 1 g of plant material
Total activity = [Ec. 2]
MIC
RESULTS AND DISCUSSION 
The broth dilution method, standardized by
the Clinical and Laboratory Standards Institute
(formerly NCCLS) allowed determining the mini-
mal inhibitory concentrations (MICs) and also
the MMC, MFC and MAC of the crude extract
and different fractions. Results showed that the
different extracts differed clearly in their antimi-
crobial activities (Table 1). 
The crude extract and ethyl acetate fraction
showed a discrete activity against S. aureus with
MIC and MMC at 1250 µg.mL–1 whereas only the
dichloromethane fraction presented MIC and
MMC at 1250 µg.mL–1, against B. subtilis. An-
tibacterial activity against various strains of S.
aureus has been reported for the crude ethano-
lic extract from A. brasiliana by Caetano et al. 6
at a concentration of 65 µg.mL–1 determined by
the agar diffusion technique. Although using dif-
ferent methods, the results reported here are
near 50 times better than those reported above.
Concerning the gram-negative bacteria, the re-
sults obtained were modest, with a reasonable
activity of the dichlorometane fraction against P.
aeruginosa at 1250 µg.mL–1, as this microrgan-
ism exhibits resistance to many antimicrobial
agents 13, whereas against E. coli, all samples
were ineffective even at the higher concentra-
tion tested (2500 µg.mL–1). In respect of S. cere-
visae, the crude extract and the fractions tested
were effective, with better results obtained with
crude extract (MIC and MFC at 625 µg.mL–1). 
No activity was observed for all fractions
against C. albicans and C. glabrata, with MIC
above 1250 µg.mL–1. Only the dichloromethane
fraction showed good activity against the algae
P. zopfii, with MIC and MAC at 312.5 µg.mL–1.
To our knowledge, this is the first assay involv-
ing this plant and the algae P. zopfii. Protothe-
cosis is especially difficult to eradicate with
896
PEREIRA D.F., dos SANTOS M., POZZATTI P., ALVES S.H., CAMPOS M.M.A. de & ATHAYDE M.L.
drugs and there is no defined treatment protocol
14,15. 
The discovery of new plant products with
potential antimicrobial application is of consid-
erable interest in view of the increasing antibiot-
ic resistance observed in many bacteria such as
S. aureus, E. coli, and P. aeruginosa and the use
of ethnopharmacological knowledge is one
striking way to reduce empirism and enhance
the probability of success in new drug active
development. Partition between immiscible sol-
vents is an adequate approach for the prelimi-
nary separation of complex matrices because
this procedure permit discrimination between
polar and non-polar fractions activities. The im-
portance of a preliminary fractionation could be
seeing in the case of P. zopffi, in view of the
fact that the crude extract was ineffective against
this algae and the dichloromethane fraction
showed a very good activity (Table 1). It is
mainly probable that the low concentration of
the active(s) compounds may prejudice their de-
tection in crude extracts. In this study,
dichlorometane fraction was found to be more
effective than the crude extract and ethyl acetate
and butanolic fractions against all the micror-
ganisms tested, suggesting that the active(s) sub-
stance(s) are concentrated in the less-polar frac-
tion. Total activities values revealed that the
dichloromethane fraction had a high magnitude
of antimicrobial activity, as the active compo-
nent(s) from this fraction can be diluted in ca 80
mL of solvent and still inhibits growth of P. zop-
fii (total activity= 79.93 mL g–1) and in ca 40 mL
for the inhibition of S. cerevisae (total activity =
39.96 mL g–1). Also, the Percent Activity (A%)
for the crude extract and fractions against the
panel of microrganisms tested was:
dichloromethane (62.5%), crude extract and
ethyl acetate fraction (37.5%) and butanolic frac-
tion (12.5%), the less active fraction.
On the other hand, it is moreover of funda-
mental importance to clarify the ethno-pharma-
cological claim regarding the plant employ in
infectious processes, because the results ob-
tained in this work, even though a reasonable
antimicrobial activity against some bacteria’s and
a algae were not sufficient to justify the plant
use in such diseases especially when we consid-
er Cos et al. 16 guiding principles and the MICs
of the antibiotics used as reference standards
(Table 1). Additionally, the local names of A.
brasiliana such as “Doril”, “Penicilina” and “Ter-
ramicina” may be conduct to a misuse of the
plant and may also encourage self medication,
because in Brazil, “Doril” is the trade name of a
pharmaceutical speciality commercialised as
analgesic (contains acetilsalicilic acid) whereas
“Penicilina” is a short name commonly used to
designed the β-lactam antibiotic benzylpenicillin
(Penicillin G) and “Terramicina” (Terramycin) is
a registered trade mark of the antibiotic oxyte-
tracycline. 
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Antimicrobial Activity of a Crude Extract and Fractions from Alternanthera brasiliana (L.) O. Kuntze Leaves

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Antimicrobial Activity of a Crude Extract and Fractions from Alternanthera brasiliana (L.) O. Kuntze Leaves

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