Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 103(1): 31-38, February 2008
31
Screening of Amazonian plants from the Adolpho Ducke forest
reserve, Manaus, state of Amazonas, Brazil, for antimicrobial activity
Ana Lúcia Basílio Carneiro, Maria Francisca Simas Teixeira1, Viviana Maria Araújo de Oliveira2,
Ormezinda Celeste Cristo Fernandes3, Gláucia Socorro de Barros Cauper4,
Adrian Martin Pohlit4/+
Departamento de Morfologia 1Laboratório de Micologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Manaus,
AM, Brasil 2Fundação de Hematologia e Hemoterapia do Amazonas-HEMOAM, Manaus, AM, Brasil 3Centro de Pesquisa Leônidas e
Maria Deane-Fiocruz, Manaus, AM, Brasil 4Laboratório de Princípios Ativos da Amazônia-LAPAAM, Coordenação de Pesquisa em
Produtos Naturais-CPPN, Instituto Nacional de Pesquisa da Amazônia-INPA, Av. André Araújo 2936, 69011-970 Manaus, AM, Brasil
Tropical forests are species-rich reserves for the discovery and development of antimicrobial drugs. The aim of
this work is to investigate the in vitro antimicrobial potential of Amazon plants found within the National Institute
on Amazon Research’s Adolpho Ducke forest reserve, located in Manaus, state of Amazonas, Brazil. 75 methanol,
chloroform and water extracts representing 12 plant species were tested for antimicrobial activity towards strains of
Mycobacterium smegmatis, Escherichia coli, Streptococcus sanguis, Streptococcus oralis, Staphylococcus aureus and
Candida albicans using the gel-diffusion method. Active extracts were further evaluated to establish minimum inhibitory concentrations (MIC) and antimicrobial profiles using bioautography on normal-phase thin-layer chromatography plates. Diclinanona calycina presented extracts with good antimicrobial activity and S. oralis and M. smegmatis
were the most sensitive bacteria. D. calycina and Lacmellea gracilis presented extracts with the lowest MIC (48.8 µg/ml).
D. calycina methanol and chloroform leaf extracts presented the best overall antimicrobial activity. All test organisms
were sensitive to D. calycina branch chloroform extract in the bioautography assay. This is the first evaluation of the
biological activity of these plant species and significant in vitro antimicrobial activity was detected in extracts and
components from two species, D. calycina and L. gracilis.
Key words: plant extract - antibacterial - antifungal - Diclinanona calycina - Lacmellea gracilis - bioautography
The Amazon region is the largest tropical forest in the
world. It occupies almost half the South American continent and is a center of biological diversity. Aside from
its importance in the global ecological equilibrium and
as a location of increasing ecological tourism, this region
is a very rich source of species for agriculture, plant domestication and medicinal applications. Approximately
125,000 plant species are in tropical forests which continue to be a great reservatory for the discovery of new
bioactive molecules and phytotherapeutic agents. However, the pharmacological potential of only about 1%
of all tropical plant species has been evaluated. Also,
Brazil has catalogued only about 0.4% of its flora. It is
estimated that ca. 60% of all commercially available or
clinical-phase antitumor and antimicrobial drugs are of
natural origin (Montanari & Bolzani 2001, Gurib-Fakim
2006, Turolla & Nascimento 2006).
Despite the existence of publications on the traditional (Fenner et al. 2006, Giorgetti et al. 2007) and pharmaceutical (Moreira et al. 2006) uses of plant species found
Finnancial support: PPG-7/CNPq (557106/2005-2), PPBio/MCT/INPA
+ Corresponding author: ampohlit@inpa.gov.br
Received 15 May 2007
Accepted 12 February 2008
in Brazil there are still relatively few scientific studies
on the pharmacological and toxicological properties of
plants from Brazil. There are also very few examples of
the discovery of bioactive substances from Brazilian biodiversity which have served as prototypes for development of phytotherapeutic agents and pharmaceuticals in
Brazil. Furthermore, many active substances present in
native plant extracts from Brazil have still not been identified so this is an area which needs to be explored (Moreira et al. 2006). As examples of Brazilian plant-derived
commercial products one should mention Ierobina®,
which is used in the treatment of indigestion (Botion et
al. 2005), and the anti-inflammatory phytotherapeutic
agent Acheflan® (Giorgetti et al. 2007). The relatively
scarce interaction between mainly public universities and
companies in Brazil as well as Brazilian patent law have
been drawbacks for transformation of research results
into developed products and patents (Moreira et al. 2006,
Giorgetti et al. 2007).
Native Amazon forest plants have been poorly studied and there are no good estimates of the number of species which have been studied or which have application to
human health. The use of flora from this region has been
modest in relation to its strategic value in the development
of local products. Our group has been actively screening
medicinal and other plants found in the Brazilian Amazon
region for larvicidal and insecticidal (Pohlit et al. 2004)
as well as cytotoxic (Quignard et al. 2003, Quignard et al.
2004) properties, among other biological activities.
online | memorias.ioc.fiocruz.br
32
Antimicrobial Amazonian species • Ana Lúcia B Caneiro et al.
Many plant species have demonstrated antibacterial
(Koo et al. 2000, Melo et al. 2006) and antifungal (Motsei et al. 2003, Gayoso et al. 2004) properties. However,
little research has focused on the evaluation of plant species for activity against microorganisms from the oral
cavity (Pereira et al. 2005, Bandeira et al. 2006). This is
especially true for plants from the Amazon region.
Therefore, there is a general lack of scientific investigation which can provide more realistic knowledge of
the potential of Amazon biodiversity. As a means of contributing to knowledge of the pharmacological potential
of the Amazon flora, the present work evaluated 12 Amazonian plant species - having no known previous pharmacological study- for in vitro antimicrobial activity in
bacteria and a fungus species associated with the human
oral cavity diseases, as well as other diseases.
MATERIALS AND METHODS
Plant selection - Species identified by the Flora Project (Ribeiro et al. 1999) in the National Institute on Amazon Research’s Adolpho Ducke forest reserve in Manaus,
state of Amazonas, Brazil were initially contemplated
for study. These plant specimens had been previously
catalogued so that their botanic identities (based on
voucher samples at the Instituto Nacional de Pesquisa da
Amazônia-INPA Herbarium) and exact locations in the
Ducke reserve were readily available. After a literature
search, several hundred plant species for which no ethnobotanic, phytochemical, pharmacological or medicinal
information were available were identified. From this
larger group of plants, the 12 plant species were chosen
for the purposes of this study (Table I) based on: (1) the
ease of access and availability of sufficient plant materials in the Ducke reserve; and (2): their broad representative value of the local, unexplored flora (the species under study belong to 11 distinct botanic families).
Plant collection - Plants were collected between January and June 2004 in the Ducke reserve. Plant specimens
were located in the field based on the Ducke reserve trail
map (Flora Project 2006) and geographical coordinates
were obtained from the Flora Project database. Plant
materials (leaves, branches, vine or bark) were initially
dried in an air-conditioned, dehumidified room, then further dried in an oven at ca. 40ºC for a total of ca. seven
days, and then finally ground.
Preparation of extracts - Plant methanol and chloroform extracts were prepared by continuous extraction of
ground plant material in a soxhlet apparatus for 18 h (3
x 6 h) followed by rotary evaporation and freeze-drying.
Water extracts were prepared by infusion followed by filtration and total evaporation of the filtrate. Extracts were
stored in a freezer at -20ºC.
Microorganisms used - Standardized strains from
the American type culture collection (ATCC) and the
Department of Antibiotics, Federal University of Pernambuco (DAUPE) were used in bioassays. The Grampositive bacteria were Mycobacterium smegmatis (ATCC
607), Streptococcus oralis (ATCC 10557), Streptococcus sanguis (ATCC 15300) and Staphylococcus aureus
(DAUPE). The Gram-negative bacterium was Escherichia coli (DAUPE 224). Antifungal activity was evaluated using a clinical strain of Candida albicans from the
collection of the Department of Parasitology, Federal
University of Amazonas. Organisms were maintained at
4ºC on brain heart agar (bacteria) and sabouraud (SAB)
(C. albicans). For the antibacterial tests, organisms were
grown overnight in brain heart infusion followed by incubation at 37ºC. Before the test, C. albicans was cultured on SAB at 37ºC for 48 h.
Antimicrobial susceptibility testing - Evaluation of the
antimicrobial activity of plant extracts was carried out in
Petri dishes using the agar diffusion method (Alves et al.
2000, Chah et al. 2006, Melo et al. 2006) by perforating
the culture medium and charging each cavity with extract
dissolved in dimethylsulphoxide (DMSO), ethanol or sterilized distilled water at a concentration of 50 mg/ml.
For antibacterial tests, 25 ml of Mueller Hinton (MH)
culture medium was used. For antifungal tests, SAB was
used (C. albicans). Microbial suspensions were prepared
TABLE I
Families, species names and other information for the plants from the Ducke Reserve under study
Family
Species
Synonym
Common Name
Anacardiaceae
Annonaceae
Apocynaceae
Thyrsodium spruceanum Benth.
Diclinanona calycina Benoiste
Lacmellea gracilis (Mull.Arg.) Markgr.
Thyrsodium paraense Huber
Xylopia calycina Diels
Zschokkea gracilis Mull
Icacinaceae
Olacaceae
Passifloraceae
Pleurisanthes parviflora (Ducke) Howard
Chaunochiton kappleri (Sagot ex Engl.) Ducke
Dilkea johannesii Barb. Rodr.
Chaunochiton breviflorum Ducke
Dilkea ulei Harms
Polygalaceae
Rhizophoraceae
Sapotaceae
Sapotaceae
Thymelaeaceae
Violaceae
Moutabea guianensis Aubl.
Sterigmapetalum obovatum Kuhlm.
Elaeoluma nuda (Baehni) Aubrév.
Sarcaulus brasiliensis (A. Dc.) Eyma
Schoenobiblus daphnoides Mart.
Paypayrola grandiflora Tul.
Pouteria nuda
Sarcaulus macrophyllus (Mart) Radlk
Paypayrola ventricosa Tul.
Breu-de-leitea
Envireiraa
Caramurizinho,
sorvinhab
Grão de bodeb,
Pacarupiac
Guajaraa
-
References for species names: a: Ribeiro et al. (1999); b: Silva et al. (1977); c: Corrêa (1978).
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 103(1), February 2008
and the density was adjusted in the tube to 1 on the McFarland scale. After solidification, medium was inoculated with microbial suspension (100 µl) with the aid of a
swab. After 10 min, the agar was perforated so as to yield
five circular, equidistant cavities (diameter 6 mm each).
An aliquot of extract (50 µl of a 50 mg/ml solution) and
positive (rifampicin and nistatin) and negative (solvent
blank) controls were transferred to cavities. After the incubation period (24 and 48 h) at 37ºC, plates were examined and extract activity was evaluated by measurement of
inhibition zone diameters (in mm). Tests were performed
in triplicate and average halo diameters were determined.
Rifampicin (1 mg/ml) was used as positive control for
bacteria and nistatin (1 mg/ml) was used for tests involving C. albicans. The negative controls were DMSO, ethanol and sterilized, distilled water in accordance with the
solvents used to dissolve each extract. A negative control
containing culture medium was also used.
Determination of minimum inhibitory concentrations
(MIC) - Extracts considered very active in the above susceptibility tests (inhibition halo > 20 mm in the agar diffusion test) were next evaluated to determine MIC. This
was carried out by dilution in solid culture medium by
adapting techniques proposed by Melo et al. (2006). For
the test, 35 ml of agar were prepared. MH was used for
bacteria and SAB for C. albicans, using homogenization
with 1.5 ml of the microorganism suspension having a
density equal to 1 on the McFarland scale. Thirty 4 mm
diameter orifices were prepared and inoculated, in triplicate, with 15 µl of each of a series of 10 dilutions of
each crude extract stock solution (50 mg/ml). MICs correspond to the lowest concentration which inhibited the
growth of the microorganism.
Thin-layer chomatography (TLC) - Aluminum-backed
commercial TLC silica gel GF254 plates (MERCK) were
used. The extracts were applied to plates and eluted with
chloroform/acetone (90:10). Eluted TLC plates were observed under ambient lighting and illuminated with an
ultraviolet lamp at 254 and 366 nm.
Bioautography - Active extracts were evaluated
through the bioautography technique adapted from Alves
et al. (2001) and Holetz et al. (2002). Briefly, microorganism culture (1 on McFarland’s scale, 500 µl) and developing agent (2,3,5-triphenyltetrazolium chloride, 1 ml)
in aqueous solution (this reagent aids in the observation
of inhibition zones) were added to the culture medium
(25 ml) at 50ºC. After homogenization, this mixture was
poured over eluted chromatograms and these were placed
in Petri dishes and incubated at 37ºC. Plates were observed after incubation for 24 and 48 h. Inhibition was
evidenced by clear zones on the plates where no ostensible microorganism growth had occurred.
RESULTS
In the screening for antimicrobial activity, after a period of 24 h, the largest average inhibition halos resulted
from the action of the methanol and chloroform extracts
of Diclinanona calycina. S. oralis e M. smegmatis were
the most sensitive organisms in this test. Of the 75 extracts
33
evaluated for inhibitory effects on M. smegmatis, only ten
presented negative results (no inhibition halos), while 11
allowed for the formation of inhibition halos with diameters greater than 20 mm. Extracts of Pinus parviflora
presented slight or no activity towards the microorganisms
used in testing. These and other results are presented in
Table II. Antimicrobial substances used as positive controls presented inhibition halos as expected, differing from
negative controls (DMSO, water, ethanol blanks), which
in general did not present inhibition halos.
This preliminary screening of all extracts permitted
triage of promising antimicrobial extracts (presenting inhibition halo diameter ≥ 20 mm). 34 halos with diameters
≥ 20 mm, corresponding to 23 different extracts, were
observed. Active extracts were further tested in dilution
for the determination of minimum inhibition concentrations (MIC) by diffusion in solid culture medium. D.
calycina e Lacmella gracilis presented the lowest MIC
values (48.8 µg/ml). D. calycina leaf methanol and chloroform extracts presented the best inhibitory effects on
E. coli, S. oralis, S. aureus and S. sanguis. L. gracilis
branch chloroform extract presented the lowest MIC on
M. smegmatis (48.8 µg/ml). Under the experimental conditions used, none of the eight tested extracts inhibited C.
albicans growth (Table III).
Results of bioautography screening revealed several
(mainly chloroform) extracts which exhibited inhibition
zones corresponding to substances of differing polarities/
retention factors (Rfs) (Table IV). D. calycina extracts
presented the best bioautography results. All microorganisms tested in this study were sensitive to D. calycina
branch chloroform extract and most of these microorganisms (M. smegmatis, S. oralis, S. aureus and S. sanguis)
showed sensitivity to more than one chemical component
of this extract. In this way, screening and MIC determinations by diffusion in agar and bioautography permitted
identification of active extracts containing antibacterial
components. In this way, it was observed that in screening of extracts for antimicrobial activity it can be very
important to use a variety of test methods which can, as
in the present case, reveal information about the most active extracts and also reveal information on the number
of bioactive components.
DISCUSSION
Microorganisms selected for this study are important
human oral cavity pathogens or are of interest because
they represent diseases which are of interest to public
health. S. sanguis, for example, is one of the predominant colonizers of teeth. It has been isolated from human
dental plaque, tongue, saliva, root canals, periapical and
periodontal infections and it causes endocarditis. Cavities produced by S. sanguis occur principally in tooth fissures (Hamada & Slade 1980, Coykendall 1989, Piovano
1999, Wade et al. 2005).
E. coli is recommended as control group in antimicrobial susceptibility tests for enterobacteria. Also, E. coli and
S. aureus are included among the most common agents of
hospital infections and are detected in cases of meningitis and are potential respiratory pathogens (Gendron et al.
2000, Melo-Souza, 2000, Esmerino et al. 2005).
Antimicrobial Amazonian species • Ana Lúcia B Caneiro et al.
34
TABLE II
Average inhibition zone diameters (in mm) observed for plant extracts (well concentrations 50 mg/ml) in the agar diffusion
method for the different test microorganisms used
Average inhibition zone diameter (mm)
Species
Part
Solvent
Ms
Ec
Sa
So
Ss
Ca
C. kappleri
leaf
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
19
15
14
14
15
15
26
40
16
15
21
15
12
14
8
11
15
12
10
16
17
15
15
15
10
15
12
12
21
10
11
12
10
12
19
12
12
25
10
10
9
11
15
16
15
10
15
13
10
14
16
15
20
20
16
15
_
11
17
11
12
11
20
10
11
20
15
9
10
12
16
15
10
12
18
12
11
10
10
10
12
11
14
12
12
12
18
14
15
10
12
14
11
9
14
20
17
14
11
8
8
13
9
10
10
11
8
10
10
15
10
9
11
8
21
12
15
11
14
13
13
10
15
9
15
11
12
13
12
16
9
15
15
12
15
16
23
18
15
15
13
14
8
9
11
10
10
8
14
16
20
14
11
10
13
12
25
24
11
11
10
10
12
19
9
13
12
14
15
12
8
9
12
11
10
13
14
13
12
10
13
14
16
15
13
22
20
14
18
16
9
9
15
10
9
9
12
12
10
13
10
10
13
8
10
15
11
9
8
8
15
10
10
9
14
14
14
9
12
9
9
12
12
11
14
16
10
11
12
15
12
15
15
10
9
13
20
15
10
12
10
15
12
23
10
8
8
20
10
12
12
10
15
10
16
branch
D. calycina
leaf
branch
D. johannesii
leaf
branch
E. nuda
leaf
branch
bark
L. gracilis
leaf
branch
M. guianensis
leaf
branch
P. grandiflora
leaf
branch
P. parviflora
leaf
vine
S brasiliensis
leaf
branch
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 103(1), February 2008
35
Average inhibition zone diameter (mm)
Species
S. daphnoides
Part
leaf
branch
S. obovatum
leaf
branch
T. spruceanum
leaf
branch
Rifampicin
Nistatin
Solvent
Ms
Ec
Sa
So
Ss
Ca
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
CHCl3
CH3OH
H2O
H2O
H2O
24
15
12
14
18
10
14
14
11
15
15
26
25
25
11
27
25
30
nd
13
13
13
12
12
20
15
9
18
20
9
10
15
15
11
20
16
21
nd
14
11
9
11
16
15
12
11
11
12
11
12
10
10
14
13
10
11
8
16
nd
14
15
10
10
8
18
13
8
12
12
8
13
13
10
8
13
16
15
16
17
21
nd
14
12
9
11
15
13
17
13
13
15
12
12
9
14
14
15
20
16
20
nd
15
10
9
9
20
19
19
25
30
25
nd
35
Ms: M. smegmatis; Ec: E. coli; Sa: S. aureus; So: S. oralis; Ss: S. sanguis; Ca: C. albicans; - : negative result; nd: not determined.
TABLE III
Minimal inhibitory concentrations (MIC) of extracts from 12 Ducke Forest Reserve plant species
MIC (µg/ml)
Species
C. kappleri
D. calycina
D. johannesii
E. nuda
L. gracilis
M. guianensis
P. grandiflora
P. parviflora
S. brasiliensis
S. obovatum
T. spruceanum
Part
Solvent
Ms
Ec
So
Sa
Ss
Ca
Branch
Leaf
Leaf
Branch
Branch
Branch
Leaf
Leaf
Branch
Branch
Leaf
Branch
Branch
Vine
Branch
Leaf
Branch
Branch
Leaf
Leaf
Leaf
Branch
Branch
Branch
CH3OH
CH3OH
CHCl3
CH3OH
CHCl3
H2O
CH3OH
H2O
CHCl3
H2O
H2O
H2O
CH3OH
CHCl3
H2O
CHCl3
CH3OH
H2O
CH3OH
CHCl3
H2O
CH3OH
CHCl3
H2O
> 1000
195.0
781.3
> 1000
97.7
nd
> 1000
> 1000
48.8
nd
nd
nd
> 1000
> 1000
nd
nd
nd
> 1000
> 1000
390.6
781.3
> 1000
> 1000
nd
195.0
97.7
nd
781.3
> 1000
nd
> 1000
781.3
nd
nd
nd
nd
nd
nd
781.3
> 1000
nd
nd
nd
nd
> 1000
nd
nd
nd
97.7
48.8
nd
nd
nd
nd
nd
390.6
nd
nd
nd
nd
781.3
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
48.8
48.8
nd
nd
nd
nd
nd
nd
nd
> 1000
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
195.0
48.8
nd
781.3
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
> 1000
nd
nd
nd
nd
nd
nd
nd
nd
> 1000
nd
> 1000
> 1000
nd
> 1000
nd
nd
nd
nd
nd
> 1000
nd
nd
nd
> 1000
> 1000
> 1000
Ms: M. smegmatis; Ec: E. coli; Sa: S. aureus; So: S. oralis; Ss: S. sanguis; Ca: C. albicans; nd: not determined (see Table II for
extracts with inhibition halo diameter < 20 mm at 50 g/l).
36
Antimicrobial Amazonian species • Ana Lúcia B Caneiro et al.
TABLE IV
Results from bioautography applied to extracts from plants from the Ducke Forest Reserve and thin-layer chromatography (TLC)
retention factors (Rfs) of active antimicrobial spots
Rf
Antibacterial activity
Antifungal activity
Species
Part
Solvent
Ms
Ec
So
Sa
Ss
Ca
D. calycina
Leaf
Leaf
CH3OH
CHCl3
0.93
0.93
0.71
0.70
-
0.78
0.55
0.86
0.81
0.78
Branch
CH3OH
0.75
-
-
-
0.5
0.39
0.28
-
Branch
CHCl3
0.55
0.56
0.66
0.8
H2O
H2O
CHCl3
CHCl3
nd
0.46
0.52
nd
nd
-
nd
nd
0.39
P. grandiflora
Branch
Leaf
Branch
Branch
H2O
H2O
H2O
CH3OH
nd
-
nd
0.5
0.49
0.53
0.23
nd
nd
0.86
0.77
0.52
0.16
nd
nd
0.32
0.59
0.42
0.27
Branch
Leaf
Leaf
Branch
0.47
0.36
0.24
0.20
nd
nd
-
nd
nd
nd
nd
nd
nd
P. parviflora
Vine
CHCl3
-
nd
-
nd
nd
nd
nd
nd
nd
S. obovatum
T. spruceanum
Leaf
Leaf
Leaf
Leaf
Branch
Branch
Branch
CHCl3
CH3OH
CHCl3
H2O
CH3OH
CHCl3
H2O
0.27
-
nd
nd
nd
0.22
nd
nd
nd
nd
-
nd
nd
nd
nd
nd
0.40
0.16
nd
D. johannesii
E. nuda
L. gracilis
M. guianensis
0.83
0.76
0.83
0.77
-
-
Ms: M. smegmatis; Ec: E. coli; Sa: S. aureus; So: S. oralis; Ss: S. sanguis; Ca: C. albicans; -: negative result; nd: not determined.
M. smegmatis is an important test model for initial,
primary screening for antimycobacterial activity, which
is important in the search for drugs with potential antituberculosis effects. Mycobacterium tuberculosis (tuberculosis causing agent) is usually used at a later stage for
further studies. M. smegmatis (ATCC 607) has been employed in bioassays and is cited many times, erroneously,
in many publications as M. tuberculosis 607. It is chosen
as a model for tuberculosis since it does not present the
pathogenic properties ascribed to M. tuberculosis and
it exhibits rapid growth, in contrast to M. tuberculosis,
with which slow growth is normally associated (Reyrat
& Kahn 2001, Newton et al. 2002, Okunade et al. 2004,
Pauli et al. 2005).
Several screening studies have been performed to
evaluate in vitro antimycobacterial activity of plant extracts. Tosun et al. (2004), using M. tuberculosis H37Ra
as test microorganism, and an ethnobotanic approach for
plant selection, evaluated extracts from 44 plant species
belonging to 17 families and found that 23 extracts inhibited growth at concentrations from 50 to 200 µg/ml. Billo
et al. (2005) evaluated 22 plants (55 extracts) used in traditional medicine for treatment of symptoms potentially
related to tuberculosis, representing 16 families, using M.
bovis BCG as test organism, and found that five species
exhibited activity towards this test organism.
Many plant species present inhibition zones of differing diameters, however, size difference of the inhibition
zone depends primarily upon these factors: (a) diffusion
capacity of substances (present in the extracts) in the agar
medium, (b) antimicrobial activity of diffused substances,
(c) origin of microorganisms, (d) pH of substrates in plates,
(e) density of inoculation, and (f) growth and metabolic
activity of microorganisms in the medium. This suggests
that inhibitory activity is not necessarily proportional to
the inhibition zone diameter, especially when comparing
different extracts. Inhibition zone diameter can further be
associated with polarities of substances which make up
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 103(1), February 2008
the tested extracts and also with cell wall composition of
test organisms since Gram-positive bacteria present cell
walls with lower lipid levels than do Gram-negative bacteria (Pauli et al. 2005, Bandeira et al. 2006).
Mycobacterium cell walls have elevated levels of
high molecular weight lipids. This feature of cell wall
structure seems to function as a barrier to the direct absorption of polar compounds (Pauli et al. 2005) and may
be responsible for the more promising results obtained in
the present study for chloroform extracts which have the
lowest polarity among the extracts tested.
The present study made use of simple, rapid and inexpensive techniques for in vitro evaluation of antimicrobial activity. A similar method was used in recent studies
to evaluate the activity of plant extracts and fractions in
strains of S. aureus, E. coli and Pseudomonas aeruginosa
(Chah et al. 2006), Streptococcus mitis, Streptococcus
mutans and S. sanguis (Melo et al. 2006), as well as, C.
albicans and other fungi which cause opportunistic infections (Lima et al. 2005). Alves et al. (2000), using the same
method employed in the present study, namely diffusion in
agar (wells were 7 mm in diameter), considered extracts
with inhibition zones with diameters between 9 and 12
mm to be partially active, between 13 and 18 mm to be active, and greater than 18 mm to be very active. In the present study, extracts having inhibition zones with diameters
greater than 20 mm were considered to be very active.
Bioautography revealed promising extracts from D.
calycina. These results not only confirmed the activity of
the leaf chloroform extract of this species detected in the
other assays, but also revealed the antimicrobial potential of substances present in the branchwood chloroform
extract, which were active in all microorganisms tested.
Bioautography also revealed six extracts having components with inhibitory effects towards C. albicans. This
activity towards C. albicans observed in bioautography
contrasts strikingly with the results of MIC evaluation
using this same species which revealed no significant activity for any of the extracts.
A considerable difference was shown to exist in the
results obtained using diffusion in agar and bioautography. For example, L. gracillis branch chloroform extract,
which presented the lowest MIC (48.8 µg/ml) using M.
smegmatis in the diffusion method, was inactive in bioautography. On the other hand, D. calycina branch chloroform extract, which presented inhibition zone diameter
> 20 mm and good inhibitory activity (MIC < 97.7 µg/ml)
only towards M. smegmatis, under bioautography conditions, presented active (growth inhibiting) substances
(multiple Rfs) for all microorganisms evaluated.
Based on these results, D. calycina (Annonaceae) e L.
gracilis (Apocynaceae) are promising antimicrobial plant
species which need further study to reveal the identity
of the antimicrobial substances present in their extracts.
It should be stressed that the antimicrobial activity presented for these plant species is new information, having
no basis in popular use or direct relation to previously
published laboratory studies. These results contribute to
the knowledge of bioactive species in the Amazon flora
which, through further study, can reveal new lead compounds for drug development.
37
REFERENCES
Alves TMA, Ribeiro FL, Kloos H, Zani CL 2001. Polygodial, the fungitoxic component from the brazilian medicinal plant Polygonum
puctatum. Mem Inst Oswaldo Cruz 96: 831-833.
Alves TMA, Silva AF, Brandão M, Grandi TSM, Smânia EFA, Smânia Jr A, Zani CL 2000. Biological screening of brazilian medicinal plants. Mem Inst Oswaldo Cruz 95: 367-373.
Bandeira MFCL, Teixeira MFS, Abinader CD, Parente RC, Lima PSL
2006. Avaliação in vitro da sensibilidade da Candida albicans ao
hidróxido de cálcio associado ao óleo de copaíba. Rev Dentística
6: 12-22.
Billo M, Cabalion P, Waikedre J, Fourneau C, Bouttier S, Hocquemiller R, Fournet A 2005. Screening of some new Caledonian
and Vanuatu medicinal plants for antimycobacterial activity. J
Ethnopharmacol 96: 195-200.
Botion LM, Ferreira AVM, Côrtes SF, Lemos VS, Braga FC 2005.
Effects of the Brazilian phytopharmaceutical product Ierobina®
on lipid metabolism and intestinal tonus. J Ethnopharmacol 102:
137-142.
Chah KF, Eze CA, Emuelosi CE, Esimone CO 2006. Antibacterial
and wound healing properties of methanolic extracts of some nigerian medicinal plants. J Ethnopharmacol 104: 164-167.
Corrêa MP 1984. Dicionário das Plantas Úteis do Brasil e das Exóticas
Cultivadas, Vol. 6, Ministério da Agricultura, Instituto Brasileiro de
Desenvolvimento Florestal, Imprensa Nacional, Brasília, 777 pp.
Coykendall AL1989. Classification and identification of the viridans
Streptococci. J Ethnopharmacol 2: 315-328.
Esmerino LA, Pereira AV, Schelesky ME 2005. Doseamento da potência da ciprofloxacina em comprimidos orais. Rev Bras Farm
86: 17-20.
Fenner R, Betti AH, Mentz LA, Rates SMK 2006. Plantas utilizadas
na medicina popular brasileira com potencial atividade antifúngica. Braz J Pharm Sci 42: 369-394.
Flora Project 2006. Available from: http://curupira.inpa.gov.br/projetos/ducke/ index.html [cited 2006 Dec 15].
Gayoso CW, Lima EO, Souza EL, Trajano VN, Pereira FOE, Lima IO
2004. Ação Inibitória do óleo essencial de Cinnamonum zeylanicum Blume, �-pineno
-pineno e �-pineno
-pineno sobre
sobre fungos
fungos isolados
isolados de
de onicoonicomicose. J Bras Fitomed 2: 25-29.
Gendron R, Grenier D, Maheu-Robert L 2000. The oral cavity as a
reservoir of bacterial pathogens for focal infections. Microbes
Infect 2: 897-906.
Giogetti M, Negri G, Rodrigues E 2007. Brazilian plants with possible
action on the central nervous system - a study of historical sources
from the 16th to 19th century. J Ethnopharmacol 109: 338-347.
Gurib-Fakim G 2006. Medicinal plants: traditions of yesterday and
drugs of tomorrow. Mol Aspects Med 27: 1-93.
Hamada S, Slade H 1980. Biology, immunology, and cariogenicity of
Streptococcus mutans. Microbiol Rev 44: 331-384.
Holetz FB, Pessini GL, Sanches NR, Cortez DAG, Nakamura CV,
Dias Filho BP 2002. Screening of some plants used in the brazilian folk medicine for the treatment of infections diseases. Mem
Inst Oswaldo Cruz 97: 1027-1031.
Koo H, Gomes BPFA, Rosalen PL, Ambrosano GMB, Park YK, Cury
JA 2000. In vitro antimicrobial activity of propolis and Arnica
montana against oral pathogens. Arch Oral Biol 45: 141-148.
Lima IO, Araújo R, Oliveira RAG, Lima EO, Souza EL, Farias NP,
Navarro DF 2005. Inhibitory effect of some phytochemicals in
38
Antimicrobial Amazonian species • Ana Lúcia B Caneiro et al.
the growth of yeasts potentially causing opportunistic infections.
Braz J Pharm Sci 41: 199-203.
Melo AFM, Santos EJV, Souza LFC, Carvalho AAT, Pereira MSV,
Higino JS 2006. Atividade antimicrobiana in vitro do extrato de
Anacardium occidentale L. sobre espécies de Streptococcus. Rev
Bras Farmacogn 16: 202-205.
Melo-Souza SE 2000. Tratamento das Doenças Neurológicas, Guanabara Koogan, Rio de Janeiro, 849 pp.
Montanari CA, Bolzani VS 2001. Planejamento racional de fármacos
baseado em produtos naturais. Quím Nova 24: 105-111.
Moreira AC, Müller ACA, Pereira Jr N, Antunes MAS 2006. Pharmaceutical patents on plant derived materials in Brazil: policy, law
and statistics. World Patent Inf 28: 34-42.
Motsei ML, Lindsey KL, Staden JE, Jäger AK 2003. Screening of
traditionally used African plants for antifungal activity against
Candida albicans. J Ethnopharmacol 86: 235-241.
Newton SM, Lau C, Gurcha SS, Besra GS, Wright CW 2002. The
evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea
corylifolia and Sanguinaria canadensis. J Ethnopharmacol 79:
57-67.
Okunade AL, Elvin-Lewis PF, Lewis WH 2004. Natural antimcobacterial metabolites: current status. Phytochemistry 65: 1017-1032.
Pauli GF, Case RJ, Inui T, Wang Y, Cho S, Fischer NH, Franzblau SG
2005. New perspectives on natural products in TB drug research.
Life Sci 78: 485-494.
Pereira JV, Bergamo DCB, Pereira JO, França SC, Pietro RCLR,
Souza-Silva YTC 2005. Antimicrobial activity of Arctium lappa
constituents against microorganisms commonly founding endodontic infections. Braz Dent J 16: 192-196.
Piovano S 1999. Bacteriology of most frequent oral anaerobic infections. Anaerobe 5: 221-227.
Pohlit AM, Quignard ELJ, Nunomura SM, Tadei WP, Hidalgo AF,
Pinto ACS, Santos EVM, Morais SKR, Saraiva RCG, Ming LC,
Alecrim AM, Ferraz AB, Pedroso ACS, Diniz EV, Finney EK,
Gomes EO, Dias HB, Souza KS, Oliveira LCP, Don LC, Queiroz
MMA, Henrique MC, Santos M, Lacerda Júnior OS, Pinto PS,
Silva SG, Graça YR 2004. Screening of plants found in the State
of Amazonas, Brazil for larvicial activity against Aedes aegypti
larvae. Acta Amazônica 34: 97-105.
Quignard ELJ, Nunomura SM, Pohlit AM, Alecrim AM, Pinto ACS,
Portela CN, Oliveira LCP, Don LC, Silva LFR, Henrique MC,
Santos M, Pinto PS, Silva SG 2004. Median Lethal Concentrations of Amazonian Plant Extracts in the Brine Shrimp Assay.
Pharm Biol 42: 253-257.
Quignard ELJ, Pohlit AM, Nunomura SM, Pinto ACS, Santos EVM,
Morais SKR, Alecrim AM, Pedroso ACS, Cyrino BRB, Melo CS,
Finney EK, Gomes EO, Souza KS, Oliveira LCP, Don LC, Silva
LFR, Queiroz MMA, Henrique MC, Santos M, Pinto OS, Silva SG
2003. Screening plants found Amazonas state for lethality towards
brine shrimp Artemia franciscana. Acta Amazônica 33: 93-104.
Reyrat JM, Kahn D 2001. Mycobacterium smegmatis: an absurd model for tuberculosis? Trends Microbiol 9: 472-473.
Ribeiro JELS, Hopkins MJG, Vicentini A, Sothers CA, Costa MAS,
Brito JM, Souza MAD, Martins LHP, Lohmann LG, Assunção
PACL, Pereira EC, Silva CF, Mesquita MR, Procópio LC 1999.
Flora da Reserva Ducke: guia de identificação das plantas vasculares de uma floresta de terra-firme na Amazônia Central,
INPA, Manaus, 816 pp.
Silva MF, Lisbôa PLB, Lisbôa RCL 1977. Nomes Vulgares de Plantas
Amazônicas, INPA, Belém, 222 pp.
Tosun F, Kizilay ÇA, Sener B, Vural M, Palittapongarnpim P 2004.
Antimycobacterial screening of some Turkish plants. J Ethnopharmacol 95: 273-275.
Turolla MSR, Nascimento ES 2006. Informações toxicológicas de
alguns fitofármacos utilizados no Brasil. Rev Bras Cien Farm
42: 289-306.
Wade WG, Munson MA, Lillo A, Weightman AJ 2005. Specificity of
the oral microflora in dentinal caries, endodontic infections and
periodontitis. Int Congr Ser 1284: 150-157.