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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 97, Num. 7, 2002, pp. 1027-1031
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Mem Inst Oswaldo Cruz, Rio de
Janeiro, Vol. 97(7), October
2002, pp. 1027-1031
Screening of Some
Plants Used in the Brazilian Folk Medicine for the Treatment of Infectious Diseases
Fabíola Barbiéri Holetz,
Greisiele Lorena Pessini, Neviton Rogério Sanches, Diógenes Aparício
Garcia Cortez*, Celso Vataru Nakamura**, Benedito Prado Dias Filho**/+
Programa de Pós-graduação
em Ciências Farmacêuticas *Departamento de Farmácia e Farmacologia
**Departamento de Análises Clínicas, Universidade Estadual de
Maringá, Av. Colombo 5790, 87020-900 Maringá, PR Brasil
+Corresponding author. Fax: +55-44-261.4490. E-mail:
bpdfilho@uem.br
This study was
supported by grants from the Conselho Nacional de Desenvolvimento Científico
e Tecnológico, CNPq, Capacitação de Aperfeiçoamento
de Pessoal de Nível Superior, Capes, and Programa de Pós-graduação
em Ciências Farmacêuticas da Universidade Estadual de Maringá.
Received 1 October 2001
Accepted 6 September 2002
Code Number: oc02229
Extracts of 13 Brazilian medicinal
plants were screened for their antimicrobial activity against bacteria and yeasts.
Of these, 10 plant extracts showed varied levels of antibacterial activity.
Piper regnellii presented a good activity against Staphylococus aureus
and Bacillus subtilis, a moderate activity on Pseudomonas aeruginosa,
and a weak activity against Escherichia coli. Punica granatum
showed good activity on S. aureus and was inactive against the other
standard strains. Eugenia uniflora presented moderate activity on both
S. aureus and E. coli. Psidium guajava,Tanacetum vulgare,
Arctium lappa, Mikania glomerata, Sambucus canadensis,
Plantago major and Erythrina speciosa presented some degree of
antibacterial activity. Spilanthes acmella, Lippia alba, and Achillea
millefolium were considered inactive. Five of the plant extracts presented
compounds with Rf values similar to the antibacterial compounds visible on bioautogram.
Of these, three plants belong to the Asteraceae family. This may mean that the
same compounds are responsible for the antibacterial activity in these plants.
Anticandidal activity was detected in nine plant extracts (P. guajava,
E. uniflora, P. granatum, A. lappa, T. vulgare,
M. glomerata, L. alba, P. regnellii, and P. major).
The results might explain the ethnobotanical use of the studied species for
the treatment of various infectious diseases.
Key words: medicinal plants - antibacterial
activity - anticandidal activity - bioautography
The use of medicinal plants in the
world, and especially in South America, contributes significantly to primary
health care. Many plants are used in Brazil in the form of crude extracts, infusions
or plasters to treat common infections without any scientific evidence of efficacy.
Pharmacological studies done with essential oils from 15 species of aromatic
plants obtained in Northeast of Brazil have shown activity coherent with the
use of these plants in folk medicine. These studies have dealt with the effect
of these oils on muscle contraction and with their antispasmodic, analgesic,
anti-inflammatory, anticonvulsant and antibacterial activity (Leal-Cardoso &
Fonteles 1999).
Recently, Nakamura et al. (1999)
reported the antibacterial activity of both essential oil and purified active
compound of Ocimum gratissimum, traditionally used in Brazilian folk
medicine to treat different diseases, e. g. upper respiratory tract infections,
diarrhoea, skin diseases, pneumonia, and also as a treatment for cough, fever
and conjunctivitis. The compound that showed antibacterial activity for gram-positive
and gram-negative bacteria in the essential oil was identified as eugenol by
spectral data.
More recently, Alves et al. (2000)
had screened 60 medicinal plant species from the Brazilian savanna for molluscicidal
activity, toxicity to brine shrimp, antifungal activity and antibacterial activity.
In this study, 42 species afforded extracts that showed some degree of activity
in one or more of these bioassays.
Plants which have been used as medicines
over hundreds of years, constitute an obvious choice for study. It is interesting
to determine whether their traditional uses are supported by actual pharmacological
effects or merely based on folklore. In the present study, we chose 13 plants
currently used in the folk medicine in our region. All of them have been used
in the treatment of infectious disease, so that the in vitro antimicrobial activity
was investigated.
MATERIALS AND METHODS
Plant collection - The plants
were collected in March 2001, in Maringá, PR, Brazil. The plants were
identified by the same researchers that accomplished the collection, deposited
and authenticated at Herbarium of State University of Maringá, Maringá,
PR, Brazil (Table I).
Preparation of extracts -The
plant parts selected were ground, submitted to a maceration process with ethanol-water
(90-10%) for 48 h at 25oC and protected from sunlight. The hydroalcoholic
extract obtained was evaporated under vacuum, lyophilised and the residue directly
assayed against the microorganisms presented below.
Microorganisms used and growth
conditions -The test organisms included the bacteria Escherichia coli
ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Bacillus subtilis
ATCC 6623, and Staphylococcus aureus ATCC 25923. The yeasts Candida
albicans, C. krusei, C. parapsilosis, and C. tropicalis were obtained
from local clinical microbiology laboratories. The bacteria were grown
in nutrient broth (Difco Laboratories, Detroit, MI) at 37°C and maintained
on nutrient agar slants at 4°C. The yeasts were grown and maintained on
Sabouraud-dextrose agar (Merck SA, São Paulo, Brazil).
Antimicrobial susceptibility testing
-The minimum inhibitory concentrations (MICs) of all extracts and reference
antibiotics (tetracycline, vancomycin, penicillin, and nistatin; Sigma Chemical
Co., St. Louis, MO) were determined by microdilution techniques in Mueller-Hinton
broth (Merck) for bacteria and RPMI-1640 (Sigma Chemical Co.) for fungi (NCCSL
1999, 2000). Each extract (2 mg/ml) was aseptically mixed with inoculum prepared
in the same media at a density adjusted to the tube 0.5 of McFarland' scale
(106 yeasts or 108 bacterial cells), and diluted 1:10
for the broth microdilution procedure. Microtiter trays were incubated at 37oC
and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints
were recorded for each isolated. The MIC was defined as the lowest concentration
of compounds that produced an 80% reduction in visible growth compared with
control. MBCs were defined as the lowest concentration yielding negative subcultures
or only one colony.
Thin layer chromatography -
Kieselgel GF254 plates, 20 x 20 cm, 1 mm thick, were used. Plant
extracts were applied and the chromatogram developed using CHCl3/MeOH/H2O
(65:35:5) as solvent. TLC plates were run in duplicate and one set was used
as the reference chromatogram. Spots and bands were visualized by UV irradiation
(254 and 366 nm) and H2SO4 spray reagent. The other set
was used for bioautography.
Bioautography - Chromatogram
developed as described above was placed in plate square with cover and a inoculum
of S. aureus containing 106 CFU/ml in molten Mueller-Hinton
agar was distributed over the plates. After solidification of the medium, the
TLC plate was incubated overnight at 37oC. Subsequently bioautogram
was sprayed with an aqueous solution of 2,3,5-triphenyltetra-zolium chloride
(TTC) and incubated at 37oC for 4 h. Inhibition zones indicated the
presence of active compounds.
RESULTS AND DISCUSSION
The ethnobotanical screening tests
of hydroalcoholic extracts of 13 plants against both gram-positive and gram-negative
bacteria and yeastsby using the microdilution technique are given in Table
II. It was considered that if the extracts displayed an MIC less than 100
µg/ml, the antimicrobial activity was good; from 100 to 500 µg/ml
the antimicrobial activity was moderate; from 500 to 1000 µg/ml the antimicrobial
activity was weak; over 1000 µg/ml the extract was considered inactive.
Piper regnellii presented a good activity against the gram-positive bacteria
S. aureus and B. subtilis with MIC of 7.8 and 15.6 µg/ml,
respectively, a moderated activity on the gram-negative P. aeruginosa with
MIC of 250 µg/ml, and a weak activity against the gram-negative E. coli
with MIC 1000 µg/ml. Punica granatum showed good activity on
S. aureus with MIC of 62.5 µg/ml and was inactive against the other
standard strains at concentrations ³ 1000 µg/ml.
On the other hand, Psidium guajava afforded extract with moderate activity
against gram-positive bacteria and on the gram-negative bacteria E. coli,
while the extracts of Arctium lappa, Tanacetum vulgare and
Mikania glomerata presented some degree of activity against both gram-positive
and gram-negative bacteria. Eugenia uniflora presented moderate activity
on both S. aureus and E. coli. Sambucus canadensis shown moderate activity
against B. subtilis, and Plantago major was weakly active on S.
aureus. Spilanthes acmella, Lippia alba, and Achillea millefolium
were considered inactive. The minimal bactericidal concentrations were within
two-twofold dilution of the MICs for these organisms. The most susceptible bacterium
was S. aureus.
Different results were obtained for
the 13 studied species for antifungal activity against the yeasts C. albicans,
C. krusei, C. parapsilosis, and C. tropicalis. In the anti-yeast
assay, P. guajava extract displayed good activity against all the yeasts
tested at concentrations of 15.6 to 125 µg/ml. Both E. uniflora
and P. granatum afforded extracts with good activity against some yeast,
but were inactive on C. albicans. The extracts of L. alba and
P. major showed moderate response against C. krusei, and a weak
activity on C. tropicalis, but did not show any activity against C.
albicans and C. parapsilosis. M. glomerata and P. regnellii yielded
extracts with moderate activity against C. krusei and C. tropicalis,
while A. millefolium extractwas weakly active against C. krusei.
E. speciosa, S. acmella, and S. canadensis were inactive for all
the yeasts tested.
To obtain some information on the
active components, plant extracts were analyzed by TLC on silica gel. TLC plates
were run in duplicate and one set was used as the reference chromatogram, and
the other set was assayed for bioautography (Figure).
Panel A shows the chromatogram of plant extracts sprayed with Vanillin/Sulphuric
acid. Panel B shows the appearance of same chromatogram after treatment with
bacterial inoculum, indicating the location of bacterial inhibition zone. Two
antibacterial compounds were detected in the origin. These compounds were present
in the extracts from E. uniflora (Line 10) and P. granatum (Line
11). P. guajava presented antibacterial compound (s) with Rf of
0.68 (Line 4). All other antibacterial compounds presented similar Rf values
(0.85-0.93), and were detected in the extracts from A. lappa (Line 1),
T. vulgare (Line 2), E. speciosa (Line 3), M. glomerata
(Line 5) and P. regnellii (Line 9). In the solvent system used for screening,
it forms a streak which can extend over more than one Rf unit, which
may overlap with other active compounds in the extracts. Also, due to clustering
of bioautography zones detected in the origin, it was difficult to distinguish
individual compounds. Use of additional, more polar solvent system to develop
TLC plates in future work on individual plants will allow better separation
on these zones. In the Mirtaceae family there is a large variety of active principles
against microorganisms including essential oils, flavonoids, (Slowing et al.
1994, Lis-Balchin et al. 2000, Hernández et al. 2000) and tannins (Scalbert
1991, Djipa et al. 2000). The presence of flavonoids and tannins in Punicaceae
family can be responsible by antimicrobial activity (Sahar et al. 1997). Since
the antimicrobial activity in species of the Piperaceae family has been found
with the amides, essential oil, lignans, phenylpropanoids, alkaloids, neolignans
and chromene (Masuda et al. 1991, Benevides et al. 1999, Costantin et al. 2001,
Dorman & Deans 2000), its is possible that these compounds could be responsible
by the antimicrobial properties reported here.
Under the conditions employed here,
all test samples have more potent inhibitory effects on gram-positive bacteria
in comparison to gram-negative bacteria. According to the TLC separation, five
plant extracts yielded components with Rf values similar to the antibacterial
compounds visible on bioautograms. In addition, many of these inhibition zones
were associated with dark blue spots which had been detected under UV radiation
(data not shown). Of these, three plants belong to the Asteraceaefamily. This
may mean that the same compounds are responsible for the antibacterial activity
in these plants.
In the present study hydroalcoholic
extracts of some Brazilian medicinal plants were tested against bacteria and
yeasts. Ten out of 13 plant extracts from eight families displayed some degree
of antibacterial activity, in particular against the gram-positive bacteria
S. aureus and B. subtilis, representing more than 77% of the screened
extracts. This is to be expected because the outer membrane of gram-negative
bacteria is known to present barrier to penetration of numerous antibiotic molecules,
and the periplasmic space contains enzymes, which are able of breaking down
foreign molecules introduced from outside (Duffy & Power 2001). It was interesting
that the extracts of P. regnellii showed response on both gram-positive
and gram-negative bacteria and yeasts. In spite of P. guajava exhibited
good activity against all the yeasts and a moderate activity against gram-positive
bacteria, the inhibitory activity against gram-negative bacteria displayed by
the extract was low.
Although this study investigated
the in vitro antimicrobial activity, the results substantiate the ethnobotanical
use of the 13 studied species for the treatment of various bacteria-related
diseases. However, in vivo data may be helpful in determining the potential
usefulness of these plants for treatment of infectious diseases. In terms of
conservation, the results show that leaf material is useful for antimicrobial
uses, and it could be used without any detrimental effect on the plant.
ACKNOWLEDGMENTS
To Marinete Martinez for help in
the experiments.
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Copyright 2002 Instituto Oswaldo
Cruz - Fiocruz
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