ISSN 1516-8913 Printed in Brazil
, Diógenes Aparício Garcia Cortez
, Michelle Simone Schiavini
, Benedito Prado Dias Filho
g/ml, respectively) and
Bacillus subtilis (MICs=500
g/ml), and virtually inactive against the gram-negative bacteria Escherichia coli and
Pseudomonas aeruginosa (MICs >1000
g/ml). The ethanol:water extracts showed higher antimicrobial activity as
-amyrin) and sterol (
-sitosterol was inactive for all the bacteria tested.
Key words: Psidium guajava, antibacterial activities, flavonoids,
Author for correspondence
the family Myrtaceae, is a native plant of tropical
America. Different parts of the plant are used in
the indigenous system of medicine for the
treatment of various human ailments such as
wounds, ulcers, bowels and cholera (Begum et al.,
2002). Pharmacological investigations indicated
that its bark, fruit, and leaves possess antibacterial,
hypoglycemic, anti-inflammatory, analgesic,
antipyretic, spasmolytic, and CNS depressant
activities (Begum et al., 2002). In Mexico, P.
guajava leaves are extensively used to stop
diarrhea, and the quercetin and its glycosides were
its active compounds. The water, alcohol and
chloroform extracts of leaves were effective
against Aeromonas hydrophila, Shigella spp. and
Vibrio spp, Staphylococcus aureus, Sarcina lutea
and Mycobacterium phlei (Jaiarj et al., 1999). The
leaves of P. guajava contain an essential oil rich in
cineol, tannins, triterpenes and flavonoids (Olajide
et al., 1999).
Recently, Holetz et al. (2002) had screened 13
Brazilian medicinal plants for antimicrobial
activity against bacteria and yeasts. In this study,
antimicrobial activity and presented compound (s)
Brazilian Archives of Biology and Technology
with Rf value of 0.68 similar to the antibacterial
The present study was undertaken to investigate
the in vitro antibacterial activity of aqueous and
ethanol:water extracts from leaves, roots and stem
barks of Psidium guajava. We are reporting our
results of the antibacterial activity of flavonoid
mixture obtained by bioassay-guided fractionation
of the ethanol:water extract of the leaves of
The extract from leaves, roots and stem bark of
plant were prepared by maceration adding 40 g of
powder plant to ethanol:water 1:1, 7:3 and 9:1 at
room temperature and to 200 ml of distilled
deionized water and heated to about 100
C for 10
The solvent was removed under
C and the aqueous and
extracts were assayed against gram-positive and
gram-negative bacteria by broth microdilution
assay to determine the MICs as described below.
Most studies were performed with Escherichia
ATCC 15442, Bacillus subtilis ATCC 6623, and
from the American Type Culture Collection
(ATCC, Rockville, Md.). The bacteria
Enterococcus faecalis ATCC 29212,
Streptococcus pyogenes ATCC 19615,
Staphylococcus epidermidis ATCC 12228,
Klebsiella pneumoniae ATCC 13883,
Enterobacter cloacae ATCC 13047, Proteus
mirabilis ATCC 25933, and Shigella flexneri
ATCC 12022 were provided by Instituto Nacional
de Controle de Qualidade em Saúde, Fundação
Oswaldo Cruz (Rio de Janeiro, RJ). The
microorganisms were grown in nutrient broth
(Difco Laboratories, Detroit, MI) at 37°C and
maintained on nutrient agar slants at 4°C.
Antibacterial susceptibility testing
The minimal inhibitory concentrations (MICs) of
all the extracts and reference antibiotics
(tetracycline, vancomycin and penicillin - Sigma
Chemical Co., St. Louis, MO, US) were
determined by microdilution techniques in
Mueller-Hinton broth (Merck) according to
NCCLS (2000). Inoculates were prepared in the
same medium at a density adjusted to a 0.5
McFarland turbidity standard [10
microdilution procedure. Microtiter plates were
incubated at 37
C and the MICs were recorded
endpoints were recorded for each isolated. The
MIC was defined as the lowest concentration of
compounds at which the microorganism tested did
not demonstrate visible growth. Minimum
bactericidal concentration (MBC) was defined as
the lowest concentration yielding negative
subcultures or only one colony.
For detection of antimicrobial activity, a
sensitive radial diffusion technique was used
as described earlier (Lehrer, 1991). Flavonoid
mixtures were tested against S. aureus using a
solid agarose medium. The agarose layer
consisted of 30 mg/100 ml Tryptic Soy Broth
system (BBL). in 10 mM phosphate buffer,
pH 7.2 with 0.02% Tween 20 and 0.8% GTG
agarose phase (Sigma Chemical Co.). The
plates were incubated at 37
C for 16 h until
The diameter of the clear zone was measured
and expressed in arbitrary units (0.1 mm = 1
U) after subtraction of the diameter of the well
Thin layer chromatography
plates, 20 x 20 cm, 1 mm thick,
l) and the chromatogram developed using
ethyl acetate:methanol (90:10) 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 365
nm) and vanillin/sulphuric acid (2%) spray
The other set was used for bioautography.
g) (Bristol Myers Squibb) was
Chromatograms developed as described above
were placed in a square plate with cover and an
inoculum of S. aureus containing 10
molten Mueller-Hinton agar was distributed over
TLC plate was incubated overnight at 37
Subsequently the bioautogram was sprayed with
chloride (TTC) and incubated at 37
C for 4 h.
The active ethanol:water extract (7:3) from leaves
of P. guajava (14 g) was submitted to vacuum
chromatography over on silica gel (32 g) eluted
with hexane, hexane- dichlomethane (1:1),
dichlomethane:ethyl acetate (95:5),
dichlomethane:ethyl acetate (80:20),
dichlomethane:ethyl acetate (50:50) ethyl acetate,
methanol, and methanol -water (9:1) to gave ten
fractions F1 (154.0 mg), F2 (405.6 mg), F3 (158.5
mg), F4 (42.7 mg), F5 (60.2 mg), F6 (117.3 mg), F7
(170.7 mg), F8 (807.1 mg), F9 (9,100.5 mg) and
F10 (111 mg). The fractions F1 to F10 were
assayed against S. aureus by bioautography.
Combined fractions F3 to F5 (261.4 mg) was
chromatographed on a column by gel filtration over
Sephadex LH-20 eluted with methanol to give 36
sub-fractions. The sub-fraction 15, which exhibited
antibacterial properties, was further fractionated by
column chromatography on silica gel (230-400
mesh) with ethyl acetate, ethyl acetate: methanol
(95:5, 90:10, 70:30) and methanol to yield a
flavonoids mixture (1, 22.0 mg). The fraction F2
(405.6 mg) was chromatographed by Sephadex LH-
20 and eluted with methanol to give 20 sub-
fractions. Combined sub-fractions 6-12 from
column were rechromatographed on silica gel (230-
400 mesh) using ethyl acetate, ethyl
acetate:methanol (95:5, 70:30) furnished a mixture
-amyrin (2, 5.8 mg). The fraction F6
(117.5 mg) was chromatographed on silica gel (70-
230 mesh) using ethyl acetate:methanol (90:10,
70:30, 50:50), methanol and methanol:water (95:5)
afforded 50 sub-fractions. Combined sub-fractions
12-18 was purified on silica gel (230-400 mesh)
using hexane, hexane:ethyl acetate (90:10, 80:20,
70:30, 50:50) and ethyl acetate to give
The NMR spectra were obtained in a BRUKER
DRX400 (9.4 T) and VARIAN GEMINI300
(7.05T), using deuterated solvent for field
homogeneity, TMS as internal standard and 298K.
ESI-MS: low resolution on a triple quadrupole was
recorded on a Micromass Quattro LC instrument
equipped with a “Z-spray” ion source, CC: silica
gel 60 (70-230 and 230-400 mesh) and gel
filtration on Sephadex LH-20. TLC: silica gel
The results of antimicrobial activities of the
extracts by using both microdilution assay are
summarised in Table 1.
ethanol:water extracts of leaves, stem bark and roots from Psidium guajava
Different results were obtained for the studied
bacteria. The ethanol:water extracts of P. guajava
leaves, stem bark and roots were active against the
gram-positive bacteria Staphylococcus aureus
(MICs=125, 62.5 and 125
g/ml, respectively) and
stronger antimicrobial activity as compared to
aqueous extracts. All extracts were virtually
inactive against the gram-negative bacteria
g/ml). Although the differences
extracts tended to be more active (i.e. have a lower
MIC) than the leaves and roots extracts. The
results showed that leaf material could be useful
for antibacterial uses, and it could be used without
any detrimental effect on the plant.
On the basis of this finding, the dose-response
effect of the ethanol:water (70:30) extract of P.
enterococci (Fig 1). The EC
, defined as the drug
effect, was 150
g/ml and 600
The ethanol:water extract of leaves from P.
chromatography on silica gel and the resulting
sub-fractions were analysed by TLC on silica gel.
Fig. 2 presents the results of TLC-bioautography
screening. Panel A shows
the chromatogram of
acid. Panel B shows the appearance of same
chromatogram after treatment with bacterial
inoculum, indicating the location of bacterial
inhibition zone at Rf values of 0.15, 0.44 and 0.70.
In the solvent system used for screening, it formed
a streak, which can extend over more than one Rf
unit, which might overlap with other active
compounds in the extracts. Also, due to clustering
of bioautography zones detected near the origin, it
was difficult to distinguish individual compounds.
Repeated silica gel and Sephadex LH-20 column
chromatography of the active extracts of P.
guajava afforded a mixture of flavonoids (1),
triterpenes (2) and sterol (3). The triterpene (
-amyrin) (Maia et al., 2000) and sterol (
sitosterol) (Goular et al., 1993) were identified by
The flavonoids group (1) could be easily detected
by spraying the TLC plates with NP/PEG (natural
products-polyethylenglycol reagent) showing
yellow colour for a monohydroxylated systems in
B ring (Wagner et al., 1984). In addition, the UV
(270, 331, 392 nm) spectra data of 1 was typical of
Further confirmation of our results was obtained
by a radial diffusion assay with S. aureus. The
activity of flavonoid mixture (1) against tested
bacterium was assessed in agarose gel. A
representative view of a gel in this assay is shown
in Fig. 3A. In Fig. 3B, the activity of 1 is
expressed in units as described in Material and
Methods. The minimal concentration of 1 that
resulted in inhibition of staphylococci growth was
g. This assay was designed to achieve
maximal sensitivity with minimal consumption of
reagents and it could be used in a bioassay-guided
Triterpene, flavonoid and sterol obtained from P.
among the tested microrganisms. In contrast to the
relatively low MICs for gram-positive bacteria,
gram-negative bacteria tested were not inhibited
by flavonoid mixture,
-sitosterol (Table 2). The results obtained
homogeneous, and both S. aureus and E. faecalis
were considered susceptible with MICs of 25
g/ml and 50
g/ml, respectively. The MICs of the
reference drugs used in this study were similar to
those presented in other reports.
other set was used for bioautography with S. aureus (B and D) Panels A and B show the chromatogram
of fractions 1-10. Panels C and D show
the chromatogram of flavonoid mixture (1),
-sitosterol (3) and penicillin (C).
are a large group of compounds naturally
occurring in higher and lower plants. Flavonoids
have been shown to be able to affect various
biological functions: capillary permeability,
cellular secretory processes involved in the
inflammatory response and inhibition of enzymes,
receptors and carriers (Torel, 1983; Affany, et al.,
1987; Middleton, 1988; Afanas'ev et al., 1989).
The inhibitory activities of flavonoids against
bacteria and yeast have been investigated by a
number of researchers, especially in Latin America
(Egil et al., 1985; Mendonza et. al., 1997;
Tereschuk et al., 1997; Hernandez et al., 2000;
Jussi-Pekka et al., 2000; Fukai et al., 2002).
Figure 3 - Radial diffusion assay with S. aureus. A representative view of a gel in this assay
is shown in A. Each well was loaded with 10
l sample containing various
g, C-and C+ negative and positive controls, respectively). In B, the activity of
flavonoid mixture (1) is expressed in units as described in Material and Methods.
The minimal concentration of flavonoid mixture (circle) that resulted in
inhibition (tendency line) of staphylococci growth was 2.0
ethanol:water (7:3) extracts of leaves, stem bark and roots from Psidium guajava
Ethanol:water extracts from
Olajide and Makinde (1999) reported that leaves
of P. guajava contain an essential oil rich in
cineol, tannins and triterpenes. In this study, the
analgesic and anti-inflammatory effects of
methanol extract from leaves were probably due to
the essential oils present in the plant. According to
these authors, the flavonoid content on the plant
inflammatory activity exhibited by the extract.
Gnan and Demello (1999) reported a complete
inhibition of growth of S. aureus, S. epidermidis
and S. typhimurium caused by aqueous guava leaf
extract at a concentration of 8 mg/ml. Vieira et al.
(2001) reported the microbiocidal effect of guava
sprout extract (ethanol, acetone and water) upon
toxigenic S. aureus and E. coli, performed using
radial diffusion. Extracts prepared with 60%
alcohol and 60% acetone produced the largest
halos for both species of bacteria. Abdelrahim et
al. (2002) also reported a complete inhibition of B.
subtilis, S. aureus, E. coli, and P. aeruginosa with
extract of the guava bark.
As described above, bioassay-guided fractionation
of ethanol:water extracts of leaves from P.
extracts, semi-purified fractions and compounds
from leaves of P. guajava showed considerable
activity against gram-positive bacteria but not
against gram-negative species. This could to be
expected because the outer membrane of gram-
negative bacteria is known to present a 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 and Power, 2001).
In conclusion, the results of the antibacterial
property of P. guajava extracts showed a good
correlation between reported uses of this plant in
Brazilian folk medicine against infectious diseases
and the experimental data of such extracts toward
the most common pathogens. However, the
extracts and active compound isolated from P.
models for in vitro efficacy and toxicity. In term of
conservation, the results showed that leaf material
could be useful for antimicrobial uses, and it could
be used without any detrimental effect on the
This study was supported by grants from the
Conselho Nacional de Desenvolvimento Científico
e Tecnológico, CNPq and Programa de Pós-
graduação em Ciências Farmacêuticas de
Universidade Estadual de Maringá. We are
grateful to Marinete Martinez Vicentim for help in
O presente estudo foi conduzido para avaliar a
atividade antibacteriana dos extratos etanol:água e
aquoso das folhas, raízes e casca do caule de
dos extratos contra as bactérias foram testados
usando o ensaio de microdiluição em caldo. O
extrato aquoso das folhas, raízes e casca do caule
de P. guajava foram ativos contra as bactérias
Gram-positivas Staphylococcus aureus
(CIMs=500, 125 e 250
g/ml, respectivamente) e
g/ml), e foram
g/ml). Os extratos etanol:água
com os extratos aquosos. Com base nestes
resultados, o extrato de folhas de P. guajava foi
fracionado em cromatografia em coluna de sílica
gel em um bioensaio de fracionamento
direcionado, produzindo uma mistura de
flavonoides, uma mistura de
sitosterol. A mistura de flavonoides mostrou boa
-sitosterol foi inativo para todas as bactérias
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Received: December 28, 2003;
Revised: April 20, 2004;
Accepted: September 24, 2004.