Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol.

101(4): 387-390, June 2006 387

Synergism between plant extract and antimicrobial drugs used on

Staphylococcus aureus diseases
Joyce Elaine Cristina Betoni, Rebeca Passarelli Mantovani, Lidiane Nunes Barbosa,
Luiz Claudio Di Stasi*, Ary Fernandes Junior/+
Departamento de Microbiologia e Imunologia * Departamento de Farmacologia, Instituto de Biociências,
Universidade Estadual Paulista Julio de Mesquita Filho, 18618-000 Botucatu, SP, Brasil

Searches for substances with antimicrobial activity are frequent, and medicinal plants have been considered
interesting by some researchers since they are frequently used in popular medicine as remedies for many infectious
diseases. The aim of this study was to verify the synergism between 13 antimicrobial drugs and 8 plant extracts –
“guaco” (Mikania glomerata), guava (Psidium guajava), clove (Syzygium aromaticum), garlic (Allium sativum), lem-
ongrass (Cymbopogon citratus), ginger (Zingiber officinale), “carqueja” (Baccharis trimera), and mint (Mentha piperita)
– against Staphylococcus aureus strains, and for this purpose, the disk method was the antimicrobial susceptibility
test performed. Petri dishes were prepared with or without dilution of plant extracts at sub-inhibitory concentra-
tions in Mueller-Hinton Agar (MHA), and the inhibitory zones were recorded in millimeters. In vitro anti-Staphylo-
coccus aureus activities of the extracts were confirmed, and synergism was verified for all the extracts; clove, guava,
and lemongrass presented the highest synergism rate with antimicrobial drugs, while ginger and garlic showed
limited synergistic capacity.
Key words: medicinal plants - Staphylococcus aureus - antimicrobial drugs - synergism - Kirby & Bauer method

In a constant attempt to improve their quality of life, aromaticum (L) Merrill & Perry (clove) (Lopez et al. 2005),
men have used plants as source of food, shelter, clothing, Allium sativum L (garlic) (Ankri & Mirelman 1999, Ahmad
medicine, cosmetics, and for seeking relief from hardship & Beg 2000, Srinivasan et al. 2001, Benkeblia 2004),
of life. Some plants are known as medicinal because they Zingiber officinale Roscoe (ginger) (Silva Junior et al.
contain active substances that cause certain reactions, 1994, Konning et al. 2004), Cymbopogon citratus (DC)
from relenting to the cure of diseases, on the human or- Stapf (lemongrass) (Cimanga et al. 2002, Di Stasi & Hiruma-
ganism (Silva Junior et al. 1994). Knowledge on medicinal Lima 2002), Mentha piperita L (mint) (Silva Junior et al.
plants sometimes means the only therapeutic resource of 1994, Tassou et al. 2000), and Baccharis trimera (Less.)
some communities and ethnic groups (Di Stasi 1996); and DC (“carqueja”) (Avancini et al. 2000).
their use, especially in South America, contributes sig- A recent paper on medicinal plants and antimicrobial
nificantly to primary health care (Holetz et al. 2002). Infec- activity whose objective was to analyze past, present,
tious diseases still represent an important cause of mor- and future of medicinal plants to suggested as fundamen-
bidity and mortality among humans, especially in devel- tal the research on plant extract mechanism of action, in-
oping countries. Even though pharmaceutical industries teractions with antibiotics or with other medicinal plants,
have produced a number of new antimicrobial drugs in and extracts pharmacokinetic profile (Ríos & Recio 2005).
the last years, resistance to these drugs by microorgan- Research on synergism is very limited and few studies
isms has increased. In general, bacteria have the genetic have been reported (Nascimento et al. 2000, Aburjai et al.
ability to transmit and acquire resistance to drugs used as 2001, Aqil et al. 2005). Thus, in our research, we evaluated
therapeutic agents (Nascimento et al. 2000). in vitro synergism between extracts of M. glomerata, P.
In vitro studies on plants used in traditional medicine guajava, S. aromaticum, A. sativum, C. citratus, Z.
have been carried out in the field of microbiology, espe- officinale, B. trimera, and M. piperita and antimicrobial
cially on pathogenic bacterial growth; and some of these drugs utilized against S. aureus strains by using the Kirby
studies were about the antimicrobial activity of Mikania & Bauer method.
glomerata Spreng (“guaco”) (Boyayan 2002, Holetz et al. MATERIALS AND METHODS
2002), Psidium guajava L (guava) (Gnan & Demello 1999,
Jaiarj et al. 1999, Nascimento et al. 2000, Ahmad & Beg Plant samples - M. glomerata, P. guajava, B. trimera,
2001, Abdelrahim et al. 2002, Holetz et al. 2002, Vora- M. piperita, and C. citratus samples were collected in
vuthikunchai et al. 2004, Qadan et al. 2005), Syzygium 2004 from an experimental field of the School of
Agronomical Sciences, Unesp, Botucatu, São Paulo, Bra-
zil, and the voucher specimens were deposited at the Her-
barium of the Department of Botany, Institute of Bio-
sciences, Unesp. Their leaves were dried at 40oC and tritu-
Financial support: Fapesp rated in a mechanical mill. A. sativum, S. aromaticum, and
+Corresponding author: [email protected] Z. officinale samples were obtained from the local com-
Received 21 January 2006 merce in the same year and were used in natura for the
Accepted 8 May 2006 extracts preparation.
388 Antimicrobial drugs and plants synergism • Joyce Elaine Cristina Betoni et al.

Preparation of plant extracts - Plant material, dried and in plates containing MHA plus one-fourth the MIC
(M. glomerata, P. guajava, B. trimera, M. piperita, C. 90% of the respective extracts. The diameters (mm) of
citratus) or not (A. sativum, S. aromaticum, Z. officinale) the each inhibitory zone were recorded after incuba-
was ground, extracted with 70% methanol and filtered af- tion at 37oC/18 h.
ter 48 h. The plant residue was re-extracted with addition Statistical analysis - Results from the synergism as-
of 70% methanol, and after 24 h it was filtered again. Com- says were subjected to the Wilcoxon nonparametric test
bined filtrates were concentrated on a rotary evaporator to compare the values (mm) of the inhibitory zones ob-
at 45oC for methanol elimination, and the extracts were tained by the disk diffusion method (Minitab Statistical
kept in sterile bottles under refrigerated conditions until Software version 13.32). Results were considered signifi-
use. The extracts’ dry weight was obtained by the solvent cant when p < 0.05.
evaporation and used to determine concentration in
mg/ml. RESULTS AND DISCUSSION

Bacterial strains - Thirty-two S. aureus strains were Characteristics, MIC 90% (mg/ml) against 32 S. aureus
isolated from clinical specimens of newborns admitted to strains, and one-fourth the MIC 90% values obtained in
the Neonatal Unit of the Hospital of the School of Medi- the synergism assays for the plants and their respective
cine, Botucatu, SP, Brazil. Strains were isolated in sheep extracts are presented in Table I. Anti-S. aureus activity
blood agar and, after identification (Koneman et al. 2005), was verified for all the plants. S. aromaticum showed the
they were stored in brain heart infusion (BHI) plus agar. highest activity, followed by P. guajava; the lowest activ-
ity was recorded for lemongrass. The MIC 90% range was
Antimicrobial tests - Before the synergism assays 0.36 mg/ml for clove and 17.84 mg/ml for C. citratus and it
between the plant extracts and the antimicrobial drugs is not surprising the differences in the antimicrobial activ-
were evaluated, the minimal inhibitory concentration (MIC) ity of plants tested, due to phytochemical properties and
of the extracts was determined for 32 S. aureus strains by differences among species. Although the antimicrobial
diluting the extracts in Mueller Hinton agar (MHA) media activities of C. citratus, B. trimera, and Z. afficinale, have
(NCCLS 2004a,b). Petri dishes, controls and with differ- not been relatively high, synergism assays were carried
ent concentrations of plant extracts (mg/ml), were inocu- out for them and the synergism rate of C. citratus was as
lated with S. aureus strains (104 CFU) using a Steer’s high as that of S. aromaticum (Table II).
replicator and were incubated at 37oC/24 h. The concen- Antimicrobial mechanisms of the drugs used here were
tration that inhibited visible growth of each strain (MIC) variable and the protein synthesis inhibitors were those
was recorded, and the MIC 90% was calculated. One- that presented strongest synergistic effect (5.2 extracts/
fourth the MIC 90% was considered as the sub-inhibitory drug) together with folic acid (4 extracts/drug) and bacte-
concentration of the plant extracts in the synergism as- rial cell wall synthesis (3.8 extracts/drug) inhibitors. In-
says (Mahon & Manuselis 1995), which were carried out hibitors of the nucleic acid synthesis (2 extracts/drug)
on 15 S. aureus strains, including the ATCC 13565 strain showed weak synergism with plant extracts. Among the
by the disk diffusion method (Kirby & Bauer method) protein synthesis inhibitors, tetracycline showed syner-
gism with all the extracts, followed by chloramphenicol
(NCCLS 2004) on MHA media. Thirteen drugs were evalu-
and netilmicim. The synergistic capacity was promising
ated: penicillin (PEN; 10 IU), oxacillin (OXA; 1 µg), vanco-
for the extracts of some plants such as S. aromaticum, C.
mycin (VAN; 30 µg), ampicillin (AMP; 10 µg), cephalothin citrates, and P. guajava, which presented synergism with
(CFL; 30 µg), cefoxitin (CFO; 30 µg), chloramphenicol 11, 11, and 9 drugs, respectively; while garlic and ginger
(CLO; 30 µg), gentamicin (GEN; 10 µg), netilmicin (NET; showed synergism with only 3 and 2 drugs, respectively.
30 µg), tetracycline (TET; 30 µg), erythromycin (ERI; 15 The high synergism rate of protein synthesis inhibi-
µg), cotrimoxazole (SUT; 25 µg), and ofloxacin (OFX; 5 tors, although an important data, shows the need for more
µg). Two antibiogram sets were performed in duplicate for studies concerning the molecular basis of these interac-
each S. aureus strain in control plates, with plain MHA, tions. Similar results with synergism of protein synthesis

TABLE I
Characteristics, minimal inhibitory concentration 90% (MIC 90%) values, and sub-inhibitory one-fourth the MIC 90% values
obtained in the synergism assays for the plants and their extracts used
Scientific Common Part of the Extracts’ dry MIC 90% ¼ MIC 90%
name name plant used Efficacy (%) Weight (mg/ml) (mg/ml) (mg/ml)
Allium sativum Garlic Bulbs - 94.12 5.05 1.26
Baccharis trimera “Carqueja” Leaves 55.07 52.75 7.23 1.80
Cymbopogon citratus Lemongrass Leaves 25.62 63.87 17.84 4.46
Mikania glomerata “Guaco” Leaves 28.48 59.62 3.80 0.95
Psidium guajava Guava Leaves 47.68 131.75 0.52 0.13
Syzygium aromaticum Clove Flower buds - 58.75 0.36 0.09
Zingiber officinale Ginger Rhizomes - 11.75 3.56 0.89
Mentha piperita Mint Leaves 20.83 11.0 2.20 0.55
(-): for non-dried plants the efficacy was considered 100%.
 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101(4), June 2006 389

TABLE II
Synergism rate between antimicrobial drugs and plant extracts against 15 Staphylococcus aureus strains by the Kirby and
Bauer method
Psidium Syzygium Allium Mikania Baccharis Zingiber MenthaCymbopogon Synergism rate
Drug target Drugguajava aromaticum sativum glomerata trimera officinale piperita citratus (extract/drug)
MEAN
Protein synthesis TET x x x x x x x x 8 5.2
CLO x x - x x - x x 6
NET - - - x x x x x 5
ERI x x - - x - x - 4
GEN - - x x - - x - 3
cell wall synthesis VAN x x - x x - - x 5 3.8
PEN - x - x x - - x 4
OXA x x - - - - x x 4
CFL x x - x - - - x 4
AMP x x - - - - - x 3
CFO x x - - - - - x 3
Folic acid SUT x x x - - - - x 4 4
Nucleic acids OFX - x - - - - - x 2 2
Total 13 9 11 3 6 6 2 6 11
x: synergism when p ≤ 0.05; (-) no synergism; TET: tetracycline; CLO: chloramphenicol; NET: netilmicin; ERI: erythromycin; GEN:
gentamicin; VAN: vancomycin; PEN:penicillin; OXA: oxacillin; CFL: cephalothin; AMP: ampicillin; CFO: cefoxitin; SUT:
cotrimoxazole; OFX: ofloxacin.

inhibitors and propolis ethanolic extract of Apis mellifera ACKNOWLEDGMENTS

by E-test and disk diffusion methods were reported by To Dr Lin Chau Ming (FCA/Unesp/Botucatu) for provid-
Fernandes Junior et al. (2005). ing the plant specimens supply; Dr Maria de Lourdes Ribeiro
The synergism recorded here to plant extracts with de Souza da Cunha for S. aureus strains and Dr Lidia Raquel de
weak action on S. aureus growth, such as lemongrass, is Carvalho (IBB/UNESP/Botucatu) for the statistical analysis.
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