1

SUPPLEMENTARY MATERIAL

Antimicrobial activity and phytochemical analysis of crude extracts and essential oils
from medicinal plants

N.C.C. Silva
a
, L. Barbosa
b
, L.N. Seito
c
and A. Fernandes Jnior
a
*


a
Department of Microbiology and Immunology, Institute of Biosciences, So Paulo State
University, UNESP, Botucatu, So Paulo, Brazil;
b
Department of Biostatistics, Institute of
Biosciences, So Paulo State University, UNESP, Botucatu, So Paulo, Brazil;
c
Department of Pharmacology, Institute of Biosciences, So Paulo State University,
UNESP, Botucatu, So Paulo, Brazil

*Corresponding author. Email: ary@ibb.unesp.br

We aimed to establish a phytochemical analysis of the crude extracts and
performed GC-MS of the essential oils (EOs) of Eugenia uniflora L.
(Myrtaceae) and Asteraceae species Baccharis dracunculifolia DC, Matricaria
chamomilla L. and Vernonia polyanthes Less, as well determining their
antimicrobial activity. Establishment of the minimal inhibitory concentrations
of the crude extracts and EOs against 16 Staphylococcus aureus and 16
Escherichia coli strains from human specimens was carried out using the
dilution method in MuellerHinton agar. Some phenolic compounds with
antimicrobial properties were established, and all EOs had a higher
antimicrobial activity than the extracts. M. chamomilla extract and E. uniflora
EO were efficient against S. aureus strains, while E. uniflora and V. polyanthes
extracts and V. polyanthes EO showed the best antimicrobial activity against E.
coli strains. Staphylococcus aureus strains were more susceptible to the tested
plant products than E. coli, but all natural products promoted antimicrobial
growth inhibition.

Keywords: plant crude extracts; essential oils; medicinal plants; minimal
inhibitory concentration; antibacterial activity; phytochemical analysis

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Experimental
Plants, crude methanolic extracts and EO
Methanol extracts and EO were achieved from B. dracunculifolia and V. polyanthes
(leaves) collected near the Campus of the University at first hours of the day. The sample
of M. chamomilla (dried flowers) was obtained from Centro Flora-Anidro, Botucatu, So
Paulo State, Brazil. The E. uniflora samples (leaves) were obtained from an EO producer
and their crude methanolic extract was further achieved. The taxonomic identification and
voucher deposit was stored at the Herbarium of the Department of Botany, UNESP (Botu
25794 M. chamomilla, Botu 25795 B. dracunculifolia, Botu 25796 E. uniflora and
Botu 25797 V. polyanthes)
Dried plants samples (50C at greenhouse with a forced circulation of air) were
powdered in a blender and submitted to maceration for 48 h in 70% methanol solution at
refrigerator temperature ( 4C). Afterwards, extracts were filtered, the solvent was
evaporated using a rotaevaporator at 45C and the dry weight of each extracts was
calculated after complete evaporation of the solvent (Betoni, Mantovani, Barbosa, Di Stasi,
& Fernandes Jnior, 2006).
The M. chamomilla, B. dracunculifolia and V. polyanthes EO were prepared by steam
distillation in Clevenger apparatus (Marconi model M480) (Souza, Pereira, Anglico, &
Pimenta, 2004), and after measurement of EO volumes, the raw material was stored at 4C.
The E. uniflora EO was achieved from EO producer by steam distillation. The density of
the EO was calculated according to Fonseca and Librandi (2008): P1 is weight of the
container without essential oil and P2 with weight sample of 1 ml (V) of essential oil.
mL
mg
V
P P
D
1 2
.

Qualitative phytochemical analysis of crude methanol extracts and essential oils
The qualitative phytochemical analysis of crude extracts included steroids, triterpenes,
saponins, fixed strong acids, phenolic compounds, quaternary amines and alkaloids
(Matos, 1988). EO chemical analysis was performed using gas chromatography-mass
spectrometry- (GC-MS) (Shimadzu, model QP5050A), with a capillary column CBP-5,
50m length, 0.25mm of diameter and 0.25 m film thickness. The temperature of the
injector and of the interface was 250
0
C; the detector operated in mode EI at 70eV and He
was the carrier gas. Chromatographic conditions were: M. chamomilla: initial temperature
of 60C, heating at a rate of 4C min
1
up to 100
0
C, heating at 6C min
1
up to 200C,
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keeping this temperature for 3 min, heating at 20C min
1
up to 220C, keeping this
temperature for 25 min; B. dracunculifolia: initial temperature of 60C, heating at a rate of
5C min
1
up to 160C, heating at 15C min
1
up to 220C, keeping this temperature for 25
min, heating at 20C min
1
up to 220C, keeping this temperature for 25 min; V.
polyanthes: initial temperature of 60C, heating at a rate of 9C min
1
up to 180C, heating
at 15C min
1
up to 220C, keeping this temperature for 4 min, heating at 3C min
1
up to
220C, keeping this temperature for 4 min, heating at 5C.min
1
up to 220C, keeping this
temperature for 10 min; E. uniflora: initial temperature of 60C, heating at a rate of 3C
min
1
up to 160C, heating at 15C min
1
up to 220C, keeping this temperature for 20 min.
EO compounds were identified by NIST mass spectral library (National Institute of
Standards and Technology), mass spectra and data available in literature (Adams, 1989).

Bacterial strains
Sixteen S. aureus and sixteen E. coli strains isolated from human specimens in the Clinical
Hospital of Botucatu Medical School, UNESP were carried out in the susceptibility assays.
The strains were identified (Koneman, Allen, Janda, Sherechkenberger, & Winn, 2001)
and kept in Nutrient Agar (Difco, Sparks, USA) and the use of bacterial strains was
approved by the Ethics Committee on Research of Botucatu Medical School, UNESP
(protocol number 3.152-2009). These species were chosen for the study aimed to
determine differences in susceptibility between Gram positive and Gram negative bacterial
strains.

Susceptibility assays (minimal inhibitory concentration, MI C)
The susceptibility assays were carried out against 32 strains using agar dilution method of
natural products in Agar Mueller Hinton (MHA) (Difco, Sparks, USA) plus 0.2% Tween
80, totaling 20 mL of MHA in the Petri plates. The concentrations were initially
established as %v/v (ranging from 2 to 44%v/v for crude extracts and from 0.05 to 3 %v/v
for EO) and converted to mg/mL using extracts dry weight and EO density. Positive
control Petri plates were prepared for bacterial normal growth and assays were carried out
in duplicate (Betoni et al, 2006; Silva, Ushimaru, Barbosa, Cunha, & Fernandes Jnior,
2009). The strains were inoculated using a Steers replicator with a standardized bacterial
suspension (0.5 McFarland Scale) of 10
5
10
6
CFU/mL and incubation at 37C/1824 h.
MIC values were recorded by presence or not of colonies (Clinical and Laboratory
Standards Institute [CLSI], 2005) and the MIC90% were calculated.
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Statistical analysis
MIC results were analysed using the nonparametric test Kruskal-Wallis to compare
independent treatments, with significant analysis (p < 0.05 and p < 0.001). Dunn and
StudentNewmanKeuls tests were applied for multiple comparisons between treatments
(SAS for Windows, Version 9.1.3).


References

Adams, R.P. (1989). Identification of essential oils by ion trap mass spectroscopy. San
Diego, California: Academic Press.
Betoni, J.E.C., Mantovani, R.P., Barbosa, L.N., Di Stasi, L.C., & Fernandes Jnior, A.
(2006). Synergism between plant extract and antimicrobial drugs used on
Staphylococcus aureus diseases. Memrias do Instituto Oswaldo Cruz, 101, 387
390.
Clinical and Laboratory Standards Institute/National Comitee for Clinical Laboratory
Standards (CLSI/NCCLS) (2005). Performance standards for antimicrobial
susceptibility testing; Fifteenth Information Supplement. CLSI/NCCLS Document M
100-S15. Wayne, PA.
Fonseca, P., & Librand, A.P.L. (2008). Evaluation of physico-chemical and phytochemical
characteristics of different tinctures of barbatimo (Stryphnodendron barbatiman).
Brazilian Journal of Pharmaceutical Sciences, 44, 271277.
Koneman, E.W., Allen, S.D., Janda, N.M., Sherechkenberger, P.C., & Winn, J.R. (2001).
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Matos, F.J.A. (1988). Introduo fitoqumica experimental (1st ed.). Fortaleza, Brasil:
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Silva, M.T.N., Ushimaru, P.I., Barbosa, L.N., Cunha, M.L.R.S., & Fernandes Jnior, A.
(2009). Antibacterial activity of plant essential oils against Staphylococcus aureus
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