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Antibacterial activity of high safrole contain essential oils from Piper

xylosteoides (Kunth) Steudel

Article  in  Journal of Essential Oil Research · July 2012

DOI: 10.1080/10412905.2012.676768

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Antibacterial activity of high safrole contain essential

oils from Piper xylosteoides (Kunth) Steudel
a a a a
Jocinei Dognini , Emanuelle K. Meneghetti , Morgana N. Teske , Iêda M. Begnini ,
a b c c
Ricardo A. Rebelo , Eduardo M. Dalmarco , Marcio Verdi & André L. de Gasper
a
Department of Chemistry, Regional University of Blumenau, Blumenau-SC, Brazil
b
Department of Pharmaceutical Sciences, Regional University of Blumenau, Blumenau-SC,
Brazil
c
Department of Natural Sciences, Regional University of Blumenau, Blumenau-SC, Brazil

Available online: 11 May 2012

To cite this article: Jocinei Dognini, Emanuelle K. Meneghetti, Morgana N. Teske, Iêda M. Begnini, Ricardo A. Rebelo,
Eduardo M. Dalmarco, Marcio Verdi & André L. de Gasper (2012): Antibacterial activity of high safrole contain essential oils
from Piper xylosteoides (Kunth) Steudel, Journal of Essential Oil Research, 24:3, 241-244

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 The Journal of Essential Oil Research
Vol. 24, No. 3, June 2012, 241–244

Antibacterial activity of high safrole contain essential oils from Piper xylosteoides (Kunth)
Steudel
Jocinei Dogninia, Emanuelle K. Meneghettia, Morgana N. Teskea, Iêda M. Begninia, Ricardo A. Rebeloa*, Eduardo
M. Dalmarcob, Marcio Verdic and André L. de Gasperc
a
Department of Chemistry, Regional University of Blumenau, Blumenau-SC, Brazil; bDepartment of Pharmaceutical Sciences,
Regional University of Blumenau, Blumenau-SC, Brazil; cDepartment of Natural Sciences, Regional University of Blumenau,
Blumenau-SC, Brazil
(Received 31 March 2011; final form 26 August 2011)

In our continuous search for naturally occurring compounds of scientific and technological relevance, the chemical
composition and antibacterial activity of the essential oils of two populations of Piper xylosteoides from Santa
Catarina, Brazil, is described. Both oils showed a very high safrole concentration, being the highest value associated
to the sample collected in Orleans (84.1%). As the second major compound, the monoterpene α-pinene was present
Downloaded by [Ricardo Rebelo] at 17:20 11 May 2012

with the highest concentration (15.3%) in the São Bonifácio sample. The antibacterial activity was examined towards
both Gram-positive and Gram-negative bacteria. The oils showed weak activity against the bacteria tested. This
species can be considered a new economically important natural source of the arylpropanoid safrole.
Keywords: Piper xylosteoides; essential oil; safrole; antibacterial activity

Introduction without safrole in its composition (18). Presently, the

Safrole is an arylpropanoid known for its nephro-
toxic, hepatotoxic and cancerous effects and forbid-
den in many countries as additive in foods and
perfumery (1, 2). It is also a precursor-compound in
the production of psychotropic MDMA (‘Ecstasy’) a
drug with no legitimate medical purpose (3). Industri-
ally, safrole is an important starting material for the
synthesis of heliotropine (piperonal), a fragrance sub-
stance, and piperonyl butoxide, a synergist for pyre-
thrum-based insecticides (4). Furthermore, it has been
the precursor of several bioactive molecules (5–11).
The main natural sources of safrole are the root-bark
of Sassafras albidum growing in North America (12),
Piper auritum in Central America (13) and Ocotea
pretiosa growing in South America (14). Its natural
occurrence in the Brazilian territory is intimately
associated to the genus Piper where it has been
found in considerable concentration (>60%) in the
essential oils of P. hispidinervium, P. marginatum, P.
cavalcantei and P. callosum, which occurs in the Bra-
zilian Amazon rainforest (15), and P. mikanianum,
from the Atlantic rainforest (16). Recently, the pres-
ence of this secondary metabolite was also described
in leaf essential oil of Piper xylosteoides found in
Rio Grande do Sul, South Brazil, with a concentra-
tion of about 48% (17). The same species was
described as occurring in Central area of Brazil but
chemical composition and antibacterial activity of
essential oils from two populations of P. xylosteoides
(Kunth) Steudel occurring in Santa Catarina, Southern
Region of Brazil, is described.
Experimental
Plant material
Leaves of P. xylosteoides were collected in Orleans (49°
19’ 09” W, 28° 15’ 39” S, 314 m) on February 22,
2010, and São Bonifácio (48° 57’ 36” W, 27° 59’ 38”
S, 339 m) on June 30, 2010. The collecting sites are
inserted within the phytoecological region of the Dense
Ombrophilous Forest, in Santa Catarina State. Exsic-
cates were deposited in the Herbarium Dr. Roberto
Miguel Klein of the Universidade Regional de Blume-
nau-FURB under the numbers 19632 and 27949,
respectively. Their identification was made by compari-
son with an authentic sample deposited in the same
herbarium under the number 17887.
Essential oil isolation
This was obtained from dried leaves by hydrodistilla-
tion for 2 hours in a Clevenger-type apparatus, under
nitrogen atmosphere, using a 8:1 proportion of distilled
water:dry leaves (v/m).

*Corresponding author. Email: ricardorebelo@furb.br; ricardorebelo@pq.cnpq.br

ISSN 1041-2905 print/ISSN 2163-8152 online

Ó 2012 Taylor & Francis
http://dx.doi.org/10.1080/10412905.2012.676768
http://www.tandfonline.com
 242 J. Dognini et al.
Density
This was determined at 22°C by the capillary method
as described by Mayo (19) and was expressed as a
medium of three measurements.
GC–FID and GC/MS analysis
Oil samples diluted in bidistilled hexane at 0.01 g/mL
concentrations were analyzed in a Shimadzu GC-14B
equipped with a DB-5 fused silica capillary column
(Simplicity-1, 30 m × 0.25 mm, 0.25 μm film thickness).
Injector and detector temperatures were set at 220° and
250°C, respectively. Oven temperature was pro-
grammed as follows: 60–195°C at 3°C/minute, rising to
235°C at 20°C/minute, then held for 5 minutes, rising
to 240°C at 5°C/minute and then kept at 240°C for
2 minutes. The carrier gas was He used at a flow rate of
1.0 mL/minute. The injection volume was 0.5 μL in
split mode (1:100).
Downloaded by [Ricardo Rebelo] at 17:20 11 May 2012
Gas chromatography/mass spectrometry (GC/MS)
analysis was performed on a Varian-CP 3800/Saturn-
2000 using electron impact ionization at 70 eV and the
same chromatographic conditions as described above.
The identification of components was made by
computer library search based on matching of MS
spectra (NIST 2002), comparison with literature data
(20) and experimental arithmetic indices (AI) (21),
which were calculated by using a homologous series of
linear alkanes analyzed under the same GC-flame ioni-
zation detector (FID) conditions previously described.
The components quantification was based on their GC
peaks areas without correcting for response factors.
Antibacterial activity
The bacterial strains were acquired from the American
Type Culture Collection (ATCC). Tests were carried out
in duplicate with strains of Gram-positive bacteria Staph-
ylococcus aureus (ATCC 25923) and Bacillus cereus
(ATCC 11778), Escherichia coli (ATCC 25922) and
Pseudomonas aeruginosa (ATCC 27853). The identifica-
tion of strains was confirmed by the use of biochemical
profiles according to the recommendations of the Man-
ual of Clinical Microbiology (22).
The direct antibacterial effect was evaluated by
the broth microdilution method as recommended by
the Clinical and Laboratory Standards Institute (23)
for determination of the MIC (minimal inhibitory
concentration) and MBC (minimal bactericidal
concentration) for the essential oils from leaves of
P. xylosteoides. The essential oil was dissolved in an
aqueous solution of dimethyl sulfoxide (DMSO; 20%,
v/v) to give a 16 μL/mL concentration. This solution
was transferred to 96-well plates (100 μL/well) and
was also diluted with sterile Mueller–Hinton broth
(100 μL/well). Then serial dilutions were made result-
ing in concentrations of 8000 to 62.5 μL/mL. The
inoculum (5 μL) containing 1.5 × 108 colony-forming
units/mL (CFU/mL; 0.5 McFarland scale) of each
microorganism was added to each well. A number of
wells were reserved on each plate for sterility control
(no inoculum added), inoculum viability (no essential
oil added) and the positive control (gentamicin added
in different concentrations) (24). Plates were aerobi-
cally incubated for 18–24 hours at 35°C, and 10 μL
of methanol solution (5 mg/mL) of 2,3,5 triphenyl-
tetrazolium chloride (TTC; Vetec, São Paulo, Brazil)
was added to each well to detect the active bacterial
metabolism. The MIC was defined as the lowest con-
centration of essential oil that visibly inhibited the
growth of bacterial spots detected with TTC (25).
The assays were performed in duplicate to ensure
reproducible results.
To determine the MBC, 10 μL of aliquots broth
were taken from each well and plated in Mueller–
Hinton agar for 24 hours at 37°C. MBC represents the
concentration necessary to kill 99.9% or more of the
initial inoculum (26).
The oil density values were used to convert μL/mL
into μg/mL. The latter was used to express MIC and
MBC.
Results and discussion
The oil yields from dry leaves of P. xylosteoides col-
lected in Orleans and São Bonifácio were 2.3% and
3.2% (w/w), respectively. They are superior to the one
observed by Ferraz et al. (17) using similar experimen-
tal conditions (1.8%, w/w). The oils densities at 22°C
were similar, 1.048±0.018 g/mL for Orleans and 1.048
±0.023 g/mL for São Bonifácio.
Eleven compounds were identified in the essential
oil from Orleans, representing 98.9% of the total oil
composition. On the other hand, eight compounds were
identified in the São Bonifácio sample, representing
99.8% of the total oil (Table 1). Six of the constituents
of São Bonifácio sample (96.3%) were also present in
the Orleans essential oil. Safrole was the major constitu-
ent in both samples, with the highest value of 84.1% in
the Orleans sample, followed by α-pinene, which
showed the highest value of 15.3% in the São Bonifácio
sample.
The concentration of safrole was higher than that
observed by Ferraz et al. (17) on material collected in
São Francisco de Paula, Rio Grande do Sul, Brazil
(47.8%). An important aspect related to the oil compo-
sition of this species occurring in Santa Catarina is the
low concentration of sesquiterpenes, less than 5%. This
justify the short distillation time for the complete essen-
tial oil extraction, which is commonly made in a 4-hour
period (15–17). Another important aspect, related to the
low sesquiterpene concentration and the presence of
 The Journal of Essential Oil Research 243

Table 1. Chemical composition of Piper xylosteoides leaf oils from Orleans and São Bonifácio, Santa Catarina, Brazil.
Orleans São Bonifácio
No. Compound RILit RIExp % RIExp %
Monoterpene 11.2 19.4
1 α-Pinene 932 930 7.7 930 15.3
2 β-Pinene 974 974 0.7 974 1.2
3 Myrcene 988 990 0.9 – –
4 Limonene 1024 1027 1.4 1027 0.6
5 cis-β-Ocimene 1032 – – 1035 1.6
6 tran-β-Ocimene 1044 1046 0.5 1046 0.7
Phenylpropanoids 84.1 75.8
7 Safrole 1285 1291 84.1 1291 75.8
Sesquiterpenes 4.5 4.6
8 β-Elemene 1389 – – 1388 1.9
9 trans-Caryophyllene 1417 1416 1.1 – –
10 Alloaromadendrene 1458 1460 0.6 – –
11 cis-β-Guaiene 1492 1489 <0.1 – –
12 γ-Amorphene 1495 1494 0.6 – –
13 Bicyclogermacrene 1500 1498 1.3 1500 2.7
% of identified compounds 98.9 99.8
Downloaded by [Ricardo Rebelo] at 17:20 11 May 2012

Notes: RIExp, experimental retention index; RILit, literature retention index.

Table 2. Antibacterial activity of Piper xylosteoides leaf oils References

from Orleans and São Bonifácio.
Bacteria Orleans
MIC/ MBC
S. aureus 2091/ 2091
B. cereus 2091/ 4182
P. aeruginosa 4182/ 8364
E. coli 4182/ 8364
hydrocarbon monoterpenes as the second main constitu-
ents, is that one may consider the selective removal of
the latter chemical group during the drying process.
This will provide a more marketable product in terms
of safrole concentration.
Table 2 shows the results of antibacterial activity,
which indicate weak activity of both oils against the
tested bacteria, Gram-positive and Gram-negative. This
result is similar to the one observed by Lobato et al.
(27) for P. hispidinervium, presenting a very high saf-
role concentration where the oil was inactive against
the Gram-negative P. aeruginosa and E. coli, but active
against the Gram-positive S. aureus.
Piper xylosteoides from Santa Catarina, Brazil, can
be considered an economically important species due to
its high safrole content. The antibacterial activity of its
essential oil determined using the microdilution method
was weak for the tested bacteria.
Acknowledgements
The authors acknowledge a grant from FAPESC and the
financial support from FURB. Thanks are due to Marina
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