Pharmacology & Pharmacy, 2012, 3, 433-438
http://dx.doi.org/10.4236/pp.2012.34058 Published Online October 2012 (http://www.SciRP.org/journal/pp) 1
Antibacterial Activity of Bougainvillea glabra, Eucalyptus
globulus, Gnaphalium attenuatum, and Propolis Collected
in Mexico
Oswaldo Javier Enciso-Díaz1, Alfonso Méndez-Gutiérrez2, Lourdes Hernández De Jesús3,
Ashutosh Sharma1, María Luisa Villarreal1, Alexandre Cardoso Taketa1*
1Biotechnology Research Center (CEIB), Autonomous University of Morelos State (UAEM), Morelos, Mexico; 2Cente r fo r Research
and Teaching in Humanities of the State of Morelos (CIDHEM), Morelos, Mexico; 3Department of Pharmacy, National School of
Biological Sciences, National Polytechnic Institute (IPN), Mexico City, Mexico.
Email: *ataketa@uaem.mx
Received July 4th, 2012; revised August 12th, 2012; accepted September 14th, 2012
ABSTRACT
Bougainvillea g labra C., Eucalyptus globulus Labill., and Gnaphalium attenuatum DC., as well as propolis, have been
used in Mexican trad itional medicine as a remedy to treat respiratory illnesses. There are few biological reports of such
material collected in Mexico, despite the high demand for them as raw material for popular and industrial uses. The
antibacterial activity of the plants and propolis studied here were evaluated against a panel of bacteria using three dif-
ferent methodologies: agar disc diffusion, macro and micro dilution methods. E. globulus and G. attenuatum extracts
showed the strongest active values (P > 0.05) in the agar disc diffusion method with a range of 8 - 22 mm inhibition
zone, MIC values ranging from 25 - 250 g/mL, and MBC values of 25 - 500 g/mL obtained by macro and micro di-
lution methods. B. glabra extracts were active against E. coli, S. typhi, K. pneumoniae, S. aureus and S. agalactiae, with
8 - 13 mm inhibition zone, MICs ranging from 500 - 3000 g/mL, and MBCs of 1000 - 3000 g/mL. The propolis ex-
tract turned out to be active against E. coli, K. pneumoniae, S. aureus and S. a g alactiae, with values of 7 - 12 mm, MICs
of 1000 - 2000 g/mL, and MBCs of 2000 - 2500 g/mL. E. globulus and G. attenuatum extracts were the most active
in the three methodologies assayed. This is also the first time that the antibacterial activity of G. attenuatum has been
experimentally demonstrated. The microdilution method showed to be more sensitive, less expensive and minor
time-consuming technique compared with the other two.
Keywords: Antibacterial Activity; Bougainvillea glabra; Eucalyptus globulus; Gnaphalium attenuatum; Mexican
Ethnomedicine; Propolis
1. Introduction
Acute respiratory infections represent a serious health
problem worldwide, and in Mexico, in the 2008 national
statistics list, these infections occupied the first position
in the top 20 illness affecting more than 24 million
people, especially young children between 1 and 4 years
[1]. Mexico has an ancient tradition in the use of
medicinal plants as first aid remedies, which actually
remains in use now. An important number of products
derived from plants are used to manufacture a broad
range of herbal formulas for the treatment of respiratory
diseases, which are commercialized in such different
forms, as infusions, plasters, syrups, pills, and candies.
These products are usually registered as supplements or
dietary aids in order to avoid the more strict governmental
health policies [2]. The local market is flooded with a
variety of herbal products lacking any kind of quality
control guarantying efficacy and safety. The most im-
portant missing issue is a scientific approach to the speci-
fication of the concentration levels of the crude extracts,
as well as the pharmacological activity and safety. Some
medici nal plants such as Bouga invillea glabra C. (Nyctagi-
naceae), Eucalyptus globulus Labill. (Myrtaceae) and
Gnaphalium attenuatum DC. (Compositae), as well as
extracts from propolis, are popularly employed alone or
in combination. Neglected is the importance of cha r a c t eri z-
ing a plant extract in terms of the origin of the raw material:
local, seasonality and ontogenetic variations depending
on its site of collection for. It has been demonstrated that
the metabolic profiling of a plant species can change
dramatically affecting the pharmacological response in
relation to different environments [3,4]. In the same way,
*Corresponding a uthor.
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Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium
attenuatum, and Propolis Collected in Mexico
434
propolis, a resin with a highly complex composition, can
present different antimicrobial effectiveness depending
on the solvent used in the extraction process, as well as
the origin of the resin [5].
Although there are guidelines, the reality is that
researchers adopt a wide range of criteria which depends
on individual situations and conditions to define such
parameters as plant material, techniques employed, growth
medium and tested microorganisms in the antibacterial
studies of medicinal plants [6]. This complex issue alerts
us to the need to define the pharmacological properties of
a plant material in time and space. Even though there are
a number of antibacterial studies of Bougainvillea glabra
[7,8], Eucalyptus globulus [9-11], Gnaphalium sp . [12,
13], and propolis [5,14 ], each one was based on only one
methodology. On the contrary, we are reporting the acti-
vity of these same plants, collected in the state of More-
los, as well as propolis fro m Oaxaca State, Mexico, which
we subjected to three different methodologies: agar disc
diffusion, macro and micro dilution methods. The panel
of Gram positive bacteria included Staphylococcus aureus,
Streptococcus agalactiae, Listeria monocytogenes and
Bacillus subtilis; and the Gram negative bacteria were
Escherichia coli, Klebisiella p neumoniae and Salmonella
typhi.
2. Materials and Methods
2.1. Plant Material
Plants were collected in Morelos State, Mexico, in the
year 2007, and propolis samples were collected from
beehives in March, 2007, in Oaxaca State, Mexico.
Voucher specimens were authenticated by Juan Carlos
Juárez and then deposited at the HUMO Herbarium,
CEAMISH (Centro de Educación Ambiental e Investi-
gación Sierra de Huautla) UAEM, under the numbers
26348 (B. glabra), 26347 (E. globulus), and 26346 (G.
attenuatum).
2.2. Preparation of Plant Extracts
Dried aerial part of the plants (200 g) were powdered and
put into a container with 300 mL of cold water, and then
heated to boiling. After cooling down the mixture decoction
to room temperature, 700 mL of EtOH was added, and
then macerated for 2 weeks in a dark and cool place. This
procedure has been used by some industries to prepare a
mother tincture. In this study, these macerates were
filtered and the solvent was evaporated under reduced
pressure to obtain an ex tract that was used to prepare the
dilutions employed for th e biological testing. Plant crude
extracts and propolis resin were dissolved in ethanol or
DMSO prior to use in the antibacterial assay.
2.3. Test for Antibacterial Activity
The minimal inhibitory concentration (MIC) and the
minimal bactericidal concentration (MBC) were established
for the hydroalcoholic extracts for each species, and for
the propolis extract, us ing both macro and micro dilution
methods with tetrazolium chloride dyeing agent. Ad-
ditionally, a disc diffusion method was applied to record
the diameter of inhibitory zone promoted by the extracts.
The antibacterial tests were adapted from the National
Committee for Clinical Laboratory Standards (CLSI),
formerly NCCLS, gu idelines f or document M26-A ( 1999)
[15] and M38-A (2002) [16].
2.4. Microorganisms
The b acteria u sed in the pr esent study were Gram positive:
Staphylococcus aureus (ATCC 25923), Streptococcus
agalactiae (ATCC 25924), Listeria monocytogenes
(ATCC 244), and Bacillus subtilis (ATCC 6633); and
Gram negative: Escherichia coli (ATCC 25922), Klebi-
siella pneumoniae (ATCC 10031) and Salmonella typhi
(ATCC 6539). The b acteria were provided from th e Micro-
biology Laboratory Culture Collection, National School
of Biological Sciences, National Polytechnic Institute,
Mexico. The microorganisms wer e first inc ubated at 37˚C
± 0.1˚C for 24 h in the nutrient broth (Mueller-Hinton).
The bacterial suspensions were prepared and adjusted by
comparison against 0.5 of the MacFarland turbidity
standard (5 × 107 cells/mL) tubes, and further diluted to
obtain a final concentration of 5 × 106 cells/mL that was
standardized in our laboratory by a turbidity measure at
420 nm wavel ength prior to use.
2.5. Agar-Disc Diffusion Method
Sterilized filter papers discs with 6 mm diameter were
impregnated with 2 mg extracts, and then placed into
Petri dishes (9 cm) with Mueller-Hinton agar (Oxoid),
which were previously streaked with a swab containing
the bacteria. The plates were incubated at 37˚C ± 0.1˚C
for 24 h. Diameter of inhibition zones appearing around
the discs were measured and recorded in mm. Chloram-
phenicol (2 g/disc) was used as a positive control, and
negative control discs were prepared with ethanol only.
2.6. Microdilution Method (MiD)
Dilutions of the extracts solubilized in DMSO were
prepared and dispensed into 96 microdilution wells to
obtain a final volume of 200 L at a concentration range
of 25 - 3000 g/mL of inoculum suspension. Microdilution
trays were incubated at 37˚C ± 0.1˚C for 24 h to
determine the MIC and the MBC. To establish the MIC
values, 20 l of tetrazolium chloride at 20 mg/mL in
Copyright © 2012 SciRes. PP
Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium
attenuatum, and Propolis Collected in Mexico
Copyright © 2012 SciRes. PP
435
water was added to each well. After incubation at 37˚C ±
0.1˚C for 30 min, the presence of live bacteria was
recorded by visual inspection using the red formazan
derivative. Before the addition of tetrazolium chloride, an
aliquot was taken from the wells, where the turbidity was
absent, and then inoculated in a tube with broth medium.
The concentration point in which the bacterial growth
was not observed was defined as the MBC value.
2.7. Macrodilution Method (MaD)
Extracts were assayed at the same concentration and
inoculation conditions as those used in the MiD method;
however, a volume of 2 mL inoculums per tube was
employed. The determination of MIC and MBC were
carried out in the same way explained above for the MiD
method.
3. Results
Extraction procedures yielded 2.89%, 6.11%, and 2.60%
w/w of t he hy dr oalc ohol ic extra cts of B. glabr a, E. globulus
and G. attenuatum, respectively. The results of bacterial
susceptibility testing for these extracts, as well as for
propolis, are reported in Table 1. T he cr ude ex trac ts of E.
globulus showed to be the most active one, with MIC
values lower than 25 g/mL and up to 50 g/mL (MiD),
50 - 100 g/mL (MaD), and MBC values of <25 - 100
g/mL (MiD), <25 - 250 g/mL (MaD), and diameter of
inhibitory zone at 8 - 22.5 mm, against all bacteria
assayed (P > 0.05). G. attenuatum turned out to be the
second most active extract, with a broad spectral action
against all tested strains, and with MIC values of 50 -
200 g/mL, MBC of 100 - 250 g/mL, and diameter of
inhibitory zone of 8 - 21.5 mm.
Table 1. Antibacterial activity of crude extracts of B. glabra, E. globulus, G. attenuatum, and propolis using different method-
ologies.
Extacts Tests S. aur. S. aga. L. mon. B. sub. E. coli K. pneu. S. typ .
DD 13(**) 13(***) 0
(***) 0
(**) 8
(*) 0
(****) 8
(**)
MaDMIC 1000(C) 1000(C) n.a. n.a. 1500(B) n.a. 1000(B)
MaDMBC 2000(c) 2000(a) n.a. n.a. 1500(c) n.a. 2000(a)
MiDMIC 2500(A) 3000(A) n.a. n.a. 500(C) 2500(A) 1500(A)
Bougainvillea glabra
MiDMBC 3000(a) n.a. n.a. n.a. 1000(d) 3000(a) 2000(a)
DD 22.5(*) 16.5(**) 15(*) 15(*) 8
(*) 15(*) 14.5(*)
MaDMIC 100(D) 50(E) 50(B) 100(A) 100(F) 50(E) 100(D)
MaDMBC 250(d) 100(c) 100(b) 250(a) 250(f) 100(e) 250(c)
MiDMIC 50(E) <25(F) 50(B) <25(C) 50(G) 50(E) 50(E)
Eucalyptus globulus
MiDMBC 100(f) <25(d) 100(b) 25(c) 100(g) 100(e) 100(d)
DD 20(*) 21.5(*) 12(**) 16
(*) 8
(*) 12(**) 8
(**)
MaDMIC 100(D) 250(D) 100(A) 100(A) 250(D) 100(D) 250(C)
MaDMBC 250(d) 250(b) 250(a) 250(a) 500(e) 250(d) 500(b)
MiDMIC 100(D) <25(F) 50(B) 50(B) 200(E) 50(E) 50(E)
Gnaphalium attenuatum
MiDMBC 150(e) <25(d) 100(b) 100(b) 250(f) 100(e) 100(d)
DD 10(***) 12*** 0
(***) 0
(**) 7
(**) 7
(***) 0
(***)
MaDMIC n.a. n.a. n.a. n.a. 2000(A) 1000(C) n.a.
MaDMBC n.a. n.a. n.a. n.a. 2000(b) 1000(c) n.a.
MiDMIC 2000(B) 1500(B) n.a. n.a. 2000(A) 2000(B) n.a.
Propolis
MiDMBC 2500(b) 2000(a) n.a. n.a. 2500(a) 2500(b) n.a.
DD 12 16 15 11 16 19 13
MaDMIC 16 8 12 12 16 12 16
Chloramphenicol
MiDMIC 8 4 4 4 8 12 4
DD: disc diffusion method (mm); MaD: macrodilution method (g/mL); MiD: microdilution method with a 96-well microtiter plate (g/mL); S. aur. (Staphy-
lococcus aureus), S. aga. (Streptococcus agalactiae), L. mon. (Listeria mono cytogenes), B. sub. (Bacillus subtilis), E. coli (Escher ichia coli), K. pneu. (Kleb-
siella pn eumoni a), and S. typ. (Salmonella typhi); n.a.: no active at a concentration of 3000 g/mL. Values pres ented are av erages of t hree r epl icates. V alue s in
parentheses in the same column following different asterisk, capital, and lower case letters designate a significant difference, in the DD, MICs, and MBCs
values, respectively, by Duncan’s multiple range test, P < 0.05.
Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium
attenuatum, and Propolis Collected in Mexico
436
B. glabra crude extract was mildly active against S.
aureus, S. agalactiae, E. coli, and S. typhi, with MIC
values of 500 - 3000 g/mL (MiD), 1000 - 1500 g/mL
(MaD), and MBC of 1000 - 1500 g/mL (MiD) and 1500 -
2000 g/mL (MiD), and diameter of inhibitory zone at 8 -
13 mm (P > 0.05). This extract was not active against L.
monocytogenes and B. subtilis at the maximal dosis
assessed of 3000 g/mL. Propolis extract resulted to
posses a weak activity against S. aureus, S. agalactiae, E.
coli, and K. pneumoniae, with MIC values of 1500 -
2000 g/mL, MBC of 2000 - 2500 g/mL, and diameter
of inhibi t ory zone of 7 - 1 2 m m.
4. Discussion
The microplate and tube macro dilution methods em-
ploying tetrazolium chloride dyeing agent, as well as the
disc diffusion assay, are currently used methods applied
to determine the antibacterial potency of crude extracts
derived from natural products [17,18]. In the present
work, the three methods were used to compare the
activity of some important plants employed in Mexican
folk medicine to treat respiratory diseases. These three
methods covered a broad intrinsic variability of each
technique, and extract complexity, and also permitted the
comparison of the data obtained in this study with
previously reported literature. In general, the MiD
method was more sensitive and quick than the MaD
assay, requiring a smaller sample amount. Additionally,
we performed the disc diffusion method to compare our
results with data from literature. This method is largely
used due to it is a reliable, easy and inexpensive suscep-
tibility test. In some occasions, the most active extract
employing the disc diffusion assay did not agree with
that determined by MiD test.
The genus Bougainvillae comprises 18 species, and
some of them have been used in traditional medicine to
treat such disorders as diarrhea, stomach acidity, cough,
sore throat, leucorrohea, hepatitis, and as an anti-inflam-
matory, antiviral and antibacterial agent [7,8]. Leaves
and inflorescence of Bougainvillaea g labra C. have been
used in Mexican traditional medicine as a remedy for
such respiratory illnesses as cough, cold, bronchitis and
asthma [18,19]. Its antimicrobi al effect can be associated
with the presence of betalains pigments [20], as well as
steroidal compounds with anti-inflammator y activity [2 1].
Gupta et al. (2009) found that a 500 g/disc hydroalco-
holic extract from leaves of B. glabra was active against
S. aureus (10 mm), B. subtilis (15 mm), E. coli (12 mm),
S. typhi (14 mm), and K. pneumoniae (16 mm). These
results confirmed the level of activity reported in the
present study ag ainst S. aureus (13 mm), E. coli (8 mm),
and S. typhi (8 mm). However, the extracts were not
active against B. subtilis and K. pneumonia, the most
susceptible bacteria reported by Gupta when tested by
disc diffusion assay. These differences could be attri buted
to a chemical profiling v ariation associated with ch anges
in the plant environment.
The leaves extract and essen tial oil from Eucalyptus sp.
have been used in the traditional medicine of different
countries to treat patien ts with respiratory tract infections
[10,11]. A study p erformed with E. globulus collected in
Morelos, Mexico, using the methanolic extract from the
leaves, reported MIC values of 5000 g/mL against S.
aureus and 10,000 g/mL against E. coli [9]. These con-
centrations are far higher than those MIC values we
found in this investigation (S. aureus of 100 g/mL and
to E. coli of 100 g/mL, both in the MaD method; and S.
aureus of 50 g/mL and to E. coli of 50 g/mL, both in
the MiD method). These huge differences can be e x p la i n e d
due to intrinsic factors associated w i th th e collec ted plants,
as seasonality and particular environments, that cause a
change in the metabolic profiling, and consequently, in
its activity.
Gnaphalium sp. infusion is the most common herbal
remedy used in Mexican ethnomedicine in the treatment
of both adult and children’s cough and upper respiratory
tract infections [12,13]. It has been reported that extracts
from aerial parts of G. oxyphyllum, G. liebmannii and G.
viscosum, exhibited a wider spectrum of activity against
S. aureous, B. cereus, S. typhimurium, and E. coli. As far
as we know, there are no antibacterial activities reported
for G. attenuatum, a very important medicinal plant
found from Mexico to Panama [22]. In our study, this
plant was active against all tested bacteria, showing the
wider spectrum of action, with high inhibition zones (8 -
21.5 mm) and low MICs ranging between <25 - 200
g/mL (MiD) and 100 - 250 g/mL (MaD); and MBC
values of <25 - 250 g/mL (MiD) and 250 - 500 g/Ml
(MaD). There are no biological or chemical studies for G.
attenuatum, however, Fajardo-Ochoa (2004) reported th at
Gnaphalium sp. may conta in sapo nin s with low n a rc o t iz i n g
action responsible for lethargy and respiratory failure in
newborns [13].
It has been demonstrated that the propolis extract from
the state of Campeche, Mexico, was active against P.
aeruginosa with a MBC of 3.33 mg/mL, followed by S.
aureus ( MBC of 4.82 mg/mL) and Streptococcus pyo ge n es
(MBC of 5.38 mg/mL). In that work, S. typhi turned out
to be the most resistant bacteria (MBC of 9.82 mg/mL)
[5]. Our study shows that propolis from Oaxaca was not
active against B. subtilis, L. monocytogenes and S. typhi,
with MIC and MBC values investigated at a maximal
concentration of 3 mg/mL. However, this propolis extract
exhibited MIC values of 1.50 - 2.00 mg/mL and MBC of
2.0 - 2.5 mg/mL against E. coli, K. pneumoniae, S.
aureus and S. agalactiae, when tested by MiD method.
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Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium
attenuatum, and Propolis Collected in Mexico 437
In the same way that the antibacterial potency of honey
varies very markedly, and depends on the flora source of
the honey [23], the activity of pro polis resins can change
depending on its content of flavonoids and aromatic
acids and esters [14]. This variation in the secondary
metabolite content can explain why propolis samples
collected from various geographical regions of Turkey
presented a marked antibacterial activity against the
Gram positive strain (Listeria monocytogenes) and a low
action against Gram negative bacteria (Salmonella en-
teritidis) [24], contrasting with our results. Although the
propolis extracts from Campe che and Oaxaca have shown
to be less potent than the plants extracts investigated in
this work, the use of propolis resin in combination with
plant extracts can be supported by the fact that propolis
have been demonstrated to potentiate the effect of certain
antimicrobial drugs [25], as well as to prevent or reduce
the development of tolerance to antibiotics [14]. The
microplate method using color detection by formation of
formazan derivative (with red color) showed to be better
than the other two, displaying high active values. It was
the most sensitive, less expensive and minor time-con-
suming technique compared with the macrodilution as-
say.
5. Conclusions
Extracts prepared from aerial parts of Bougainvillea
glabra, Eucalyptus globulus, and Gnaphalium attenuatum,
as well as propolis are frequently commercialized in
Mexico as ingredients of a variety of syrups, candies, and
other remedies destined to relieve the symptoms of upper
respiratory tract infections like cough and sore throat.
Ethnobotanical stud ies strongly support their uses, which
were confirmed by the present study, revealing that E.
globulus and G. attenuatum extracts were the most active,
showing broad spectrum activity, in contrast with B.
glabra and propolis extracts which were less active and
more selective in action. Here, we present the first report
for the antibacterial activity of the aerial parts of G.
attenuatum against a panel of Gram positive and negative
bacteria. There was a good correlation between the
macro and micro dilution methods, th e latter being faster,
more sensitive and requiring less sample amount. The
disc diffusion method correlated well, but presented the
advantages of being cheaper and simpler to read.
For the antibacterial activity of the selected plants we
obtained different quantitative and qualitative results
when compared with literature data. It evidences that the
antibacterial activities of plants does vary and depends
upon a variety of factors, indicating that the raw material
used by producers of products containing herbals must be
scrutinized under a very stricter control of quality.
6. Acknowledgements
The authors gratefully acknowledge financial support from
Conacty No. 156276 and 80980.
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