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Antimicrobial bioassay-guided fractionation of

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DOI: 10.3109/13880209.2014.948634

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 http://informahealthcare.com/phb
ISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. Pezzuto
Pharm Biol, Early Online: 1–7
! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.948634

ORIGINAL ARTICLE

Antimicrobial bioassay-guided fractionation of a methanol extract of

Eupatorium triplinerve
Tamyris Regina Matos Lopes1, Fábio Rodrigues de Oliveira1, Flávia Filocreão Malheiros2, Marcieni Ataı́de
de Andrade1, Marta Chagas Monteiro1, and Ana Cristina Baetas Gonçalves2
1
Programa de Pós-graduação em Ciências Farmacêuticas, Faculdade de Farmácia and 2Faculdade de Farmácia, Universidade Federal do
Pará/UFPA, Belém, PA, Brasil
Pharmaceutical Biology Downloaded from informahealthcare.com by 200.239.65.127 on 11/28/14

Abstract Keywords
Context: Eupatorium triplinerve Vahl (Asteraceae), popularly known as Japana, is widely used Antimicrobial action, asteraceae, biological
in folk medicine, due its analgesic, anticoagulant, antianorexic, antiparasitic, anthelmintic, products, chemical fractionation,
sedative, antifungal, and antibacterial properties. coumarins, medicinal, plants
Objective: The present study evaluated the chemical composition and antimicrobial activity of
E. triplinerve extracts from different parts of the plant and identified the extract with the highest History
antimicrobial potential.
Materials and methods: Extracts were obtained by maceration of all parts of plant, and Received 11 February 2014
subjected to bioassay-guided fractionation of methanol extract by partition column chroma- Revised 1 July 2014
tography. The major chemical groups, saponins, reducing sugars, alkaloids, steroids, triterpen- Accepted 22 July 2014
oids, phenols, tannins, flavonoids, and others were screened by standard techniques. The Published online 27 November 2014
For personal use only.

antimicrobial activity of the different extracts was performed by microdilution assay and the
minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values
were reported.
Results: Phytochemical screening of hydroalcoholic extract from all parts of E. triplinerve
identified mainly steroids, coumarins, alkaloids, saponins, tannins, depsides and absence of
polysaccharides and flavonoids. The methanol extract of leaves presented the highest content
of coumarins and lower MIC values of 62 and 75 mg/mL against Enterococcus faecalis and
Staphylococcus aureus, respectively. In addition, its non-polar fractions showed antimicrobial
activity with MIC ranging from 16 to 125 mg/mL against Gram-negative bacteria, mainly
Escherichia coli.
Discussion and conclusion: Data showed that non-polar fractions of E. triplinerve methanolic
extract has better antimicrobial activity and most likely depends on the presence of several
compounds, such as depsidones, coumarins, saponins, and triterpenes on crude extract. The
results can be exploited largely in research of new antibacterial agents.

Introduction aromatic, smooth, simple, opposite, sub-sessile, 3-nerved,

acuminate, glabrous, and lanceolate. The stems are reddish
Amazonian biodiversity is relevant to research of new plant-
brown. The many flowering heads are each 6–13 mm long and
based treatments that are based on medicinal plants used by
bear approximately 40 pink flowers (Gauvin-Bialecki &
indigenous communities either directly as folk remedies or on
Marodon, 2009).
indigenous information systems (Baptista, 2007). Eupatorium
Eupatorium triplinerve is widely used in folk medicine and
triplinerve Vahl (Asteraceae), also referred as Ayapana
its analgesic, anticoagulant, antianorexic, antiparasitic,
triplinervis (Vahl) R.M. King & H. Rob. and Eupatorium
anthelmintic, sedative, antifungal, and antibacterial properties
triplinerve Vahl by other authors (www.tropics.org), and it is
have been reported (Bose et al., 2007; Chaurasia & Kher,
native to South America, particularly the Amazon region of
1978; Garg & Nakhare, 1993; Gupta et al., 2002; Jelager
Brazil (Trang et al., 1993). It has also been found in Hawaii,
et al., 1998; Kokate et al., 1971; Verpoorte & Dihal, 1987;
India, Vietnam, and the Mascarene Islands (Gauvin-Bialecki
Yadava & Saini, 1990). In addition, the plant extract is used as
& Marodon, 2009). This plant grows up to 1 m high and is an
an antiseptic to treat ulcers, hemorrhages (Ghani, 1998), and
ornamental erect perennial herb that is semi-woody at the
also as sedative, anxiolytic, and antidepressive (Melo et al.,
base. The leaves (4.5–10.5 cm long and 0.8–1.7 cm wide) are
2013).
Although several studies have shown antibacterial and
Correspondence: Marta Chagas Monteiro, Programa de Pós-Graduação antifungal activities in various extracts from different plant
em Ciências Farmacêuticas, Universidade Federal do Pará, Av. Augusto
Correa no. 1, 66075-110 Belém, PA, Brazil. Tel. + 55 91 3201 8829. parts of E. triplinerve, the data are still quite controversial,
Fax: +55 91 3201 7201. E-mail: martachagas2@yahoo.com.br and little is known about the likely components associated
 2 T. R. M. Lopes et al.
Figure 1. Preparation of extracts and
bioassay-guided fraction.
Pharmaceutical Biology Downloaded from informahealthcare.com by 200.239.65.127 on 11/28/14
with these activities (Gupta et al., 2002; Sharma & Singh,
For personal use only.
1979; Verpoorte & Dihal, 1987). The oil of the plant has been
found to possess antimicrobial activity (Yadava & Saini,
1990). Among different plant derivatives, secondary metab-
olites have been proven to be the most important group of
compounds, with a wide range of antibacterial and antifungal
activities (Ahmad et al., 2002; Rahman et al., 1999). Thus,
this study aimed to evaluate the chemical composition and
antimicrobial activity of E. triplinerve extracts from different
plant parts and to identify the most active fraction.
Materials and methods
Collection and identification of E. triplinerve
Eupatorium triplinerve was collected from São Raimundo, a
village in Acará city (Pará state, Amazon region) in March,
2010. The region is situated at latitude 01132.6840 and
longitude 048123.9840 (geographic coordinates obtained
using global positioning system [GPS] equipment). The
botanical identification was performed by Dr. Mário Jardim,
a specialist from Emilio Goeldi Museum (Pará, Brazil), where
a sample was deposited as a voucher specimen under number
MG123913.
Preparation of hydroalcoholic extract of different
parts of E. triplinerve
The whole plant was washed with water and 10% ethanol
solution and separated into leaves, stems, leaves/stems, and
roots. The plant parts were dried at room temperature for 2 d,
dried in an oven with forced air circulation at an average
temperature of 40  C for 6 d and then crushed in a knife mill
to obtain the drug spray. After grinding, the dust was
macerated for 5 d in 70% ethanol to obtain the hydroalcoholic
Pharm Biol, Early Online: 1–7
extract of E. triplinerve. The mash was dried in a rotator
evaporator (Laborata 4000 efficient; Heidolph Instruments
GmbH & Co. KG, Schwabach, Germany) at 45  C, 1 atm
pressure and 120 rpm. For total removal of the solvent, the
mash was kept in the oven and water bath at 40  C. Leaves
and stem extracts (231 g, corresponding to 820 g of the
dried plant yield of the extract was 28.16%) were obtained
and used in animal treatments. All hydroalcoholic extracts
were analyzed to identify the plant parts with antibacterial
properties.
Different extracts of the leaf and bioguided
fractionation of the methanol extract of
E. triplinerve leaves
Eupatorium triplinerve leaves (100 g) were extracted with
500 mL of increasing polarity solvents, including hexane,
ethyl acetate, and methanol, at room temperature for a period
of 7 d each. The solvent was evaporated under reduced
pressure. The methanol extracts were subjected to partition
column chromatography using silica gel 60 (Macherey-Nagel,
Sigma-Aldrich, St. Louis, MO); were partitioned with hexane,
dichloromethane, ethyl acetate, and methanol; and were
concentrated as described. The extracts were investigated
for their phytochemical characteristics; then, the extracts and
fractions obtained from the methanol extract were evaluated
for antibacterial activity (Figure 1).
Phytochemical screening
The crude extracts were analyzed for the presence of major
chemical groups (i.e., saponins, reducing sugars, alkaloids,
steroids and triterpenoids, phenols, tannins, flavonoids, poly-
saccharides, coumarin, depsides, and depsidones) by standard
phytochemical techniques (Barbosa et al., 2001).
 DOI: 10.3109/13880209.2014.948634 Antibacterial evaluation of Eupatorium triplinerve extracts 3

In vitro antimicrobial activity
Microorganisms
Antibacterial activity was evaluated on the following standard
strains: (i) Gram-positive bacteria: Staphylococcus aureus
(ATCC 6538) and Enterococcus faecalis (ATCC 29212) and
(ii) Gram-negative bacteria: Pseudomonas aeruginosa (ATCC
25853), Escherichia coli (ATCC 8739), Klebsiella pneumonie
(ATCC 4352), and Proteus mirabilis (ATCC 15290). All
strains were obtained from the INCQS/FIOCRUZ (National
Institute for Health Quality Control, Rio de Janeiro, Brazil).
In addition, one clinical isolate of E. coli was used as a test
organism. The clinic isolate was obtained of a culture from
patient samples collected in the Microbiology Laboratory,
Pará, Brazil.
The microorganisms used in the study were maintained in
the Laboratory of Microbiology in the Pharmacy Faculty,
Pharmaceutical Biology Downloaded from informahealthcare.com by 200.239.65.127 on 11/28/14
occurred. To determine the MBC, 10 mL of broth was taken
from each well and incubated in Mueller–Hinton agar at 37  C
for 24 h. The MBC was defined as the lowest concentration of
extracts or fractions that resulted in either no growth or fewer
than three colonies (99.9% killing) as described by Quadros
et al. (2011). Each test was performed in three replicates and
repeated twice. The negative control consisted of 100 mL of
the bacterial inoculum in 100 mL of DMSO. Chloramphenicol
(50 mg/mL) and gentamicin (10 mg/mL) were used as positive
controls for Gram-positive and Gram-negative bacteria,
respectively.
Results
There were no qualitative differences in the chemical
composition of the hydroalcoholic extracts from different
parts of E. triplinerve. Table 1 shows the presence of saponin,
reducing sugars, alkaloids, phenols, tannins, steroids/triter-

Federal University of Pará/UFPA. The standard strains were

penoids, depsides/depsidones, coumarin derivatives, and
kept in nutrient agar at room temperature. For the tests, all
sesquiterpene lactones and the absence of polysaccharides
strains were grown in Petri dishes containing media specific
and flavonoids in all the hydroalcoholic plant extracts.
to each bacterium. For S. aureus, mannitol salt agar medium
Table 2 shows that all extracts from the different parts of
was used; for E. faecalis, nutrient agar was used; for
E. triplinerve were effective in inhibiting the growth of Gram-
P. aeruginosa, cetrimide agar was used; and for E. coli,
negative bacteria, particularly the leaves extract. The data
K. pneumonia, and P. mirabilis, MacConkey agar was used.
showed that the MIC values of the leaves plus stems, stems, or
All plates were incubated at 37  C for 24 h to induce
roots extracts were 187 mg/mL against P. aeruginosa and
exponential growth.
E. coli, while the MIC value of the leaves extract was 94 mg/
mL against both bacteria. The MBC value for all extracts was
For personal use only.

Determination of the minimum inhibitory 375 mg/mL. Among Gram-positive bacteria, the E. faecalis
concentration (MIC) and minimum bactericidal strain was sensitive to the extract of leaves plus stems, with an
concentration (MBC) MIC value of 37 mg/mL and an MBC value of 75 mg/mL,
whereas S. aureus was more sensitive to the leaves extract,
To prepare the bacterial inoculum, strains were grown in
with an MIC value of 75 mg/mL.
the exponential phase in Mueller–Hinton broth (Merck,
The phytochemical screening of different E. triplinerve
Darmstadt, Germany) at 37  C for 18 h. The turbidity was
leaf extracts of increasing polarity showed the presence of
adjusted to 0.5 on the McFarland scale (approximately of
alkaloids in all extracts; furthermore, saponins, reducing
2  108 CFU/mL) by diluting fresh cultures and then diluted
sugars, and high concentration of coumarins were detected
to 1  103 CFU/mL as described by the Clinical and
only in the methanol extract. Low concentrations of depsides,
Laboratory Standards Institute (CLSI, 2012).
depsidones, and coumarins and high concentrations of
MIC and MBC assays were performed using the broth
triterpenoids and steroids were found in the ethyl acetate
microdilution method in the Mueller–Hinton broth as
extract, whereas in the hexane extract, only depsides,
described by the Clinical and Laboratory Standards Institute
depsidones, and alkaloids were found (Table 3).
(CLSI, 2012). The MIC is defined as the lowest concentration
of an extract or fraction that inhibits the growth of
microorganism. The E. triplinerve extract or fraction was Table 1. Phytochemical screening of the hydroalcoholic extracts of
dissolved in a solution of 50% dimethylsulfoxide (DMSO), leaves and stalks, stems, and roots of E. triplinerve.
and serial two-fold dilutions were made until a final
concentration of 16 mg/mL in 1 mL sterile test tubes contain- Plant parts
ing the Mueller–Hinton broth (MHA). Leaves plus
For the microdilution test, the inoculums (100 mL) con- Components stems Stems Roots Leaves
taining 5  108 CFU/mL were added to each well, and 100 mL Saponin +++ ++ ++ ++
of the serial dilutions was transferred into consecutive wells. Reducing sugars +++ ++ ++ ++
After 24 h of incubation, 20 mL of 3-(4,5-dimethylthiazol-2- Polysaccharides    
yl)-2,5-diphenytetrazolium bromide (MTT), a tetrazolium salt Alkaloids ++ ++ ++ ++
Phenols and tannins + + + +
that is reduced by metabolically active cells to a colored triterpenoids and steroids +++ + + +
water-soluble formazan derivative, was added to the wells to Flavonoids    
allow visual identification of metabolic activity (Mosmann, depsides and depsidones +++ +++ +++ +++
1983). The final concentration of MTT after inoculation was Coumarin derivative + + + +
sesquiterpene lactones ++ + + +
0.005% (v/v). After incubation, growth was indicated by the
development of a blue color. The MIC was read as the lowest , absent; +, slightly present; ++, moderate present; +++, heavily
concentration of the extracts at which a change in color present.
 4 T. R. M. Lopes et al. Pharm Biol, Early Online: 1–7

Table 2. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the
hydroalcoholic extracts of leaves and stalks, stems, leaves, and root E. triplinerve against the different microorganisms.

Plant parts – MIC and MBC (mg/mL)

Leav. plus stems Stems Roots Leaves
Bacteria MIC MBC MIC MBC MIC MBC MIC MBC
S. aureus ATCC 6538 4500 4500 4500 4500 4500 4500 75 4500
E. faecalis ATCC 29212 37 75 4500 4500 4500 4500 4500 4500
P. aeruginosa ATCC 25853 187 375 187 375 187 375 94 375
E. coli ATCC 8739 187 375 187 375 187 375 94 375

climatic factors such as seasonality, rainfall (affecting the

Table 3. Phytochemical screening of different extracts of increasing availability of nutrients and water), and changes in the
polarity of leaves of E. triplinerve.
circadian cycle may influence the biochemical and physio-
Components Hexane Ethyl acetate Methanol
logical processes of plants and thus changing its composition
(Czelusniak et al., 2012; Gobbo Neto et al., 2007).
Pharmaceutical Biology Downloaded from informahealthcare.com by 200.239.65.127 on 11/28/14

Saponins   + In this study, the phytochemical screening of the leaves

Reducing Sugars
Polysaccharides
Flavonoids
Phenols and tannins
Depsides and depsidones
Coumarin derivative
Sesquiterpene lactones
Alkaloids
Triterpenoids and steroids
, absent; +, slightly present; ++, high present.
For personal use only.
 
 
 
 
+ +
 +
 
+ +
 ++
+
 extract of E. triplinerve showed the presence of steroids,
 coumarins, alkaloids, and saponins. The methanol extract of
 the leaves presented better antimicrobial activity compared

with other extracts with increasing polarity, and the non-polar
++
 leaf fractions showed good antimicrobial activity against
+ Gram-negative bacteria.
+ The initial phytochemical screening of the hydroalcoholic
extract of E. triplinerve showed the presence of saponins,
reducing sugars, alkaloids, coumarins, phenols, steroids, and

depsides. However, there was no qualitative difference in the

The results demonstrate that the methanol extract of
E. triplinerve presented the best antimicrobial activity with
MIC values of 62 and 75 mg/mL against Gram-positive
bacteria E. faecalis and S. aureus, respectively. In addition,
the MIC values against Gram-negative bacteria were at least
125 mg/mL. Other extracts showed antimicrobial activity with
MIC values at least 125 mg/mL against Gram-negative
bacteria and 259 mg/mL against Gram-positive bacteria
(Table 4).
The bioactivity-guided fractionation of the methanol
extract of E. triplinerve showed that the active antimicrobial
components were mainly found in the hexane, dichloro-
methane, and dichloromethane/ethyl acetate fractions. The
hexane and dichloromethane/ethyl acetate fractions exhibited
the best MIC and CBM values against E. coli ATCC at 16
and 31 mg/mL, respectively. The dichloromethane fraction
was more effective against the E. coli clinic isolate and
P. aeruginosa, with MIC values of 31 and 62 mg/mL,
respectively, and the CBM value was at least 125 mg/mL
(Table 5).
Discussion
The purpose of this study was to evaluate the chemical
composition and antimicrobial activity of different extracts of
E. triplinerve, a medicinal plant used by indigenous
communities from Amazon region as hemostatic, cardiotonic,
diaphoretic, emetic, antiseptic, antimicrobial, hepatoprotec-
tive, antidepressive, and anxiolytic (Melo et al., 2013). Even
though the chemical composition of E. triplinerve has already
been studied by other authors, here we also investigated the
presence of the major secondary metabolites, because
metabolites present in different plant parts (Table 1). The
presence of these metabolites in the Eupatorium genus has
been reported previously (Bose & Roy, 1936; Inya-agha
et al., 1987; Jukic et al., 2007; Maas & Hensel, 2008; Maas
et al., 2009; Zhang et al., 2008).
The hydroalcoholic leaf extract showed better antimicro-
bial activity with lower MIC values (94 mg/mL) against Gram-
negative bacteria. Therefore, the leaf extract was selected for
fractionation into daughter fractions of different polarities.
Gupta et al. (2002) reported the broad-spectrum antimicro-
bial activity of the petroleum ether and methanol extracts
from leaves of E. triplinerve against Gram-positive and
Gram-negative bacteria at concentrations of 250–1000 mg/
mL. A similar antibacterial activity was also reported by
Rahman and Junaid (2008), who showed that the crude leaf
extract of E. triplinerve has antibacterial and antifungal
properties and can be used as a novel antimicrobial agent.
Taking into account the different leave extract fractions,
the methanol extract exhibited the highest antimicrobial
activity against Gram-positive and Gram-negative bacteria
and contained traces of coumarins (Tables 3 and 4, respect-
ively). In addition, coumarins have shown higher activity
against Gram-negative bacteria than Gram-positive bacteria
when isolated from other plants (Basile et al., 1997, 1999,
2009; Souza et al., 2005). The polarity of the solvent can
influence the interaction between bacterial cell wall compo-
nents and polar constituents present in methanol extracts, such
as coumarin derivatives, saponins, reducing sugars, sesqui-
terpene lactones, and depsides as revealed by this phyto-
chemical screening (Table 3). The bioactivity-guided assay
led to the identification of the dichloromethane fraction of the
methanolic extract as a bioactive fraction with good
 DOI: 10.3109/13880209.2014.948634 Antibacterial evaluation of Eupatorium triplinerve extracts 5
Table 4. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of different extracts
of increasing polarity of leaves of E. triplinerve against different bacteria.

Leaves extracts – MIC and MBC (mg/mL)

H EA MT
Bacteria MIC MBC MIC MBC MIC MBC
S. aureus ATCC 6538 4500 4500 4500 4500 75 150
E. faecalis ATCC 29212 250 4500 250 4500 62 4250
P. aeruginosa ATCC 25853 4500 4500 4500 4500 5125 4500
E. coli ATCC 8739 125 500 5125 125 125 500

Extracts: H, nhexanes; EA, ethyl acetate; MT, methanol.

Table 5. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in mg/mL of fractions from methanolic extract
of the leaves of E. triplinerve against the different microorganisms.
Pharmaceutical Biology Downloaded from informahealthcare.com by 200.239.65.127 on 11/28/14

Methanolic extract fraction – MIC and MBC (mg/mL)

H DC DC / EA (1:1) EA EA /MT (1:1) MT MT/W (1:1)
Bacteria MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
P. aeruginosa ATCC 25853 4500 4500 62 4125 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500
Klebsiella pneumonia ATCC 4352 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500
Proteus mirabilis ATCC 15290 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500
E. coli ATCC 8739 16 31 125 4125 16 31 4500 4500 4500 4500 4500 4500 4500 4500
E.coli clinic isolate 4500 (500 31 125 4500 4500 4500 4500 4500 4500 4500 4500 4500 4500

Extracts: H, nhexanes; DC, dichloromethane; EA, ethyl acetate; MT, methanol; W, water.
For personal use only.

antibacterial activity. More studies are necessary to identify
the component(s) responsible for this activity to facilitate its
possible use as a new phytomedicine.
According to previous phytochemical studies, the most
characteristic secondary metabolites of this plant are couma-
rins. Ayapanin (or herniarin), ayapin, daphnetin, daphnetin
dimethyl ether, daphnetin-7-methyl ether, hydrangetin, and
umbelliferone have been identified in the plant (Gauvin-
Bialecki & Marodon, 2009). This plant has an odor that is
slightly coumarin like, and the taste is mildly astringent and
aromatic. The leaves are recommended to treat indigestion,
pectoral complaints, and cholera, and they are employed
externally and internally during the treatment. Several studies
of E. triplinerve leaves have identified the presence
of 7-ethoxycoumarin (ayapanin), 6,7-dimethoxycoumarin
(ayapin), carotene, vitamin C, and stigmasterol (Bose &
Roy, 1936; Chaturvedi & Mulchandani, 1989; Natarajan &
Natarajan, 1979; Trang, 1992; Trang et al., 1993). Other
important compounds include thymohydroquinone and ter-
penoids, which are also present in high percentages (Jukic
et al., 2007). Coumarins are components involved in defense
response of plants to abiotic and biotic stresses, providing
antimicrobial or anti-inflammatory activity, acting as inhibi-
tors of numerous enzyme systems (Murray et al., 1982).
Some studies reported that coumarins are responsible for
many biological activities such as antithrombotic, anti-
inflammatory, vasodilator, and antimicrobial activities (Bose
& Roy, 1936; Chaturvedi & Mulchandani, 1989; Murray
et al., 1982; Späth et al., 1937; Trang, 1992; Trang et al.,
1993). Recently, our group showed that leaf and stem extracts
of Ayapana triplinervis exert mild sedative, anxiolytic, and
antidepressive behavioral effects in the central nervous system
(CNS) of rat and antinociceptive and antioxidant activities in
animal models (Melo et al., 2013).
Kayser and Kolodziej (1999) suggested that the fairly high
antibacterial activity of coumarin is due to both its lipophilic
character and planar molecular structure, which contribute to
its penetration through the bacterial cell membrane or cell
walls. These authors also suggested that the antibacterial
activity of oxygenated coumarins depends on the position
of polar (OH) and less polar (OMe, Me) functions at the
aromatic nucleus of the coumarin structure. In addition, Tegos
et al. (2002) reported that scopoletin, a coumarin, displays
better antibacterial activity against Gram-positive bacteria
than Gram-negative bacteria, perhaps because of an efficient
efflux pumps on Gram-negative bacteria. Efflux pumps
extrude the drug from the cell before they attain an adequate
concentration at the site of action (Tenover, 2006).
Another major component of the majority of extracts is the
saponins, which are widely distributed in the plant kingdom
and have several biological properties such as antimicrobial
action (Avato et al., 2006; Oleszek, 1996). Studies have
shown that leaf extracts of Eupatorium odoratum suppress
the growth of Propionibacterium acne (Chomnaweng et al.,
2005). Triterpenes and steroids were found in the phyto-
chemical screening, and these substances are found in
essential oils, also called the fifth essence and are responsible
for the fragrance of plants (Cavalcanti et al., 2004). Certain
representatives of this group, such as capsaicin, have an
antimicrobial effect through disruption of the plasma mem-
brane (Cowan, 1999). Coumarins, saponins, and triterpenes
were found in the methanol extract and might be involved in
the antibacterial activity of this fraction due to a synergistic
interaction between them.
 6 T. R. M. Lopes et al.
Depsides have been shown to reduce bacterial and fungal
growth (Manojlovic et al., 2012; Schmeda-Hirschmann
et al., 2008; Vartia, 1973) and have other properties such as
antioxidant (Hidalgo et al., 1994), antiviral (Neamati et al.,
1997), antitumor (Yamamoto et al., 1995), analgesic, and
antipyretic (Okuyama et al., 1995). These compounds were
only found in the hexane and ethyl acetate extracts.
Some criteria should be considered to avoid incorrect
analysis of the antimicrobial data and false positive results
such as the use of high concentrations of crude extracts or
isolated compounds. Concentrations of 100 mg/mL and
25 mM are considered optimal but the type of microorganism
being tested must be taken into consideration (Cos et al.,
2006). The antimicrobial activity of E. triplinerve is most
likely due to the presence of depsidones, coumarins, saponins,
and triterpenes, which are important antimicrobial com-
pounds present in the methanol extracts, as reported in this
Pharmaceutical Biology Downloaded from informahealthcare.com by 200.239.65.127 on 11/28/14
study.
Our previous and current studies indicated that
E. triplinerve contains steroids, coumarins, alkaloids, and
saponins. The leaf methanol extract showed the best anti-
microbial activity, and its non-polar fractions showed good
antimicrobial activity against Gram-negative bacteria. Studies
to isolate and identify these compounds should be performed.
Acknowledgements
For personal use only.
We are grateful to Dr. Mario Jardim and the Emilio Goeldi
Museum for identifying the plant and production of voucher
specimen and to FAPESPA and the Federal University of Pará
for financial support.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article. The
authors thank the Federal University of Pará for financial
support.
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