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Antimicrobial bioassay-guided fractionation of


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Article in Pharmaceutical Biology · November 2014


DOI: 10.3109/13880209.2014.948634

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! 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
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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. Pharm Biol, Early Online: 1–7

Figure 1. Preparation of extracts and


bioassay-guided fraction.
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with these activities (Gupta et al., 2002; Sharma & Singh, extract of E. triplinerve. The mash was dried in a rotator
For personal use only.

1979; Verpoorte & Dihal, 1987). The oil of the plant has been evaporator (Laborata 4000 efficient; Heidolph Instruments
found to possess antimicrobial activity (Yadava & Saini, GmbH & Co. KG, Schwabach, Germany) at 45  C, 1 atm
1990). Among different plant derivatives, secondary metab- pressure and 120 rpm. For total removal of the solvent, the
olites have been proven to be the most important group of mash was kept in the oven and water bath at 40  C. Leaves
compounds, with a wide range of antibacterial and antifungal and stem extracts (231 g, corresponding to 820 g of the
activities (Ahmad et al., 2002; Rahman et al., 1999). Thus, dried plant yield of the extract was 28.16%) were obtained
this study aimed to evaluate the chemical composition and and used in animal treatments. All hydroalcoholic extracts
antimicrobial activity of E. triplinerve extracts from different were analyzed to identify the plant parts with antibacterial
plant parts and to identify the most active fraction. properties.

Materials and methods Different extracts of the leaf and bioguided


fractionation of the methanol extract of
Collection and identification of E. triplinerve E. triplinerve leaves
Eupatorium triplinerve was collected from São Raimundo, a Eupatorium triplinerve leaves (100 g) were extracted with
village in Acará city (Pará state, Amazon region) in March, 500 mL of increasing polarity solvents, including hexane,
2010. The region is situated at latitude 01132.6840 and ethyl acetate, and methanol, at room temperature for a period
longitude 048123.9840 (geographic coordinates obtained of 7 d each. The solvent was evaporated under reduced
using global positioning system [GPS] equipment). The pressure. The methanol extracts were subjected to partition
botanical identification was performed by Dr. Mário Jardim, column chromatography using silica gel 60 (Macherey-Nagel,
a specialist from Emilio Goeldi Museum (Pará, Brazil), where Sigma-Aldrich, St. Louis, MO); were partitioned with hexane,
a sample was deposited as a voucher specimen under number dichloromethane, ethyl acetate, and methanol; and were
MG123913. concentrated as described. The extracts were investigated
for their phytochemical characteristics; then, the extracts and
Preparation of hydroalcoholic extract of different fractions obtained from the methanol extract were evaluated
parts of E. triplinerve for antibacterial activity (Figure 1).
The whole plant was washed with water and 10% ethanol
solution and separated into leaves, stems, leaves/stems, and Phytochemical screening
roots. The plant parts were dried at room temperature for 2 d, The crude extracts were analyzed for the presence of major
dried in an oven with forced air circulation at an average chemical groups (i.e., saponins, reducing sugars, alkaloids,
temperature of 40  C for 6 d and then crushed in a knife mill steroids and triterpenoids, phenols, tannins, flavonoids, poly-
to obtain the drug spray. After grinding, the dust was saccharides, coumarin, depsides, and depsidones) by standard
macerated for 5 d in 70% ethanol to obtain the hydroalcoholic phytochemical techniques (Barbosa et al., 2001).
DOI: 10.3109/13880209.2014.948634 Antibacterial evaluation of Eupatorium triplinerve extracts 3

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

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


Reducing Sugars   +
Polysaccharides    extract of E. triplinerve showed the presence of steroids,
Flavonoids    coumarins, alkaloids, and saponins. The methanol extract of
Phenols and tannins    the leaves presented better antimicrobial activity compared
Depsides and depsidones + + 
with other extracts with increasing polarity, and the non-polar
Coumarin derivative  + ++
Sesquiterpene lactones    leaf fractions showed good antimicrobial activity against
Alkaloids + + + Gram-negative bacteria.
Triterpenoids and steroids  ++ + The initial phytochemical screening of the hydroalcoholic
, absent; +, slightly present; ++, high present. extract of E. triplinerve showed the presence of saponins,
reducing sugars, alkaloids, coumarins, phenols, steroids, and
For personal use only.

depsides. However, there was no qualitative difference in the


The results demonstrate that the methanol extract of metabolites present in different plant parts (Table 1). The
E. triplinerve presented the best antimicrobial activity with presence of these metabolites in the Eupatorium genus has
MIC values of 62 and 75 mg/mL against Gram-positive been reported previously (Bose & Roy, 1936; Inya-agha
bacteria E. faecalis and S. aureus, respectively. In addition, et al., 1987; Jukic et al., 2007; Maas & Hensel, 2008; Maas
the MIC values against Gram-negative bacteria were at least et al., 2009; Zhang et al., 2008).
125 mg/mL. Other extracts showed antimicrobial activity with The hydroalcoholic leaf extract showed better antimicro-
MIC values at least 125 mg/mL against Gram-negative bial activity with lower MIC values (94 mg/mL) against Gram-
bacteria and 259 mg/mL against Gram-positive bacteria negative bacteria. Therefore, the leaf extract was selected for
(Table 4). fractionation into daughter fractions of different polarities.
The bioactivity-guided fractionation of the methanol Gupta et al. (2002) reported the broad-spectrum antimicro-
extract of E. triplinerve showed that the active antimicrobial bial activity of the petroleum ether and methanol extracts
components were mainly found in the hexane, dichloro- from leaves of E. triplinerve against Gram-positive and
methane, and dichloromethane/ethyl acetate fractions. The Gram-negative bacteria at concentrations of 250–1000 mg/
hexane and dichloromethane/ethyl acetate fractions exhibited mL. A similar antibacterial activity was also reported by
the best MIC and CBM values against E. coli ATCC at 16 Rahman and Junaid (2008), who showed that the crude leaf
and 31 mg/mL, respectively. The dichloromethane fraction extract of E. triplinerve has antibacterial and antifungal
was more effective against the E. coli clinic isolate and properties and can be used as a novel antimicrobial agent.
P. aeruginosa, with MIC values of 31 and 62 mg/mL, Taking into account the different leave extract fractions,
respectively, and the CBM value was at least 125 mg/mL the methanol extract exhibited the highest antimicrobial
(Table 5). 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
Discussion
against Gram-negative bacteria than Gram-positive bacteria
The purpose of this study was to evaluate the chemical when isolated from other plants (Basile et al., 1997, 1999,
composition and antimicrobial activity of different extracts of 2009; Souza et al., 2005). The polarity of the solvent can
E. triplinerve, a medicinal plant used by indigenous influence the interaction between bacterial cell wall compo-
communities from Amazon region as hemostatic, cardiotonic, nents and polar constituents present in methanol extracts, such
diaphoretic, emetic, antiseptic, antimicrobial, hepatoprotec- as coumarin derivatives, saponins, reducing sugars, sesqui-
tive, antidepressive, and anxiolytic (Melo et al., 2013). Even terpene lactones, and depsides as revealed by this phyto-
though the chemical composition of E. triplinerve has already chemical screening (Table 3). The bioactivity-guided assay
been studied by other authors, here we also investigated the led to the identification of the dichloromethane fraction of the
presence of the major secondary metabolites, because 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.
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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 (CNS) of rat and antinociceptive and antioxidant activities in
the component(s) responsible for this activity to facilitate its animal models (Melo et al., 2013).
possible use as a new phytomedicine. Kayser and Kolodziej (1999) suggested that the fairly high
According to previous phytochemical studies, the most antibacterial activity of coumarin is due to both its lipophilic
characteristic secondary metabolites of this plant are couma- character and planar molecular structure, which contribute to
rins. Ayapanin (or herniarin), ayapin, daphnetin, daphnetin its penetration through the bacterial cell membrane or cell
dimethyl ether, daphnetin-7-methyl ether, hydrangetin, and walls. These authors also suggested that the antibacterial
umbelliferone have been identified in the plant (Gauvin- activity of oxygenated coumarins depends on the position
Bialecki & Marodon, 2009). This plant has an odor that is of polar (OH) and less polar (OMe, Me) functions at the
slightly coumarin like, and the taste is mildly astringent and aromatic nucleus of the coumarin structure. In addition, Tegos
aromatic. The leaves are recommended to treat indigestion, et al. (2002) reported that scopoletin, a coumarin, displays
pectoral complaints, and cholera, and they are employed better antibacterial activity against Gram-positive bacteria
externally and internally during the treatment. Several studies than Gram-negative bacteria, perhaps because of an efficient
of E. triplinerve leaves have identified the presence efflux pumps on Gram-negative bacteria. Efflux pumps
of 7-ethoxycoumarin (ayapanin), 6,7-dimethoxycoumarin extrude the drug from the cell before they attain an adequate
(ayapin), carotene, vitamin C, and stigmasterol (Bose & concentration at the site of action (Tenover, 2006).
Roy, 1936; Chaturvedi & Mulchandani, 1989; Natarajan & Another major component of the majority of extracts is the
Natarajan, 1979; Trang, 1992; Trang et al., 1993). Other saponins, which are widely distributed in the plant kingdom
important compounds include thymohydroquinone and ter- and have several biological properties such as antimicrobial
penoids, which are also present in high percentages (Jukic action (Avato et al., 2006; Oleszek, 1996). Studies have
et al., 2007). Coumarins are components involved in defense shown that leaf extracts of Eupatorium odoratum suppress
response of plants to abiotic and biotic stresses, providing the growth of Propionibacterium acne (Chomnaweng et al.,
antimicrobial or anti-inflammatory activity, acting as inhibi- 2005). Triterpenes and steroids were found in the phyto-
tors of numerous enzyme systems (Murray et al., 1982). chemical screening, and these substances are found in
Some studies reported that coumarins are responsible for essential oils, also called the fifth essence and are responsible
many biological activities such as antithrombotic, anti- for the fragrance of plants (Cavalcanti et al., 2004). Certain
inflammatory, vasodilator, and antimicrobial activities (Bose representatives of this group, such as capsaicin, have an
& Roy, 1936; Chaturvedi & Mulchandani, 1989; Murray antimicrobial effect through disruption of the plasma mem-
et al., 1982; Späth et al., 1937; Trang, 1992; Trang et al., brane (Cowan, 1999). Coumarins, saponins, and triterpenes
1993). Recently, our group showed that leaf and stem extracts were found in the methanol extract and might be involved in
of Ayapana triplinervis exert mild sedative, anxiolytic, and the antibacterial activity of this fraction due to a synergistic
antidepressive behavioral effects in the central nervous system interaction between them.
6 T. R. M. Lopes et al. Pharm Biol, Early Online: 1–7

Depsides have been shown to reduce bacterial and fungal Cavalcanti ES, Morais SM, Lima MA, Santana EW. (2004). Larvicidal
activity of essential oils from brazilian plants against Aedes aegypti L.
growth (Manojlovic et al., 2012; Schmeda-Hirschmann Mem Inst Oswaldo Cruz 99:541–4.
et al., 2008; Vartia, 1973) and have other properties such as Chaturvedi R, Mulchandani NB. (1989). Coumarins from Eupatorium
antioxidant (Hidalgo et al., 1994), antiviral (Neamati et al., ayapana. J Ind Chem Soc 66:286–7.
1997), antitumor (Yamamoto et al., 1995), analgesic, and Chaurasia SC, Kher A. (1978). Activity of essential oils of three
medicinal plants against various pathogenic and nonpathogenic fungi.
antipyretic (Okuyama et al., 1995). These compounds were Indian J Hosp Pharm 15:139–41.
only found in the hexane and ethyl acetate extracts. CLSI. (2012). Methods for dilution antimicrobial susceptibility tests for
Some criteria should be considered to avoid incorrect bacteria that grow aerobically; Approved Standard–Ninth Edition.
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[ISBN 1-56238-783-9] USA.
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isolated compounds. Concentrations of 100 mg/mL and Antimicrobial effects of Thai medicinal plants against acne-inducing
25 mM are considered optimal but the type of microorganism bacteria. J Ethnopharmacol 101:330–3.
being tested must be taken into consideration (Cos et al., Cos P, Vlietinck AJ, Berghe DV, Maes L. (2006). Anti-infective potential
of natural products: How to develop a stronger in vitro ‘proof-of-
2006). The antimicrobial activity of E. triplinerve is most concept’. J Ethnopharmacol 106:290–302.
likely due to the presence of depsidones, coumarins, saponins, Cowan MM. (1999). Plants products as antimicrobial agents. Clin
and triterpenes, which are important antimicrobial com- Microbiol Rev 12:564–82.
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Farmacobotânica, fitoquı́mica e farmacologia do Guaco: Revisão


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Our previous and current studies indicated that Schulyz Bip. ex Baker. Rev Bras Plantas Med 14:400–9.
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saponins. The leaf methanol extract showed the best anti- of Eupatorium triplinerve. Índian Perfumer 37:318–23.
Gauvin-Bialecki A, Marodon C. (2009). Essential oil of Ayapana
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Acknowledgements influência no conteúdo de metabólitos secundários. Quim Nova 30:
374–81.
For personal use only.

We are grateful to Dr. Mario Jardim and the Emilio Goeldi Gupta M, Mazumder UK, Chaudhuri I, et al. (2002). Antimicrobial
Museum for identifying the plant and production of voucher activity of Eupatorium ayapana. Fitoterapia 73:168–70.
specimen and to FAPESPA and the Federal University of Pará Hidalgo ME, Fernandez E, Quilhot W, Lissi E. (1994). Antioxidant
for financial support. activity of depsides and depsidones. Phytochemistry 37:1585–7.
Inya-agha SI, Oguntimein BO, Sofowora A, Benjamin TV. (1987).
Phytochemical and antibacterial studies on the essential oil of
Declaration of interest Eupatorium odoratum. Pharm Biol 25:49–52.
Jelager L, Gurib-Fakim A, Andersen A. (1998). Antibacterial and
The authors report no conflicts of interest. The authors alone antifungal activity of medicinal plants of Mauritius. Pharm Biol 36:
are responsible for the content and writing of this article. The 153–61.
Jukic M, Politeo O, Maksimovic M, et al. (2007). In vitro acetylcholin-
authors thank the Federal University of Pará for financial
esterase inhibitory properties of thymol, carvacrol and their deriva-
support. tives thymoquinone and thymohydroquinone. Phytother Res 21:
259–61.
Kayser O, Kolodziej H. (1999). Antibacterial activity of simple
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