Isolation of flavonoids from Anemopaegma arvense (Vell) Stellf. ex
de Souza and their antifungal activity against Trichophyton rubrum
Camila Di Giovane Costanzo, Vanessa Colnaghi Fernandes, Sônia Zingaretti,
Rene Oliveira Beleboni, Ana Maria Soares Pereira, Mozart Marins,
Sílvia Helena Taleb-Contini, Paulo Sérgio Pereira, Ana Lúcia Fachin*
Biotechnology Division, University of Ribeirão Preto, Ribeirão Preto, SP, Brazil
Anemopaegma arvense (Vell) Stellf. ex de Souza belongs to the family Bignoniaceae, and is popularly
known as catuaba. To evaluate the cytotoxic and antimicrobial activity of A. arvense, fraction F3 and
flavonoids 1 (quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside) (rutin) and flavonoid 2
(quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside) were isolated from the leaves of this
plant. Fraction F3 and flavonoids 1 and 2 exhibited no antibacterial activity. Furthermore, no cytotoxic
activity of fraction 3 or flavonoids 1 and 2 was observed against the tumor cells tested. However, analysis
of the antifungal activity of flavonoids 1 and 2 revealed minimum inhibitory concentrations of 0.5 and
0.25 mg/mL, respectively, against the Trichophyton rubrum strains tested (wild type and mutant). This
study demonstrates for the first time the antifungal activity of isolated flavonoids, validating the same
activity for A. arvense.
Uniterms: Bignoniaceae. Anemopaegma arvense/phytochemistry. Catuaba/phytochemistry. Flavonoids/
antifungal activity. Plant extract/evaluation.
Anemopaegma arvense pertence à família Bignoniaceae, sendo conhecida popularmente como Catuaba.
Para avaliação de sua atividade citotóxica e antimicrobiana, a fração cromatográfica F3 e os flavonoides
1 (quercetina 3-O-α-L-ramnopiranosil-(1→6)-β-D-glucopiranosídeo) (rutina) e flavonoide 2 (quercetina
3-O-α-L-ramnopiranosil-(1→6)-β-D-galactopiranosídeo) foram isolados das folhas de A. arvense. A
fração 3 e os flavonoides não apresentaram atividade antibacteriana. Nenhuma atividade citotóxica foi
observada para a fração F3 e para os flavonoides, quando avaliados contra as células tumorais em teste.
Entretanto, e considerando a atividade antifúngica, o flavonóide 1 apresentou valor de concentração
inibitória mínima (CIM) de 0,5 mg/mL, enquanto o flavonóide 2, CIM de 0,25 mg/mL contra as cepas
selvagem e mutante de Trichophyton rubrum, demonstrando, pela primeira vez, que os flavonoides
isolados possuem atividade antifúngica, o que valida a mesma atividade para A. arvense.
Unitermos: Bignoniaceae/fitoquímica. Anemopaegma arvense/fitoquímica. Catuaba/fitoquímica.
Flavonóides/atividade antifúngica. Extrato vegetal/avaliação.
INTRODUCTION
The family Bignoniaceae comprises about 800 plant
species that are found mainly in the Neotropical region
(Gentry, 1980). In Brazil, several plants of this family
are used in folk medicine as astringent and against fever,
*Correspondence: A. L. Fachin. Unidade de Biotecnologia, Universidade de
Ribeirão Preto. Av. Costábile Romano, 2201 - 14096-900 - Ribeirão Preto – SP,
Brasil. E-mail: asaltoratto@unaerp.br
rheumatism, diarrhea, cancer and microbial infections (Pio
Côrrea, Penna, 1969; Fenner et al., 2006). Anemopaegma
arvense (Vell) Stellf. ex de Souza is a species of the family
Bignoniaceae, which is popularly known as “catuaba”.
Commercially available formulations of this plant are
used as aphrodisiac (Manabe et al., 1992). The major
components identified in A. arvense are flavonoids,
catuabins, alkaloids, tannins, and resins (Charam, 1987;
Zanolari et al., 2005; Tabanca et al., 2007). Flavonoids
are becoming the subject of anti-infective research and
many groups have isolated and identified the structures
Article
Brazilian Journal of
Pharmaceutical Sciences
vol. 49, n. 3, jul./sep., 2013
560
C. D. G. Costanzo, V. C. Fernandes, S. Zingaretti, R. O. Beleboni, A. M. S. Pereira, M. Marins, S. H. Taleb-Contini, P. S. Pereira, A. L. Fachin
of flavonoids with antifungal, antiviral and antibacterial
activity (Cushnie, Lamb, 2005).
There is an urgent need to develop new and more
effective antifungal drugs because of the increased
resistance of fungi to the drugs currently used in clinical
practices (Rahalison et al., 1994). Plant secondary
metabolites represent a good source of novel antimicrobial
molecules. Furthermore, there has been an almost
exponential rise in cancer-related mortality over recent
years, which has led to an increase in the search for new
medicines, including those derived from natural products,
able to treat the various types of the diseases (Patocka,
2003; Aziz, 2004; Diwanay et al., 2005).
We investigated the cytotoxic and antimicrobial
activity of chromatographic fraction F3 and flavonoids
isolated from A. arvense in order to provide better
understanding of the biological activities of this plant.
MATERIAL AND METHODS
Plant material
Leaves of A. arvense (Vell) Stellf. ex de Souza
(Bignoniaceae) were collected in Sacramento, MG, Brazil,
in August 2007 (IBAMA License No. 02001.005076/201116) and identified by Professor Lúcia G. Lohmann,
Department of Botany, São Paulo University. Voucher
specimens (N HPMU-1333) were deposited at the
herbarium of the Ribeirão Preto University.
Extract preparation and purification
Dried and pulverized A. arvense leaves (100 g)
were extracted by maceration with MeOH (0.5 L x 3)
at room temperature. After filtration and evaporation
of the solvent under reduced pressure, the methanolic
extract (5 g) was chromatographed over a Sephadex
LH-20 column (3 x 64 cm) using MeOH as the mobile
phase, yielding three fractions: F1 (177 mL), F2
(122 mL), and F3 (150 mL). Fraction F3 (0.7 g), rich
in flavonoids, was submitted to preparative HPLC
separation on a RP-18 column (Supelcosil TM RP-18,
250 x 10 mm i.d., 5 µm) using a Shimadzu LC10A
system coupled to a diode array detector (280 nm).
The following gradient program was used: MeOH:H2O
(0-100 min: 0-60% MeOH; 100-110 min: 60-80% MeOH;
110-112 min: 80-0% MeOH; 112-120 min: 0% MeOH).
The flow rate was 2.0 mL/min and the sample injection
volume was 400 μL at a concentration of 100 mg/mL.
Six subfractions were obtained after purification. The
F3.3 (59 mg) and F3.4 (36 mg) subfractions were pooled
and purified on a Sephadex LH-20 column (2.2 x 40 cm)
using acetone:water (7:3, v/v) as the mobile phase,
yielding two flavonoids: 1 (6 mg) and 2 (12 mg).
Identification of flavonoids
The identity of the flavonoids was confirmed based
on 1H and 13C NMR spectral data and by comparison with
the literature (Jaramillo et al., 2011). The position of the
interglycosidic linkage was provided by 13C NMR and
was confirmed by HMBC and HMQC experiments. 1H
NMR (300 MHz) and 13C NMR (75 MHz) spectra were
recorded with a Bruker Avance DPX 300 spectrometer in
DMSO-d6 using TMS as internal standard. HPLC analysis
was performed using a Shimadzu LC10ADvp system
equipped with a Supelco LC18 column (SupelcosilTM RP18, 250 x 4.6 mm i.d., 5 µm) and coupled to a diode array
detector, monitored at 340 nm. The following gradient
program was used: MeOH:H 2O (0-32 min: 10-66%
MeOH; 32-35 min: 66-10% MeOH; 35-40 min: 10%
MeOH). The flow rate was 1.0 mL/min and the sample
injection volume was 20 μL at a concentration of 1 mg/mL.
Antimicrobial activity
Trichophyton rubrum
The clinical strain of T. rubrum (ATCC MYA3108)
was kindly provided by Dr. Nilce M. Martinez-Rossi.
The TruMDR2 mutant strain was obtained by disruption
of the TruMDR2 gene of strain MYA3108 (Fachin et
al., 2006). Standard techniques of manipulation and
growth as described previously (Fachin et al., 2001)
were used. Susceptibility of the MYA3108 (wild
type) and TruMDR2 (mutant) strains was tested by
determining the minimum inhibitory concentration (MIC)
of fraction F3 and of flavonoids 1 and 2 using the M38-A
microdilution technique proposed by the Clinical and
Laboratory Standards Institute (CLSI, 2002). Fraction
F3 and flavonoids were diluted in 10% DMSO and the
final concentration of DMSO in the antifungal assay
was less than 1%. Colonies obtained by growth of the
strains on Sabouraud agar plates at 28 °C for 15 days
were harvested by sterile scraping and mixed with sterile
saline and the solution was filtered through glass wool.
The resulting mixture was transferred to a sterile tube
and adjusted spectrophotometrically at a wavelength of
530 nm, ranging from 70 to 75% transmittance. These
conidial suspensions were diluted 1:50 in RPMI 1640
(Sigma, St. Louis, MO, USA) buffered with MOPS,
corresponding to twice the density needed for the test of
approximately 3-5 × 105 CFU/mL. Growth, solvent and
Isolation of flavonoids from Anemopaegma arvense (Vell) Stellf. ex de Souza and their antifungal activity against Trichophyton rubrum
sterility controls were included. Microtiter plates were
incubated at 28 °C for 7 days. The MIC100 was defined
as the lowest concentration of the fraction or flavonoid
that resulted in the complete inhibition of fungal growth.
The range of concentrations tested was 2.5-0.019 mg/mL
and 0.500-0.019 mg/mL for fraction F3 and the flavonoids
(1 and 2), respectively. The assays were carried out in
triplicate in three independent experiments. Fluconazole
and griseofulvin were used as positive controls.
Bacteria
The following strains were used as test organisms:
Staphylococcus aureus (ATCC 6538), Staphylococcus
epidermides (ATCC 2228), Escherichia coli (ATCC
25922), and Pseudomonas aeruginosa (ATCC 27853). The
antimicrobial activity of fraction F3 and flavonoids 1 and
2 was evaluated using the microdilution method according
to CLSI M7-A7 (2006). The test strains were incubated in
BHI medium for 24 h at 37 oC. The crude extract, fraction
F3 and flavonoids were diluted in 10% DMSO and the
final concentration of DMSO in the antibacterial assay was
less than 1%. The crude extract and fraction were assayed
at concentrations of 2.5, 1.25, 0.625, 0.312, 0.156, 0.078,
0.039, and 0.019 mg/mL. The flavonoids were assayed
at a concentration range of 0.500 to 0.039 mg/mL. The
assays were carried out in triplicate in three independent
experiments. Ampicillin and chloramphenicol were used
as positive controls.
561
RESULTS AND DISCUSSION
The aglycons were identified as quercetin by 1H and
C NMR. The 1H NMR spectra indicated the presence
of a rhamnosyl and a glucosyl group at δ 5.34 and δ
4.41 in flavonoid 1 and of a rhamnosyl and a galactosyl
group at δ 5.30 and δ 4.36 in flavonoid 2. The relatively
deshielded rhamnosyl protons suggested that they are
not directly attached to the aglycone, but that there is
a sugar-sugar linkage. The position of the sugar in the
aglycons provided by HMBC was demonstrated by
cross-peaks between H-1’’ and C-3. The position of the
interglycosidic linkage was provided by the 13C-NMR
downfield shift of C-6’’ at δ 67.5 and δ 65.7 in flavonoids
1 and 2, respectively. This fact was confirmed by HMBC
and HMQC experiments. On the basis of the spectroscopic
data, flavonoids 1 and 2 were characterized as quercetin
3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside
(rutin) and quercetin 3-O-α-L-rhamnopyranosyl-(1→6)β-D-galactopyranoside, respectively (Figure 1). HPLC
analysis of fraction F3 revealed the presence of six
flavonoids (Figure 2A). Flavonoids 1 and 2 isolated from
A. arvense were detected at 24.1 and 23.2 min, respectively
(Figure 2B and 2C).
13
Cytotoxic activity
The following cells were cultured in Dulbecco’s
modified Eagle’s medium (Life Technologies, Inc.,
Gaithersburg, MD, USA) supplemented with 15% fetal
calf serum (Life Technologies Inc.) at 37 °C in a humidified
atmosphere of 5% CO2: HeLa (human cervix tumor), 3T3
(mouse embryonic fibroblasts), and U343MG-a (human
glioblastoma cell line). Penicillin (100 U/mL) and
streptomycin (0.1 mg/mL) were added to the medium to
prevent bacterial growth. A stock solution (20 mg/mL) was
prepared by dissolving fraction F3 and flavonoids 1 and
2 in 10% DMSO (v/v). The final concentration of these
compounds (0.2, 0.02 and 0.002 mg/mL, respectively) was
obtained by direct dilution in the culture medium. The final
concentration of DMSO in the control and experimental
groups was 1%. The cells (105 cells/well) were seeded
into a 96-well plate 24 h prior to the beginning of the
experiment. Actinomycin D (Sigma) was used as positive
control. The cells were incubated for 48 h with the fraction
or flavonoids and analyzed by the MTT assay (Mosmann,
1983; Rubinstein et al., 1990).
FIGURE 1 - Chemical structure of flavonoids 1 and 2 isolated
from Anemopaegma arvense. Flavonoid 1: quercetin 3-O-αL-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (rutin);
flavonoid 2: quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-Dgalactopyranoside.
Analysis of the bioactivity of A. arvense showed no
cytotoxic activity of fraction F3 or of the isolated flavonoids
at a dose of 0.2 mg/mL against the cell lines tested. In
addition, at a dose of 2.5 mg/mL, the crude extract, fraction
F3 and the flavonoids exhibited no antibacterial activity
against the strains tested (data not shown). These results
partially agree with those reported by Tabanca et al. (2007)
562
C. D. G. Costanzo, V. C. Fernandes, S. Zingaretti, R. O. Beleboni, A. M. S. Pereira, M. Marins, S. H. Taleb-Contini, P. S. Pereira, A. L. Fachin
FIGURE 2 - HPLC profile of fraction F3 (A), flavonoid 1 (B), and flavonoid 2 (C) isolated from Anemopaegma arvense.
who isolated a new catuabin (catuabin A) and three known
flavan-type phenylpropanoids (cinchonain Ia, cinchonain
IIa, and candelin A1) from A. arvense. These compounds
possessed no anti-inflammatory, cytotoxic, antimicrobial
or antimalarial property, but exhibited antioxidant activity.
In the present study, fraction F3 exhibited antifungal
activity against the mutant and wild-type strains of
T. rubrum at concentrations of 1.25 and 0.625 mg/mL,
respectively. Isolated flavonoid 1 showed weak antifungal
activity, with an MIC of 0.5 mg/mL against the two fungal
strains tested, whereas flavonoid 2 exhibited moderate
antifungal activity, with an MIC of 0.25 mg/mL (Table I).
Antifungal activity of ethanol extracts of Hyptis ovalifolia
and Eugenia uniflora has been demonstrated by Souza et
Isolation of flavonoids from Anemopaegma arvense (Vell) Stellf. ex de Souza and their antifungal activity against Trichophyton rubrum
al. (2002). These extract completely inhibited the growth of
the 30 dermatophytes tested. The MIC of the H. ovalifolia
extract against T. rubrum strains was 0.25 mg/mL and the
E. uniflora extract exhibited antifungal activity against 19
of the 30 isolates at a concentration of 0.5 mg/mL. Rocha et
al. (2004) demonstrated antifungal activity of Clytostoma
ramentaceum and Mansoa hirsuta (Bignoniaceae) when
testing the low and medium polar fractions at concentrations
of 0.1 to 0.3 mg/mL. Pacciaroni et al. (2008) isolated
several flavonoids from the aerial parts of Heterothalamus
alienus and tested these compounds against clinical isolates
of dermatophytes. The flavanones showed very good
fungicidal activity against standard (MIC: 31.2 μg/mL) and
clinical isolates of T. rubrum and T. mentagrophytes (MIC:
31.2-62.5 and 31.2-125 μg/mL, respectively). However,
rutin, spathulenol (1) and two of the 3-acetylated flavanones
were inactive or marginally active against the fungal strains
(MIC > 250 µg/mL).
Methods for antimicrobial assessment of natural
products and effective MIC values are not well established
in the literature. Holetz et al. (2002), who screened
hydroalcoholic extracts from 13 Brazilian plants using the
microdilution technique, defined an MIC < 0.1 mg/mL as
good antimicrobial activity, MIC of 0.1 to 0.5 mg/mL as
moderate antimicrobial activity, and MIC of 0.5 to 1 mg/mL
as weak antimicrobial activity. Extracts exhibiting MIC
higher than 1 mg/mL are considered to be ineffective.
Reports of activity in the field of antibacterial flavonoid
TABLE I - Minimum inhibitory concentration (mg/mL) of
fraction F3 and flavonoids isolated from Anemopaegma
arvense against Trichophyton rubrum strains (MYA-3108 and
ΔTruMDR2)
Compound
MYA-3108
ΔTruMDR2
Fraction F3
0.625
1.250
Flavonoid 1
0.500
0.500
Flavonoid 2
0.250
0.250
Griseofulvin
0.0005
0.0005
Fluconazole
0.075
0.075
The minimum inhibitory concentration corresponds to the
lowest concentration of the fraction or flavonoid that resulted
in 100% inhibition of visible fungal compared to control. The
results are representative of three independent experiments
performed in triplicate.
Flavonoid 1: quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-βD-glucopyranoside (rutin); flavonoid 2: quercetin 3-O-α-Lrhamnopyranosyl-(1→6)-β-D-galactopyranoside.
Fluconazole and griseofulvin were used as positive controls.
Fraction F3: concentration range of 2.5-0.019 mg/mL.
Flavonoids: concentration range of 0.500-0.019 mg/mL.
563
research are widely conflicting, probably because of interand intra-assay variation in susceptibility testing (Cushnie,
Lamb, 2005).
Membrane transporters, especially efflux transporters,
affect the adsorption and bioavailability of drugs. MIC
of flavonoids 1 and 2 was the same for the wild-type
and mutant strain of T. rubrum (in which the ABC gene
is disrupted). This finding may be explained by the fact
that ABC transporters are not involved in the transport of
flavonoids (Walgren et al., 2000). The TruMDR2 gene was
disrupted in the mutant strain of T. rubrum and this strain has
been shown to be susceptible to several compounds (Fachin
et al., 2006). Therefore, despite relatively high MIC,
flavonoid-based inhibitors of fungi may be an alternative
for the treatment of multidrug-resistant strains since efflux
pumps do not transport these compounds.
In the present study, we were able to isolate and
identify two quercetin-derived glycosylated flavonoids
from A. arvense, which showed antifungal activity.
Quercetin is a substance widely distributed in the plant
kingdom. However, this study describes for the first time
the antidermatophyte activity of A. arvense, which could
be attributed to the presence of quercetin. In fact, the
antifungal activity of quercetin and its derivatives has
been described in other medicinal plants. Semwal et al.
(2009) demonstrated the antifungal activity of an ethanol
extract of Boehmeria rugulosa leaves and of three new
flavonoid glycosides against T. rubrum, Microsporum
canis and Microsporum gypseum, with MIC of
100 µg/mL. Pereira et al. (2008) isolated the flavonoid
rutin from the aerial parts of Solanum palinacanthum and
evaluation of its antimicrobial activity showed an MIC of
35 μg/mL against the fungus Aspergillus ochraceus.
Human mycoses are not always treated effectively.
The most important causes of treatment failure are the
recurrence of infections, drug resistance of pathogens, and
toxicity of currently available antifungal agents (Turel,
2011; Butts, Krysan 2012). Therefore, the continual search
for new and more effective antifungal drugs, which should
also be safer than currently used agents, is important
(Zacchino, 2001). The increasing prevalence of multidrugresistant pathogens requires the identification of new
antimicrobial agents as alternative therapies in difficultto-treat infections (Pereira et al., 2006). In conclusion, the
flavonoids isolated from A. arvense were bioactive against
T. rubrum and may be a promising target in studies on new
antifungal agents.
ACKNOWLEDGEMENTS
The authors thank the state funding agency Fundação
564
C. D. G. Costanzo, V. C. Fernandes, S. Zingaretti, R. O. Beleboni, A. M. S. Pereira, M. Marins, S. H. Taleb-Contini, P. S. Pereira, A. L. Fachin
de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
for financial support.
REFERENCES
FENNER, R.; BETTI, A.H.; MENTZ, L.A.; RATES, S.M.K.
Plantas utilizadas na medicina popular brasileira com
potencial atividade antifúngica. Braz. J. Pharm. Sci., v.42,
n.3, p.369-394, 2006.
AZIZ, Z. Herbal medicines: predictors of recommendation by
physicians. J. Clin. Pharm. Ther., v.29, n.3, p.241-246,
2004.
GENTRY, A.A. Flora neotropical monograph. In:__
Bignoniaceae. Part I, v.25. New York: The New York
Botanical Garden, 1980. v.25, n.2, p.1-130, 1992. p1-150
BUTTS A, KRYSAN D.J. Antifungal drug discovery: something
old and something new. PLoS Pathog., 8(9):e1002870,
2012.
HOLETZ, F.B.; PESSINI, G.L.; SANCHES, N.R.; CORTEZ,
D.A.; NAKAMURA, C.V. Screening of some plants used
in the Brazilian folk medicine for the treatment of infectious
diseases. Mem. Inst. Oswaldo Cruz., v.97, n.7, p.1027-1031,
2002.
CHARAM, I. Há ações afrodisíacas nas plantas medicinais do
Brasil? Folha Méd., v.94, n.5, p.303-309, 1987.
C L I N I C A L A N D L A B O R AT O RY S TA N D A R D S
INSTITUTE. In methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically:
approved standard. 7th ed. Wayne: Clinical and Laboratory
Standards Institute, 2006. p.19087-1898.
C L I N I C A L A N D L A B O R AT O RY S TA N D A R D S
INSTITUTE. In reference method for broth dilution
antifungal susceptibility testing of filamentous fungi,
approved standard M38-A. Wayne: Clinical and Laboratory
Standards Institute, 2002.
CUSHNIE, T.P.; LAMB, A.I. Antimicrobial activity of
flavonoids. Int. J. Antimicrob. Agents., v.26, n.5, p.343356, 2005.
DIWANAY, S.; CHISTRE, D.; PATAWARDHAN, B.
Immunoprotection by botanical drugs in cancer
chemotherapy. J. Ethnopharmacol., v.90, n.1, p.49-55,
2005.
FACHIN, A.L.; CONTEL, E.P.; MARTINEZ-ROSSI, N.M.
Effect of sub-MICs of antimycotics on expression of
intracellular esterase of Trichophyton rubrum. Med. Mycol.,
v.39, n.1, p.129-133, 2001.
FACHIN, A.L.; FERREIRA-NOZAWA, M.S.; MACCHERONI
JR, W.; MARTINEZ-ROSSI, N.M. Role of the ABC
transporter TruMDR2 in terbinafine, 4-nitroquinolineN-oxide (4NQO) and ethidium bromide resistance in
Trichophyton rubrum. J. Med. Microbiol., v.55, pt.8,
p.1093-1099, 2006.
JARAMILLO, K; DAWID , C; HOFMANN, T, FUJIMOTO,
Y; OSORIO, C. Identification of Antioxidative Flavonols
and Anthocyanins in Sicana odorifera Fruit Peel. J. Agric.
Food Chem., v.59, n.3, p 975-983, 2011
MANABE, H.; SAKAGAMI, H.; ISHIZONE, H.; KUSANO,
H.; FUJIMAKI, M.; KOMATSU, N.; NAKASHIMA, H.;
MURAKAMI, T.; YAMAMOTO, N. Effects of catuaba
extracts on microbial and HIV infection. In Vivo, v.6, n.2,
p.161-166, 1992.
MOSMANN, T. Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity
assays. J. Immunol. Methods, v.65, n.1-2, p.55-63, 1983.
PACCIARONI, A.V.; GETTE, M.L.; DERITA, M.; ARIZAESPINAR, L.; GIL, R.R.; ZACCHINO, S.A.; SILVA, G.L.
Antifungal activity of Heterothalamus alienus metabolites.
Phytother. Res., v.22, n.4, p.524-528, 2008.
PATOCKA, J. Biologically active pentacyclic triterpenes and
their current medicine signification. J. Appl. Biomed., v.1,
n.7-12, p.7-12, 2003.
PEREIRA, E.M.; MACHADO, T.B.; LEAL, I.C.R.; JESUS,
D.M.; DAMASO, C.R.A.; PINTO, A.V.; GIAMBIAGIDEMARVAL, M.; KUSTER, A.V.; NETTO DOS
SANTOS, K.R. Tabebuia avellanedae naphthoquinones:
activity against methicillin-resistant Staphylococcus strains,
cytotoxic activity and in vivo dermal irritability analysis.
Ann. Clin. Microbiol. Antimicrob., 22,.5:5, 2006.
Isolation of flavonoids from Anemopaegma arvense (Vell) Stellf. ex de Souza and their antifungal activity against Trichophyton rubrum
P E R E I R A , A . C . ; O L I V E I R A , D . F. ; S I LVA , G . H . ;
FIGUEIREDO, H.C.P.; CAVALHEIRO, A.J.; CARVALHO,
D.A.; SOUZA, L.P.; CHALFOUN, S.M. Identification
of the antimicrobial substances produced by Solanum
palinacanthum (Solanaceae). An. Acad. Bras. Ciênc., v.80,
n.3, p.427-432, 2008.
PIO CORRÊA, M.; PENNA, L. Dicionário das plantas úteis do
Brasil e das exóticas cultivadas. Rio de Janeiro: Ministério
da Agricultura, 1969. 765 p.
RAHALISON, L.; HAMBURGER, M.; MONOD, M.; FRENK,
E.; HOSTETTMANN, K. Antifungal tests in phytochemical
investigations: comparison of bioautographic methods
using phytopathogenic and human pathogenic fungi. Planta
Med., v.60, n.1, p.41-44, 1994.
ROCHA, A.D.; OLIVEIRA, A.B.; FILHO, J.D.S.; LOMBARDI,
J.A.; BRAGA, F.C. Antifungal constituents of Clytostoma
ramentaceum and Mansoa hirsuta. Phytother. Res., v.18,
n.6, p.463-467, 2004.
RUBINSTEIN, L.V.; SHOEMAKER, R.H.;. PAULL, K.D.;
SIMON, R.M.; TOSINI, S.; SKEHAN, P.; SCUDIERO,
D.A.; MONKS, A.; BOYD, M.R. Comparison of in vitro
anticancer-drug-screening data generated with a tetrazolium
assay versus a protein assay against a diverse panel of
human tumor cell lines. J. Natl. Cancer. Inst., v.82, n.13,
p.1113-1117, 1990.
SEMWAL, D.K.; RAWAT, U.; SEMWAL, R.; SINGH, R.;
KRISHAN, P.; SINGH, M.; SINGH, G.J.P. Chemical
constituents from the leaves of Boehmeria rugulosa with
antidiabetic and antimicrobial activities. J. Asian Nat. Prod.
Res., v.11, n.12, p.1045-1055, 2009.
565
SOUZA, L.K.H.; OLIVEIRA, C.M.A.; FERRI, P.H.; SANTOS,
S.C.; OLIVEIRA JÚNIOR, J.G.; MIRANDA, A.T.B.;
LIÃO, L.M.; SILVA, M.R.R. Antifungal properties of
Brazilian cerrado plants. Braz. J. Microbiol., v.33, n.3,
p.247-249, 2002.
TABANCA, N.; PAWAR, R.S.; FERREIRA, D.; MORAIS, J.P.;
KHAN, S.I.; JOSHI, V.; WEDGE, D.E.; KHAN, I.A. Flavan
3-ol-phenylpropanoid conjugates from Anemopaegma
arvense and their antioxidant activities. Planta Med., v.73,
p.1107-1111, 2007.
TÜREL O. Newer antifungal agents. Expert Rev. Anti. Infect.
Ther. v.9, n.3, p. 325-38, 2011.
WALGREN, R.A.; KARNAKY, K.J.; LINDENMAYER, G.E.;
WALLE, T. Efflux of dietary flavonoid quercetin 4’-betaglucoside across human intestinal Caco-2 cell monolayers
by apical multidrug resistance-associated protein-2. J.
Pharmacol. Exp. Ther., v.294, n.3, p.830-836, 2000.
ZACCHINO, S. Estratégia para a descoberta de novos agentes
antifúngicos, In: YUNES, R.A.; CALIXTO, J.B.(Eds.)
Plantas medicinais sob a ótica da química medicinal
moderna. Chapecó: Argos, 2001. p.435-479.
ZANOLARI, B.; GUILET, D.; MARSTON, A.; QUEIROZ,
E.F.; PAULO, M.Q.; HOSTETTMANN, K. Methylpyrrole
tropane alkaloids from the bark of Erythroxylum
vacciniifolium. J. Nat. Prod., v.68, p.1153-1158, 2005.
Received for publication on 13th November 2012
Accepted for publication on 12th April 2013