Spindola et al. Parasites & Vectors 2014, 7:537
http://www.parasitesandvectors.com/content/7/1/537
RESEARCH
Open Access
Anti-Mayaro virus activity of Cassia australis
extracts (Fabaceae, Leguminosae)
Kassia C W Spindola1*, Naomi K Simas2, Tiago S Salles4, Marcelo D F de Meneses3, Alice Sato5, Davis Ferreira3,
Wanderson Romão6 and Ricardo M Kuster1
Abstract
Background: The arthropod-borne Mayaro virus (MAYV) causes ‘Mayaro fever’, a disease of medical significance,
primarily affecting individuals in permanent contact with forested areas in tropical South America. Studies showed
that the virus could also be transmitted by the mosquito Aedes aegypti. Recently, MAYV has attracted attention due
to its likely urbanization. To date, there are no drugs that can treat this illness.
Methods: Fractions and compounds were obtained by chromatography from leaf extracts of C. australis and
chemically identified as flavonoids and condensed tannins using spectroscopic and spectrometric techniques (UV,
NMR, and ESI-FT-ICR MS). Cytotoxicity of EtOAc, n-BuOH and EtOAc-Pp fractions were measured by the dye-uptake
assay while their antiviral activity was evaluated by a virus yield inhibition assay. Larvicidal activity was measured by
the procedures recommended by the WHO expert committee for determining acute toxicity.
Results: The following group of substances was identified from EtOAc, n-BuOH and EtOAc-Pp fractions: flavones,
flavonols, and their glycosides and condensed tannins. EtOAc and n-BuOH fractions inhibited MAYV production,
respectively, by more than 70% and 85% at 25 μg/mL. EtOAc-Pp fraction inhibited MAYV production by more than
90% at 10 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. This fraction had an excellent
antiviral effect (IC90 = 4.7 ± 0.3 μg/mL), while EtOAc and n-BuOH fractions were less active (IC90 = 89.1 ± 4.4 μg/mL and
IC90 = 40.9 ± 5.7 μg/mL, respectively).
Conclusions: C. australis can be used as a source of compounds with anti-Mayaro virus activity. This is the first report
on the biological activity of C. australis.
Keywords: Cassia australis, Flavonoids, Tannins, Antiviral, MAYV, Larvicidal activity, Aedes aegypti
Background
In Brazil, MAYV is endemic in the Amazon region, but
Mayaro fever outbreaks have occurred in other regions
in Brazil [1,2]. Most arboviruses isolated in the Amazon
region are maintained in nature by different life cycles,
involving different vectors and vertebrate hosts. Oropouche virus, for example, is transmitted to humans in
urban areas by the midges Culicoides paraensis, and vertebrates such as sloths, monkeys and birds play a role in
the maintenance of the virus cycle [3]. Likewise, Mayaro
and yellow fever viruses are transmitted by the mosquito
Haemagogus janthinomys in the jungle, and monkeys are
* Correspondence: kawaldhelm@gmail.com
1
Natural Product Research Institute, Center of Health Sciences, Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil
Full list of author information is available at the end of the article
the main hosts [4]. On the other hand, dengue virus has
a simpler cycle whereby the serotypes are directly transmitted to humans by Aedes aegypti mosquito bites [5]. In
addition, imported MAYV cases in other countries from
tourists who visited the Amazon region have been described [6]. MAYV can also be transmitted by the vectors
Aedes aegypti and A. albopictus, which raises the concern
for urban areas. It is very important to point out that
MAYV is closely related to the Chikungunya virus, also
transmitted by the mosquitoes Aedes aegypti and A. albopictus, which has recently reached Europe and the Americas and is now counting nearly 800 of autochthones
infections in Brazil since the first detected case in this
country in August 2014 [7,8].
Natural products are becoming very attractive because
of their low cytotoxicity, the rapid degradation in the
© 2014 Spindola et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Spindola et al. Parasites & Vectors 2014, 7:537
http://www.parasitesandvectors.com/content/7/1/537
environment, and because of the complexity of the
chemistry in these products, that should limit resistance
and increase the applicability of use, such as vector control studies [9].
The majority of the available antiviral drugs are concentrated in a small number of viruses, such as HIV,
Herpes and Influenza [10]. Nevertheless, research efforts
to explore the potential of natural products as sources of
novel low toxicity and high selectivity antiviral substances have increased lately [11]. Because there are
many approaches for the use of natural products, the
modes of action or the active components they contain
and the metabolic pathways they interact must be studied. This can be accomplished initially by in vitro studies
such as the cell culture approach in this paper.
The genus Cassia (Fabaceae, Leguminosae) comprises
more than 600 species including shrubs, trees and herbs,
distributed in tropical and subtropical regions all over
the world. The separation of the genus Senna from the
genus Cassia has been, and still is, the subject of many
discussions [12]. The species under study was originally
classified as Cassia australis Vellozo and in 1982 by Irwin and Barneby it was transferred to Senna with the
name: Senna australis (Vell.) H.S.Irwin & Barneby.
Afterwards, this species has been renamed Senna appendiculata (Vogel) [13].
In Brazilian ecosystems, particularly in the Atlantic
forest, the genus Senna is widespread, with some species
in the Southeast greatly appreciated for the beauty of its
flowers,and therefore widely used as ornamental plants
[14]. Due to the traditional use, several species, many
already described in the literature, are medicinally used
worldwide [15-20]. Cassia australis is a medium sized
shrub and may reach up to 2.5 meters of diameter. It
occurs at Brazilian coast sandbank, mainly in Rio de
Janeiro, Espirito Santo, Bahia, Sergipe, Alagoas and Pernambuco states [21]. The genus Cassia is known for the
presence of a variety of compounds. Anthraquinones are
the main class of compounds isolated from it [17,22-25].
However, previous investigations led to the isolation of
flavonoids [26-29], piperidine alkaloids [30], stilbenoids
[30] and aliphatic esters [16]. So far, there is no paper
about the phytochemistry and biological activity of
Cassia australis.
For most mosquito-transmitted viruses, there are no
licensed antiviral drugs or vaccines available. MAYV is
an example of an arthropod-borne virus, mainly found
in South America tropical areas, which affects primarily
individuals in permanent contact with forested areas and
causes the Mayaro fever. In this study, EtOAc, n-BuOH
and EtOAc-Pp fractions containing flavonoids and other
classes of phenolics compounds were obtained from the
leaves of Cassia australis Vellozo and investigated for
their in vitro antiviral activity against MAYV replication
Page 2 of 7
in Vero cells and larvicidal activity against Aedes aegypti
larvae.
Methods
Plants, cells and viruses
Cassia australis leaves were collected in December 2008
in Rio de Janeiro State, and identified by Alice Sato.
Voucher specimens (No. 652HUNI) are deposited in the
herbarium of the University of Rio de Janeiro (UNIRIO),
Brazil.
Vero cells (African green monkey kidney, ATCC CCL81) were maintained at 37°C, 5% CO2, in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, USA)
supplemented with 5% fetal bovine serum (Cultilab, BRA),
50 IU/mL of penicillin, and 50 μg/mL of streptomycin
(Sigma-Aldrich, USA). Mayaro virus (ATCC VR-66, lineage
TR 4675) was propagated in Vero cells and viral stocks
kept at −70°C until use.
Aedes aegypti eggs were obtained at the Brazilian Army
Institute of Biology. They were kept in the tray containing tap water at optimal conditions (28 ± 1°C). After
48 hrs of incubation, the eggs were used. The 4th instar
larvae were used in the study.
Extraction, fractionation, and purification for achievement
of fractions and compounds
Air-dried leaves (850 g) were extracted with MeOH:H2O
(8:2) at room temperature by static maceration over
10 days. After concentration under reduced pressure, the
methanol extract (25 g) was suspended in MeOH:H2O
(9:1), and partitioned with hexane. After removal of the
methanol from the defatted extract, the remaining aqueous solution was partitioned successively with CH2Cl2,
EtOAc, and n-BuOH. Two grams of the EtOAc extract,
soluble in H2O:MeOH (9:1), were applied to a XAD-2
column (procedure A), and chromatographed in a stepwise gradient with H2O:MeOH (10:0/0:10). 150 ml of
each combination of solvents were eluted through the
column and fractions of 150 ml were collected. The fraction obtained with 100% of water was named EtOAc-Pp.
From H2O:MeOH (50:50) fraction, after chromatography
on Sephadex LH-20 (MeOH:H2O – 1:1, mobile phase) –
(procedure B), the flavone tricetin-4′-methoxy-3′-β-glucoside [31] was obtained. The same procedures A and B
were applied to n-BuOH extract to obtain the flavone
vicenin-2 [32].
Reverse-phase HPLC-DAD-UV, TLC, NMR and ESI-(−)-FTICR MS analyses
HPLC-DAD-UV (High Performance Liquid Chromatography with Diode Array Detector), TLC (Thin Layer Chromatography), NMR (Nuclear Magnetic Resonance) and
ESI(−)-FT-ICR MS (Electrospray ionization with Fourier
Transform Ion Cyclotron Resonance Mass Spectrometry)
Spindola et al. Parasites & Vectors 2014, 7:537
http://www.parasitesandvectors.com/content/7/1/537
were used to analyze the chemical composition of EtOAc,
n-BuOH, EtOAc-Pp fractions and compounds isolated
from them.
The mobile phase for HPLC-DAD analysis consisted of
solvent (A) 1% phosphoric acid in water and solvent (B)
1% phosphoric acid in methanol and was used under the
following gradient: 5% of B to 70% of B in A for 55 min.
The flow rate was 1 mL/min and the injection volume
20 μL. The UV–vis spectra were recorded from 210 to
400 nm and the detector focused on 254 and 365 nm.
TLC was performed on silica gel plates 60 F254 (Merck,
20×20 cm, 0.5 mm thickness), using n-butanol-wateracetic acid (4:5:1) and chloroform-methanol (9:1) as mobile phases. After elution, TLC plates were observed
under 254 nm UV light and then sprayed successively
with solutions of NP (2-aminoethyldiphenylborinate 1%
in methanol) and PEG-4000 (polyethylene glycol 5% in
ethanol) (both by Sigma-Aldrich, USA) before examination under 365 nm UV light. Cassia australis extracts
and pure compounds were analyzed by an ultra-high
resolution and accuracy mass spectrometer (model 9.4 T
Solarix, Bruker Daltonics, Bremen, Germany). Briefly, the
samples were dissolved in acetonitrile/ammonium hydroxide (99.9/0.1 v/v %) mixture to a final concentration
of 10 μg mL−1. The mass spectrometer was set to operate
in negative ion mode, ESI(−), over a mass range of m/z
200–2000. The parameters of the ESI(−) source were as
follows: nebulizer gas pressure of 0.5-1.0 bar, capillary
voltage of 3–3.5 kV, and transfer capillary temperature of
250°C. The spectrum was processed using the Compass
Data Analysis software package (Bruker Daltonics, Bremen, Germany). A resolving power, m/Δm50% ≈ 500 000,
in which Δm50% is the full peak width at half-maximum
peak height, of m/z ≈ 400 and a mass accuracy of <1 ppm
provided the unambiguous molecular formula assignments
for singly charged molecular ions. Elemental compositions
of the compounds were determined by measuring the
m/z values. NMR analysis (1H-NMR, COSY, HSQC and
HMBC) were recorded on Varian spectrometer MR-400
operating at 400 MHz. The samples were solubilized in
DMSO-d6 and TMS was used as external standard.
Final compound analysis was performed by NMR
(DMSO-d6), FT-ICR-ESI-MS, UV spectral analysis, and
by comparison with literature values.
Cytotoxicity assay
Cytotoxicity analysis was performed using the dye-uptake
method modified from Borenfreund and Puerner [33].
Vero cells grown in 96-well microplates were treated
with culture media containing different concentrations of
the substances. After 24 hours, the medium was replaced
by a solution of 50 μg/mL neutral red, the cells were incubated for 3 hours at 37°C, 5% CO2 and then fixed and
extracted with 20% formaldehyde and 50% methanol, 1%
Page 3 of 7
acetic acid. Absorbance was read at 490 nm, using a
spectrophotometer, to detect neutral red incorporation
by living cells. Absorbance results were used to calculate,
by regression analysis, the concentrations of the tested
substances capable of reducing cell viability by 50% and
90% relative to controls (CC50 and CC90, respectively).
Antiviral activity assay
For the antiviral activity, confluent Vero cell monolayers
grown in 24-well plates were infected with MAYV
(multiplicity of infection = 0.1) for 1 hour, then rinsed
with PBS and treated for 24 hours (at 37°C and 5% CO2)
with different concentrations (0–100 μg/ml) of the substances diluted in culture medium. After treatment, culture supernatants were recovered and used for titration of
extracellular infectious virus particles. Ribavirin (SigmaAldrich, USA) was used as positive control for MAYV
replication inhibition. For each substance or extract, IC50
and IC90 values were calculated and used to obtain a selectivity index (SI), expressed as the ratio CC50/IC50, and
to estimate relative potency (RP) as the ratio between ribavirin (reference substance) IC90 and the tested substance’s
IC90. Results were presented as mean inhibitory/cytotoxic
concentration ± SD, and t-tests were used to evaluate the
statistical significance of treatments relative to controls. Pvalues <0.05 were considered statistically significant.
Virus yield assay
For virus titration, confluent cell monolayers in 24-well
plates were infected with serial dilutions of recovered supernatants from the assays for 1 hour at 37°C, 5% CO2.
After inoculum removal, cells were rinsed with PBS and
the monolayer was incubated with fresh medium with
2% carboxymethylcellulose (Sigma-Aldrich, USA) for
48 hours at 37°C, 5% CO2. Finally, cells were fixed with
20% formaldehyde and stained with 0.5% crystal violet in
20% ethanol, and viral plaques were counted.
Results
Flavonoid aglycones, flavonoid glycosides and tannins
were found in extracts of C. australis leaves
HPLC-DAD-UV analysis of EtOAc, n-BuOH and EtOAcPp fractions indicated different flavonoid profiles. Flavonoid aglycones and flavonoid monoglycosides with retention time (RT) greater than 40 min., predominated in
EtOAc fraction (Figure 1A and B), while flavonoid diglycosides, more polar compounds (30 min. < RT <40 min)
(Figure 1C and D), predominated in n-BuOH fraction.
HPLC-DAD analysis of EtOAc-Pp showed more polar
compounds (20 min < RT <50 min), like tannins and flavonoid diglycosides (Figure 1E and F).
All the ESI(−)FT-ICR MS analyses (Table 1) were made
in negative ion mode. The structures were suggested
based on their ultra-high resolution and accuracy mass.
Spindola et al. Parasites & Vectors 2014, 7:537
http://www.parasitesandvectors.com/content/7/1/537
Page 4 of 7
Figure 1 HPLC-DAD analysis detects flavonoids as major compounds in fractions from C. australis. (A) EtOAc fraction from the leaves of C.
australis showing flavonoids with RT >40 min. (B) UV spectra of flavonoids in the EtOAc fraction. (C) n-BuOH fraction from the leaves of C.
australis showing flavonoids with RT <40 min. (D) UV spectra of flavonoids in the n-BuOH fraction. (E) EtOAc-Pp fraction from the leaves of C.
australis showing flavonoids and tannins with 20 min < RT <50 min. (F) UV spectra of flavonoids and tannins in the EtOAc-Pp fraction.
Molecular formula (M) and double bond equivalent
(DBE) were utilized to propose the presence of flavonols,
flavones, and their glycosides and condensed tannins
(dimer and trimer of flavan-3-ol). EtOAc fraction showed
presence of flavonols (m/z 285, 301 and 315), condensed
tannins (m/z 529, 545 and 561), flavonol glycosides (m/z
447 and 463) and flavone glycoside (m/z 477). All were
detected in deprotonated form, [M – H]− ion. From the
n-BuOH fraction were proposed flavonol glycosides (m/z
447, 463, and 609) and flavone glycosides (m/z 477 and
593) while the EtOAc-Pp showed the presence of flavonols and flavanones (m/z 285, 289, 301 and 315), flavonol
glycosides (m/z 447 and 463), flavone glycoside (m/z 477)
and condensed tannins (m/z 513, 529, 545 and 769). The
chemical structure of compounds identified is proposed
in Table 1.
Spindola et al. Parasites & Vectors 2014, 7:537
http://www.parasitesandvectors.com/content/7/1/537
Page 5 of 7
Table 1 Proposed compounds from ESI(−)FT-ICR MS analyzes
Suggested
compounds
Class of natural
product
EtOAc
fraction
n-BuOH
fraction
EtOAc-Pp
m/ztheoretical
m/zmeasured
Molecular
formula (M)
Error
(ppm)
DBE
Kaempferol
Flavonol
+
-
+
285.04046
285.04042
C15H10O6
0.41
11
-
+
301.03538
301.03538
C15H10O7
0.25
11
+
315.05103
315.05098
C16H12O7
0.33
11
Quercetin
Flavonol
+
Rhamnetin/isorhamnetin
Flavonol
+
Quercetin pentoside
Flavonol
Monoglycoside
+
+
+
447.09329
447.10361
C21H20O11
0.38
12
Quercetin hexoside
Flavonol
Monoglycoside
+
+
+
463.08820
463.08801
C21H20O12
0.38
12
Tricetin-4′-methoxy3′-â-D-glucoside1
Flavone
Monoglycoside
+
+
+
477.10385
477.10361
C22H22O12
−0.12
12
-
Flavan-3-ol dimer
+
-
+
529.15041
529.15024
C30H26O9
0.51
18
-
Flavan-3-ol dimer
+
-
+
545.14532
545.14519
C30H26O10
-
Flavan-3-ol dimer
+
-
-
561.14024
561.08601
C30H26O11
0.45
18
Flavan-3-ol dimer
+
-
-
591.11441
591.09653
C30H24O13
0.40
19
Vicenin-2 kaempferol
diglycoside1
Flavone diglycoside
-
+
+
593.15120
593.15104
C27H30O15
−0.08
13
Quercetin dihexoside
Flavonol diglycoside
-
+
+
609.14611
609.14591
C27H30O16
0.06
13
-
Flavan-3-ol trimer
-
-
+
769.22905
769.22868
C45H38O12
0.08
27
Flavan-3-ol trimer
-
-
+
785.22397
785.22349
C45H38O13
0.08
27
18
1
Purified compounds identified by NMR, DBE - double bond equivalent, + detected, − not detected, Mass error (ppm) = [(m/zmeasured – m/ztheoretical)/m/ztheoretical]*106.
Cytotoxicity and antiviral activity
EtOAc, n-BuOH and EtOAc-Pp fractions inhibited
MAYV replication in Vero cells. EtOAc and n-BuOH
fractions inhibited MAYV production, respectively, by
more than 70% and 85% at 25 μg/mL. EtOAc-Pp fraction inhibited MAYV production by more than 90% at
10 μg/mL. The antiviral ribavirin were much less potent
inhibitors of MAYV replication, with IC90 values above
100 μg/mL (Table 2 and Figure 2).
The Selectivity Index (SI) and the Relative Potency are
important indexes that can represent how suitable a substance is for further studies. EtOAc-Pp had the highest
SI and 16 times higher Relative Potency than ribavirin.
Although this is very relevant data, further studies need
to be accomplished in order to address the use of these
compounds as antivirals.
Discussion
Since the three fractions tested have phenolic derivatives
such as flavonoids and tannins as the major compounds,
their antiviral activity can be attributed to the presence
of them. EtOAc has flavonoid aglycones and flavonoid
monoglycosides as major phenolics compounds, while in
n-BuOH flavonoid diglycosides are the major ones. For
the fraction EtOAc-Pp, beyond flavonoid mono and
diglycosides, condensed tannins are present.
The presence of condensed tannins (flavan-3-ol, dimers
and trimers) may be one of the factors responsible for
antiviral activity. Tannins are known for their property of
complexing with proteins, including lipo- and glycoproteins. Previous studies have reported that the binding of
polymeric condensed tannins with protein was stronger
than that of low molecular weight oligomers and
Table 2 Cytotoxicity and anti-MAYV activity of EtOAc, n-BuOH and EtOAc-Pp fractions
Substance
CC50 (μg/mL)a
CC90 (μg/mL)a
IC50 (μg/mL)b
IC90 (μg/mL)b
SIc
RPd
n-BuOH
2614 ± 366
821 ± 115
7,1 ± 1,0
40,9 ± 5,7
20
10
EtOAc
457,7 ± 9,5
176,1 ± 3,5
8,2 ± 0,2
89,1 ± 4,4
2
1
EtOAc-Pp
324,1 ± 6,5
154,7 ± 3,1
2,5 ± 0,1
4,7 ± 0,3
33
16,5
ribavirin
523,1 ± 42,5
215,4 ± 6,2
62,3 ± 4,4
112,4 ± 8,2
2
nd
a
50% and 90% cytotoxic concentration.
b
50% and 90% inhibitory concentration of viral replication.
c
Selectivity Index = standard IC90/substance IC90.
d
Relative Potency = ratio between ribavirin (reference substance) IC90 and the tested substance’s IC90.
nd – Not determined.
Spindola et al. Parasites & Vectors 2014, 7:537
http://www.parasitesandvectors.com/content/7/1/537
Figure 2 Anti-MAYV activity of different fractions from C.
australis. The anti-MAYV activity of EtOAc, n-BuOH and EtOAc-Pp
fractions from C. australis was evaluated by treating MAYV-infected
cells with 0–100 μg/ml of these fractions for 24 h, and then staining
for viral plaque counting. The graph shows the results from three
independent experiments. Data are presented as mean% virus yield
(compared to untreated controls) ± SD.
monomers. It is believed that hydrogen bonding is an
important factor in the binding of condensed tannins
gelatin [34].
We know that the effect of the astringent polyphenols,
including flavonoids and tannins, is dependent on the
affinity of these substances with the protein and due to
this is greatly influenced by the composition of each protein, as well as their hydrophilicity, therefore different viruses react polyphenols to different manner. In addition,
previous works suggest the tannin-like proanthocyanidins may link the protein covalently [35].
Takechi et al. [36] concluded in his work more highly
condensed tannins have a greater antiviral activity, although the galloyl group contributes more to activity
than the degree of condensation. It is known that the
presence of o-dihydroxyphenyl group is related to the
formation of protein-polyphenol complex. Moreover, it is
thought that tannins interact with the protein particles
from the surface of the host cell of the virus, as well as to
the viral envelope [36].
In a previous study, Ferreira et al. found that the flavonoids quercetin group had a strong antiviral activity
against MAYV, suggesting that this virus has proteins
that are able to interact with phenolic substances from
the group of flavonoid envelope [37].
Condensed tannins have been tested for their antiviral
activity and exhibited antiviral activity against respiratory
syncytial virus (RSV), influenza A virus (FLU-A) and
parainfluenza virus (PIV). It also inhibited the growth of
herpes viruses types 1 and 2 (HSV-1, HSV-2) and hepatitis A and B viruses. The proposed mechanism of action
was from its connection with the viral envelope proteins,
inhibiting the binding and penetration of the virus in the
plasma membrane [38].
Yang et al. [39] compared several polyphenols derived
from tea against influenza A and B and concluded that
Page 6 of 7
condensed tannins were the most active against the influenza A virus than monomeric polyphenols: theaflavin,
procyanidin B-2 and procyanidin B-2. To evaluate the
structure-activity relationship, they concluded that the
dimers as theaflavin and procyanidin B-2, are more active
against influenza A and B than the catechin monomers,
such as (−)-EC and (±)-catechin and that galoyl group
present in theaflavindigallate and procyanidin B-2 digallate not help on antiviral effect, probably due to the steric
effect [39].
Since the trimers of tannins are only present in
EtOAc-Pp, we correlate this to the greater antiviral activity of this fraction. Previous studies have shown that
the degree of condensation is an important factor
[36,39], being more highly condensed tannins more
active; we believe that these substances are responsible
for anti-viral activity.
Conclusions
Our results show that C. australis is a valuable source of
phenolics derivates with antiviral activity against the
arbovirus MAYV. Although antiviral activity of tannins
and other phenolics derivates are very common, this is
the first report of anti-MAYV activity for these substances and this species. Our data are an important step
in the evaluation of natural products as sources of novel
drugs to be used in combination therapy, to circumvent
drug resistance, or to replace currently used antivirals
with unwanted cytotoxic effects.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RMK and DF conceived and designed the study. AS collected plant material
with KCWS. KCWS performed all phytochemical experiments and wrote the
initial draft of the manuscript. TSS performed cytotoxicity and viral yield
inhibition assays. WR made the ESI(−)FT-ICR MS analyses. MDFM, RC and NKS
revised the data and carried out statistical analyses. KCWS, RMK and DF
provided invaluable discussions on the chemical data and antiviral chemistry.
All authors read and approved the final manuscript.
Acknowledgements
The authors would like to thank CNPq and INBEB for financial support.
Author details
1
Natural Product Research Institute, Center of Health Sciences, Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil. 2Natural Product and Food
Department. Center of Health Sciences, Federal University of Rio de Janeiro,
Rio de Janeiro, Brazil. 3Microbiology Institute, Virology Department, Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil. 4Chemistry Institute,
Biochemistry Department, Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil. 5Botanical Department. Center of Biological and Health
Sciences, Federal University of the State of Rio de Janeiro, Rio de Janeiro,
Brazil. 6Petroleomic and Forensic Laboratory, Department of Chemistry,
Federal University of Espírito Santo, 29075-910 Vitória, ES, Brazil.
Received: 23 July 2014 Accepted: 12 November 2014
Spindola et al. Parasites & Vectors 2014, 7:537
http://www.parasitesandvectors.com/content/7/1/537
References
1. Vasconcelos PFC, Rosa APAT, Pinheiro FP, Shope RE, Rosa JFST, Rodrigues
SG, Dégallier N, Travassos da Rosa ES: Arboviruses pathogenic for man in
Brazil. In An overview of arbovirology in Brazil and neighbouring countries.
Edited by Rosa APAT, Vasconcelos PFC, Rosa JFST. Belém: Instituto Evandro
Chagas; 1998:72–99.
2. Coimbra TLM, Santos CLS, Suzuki A, Petrella SMC, Bisordi I, Nagamori AH,
Marti AT, Santos RN, Fialho DM, Lavigne S, Buzzar MR, Rocco IM: Mayaro
virus: imported cases of human infection in São Paulo state, Brazil. Rev
Inst Med Trop Sao Paulo 2007, 4:221–224.
3. Pinheiro FP, Travassos Da Rosa APA, Travassos Da Rosa JFS, Ishak R, Freitas
RB, Gomes MLC, Le Duc JW, Oliva OFP: Oropouche virus. I. A review of
clinical, epidemiological, and ecological findings. Am J Trop Med Hyg
1981, 30:149–160.
4. Pinheiro FP, Freitas RB, Rosa JFT, Gabbay YB, Mello WA, LeDuc JW: An
outbreak of Mayaro virus disease in Belterra, Brazil. I. Clinical and
virological findings. Am J Trop Med Hyg 1981, 30:674–681.
5. Vasconcelos PF, Travassos da Rosa AP, Rodrigues SG, Travassos da Rosa ES,
Dégallier N, Travassos da Rosa JF: Inadequate management of natural
ecosystem in the Brazilian Amazon region results in the emergence and
reemergence of arboviruses. Cad Saude Publica 2001, 17:155–164.
6. Receveur MC, Grandadam M, Pistone T, Malvy D: Infection with Mayaro
virus in a French traveller returning from the Amazon region, Brazil. Euro
Surveill 2010, 18:1–4.
7. Brasil, Ministério da Saúde. Ministério da Saúde intensifica medidas de
controle da febre Chikungunya. Accessed 11.03.2014. [http://portalsaude.
saude.gov.br/index.php/o-ministerio/principal/secretarias/svs/noticias-svs/
14667-ministerio-da-saude-intensifica-medidas-de-controle-da-febrechikungunya]
8. Brasil, Ministério da Saúde. Saúde atualiza situação do vírus Chikungunya.
Accessed 11.03.2014. [http://u.saude.gov.br/d0ya9751]
9. George DR, Finn RD, Graham KM, Sparagano OAE: Present and future
potential of plant-derived products to control arthropods of veterinary
and medical significance. Parasit Vectors 2014, 7:1–12.
10. Yasuhara-Bell J, Yuanan L: Marine compounds and their antiviral activities.
Antiviral Res 2010, 86:231–240.
11. Newman DJ, Cragg GM: Natural products as sources of new drugs over
the 30 years from 1981 to 2010. J Nat Prod 2012, 75:311–335.
12. Irwin HS, Barneby RC: The American Cassiinae: a synoptical revision of
Leguminosae tribe Cassieae subtribe Cassiinae in the New World. Mem
New York Bot Gard 1982, 35:1.
13. Wiersema JH: A new name for a Brazilian Senna (Leguminosae:
Caesalpinoideae). Taxon 1989, 38:652–652.
14. Viegas C Jr, Rezende A, Silva DHS, Castro-Gambôa I, Bolzani VS, Barreiro EJ,
Miranda ALP, Moreira MSA, Young MCM: Aspectos químicos, biológicos e
etnofarmacológicos do gênero Cassia. Quim Nova 2006, 29:1279–1286.
15. Nsonde Ntandou GF, Banzouzi JT, Mbatchi B, Elion-Itou RDG, Etou-Ossib
AW, Ramos S, Benoit-Vical F, Abena AA, Ouamba JM: Analgesic and
anti-inflammatory effects of Cassia siamea Lam. Stem bark extracts.
J Ethnopharmacol 2010, 127:108–111.
16. Guzmán E, Pérez C, Zavala MA, Acosta-Viana KY, Pérez S: Antiprotozoal
activity of (8-hydroxymethylen)-trieicosanyl acetate isolated from Senna
villosa. Phytomedicine 2008, 15:892–895.
17. Lombardo M, Kiyota S, Kaneko TM: Aspectos étnicos, biológicos e
químicos de Senna occidentalis (Fabaceae). Rev Ciênc Farm Básica Apl
2009, 30:1–9.
18. Longuefosse JL, Nossin EJ: Medical ethnobotany survey in Martinique.
J Ethnopharmacol 1996, 53:117–142.
19. Franco EAP, Barros RFM: Uso e diversidade de plantas medicinais no
Quilombo Olho D’água dos Pires, Esperantina, Piauí. Rev Bras Plantas Med
2006, 8:78–88.
20. Jones L, Bartholomew B, Latif Z, Sarker SD, Nash RJ: Constituents of Cassia
laevigata. Fitoterapia 2000, 71:580–583.
21. Silva ALG, Ormond WT, Pinheiro MCB: Biologia floral e reprodutiva de
Senna australis (Vell.) Irwin & Barneby (Fabaceae, Caesalpinioideae).
Bol Mus Nac NS Bot 2002, 121:1–11.
22. dos Santos RN, Silva MGV, Braz FR: Constituintes químicos do caule de
Senna reticulata Willd. (Leguminoseae). Quim Nova 2008, 31:1979–1981.
23. Barbosa FG, Oliveira MCF, Braz-Filho R, Silveira ER: Anthraquinones and
naphthopyrones from Senna rugosa. Biochem Syst Ecol 2004, 32:363–365.
Page 7 of 7
24. Hennebelle T, Weniger B, Joseph H, Sahpaz S, Bailleul F: Senna alata.
Fitoterapia 2009, 80:385–393.
25. Kanno M, Shibano T, Takido M, Kitanaka S: Antiallergic agent from natural
sources. 2. Structures and leukotriene release-inhibitory effect of
torososide B and torosachrysone 8-O-6″-malonyl beta-gentiobioside
from Cassia torosa Cav. Chem Pharm Bull 1999, 47:915–918.
26. Dehmlow EV, van Ree T, Guntenhöner M: 2,4-trans-,7 4′-dihydroxy-4methoxyflavan from Cassia abbreviata. Phytochemistry 1998, 49:1805–1806.
27. Coetzeea J, Mcitekaa L, Malana E, Ferreira D: Structure and synthesis of the
first procassinidin dimers based on epicatechin, and gallo- and
epigallo-catechin. Phytochemistry 2000, 53:795–804.
28. Hatano T, Mizuta S, Ito H, Yoshida T: C-Glycosidic flavonoids from Cassia
occidentalis. Phytochemistry 1999, 52:1379–1383.
29. Hatano T, Yamashita A, Hashimoto T, Ito H, Kubo N, Yoshiyama M, Shimura
S, Itoh Y, Okuda T, Yoshida T: Flavan dimers with lipase inhibitory activity
from Cassia nomame. Phytochemistry 1997, 46:893–900.
30. Viegas C Jr, Bolzani VS, Furlan M, Barreiro EJ, Young MCM, Tomazela D,
Eberlin MN: Further bioactive piperidine alkaloids from the flowers and
green fruits of Cassia spectabilis. J Nat Prod 2004, 67:908–910.
31. Yuping T, Weiping Z, Fengchang L, Yanfang L, Jinghua W: Flavone
Glycosides from the Leaves of Ginkgo biloba. J Chin Pharmaceut Sci 2009,
9:119–121.
32. Xie C, Veitch NC, Houghton PJ, Simmonds MSJ: Flavone C-Glycosides from
Viola yedoensis Makino. Chem Pharm Bull 2003, 51:1204–1207.
33. Borenfreund E, Puerner JA: Toxicity determined in vitro by morphological
alterations and neutral red absorption. Toxicol Lett 1985, 24:119–124.
34. Frazier RA, Deaville ER, Green RJ, Stringano E, Willoughby I, Plant J,
Mueller-Harvey I: Interactions of tea tannins and condensed tannins with
proteins. J Pharm Biomed Anal 2010, 51:490–495.
35. Gescher K, Kühn J, Lorentzen E, Hafezi W, Derksen A, Deters A, Hensel A:
Proanthocyanidin-enriched extract from Myrothamnus flabellifolia Welw.
exerts antiviral activity against herpes simplex virus type 1 by inhibition
of viral adsorption and penetration. J Ethnopharmacol 2011, 24:468–474.
36. Takechi M, Tanaka Y, Takehara M, Nonaka G, Nishioka I: Structure and
antiherpetic activity among the Tannins. Phytochemistry 1985, 24:2245–2250.
37. dos Santos AE, Kuster RM, Yamamoto KA, Salles TS, Campos R, de Meneses
MDF, Soares MR, Ferreira D: Quercetin and quercetin 3-Oglycosides from
Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity. Parasit
Vectors 2014, 7:130.
38. De Bruyne T, Pieters L, Deelstra H, Vlietinck A: Condensed vegetable
tannins: Biodiversity in structure and biological activities. Biochem Sys
Ecol 1999, 27:445–459.
39. Yang ZF, Bai LP, Huang WB, Li XZ, Zhao SS, Zhong NS, Jiang ZH:
Comparison of in vitro antiviral activity of tea polyphenols against
influenza A and B viruses and structure–activity relationship analysis.
Fitoterapia 2014, 93:47–53.
doi:10.1186/s13071-014-0537-z
Cite this article as: Spindola et al.: Anti-Mayaro virus activity of Cassia
australis extracts (Fabaceae, Leguminosae). Parasites & Vectors 2014 7:537.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit