South African Journal of Botany 110 (2017) 251–257

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South African Journal of Botany

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Phenolic composition and antioxidant, anticholinesterase and

antibiotic-modulating antifungal activities of Guazuma ulmifolia Lam.
(Malvaceae) ethanol extract
S.M. Morais a,b, J.T. Calixto-Júnior a,b, L.M. Ribeiro a, H.A. Sousa a, A.A.S. Silva c, F.G. Figueiredo d, E.F.F. Matias d,
A.A. Boligon e, M.L. Athayde e, M.F.B. Morais-Braga f, H.D.M. Coutinho f,⁎
a
Laboratório de Produtos Naturais, Universidade Estadual do Ceará – UECE, 60740-000 Fortaleza, CE, Brazil
b
Rede Nordeste de Biotecnologia – RENORBIO, 60740-000 Fortaleza, CE, Brazil
c
Doutorado em Farmacologia, Universidade Federal do Ceará, Campus do Pici, Bloco 710, CEP: 60455-900, Brazil
d
Faculdade Leão Sampaio (FALS), Avenida Dr. Leão Sampaio, Km 3, Lagoa Seca, CEP: 63180-000 Juazeiro do Norte, CE, Brazil
e
Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Prédio 26, Santa Maria, RS CEP: 97105-900, Brazil
f
Laboratório de Microbiologia e Biologia Molecular, Universidade Regional do Cariri – URCA, CEP: 63105-000 Crato, CE, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Tea from the bark and leaves of Guazuma ulmifolia Lam. (Malvaceae) are used in several countries in South and
Received 4 March 2016 Central America in cases of gastrointestinal and skin problems, among other diseases. The aim of this study was to
Received in revised form 31 July 2016 characterize the chemical composition and its antioxidant, anticholinesterase and antifungal activity to correlate
Accepted 12 August 2016
these with the popular use of this plant. The antioxidant activity was evaluated by DPPH method and the anticho-
Available online 28 September 2016
linesterase action by inhibiting the acetylcholinesterase enzyme, with an adaptation to thin layer chromatogra-
Edited by J. van Staden phy. In the antifungal assay, the minimum inhibitory concentration (MIC) was determined by the microdilution
broth method at concentrations ranging from 1024 to 1 μg/mL, moreover a modulated test on the antibiotic flu-
Keywords: conazole was performed at concentrations from 1024 to 8 μg/mL. Assays were performed in triplicates and plate
Anti-Candida activity readings were performed in an ELISA spectrophotometer. The ethanol extract showed antioxidant activity (EC50:
Acetylcholinesterase 119.85 ± 2.42 μg/mL), significant anticholinesterase activity (growth inhibition zone of 1.0 cm, near the physo-
DPPH stigmine standard), and low antifungal activity was observed against standard strains of Candida. Nevertheless,
Brazilian plants the extract showed a significant fluconazole modulatory effect, with potentiation of the antifungal action against
Candida tropicalis. The chemical study of the ethanol extract from Guazuma ulmifolia leaves revealed the presence
of flavonoids and phenolic acids with recognized antioxidant potential. The antioxidant activity combined with
anticholinesterase potential corroborates, in part, with the use of G. ulmifolia in the treatment of gastrointestinal
disorders.
© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
Guazuma ulmifolia Lam. (Malvaceae), commonly known as
mutamba, has been popularly used as a natural medicine in almost all
places where it occurs. Tea from the bark and leaves of mutamba are
used in many South and Central America countries in cases of gastroin-
testinal problems, kidney disorders, alopecia, cough, fever and skin
problems (Ramirez et al., 1988; Caceres et al., 1990; Rutter, 1990). Sev-
eral authors have evaluated different activities in mutamba ethanol ex-
tracts, among them, its antifungal activity (Caceres et al., 1987; Navarro
et al., 1996).
⁎ Corresponding author at: Universidade Regional do Cariri – URCA, Av. Cel. Antonio
Luis, 1161, Pimenta, Crato, CE, Brazil.
E-mail address: hdmcoutinho@gmail.com (H.D.M. Coutinho).
Many types of fungi can thrive and multiply on the surface of the
body, and the most common fungi which cause skin infections are called
Candida. There are many studies which have shown plant extracts with
anticandidal action (Höfling et al., 2010) and modulatory activity in as-
sociation with antifungal drugs, increasing its activity (Calixto Júnior
et al., 2015a). The limited arsenal of synthetic antifungal agents and
the emergence of resistant Candida strains have prompted researchers
towards the investigation of naturally occurring compounds or their
semisynthetic derivatives in order to propose new innovative hit com-
pounds or new antifungal combinations endowed with reduced toxicity
(Carradori et al., 2016).
Falé et al. (2013) reported that leaf infusions of some plants from
Portugal may be effective in the treatment of digestive problems due
to their content of acetylcholinesterase inhibitors and antioxidant com-
pounds such as phenolic acids (chlorogenic acid and cynarin) and

http://dx.doi.org/10.1016/j.sajb.2016.08.003
0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
252 S.M. Morais et al. / South African Journal of Botany 110 (2017) 251–257
glycosylated flavonoid derivatives, and did not show any toxicity under
the concentrations tested. Acetylcholinesterase (AChE) inhibitors can
be used to increase gastrointestinal motility since acetylcholine is the
major excitatory neurotransmitter responsible for peristaltic contrac-
tions (Holzer and Maggi, 1994). Conditions that may be associated
with gastrointestinal motility disturbances and treated with AChE
inhibitors include dysphagia, gastric stasis achalasia, abdominal pain,
paralytic ileus, vomiting and constipation (Holzer and Maggi, 1994).
Some gastrointestinal disturbances are associated with inflamma-
tion, such as inflammatory bowel diseases. The inflammation in these
cases is related to free radicals, such as reactive oxygen and nitrogen
species (ROS and RNS), and therefore the use of antioxidant com-
pounds, namely radical scavengers, may be helpful (Zhu and Li, 2012).
Research has demonstrated a direct protective role of dietary antioxi-
dants on intestinal mucosa through local antioxidant and anti-
inflammatory activities (Gálvez et al., 2001).
The objective of this work was to investigate the antifungal effect of
the ethanol extract from Guazuma ulmifolia leaves in combination with
a commercial drug against clinical isolates of multiresistant Candida
strains, as well as evaluating its scavenging activity of free radicals, an-
ticholinesterase activity and the phytochemical analysis of the ethanol
extracts in order to correlate these to the activities assigned to its popu-
lar use.
2. Material and methods
2.1. Chemicals, botanical material collection and ethanol extract
preparation
G. ulmifolia leaves were collected on November 2nd, 2013 (dry sea-
son), around 17:00 h, in the cerrado section of the Caatinga area, next
Fig. 1. Collection area. Lavras da Mangabeira City, Ceará State, Brazil.
to Lavras da Mangabeira city, located in a central mesoregion in south-
ern Ceará, Northeast Brazil.
The area is characterized as a relictual vegetation (Figueiredo and
Fernandes, 1987). The patch of savanna in question is located in the
hills with an altitude of 280 to 600 m, at the following coordinates:
(6° 72′ 2432′ S and 38° 97′ 7396′ W) (Fig. 1).
The botanical material was collected and deposited in the Prisco
Bezerra Herbarium of the Federal University of Ceará, under n° 54.743.
The plant material was dried at 60 °C, weighed and macerated in etha-
nol for seven days at room temperature. Afterwards, the ethanol extract
was filtered and concentrated by removing all the solvent on a rotary
evaporator under reduced pressure (40 rpm at 60 °C) and then in a
water bath at 60 °C, obtaining the crude extracts. Dry weight and yield
of the crude ethanol extracts of G. ulmifolia were: Dry matter: 590 g,
crude ethanol extract: 58.61 g, ethanol extract yields: 9.87%.
2.2. Phytochemical analysis
2.2.1. Quantification of flavonoids and total phenolics
Ethanol extracts were assayed for flavonoids according to the meth-
od proposed by Funari and Ferro (2006). A stock solution was prepared
by dissolving 1.2 mg of the extract in 10 mL of 70% methanol in a round-
bottom flask. A volume of 2 mL of this solution was transferred to anoth-
er 10 mL flask. The reaction was initiated by the addition of 1 mL of 15%
aluminum chloride (AlCl3), and the volume was brought to 10 mL with
70% methanol. The reaction mixture was allowed to stand for 30 min,
and the absorbance was read at 425 nm. This test was performed in
triplicate.
Total phenolic content was determined by a spectrophotometric
method using the Folin-Ciocalteu reagent and gallic acid as the refer-
ence standard (Sousa et al., 2007). Ethanol extracts (7.5 mg) were
 S.M. Morais et al. / South African Journal of Botany 110 (2017) 251–257
dissolved in methanol and transferred to a 25 mL volumetric flask and
the final volume was completed with methanol. An aliquot of 100 μL
of the diluted ethanol extract was shaken with 500 μL of Folin-
Ciocalteu reagent and 6 mL of distilled water for 1 min; afterwards,
2 mL of 15% Na2CO3 were added to the mixture and stirred for 30 s.
Finally, the solution was brought to 10 mL with distilled water.
After a 2 h incubation, the absorbance of the samples were measured
at 750 nm.
2.2.2. Chemical, apparatus and general procedures for HPLC analysis
All chemical were of analytical grade. Methanol, acetic acid,
chlorogenic acid and caffeic acid were purchased from Merck (Darm-
stadt, Germany). Catechin, quercetin, quercitrin, rutin and luteolin
were acquired from Sigma Chemical Co. (St. Louis, MO, USA). High per-
formance liquid chromatography (HPLC-DAD) was performed with the
HPLC system (Shimadzu, Kyoto, Japan), a Prominence Auto Sampler
(SIL-20A) equipped with Shimadzu LC-20AT reciprocating pumps con-
nected to the degasser DGU 20A5 with the integrator CBM 20A, UV–
VIS detector DAD (diode) SPD-M20A and Software LC solution 1.22 SP1.
2.2.3. Quantification of phenolic constituents by HPLC-DAD
Reverse phase chromatographic analyses were carried out under
gradient conditions using C18 column (4.6 mm × 250 mm) packed
with 5 μm diameter particles; the mobile phase was water containing
2% acetic acid (A) and methanol (B), and the composition gradient
was: 5% of B for 2 min and changed to obtain 25%, 40%, 50%, 60%, 70%
and 100% B at 10, 20, 30, 40, 50 and 80 min, respectively, following
the method described by Klimaczewski et al. (2014) with slight modifi-
cations. The G. ulmifolia ethanol extract was analysed and dissolved in
ethanol at a concentration of 10 mg/mL. The presence of seven phenolic
compounds were investigated, namely, chlorogenic acid, caffeic acid,
catechin, quercitrin, quercetin, rutin and luteolin. Identification of
these compounds were performed by comparing their retention time
and UV absorption spectrum with those of standards. The flow rate
was 0.7 mL/min, injection volume 40 μL and a wavelength of 280 nm
for catechin, 325 nm for caffeic and chlorogenic acids, and 365 nm for
quercitrin, quercetin, rutin and luteolin. The samples and mobile
phase were filtered through a 0.45 μm membrane filter (Millipore)
and then degassed by ultrasonic bath prior to use. Stock solutions of
standard references were prepared in the HPLC mobile phase at a con-
centration range of 0.020–0.350 mg/mL for catechin, luteolin, quercitrin,
quercetin and rutin; and 0.030–0.250 mg/mL for caffeic and chlorogenic
acids. The chromatography peaks were confirmed by comparing its re-
tention time with those of reference standards and by DAD spectra
(200 to 500 nm).
2.2.4. Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ were calculated based on the standard deviation of the
responses and the slope using three independent analytical curves, as
defined by Abbas et al. (2014). LOD and LOQ were calculated as 3.3
and 10 σ/S, respectively, where σ is the standard deviation of the re-
sponse and S is the slope of the calibration curve.
2.3. Evaluation of antioxidant activity
Antioxidant activity was determined by a spectrophotometric pro-
cedure (Morais et al., 2013): 0.1 mL of the methanol solution of the sam-
ple or as positive control, quercetin, at concentrations ranging from
10,000 to 1 ppm, were mixed with 3.9 mL of 6.5 × 10− 5 M DPPH in
methanol, and the absorbance of the reaction mixture was read at
515 nm. After 1.0 h, the UV absorbance was read for all solutions in a
Spekol 1100 spectrophotometer. To calculate the DPPH inhibition per-
centage (IP%), the following equation was used:
ADPPH −ASample
IP ¼  100
ADPPH
253
where ADPPH is the absorbance of the DPPH solution and ASample is the
absorbance of the solution containing the ethanol extract at a particular
concentration. The inhibitory potential (%) of each sample concentra-
tion was applied in the Origin 7.0 statistic program to calculate the
50% inhibitory concentration (IC50), by linear regression analysis (con-
centration X IP%). The results were compared with that of quercetin,
the standard antioxidant.
2.4. Assessment of anticholinesterase activity in vitro
Inhibition of acetylcholinesterase enzyme (AChE) was evaluated in
accordance with the methodology described by Ellman et al. (1961),
which was later adapted by Rhee et al. (2011). This bioassay consists
of the application of the sample to TLC plates and spraying the plate
with Ellman's reagent, which was prepared by mixing 5,5′-dithiobis-
2-nitrobenzoic acid (DTNB) and a buffer solution of acetylthiocholine
iodide (ATCI). The TLC plate was subsequently sprayed with AChE en-
zyme (3 U/mL). After 3 min, enzyme inhibition is observed by the pres-
ence of white spots on the yellow plate. The TLC enzyme test is basically
qualitative but is still significantly sensitive. The following solutions
were prepared for this test: (1) 50 mM Tris/HCl pH 8 (buffer);
(2) 50 mM Tris/HCl pH 8 containing 0.1% bovine serum albumin
(BSA); (3) 1 mM Ellman's reagent; and (4) 1 mM ACTI. The lyophilized
enzyme AChE was diluted in buffer solution (1) to prepare a 1000 U/mL
enzyme solution. 5 μL aliquots of compounds in CHCL3 (4 mg/mL) were
initially applied to TLC plates (DC-Alufolien, silica gel 60 F254, 0.2 mm
Merck). The plate was then sprayed with solutions (3) and (4). After
3 min, which is the time necessary for the solution to completely dry,
the plate was sprayed with AChE (3 U/mL). After approximately
10 min, the appearances of white spots were observed and their diam-
eters were immediately measured. Physostigmine was used as a posi-
tive control.
2.5. Susceptibility testing of microorganisms by broth microdilution method
Each ethanol extract (10 mg) was dissolved in 1 mL of
dimethylsulfoxide (DMSO - Merck, Darmstadt, Germany) and sterile
distilled water was added to obtain a concentration of 2048 μg/mL
(initial solution).
2.5.1. Strains, drugs and culture media
The microorganisms used in the sensitivity testing of natural prod-
ucts were obtained from the Mycology Laboratory (LM) of the Federal
University of Paraiba - UFPB and the Laboratory of Microbiology and
Molecular Biology (LMBM) of the Regional University of Cariri - URCA.
Three standard strains of Candida albicans (CA 77), C. krusei (CK 01)
and C. tropicalis (CT 23) were used; besides these, multiresistant clinical
isolates of the three species of cited fungi were used: CA 40006, CK
40095 and CT 40042. Fluconazole was used as the reference drug in
this assay. All tests were performed in triplicate.
The yeasts were first grown on solid medium, Sabouraud dextrose
agar (SDA) and then transferred to test tubes containing 5 mL of sterile
saline. They were diluted in saline to a concentration of 105 CFU/mL,
according to the McFarland scale (NCCLS, 1999). This procedure
provided a standard yeast suspension containing 1 × 105 cells/mL
(CLSI, 2002). Sabouraud dextrose broth (SDB) was the medium used
in the microdilution tests.
2.5.2. Detection of the minimum inhibitory concentration (MIC)
Eppendorf® tubes each containing 1.5 mL of solution of SDB with
1350 to 150 μL of fungal suspension were prepared for distribution in
microdilution plate. The plate was completed by adding 100 μL of this
solution to each well (96 well plates) and proceeding to a serial
microdilution with a solution of 100 μL of the natural product (ethanol
extract) with varying concentrations of 1024–8 μg/mL, with the last
well as growth control. The plates were put in the incubator for 24 h
254 S.M. Morais et al. / South African Journal of Botany 110 (2017) 251–257

Table 2
Anti-radical activity (EC50), total phenolics and flavonoids content in the G. ulmifolia leaf
extract.

Extracts Total phenolics Flavonoids EC50 (μg/mL)

(mg GAE/g extract) (mg QE/g extract)

Leaves 78.021 ± 0.0287 0.8055 ± 0.0025 119.85 ± 2.42

Quercetin − − 10.25 ± 1.45

(−) Substance not subjected to the test; GAE: gallic acid equivalent; QE: quercetin equiv-
alent; EC50, effective concentration of sample which scavenges 50% of DPPH radicals.

Fig. 2. Representative high performance liquid chromatography profile of G. ulmifolia
leaves, detection UV was at 325 nm. Catechin (peak 1), chlorogenic acid (peak 2), caffeic
acid (peak 3), rutin (peak 4), quercitrin (peak 5), quercetin (peak 6) and luteolin (peak 7).
at 35 °C (NCCLS, 2003), and thereafter in a spectrophotometric ELISA
(Termoplate®) device with a wavelength of 630 nm. The MIC was de-
fined as the lowest concentration at which no growth was observed.
As positive and negative controls we used the SDB medium with and
without the microorganisms assayed, producing a 100% and 0% percent
growth sample. All tests were performed in triplicate.
2.5.3. Fluconazole action modulation test
To investigate the modulatory antifungal action of mutamba
extracts, the method proposed by Coutinho et al. (2008) was used,
where the solution of the ethanol extract was tested at a subinhibitory
concentration (MIC/8). Eppendorf tubes each containing 1.5 mL of the
culture medium containing CSD, 150 μL of the fungal suspension and
the natural product at a concentration MIC/8 were prepared. For the
control, Eppendorf tubes with 1.5 mL of solution containing 1350 μL
and 150 μL CSD suspension of microorganisms were prepared. The
ELISA plate was completed by adding 100 μL of this solution to each
well. Then, 100 μL of the drug (antifungal agent) was mixed to the
first well, which then proceeded in the broth microdilution series until
the penultimate cavity. The concentrations of antifungal gradually de-
creased from 1024 to 1 μL.
2.5.4. Statistical analysis
The determinations were made in triplicate and the results normal-
ized by calculating the geometric mean. The results were compared
using analysis of variance (ANOVA), and the comparison between the
geometric means was performed according to Tukey's test, where
p b 0.05 was considered significant.
3. Results and discussion
3.1. HPLC analysis
The phytochemical analysis by HPLC revealed the presence of tan-
nins (catechin - Rt = 11.73 min; peak 1), phenolic acids (chlorogenic
acid - Rt = 20.98 min; peak 2 and caffeic acid - Rt = 25.04 min; peak
3) and the flavonoids rutin (Rt = 39.15 min; peak 4), quercitrin (Rt =
4527 min; peak 5), quercetin (Rt = 49.89 min; peak 6) and luteolin
(Rt = 54.81 min; peak 7) as present in the ethanol leaf extract of
G. ulmifolia (Fig. 2 and Table 1). Calibration curve for catechin: Y =
13,478× + 1257.9 (r = 0.9996); caffeic acid: Y = 11,975 × + 1196.2
(r = 0.9997); chlorogenic acid: Y = 11,863 × + 1275.48 (r =
0.9999); rutin: Y = 12,651 + 1189.7 (r = 0.9996); quercetin: Y =
13,571 × + 1360.9 (r = 0.9996), luteolin: Y = 11,894 × + 1283.7
(r = 0.9998) and quercitrin: Y = 12,493× + 1195.7 (r = 0.9999). All
chromatography assays were carried out at room temperature and in
triplicates.
The results of the measurements of total phenolics and flavonoids, as
well as EC50 values (sample effective concentration able to scavenger
50% DPPH radical) determined for G. ulmifolia extracts and for the pos-
itive control (quercetin) are shown in Table 2.
The antioxidant activity of G. ulmifolia leaf ethanol extract was previ-
ously studied showing relevant results (Navarro et al., 2003; Syaefudin
et al., 2014). Boligon et al. (2013) reported relevant radical scavenging
activity (EC50: 7.61 ± 0.09 μg/mL) for the G. ulmifolia leaf essential oil.
The antioxidant activity of the essential oil was attributed to the pres-
ence of known antioxidant components (thymol - 20.97%; carvacrol -
13.76% and eugenol - 10.13%). The extract of G. ulmifolia leaves in this
work revealed phenolic components in low yield (7.8%), concurrently
the antioxidant activity (119.85 ± 2.42 mg/mL) was proportionally
smaller in relation to the essential oil but nevertheless proved to be rel-
evant when compared to other herbal extracts (Morais et al., 2013).
In this work, the EEGUL (Ethanol extract of Guazuma ulmifolia
leaves) presented as its major compounds, chlorogenic acid (2.53%)
and the flavonoid quercetin (2.15%), compounds recognized as potent
antioxidants. The presence of other phenolics, such as quercetin gluco-
sides (rutin and quercitrin) also contribute for the antioxidant effect.
G. ulmifolia leaf ethanol extract showed similar results as the stan-
dard physostigmine against the enzyme acetylcholinesterase, which ac-
cording to Falé et al. (2013) the effectiveness of plant extracts in the
treatment of digestive problems is due to their content in acetylcholin-
esterase inhibitors and antioxidant compounds, such as phenolic acids
(chlorogenic acid and cynarin) and glycosylated flavonoid derivatives.
Both activities were found in the extract of G. ulmifolia and the cited
phenolic constituents can justify the popular use of this plant for treat-
ment of gastrointestinal disorders.

Table 1
Phenolic acids and flavonoid composition of G. ulmifolia leaves.
3.2. Anti-acetylcholinesterase activity

Compounds Ethanolic extract LOD LOQ The results of the in vitro anti-AChE effect of the ethanol extract of
mg/g % μg/mL μg/mL G. ulmifolia are shown in Table 3.
Catechin 9.83 ± 0.01 a 0.98 0.016 0.053
Chlorogenic acid 25.31 ± 0.03 b 2.53 0.025 0.082
Caffeic acid 7.34 ± 0.02 c 0.73 0.009 0.029 Table 3
Rutin 10.05 ± 0.01 a 1.00 0.023 0.075 Inhibition of the enzyme acetylcholinesterase activity by G. ulmifolia
Quercitrin 8.16 ± 0.01 c 0.81 0.015 0.049 ethanol extract, in thin-layer chromatography assay.
Quercetin 21.57 ± 0.03 d 2.15 0.030 0.098
Extract Zone of inhibition (cm)
Luteolin 4.68 ± 0.02 e 0.46 0.011 0.037
EEGUL 1.0
LOD: Limit of detection and LOQ: limit of quantification. Results are expressed as mean ±
Physostigmine 0.9
standard deviations (SD) of three determinations. Averages followed by different letters
differ by Tukey test at p b 0.05. EEGUL: Ethanolic extract of G. ulmifolia leaves.
 S.M. Morais et al. / South African Journal of Botany 110 (2017) 251–257 255

Fig. 3. Modulatory effect of G. ulmifolia leaves ethanolic extract in association with antifungal Fluconazole to clinical isolates of Candida tropicalis. Values represent the arithmetic mean ±
S.E.M. (standard error of the mean). Two-way ANOVA followed by the Bonferroni test. ⁎⁎⁎⁎p b 0,0001 vs antibiotic control; ⁎⁎⁎p b 0,001 vs antibiotic control; ⁎p b 0,05 vs antibiotic control;
Ant – Antagonism; ns - not significant.

The ethanol extract of G. ulmifolia showed significant result with re-
spect to anti-AChE activity, with an enzyme inhibition zone (IZ) value of
1.0. The similarity of these results with that of the physostigmine con-
trol (0.9) can be attributed to the presence of flavonoids and phenolic
acids in the extract.
In Brazil, Morais et al. (2013) investigated the correlation
between the presence of phenolics with antioxidant and anti-
acetylcholinesterase activities in extracts of medicinal species, noting
that in Malphigia glabra and Lippia alba, there was a direct correlation
between the two activities and phenolic content. Yamaguchi et al.
(2012), working with 20 species of the family Lauraceae in the Amazon,
reported that the extract of branches from Licaria martiniana showed
the highest DPPH sequestration capacity and the Ocotea minor was the
only one to have pronounced antioxidant and acetylcholinesterase in-
hibitory activities in extracts of both twigs and leaves.
3.3. Anti-Candida activity and potentialization of the antifungals
The ethanolic extract was tested against six strains of Candida show-
ing low activity with a MIC = 1024 μg/mL. This result demonstrates that
the extracts were not efficient growth inhibitors, as their inhibition per-
centages were lower than 50%. A relevant note regarding this research is
the value of the extract concentrations used in the test, which is consid-
ered high for clinical applications (Houghton et al., 2007). In the tests for
evaluating the modulatory effect against fluconazole, it was observed
that natural products only effectively potentiated the effect of the anti-
fungal against strains of C. tropicalis (Fig. 3) showing a synergism when
the extract was combined with fluconazole. An indifferent effect
(neither antagonism nor synergism) was observed in association with
fluconazole EEGUL against multidrug-resistant strains of C. albicans
and C. krusei (Figs. 4 and 5).
3.4. Statistical analysis
Statistically significant differences (P b 0.05) for both extracts were
observed for the growth inhibition values in C. albicans, C. tropicalis,
and C. krusei by Elisa spectrophotometric absorbance unit when com-
pared with fluconazole. Regarding C. krusei and C. albicans, no significant
differences were noted (P N 0.05), having a similar effect in the growth
inhibition when compared with the antifungal, which both demonstrat-
ed a MIC value ≥1024 μg/mL. Through Tukey's test (P N 0.05), statistical-
ly significant differences were shown for the C. tropicalis lineage,
indicating that the action of the antifungal agent alone was better
than when it was used with the ethanol extract. Significant statistical

Fig. 4. Modulatory effect of G. ulmifolia leaves ethanolic extract in association with antifungal Fluconazole to clinical isolates of Candida albicans. Values represent the arithmetic mean ±
S.E.M. (standard error of the mean). Two-way ANOVA followed by the Bonferroni test. ⁎⁎⁎⁎p b 0,0001 vs antibiotic control; Ant – Antagonism; ns - not significant.
256 S.M. Morais et al. / South African Journal of Botany 110 (2017) 251–257
Fig. 5. Modulatory effect of G. ulmifolia leaf ethanolic extract in association with antifungal Fluconazole to clinical isolates of Candida krusei. Values represent the arithmetic mean ± S.E.M.
(standard error of the mean). Two-way ANOVA followed by the Bonferroni test. ns - not significant.
values with P b 0.0001 (ANOVA) were also observed for the modulatory
action of C. tropicalis.
Morais et al. (2013) also observed low antifungal and modulation of
antibiotic effects by the ethanol extract of fern leaves from Lygodium
venustum against standard strains of C. albicans (ATCC 40006),
C. krusei (ATCC 2538) and C. tropicalis (ATCC 40042), all with a MIC ≥
1024 g/mL. The results of the modulation tests showed an indifferent
effect when the extract was associated with the commercial drugs:
nystatin, amphotericin B and mebendazole. The flavonoid quercetin
and chlorogenic acid were also found in greater amounts in the fern ex-
tract. Morais et al. (2013) attributed a positive effect of chlorogenic acid
against the C. krusei strain. The antibiotic activity enhancement of Nys-
tatin was explained by the authors to be due to the action of chlorogenic
acid when, after decreasing the stability of the membrane, favours the
entry of the antifungal agent.
The chemical constituents found in the highest percentage in this
work, quercetin and chlorogenic acid, do not show antifungal activity
when tested against standard strains of C. albicans and C. krusei (Veras
et al., 2011). The same occurred with rutin, which is also present in
the extract, and which according to Araruna et al. (2012) reported
that this flavonoid does not have an established effect against standard
C. albicans, C. krusei and C. tropicalis strains. However, chlorogenic acid
showed antifungal activity against Candida yeasts (Daneshtalab, 2008;
Lee et al., 2008) with a mechanism of action of disturbing the structure
of the fungal membrane (Sung and Lee, 2010). These studies indicate
that the antifungal action did not present the same potential for all test-
ed yeasts. Each organism has its genetic specificity and it is considerable
to explain the divergent degrees of activity in the results.
For Wynn et al. (2003), azole derivatives such as fluconazole have a
mechanism of action by inhibition of ergosterol biosynthesis through
the interaction with lanosterol demethylase, the enzyme responsible
for the conversion of lanosterol to ergosterol, an essential component
of the fungal membrane. Inhibition of ergosterol synthesis can cause
changes in the plasma membrane permeability and inhibition of prolif-
eration at all stages of the fungus life cycle (Urbina, 2009). Combination
of fluconazole with natural products can be an alternative to minimize
antibiotic side effects, since it leads to a synergistic association with a
significant effect, reducing the MIC of the antifungal agent, thus decreas-
ing the dose needed for their therapeutic use (Figueiredo et al., 2013).
The mechanisms of action may be related to antibiotic combination
with extracts at a sub-inhibitory concentration added directly to the
culture medium. This strategy is called “herbal-shotgun” or a “multi-
synergistic effect” and refers to the use of plants and drugs on an
approach that uses mono or multi combined extracts, which can affect
not only a single target, but several wherein the different therapeutic
components act together in a synergistic or antagonistic form
(Coutinho et al., 2009). Although the search for new substances in
plant extracts through their isolation and identification is ample, results
seem to be related to the combination of compounds contained in these
complex mixtures, which characterize these extracts (Matias et al.,
2013).
4. Conclusion
The chemical study of the ethanol extract of Guazuma ulmifolia
leaves revealed the presence of flavonoids and phenolic acids with rec-
ognized antioxidant potential, justifying the values obtained in the eval-
uation of free radical scavenging activity. The antioxidant activity
combined with the anticholiesterase potential corroborates with the
use of G. ulmifolia in the treatment of gastrointestinal disorders.
The mutamba ethanol extract has no clinically relevant antifungal activ-
ity, however, in combination with fluconazole for the evaluation of
interference in fungal resistance profile, indicates a positive synergistic
effect against multiresistant strains of C. tropicalis, representing an alter-
native source for efforts to combat skin problems related to this
microorganism.
Acknowledgments
The authors acknowledge the support and cooperation received
from FUNCAP (Foundation for Research Support Cearense),
RENORBIO/UECE (Northeast Biotechnology Network/State University
of Ceará), LPN/UECE (Laboratory of Natural Products) URCA/LMBM/
LFQM (University Regional Cariri//Laboratory of Microbiology and Mo-
lecular Biology/Laboratory of Pharmacology and Molecular Chemistry),
and UFSM (University Federal of Santa Maria-RS).
References
Abbas, S.R., Sabir, S.M., Ahmad, S.D., Boligon, A.A., Athayde, M.L., 2014. Phenolic profile,
antioxidant potential and DNA damage protecting activity of sugarcane (Saccharum
officinarum). Food Chemistry 147, 10–16.
Araruna, M.K.A., Brito, S.A., Morais-Braga, M.F.B., Santos, K.K.A., Souza, T.M., Leite, T.R.,
Costa, J.G.M., Coutinho, H.D.M., 2012. Evaluation of antibiotic & antibiotic modifying
activity of pilocarpine & rutin. Indian Journal of Medical Research 135, 252–254.
Boligon, A.A., Kubiça, T.F., Mario, D.N., 2013. Antimicrobial and antiviral activity-guided
fractionation from Scutia buxifolia Reissek extracts. Acta Physiologiae Plantarum 35,
2229–2239.
Caceres, A., Girón, L.M., Alvarado, S.R., Torres, M.F., 1987. Screening of antimicrobial
activity of plants popularly used in Guatemala for the treatment of dermatomucosal
diseases. Journal of Ethnopharmacology 20, 223–237.
Caceres, A., Cano, O., Samayoa, B., Aguilar, L., 1990. Plants used in Guatemala for the treat-
ment of gastrointestinal disorders. 1. Screening of 84 plants against enterobacteria.
Journal of Ethnopharmacology 39, 77–82.
Calixto Júnior, J.T., Morais, S.M., Martins, C.G., Vieira, L.G., Morais-Braga, M.F.B., Carneiro,
J.N.P., Machado, A.J.P., Menezes, I.A., Tintino, S.R., Coutinho, H.D.M., 2015a. Phyto-
chemical analysis and modulation of antibiotic activity by Luehea paniculata Mart. &
Zucc. (Malvaceae) in multiresistant clinical isolates of Candida spp. BioMed Research
International 2015.
 S.M. Morais et al. / South African Journal of Botany 110 (2017) 251–257
Carradori, S., Chimenti, P., Fazzari, M., Granese, A., Angiolella, L., 2016. Antimicrobial activ-
ity, synergism and inhibition of germ tube formation by Crocus sativus-derived com-
pounds against Candida spp. Journal of Enzyme Inhibition and Medicinal Chemistry
1-5.
CLSI, 2002. Clinical and Laboratory Standards Institute. Reference Method for Broth
Dilution Antifungal Susceptibility Testing of Filamentous Fungi. Approved Standard
M27-A2. Clinical and Laboratory Standards Institute, Wayne, Pa.
Coutinho, H.D.M., Costa, J.G.M., Siqueira-Júnior, J.P., 2008. In vitro anti-staphylococcal ac-
tivity of Hyptis martiusii Benth against methicillin-resistant Staphylococcus aureus-
MRSA strains. Revista Brasileira de Farmacognosia 18, 670–675.
Coutinho, H.D.M., Costa, J.G.M., Lima, E.O., 2009. Herbal therapy associated with antibiotic
therapy: potentiation of the antibiotic activity against methicillin – resistant Staphy-
lococcus aureus by Turnera ulmifolia L. BMC Complementary and Alternative Medicine
9, 13–22.
Daneshtalab, M., 2008. Discovery of chlorogenic acid-based peptidomimetics as a novel
class of antifungals. A success story in rational drug design. Journal of Pharmacy &
Pharmaceutical Sciences 11, 44–55.
Ellman, G.L., Courtney, K.D., Andre, V., 1961. Featherstone RM. A new and rapid colorimet-
ric determination of acetylcholinesterase activity. Biochemical Pharmacology 7,
88–95.
Falé, P.L., Ferreira, C., Rodrigues, A.M., Cleto, P., Madeira, P.J.A., Florêncio, M.H., Frazão, F.N.,
Serralheiro, M.L.M., 2013. Antioxidant and anti-acetylcholinesterase activity of com-
mercially available medicinal infusions after in vitro gastrointestinal digestion. Jour-
nal of Medicinal Plant Research 7, 1370–1378.
Figueiredo, M.A., Fernandes, A., 1987. Encraves de Cerrado no interior do Ceará. Ciência
Agronômica 18, 103–106.
Figueiredo, F.G., Ferreira, E.O., Lucena, B.F.F., Torres, C.M.G., Lucetti, D., Lucetti, E.C.P., Silva,
J.M.F.L., Santos, F.A.V., Medeiros, C.R., Oliveira, G.M.M., Colares, A.V., Costa, J.G.M.,
Coutinho, H.D.M., Alencar, I.M., Silva, J.C.F., Kerntopf, M.R., Figueiredo, P.R.L., Matias,
E.F.F., 2013. Modulation of the antibiotic activity by extracts from Amburana cearensis
A. C. Smith and Anadenanthera macrocarpa (Benth.) Brenan. BioMed Research Inter-
national 2013.
Funari, C.S., Ferro, V.O., 2006. Análise de própolis. Ciência e Tecnologia de Alimentos 26,
171–178.
Gálvez, J., Coelho, G., Crespo, M.E., Cruz, T., Rodríguez-Cabezas, M.E., Concha, A., Gonzalez,
M., Zarzuelo, A., 2001. Intestinal antiinflammatory activity of morin on chronic exper-
imental colitis in the rat. Alimentary Pharmacology & Therapeutics 15, 2027–2039.
Höfling, J.F., Anibal, P.C., Obando-Pereda, G.A., Peixoto, I.A.T., Furletti, V.F., Foglio, M.A.,
Gonçalves, R.B., 2010. Antimicrobial potential of some plant extracts against Candida
species. Brazilian Journal of Biology 70, 1065–1068.
Holzer, P., Maggi, C.A., 1994. Synergistic role of muscarinic acetylcholine and tachykinin
NK2 receptors in intestinal peristalsis. Naunyn-Schmiedeberg's Archives of Pharma-
cology 349, 194–201.
Houghton, P.J., Howes, M.J., Lee, E.E., Steventon, G., 2007. Uses and abuses of in vitro tests
in ethnopharmacology: visualising an elephant. Journal of Ethnopharmacology 110,
391–400.
Klimaczewski, C.V., Saraiva, R.A., Roos, D.H., Boligon, A.A., Athayde, M.L., Kamdem, J.P.,
Barbosa, N.V., Rocha, J.B.T., 2014. Antioxidant activity of Peumus boldus extract and
alkaloid boldine against damage induced by Fe (II)-citrate in rat liver mitochondria
in vitro. Industrial Crops and Products 54, 240–247.
257
Lee, J.H., Yang, H.Y., Lee, H.S., Hong, S.K., 2008. Chemical composition and antimicrobial
activity of essential oil from cones of Pinus koraiensis. Journal of Microbiology and
Biotechnology 18, 497–502.
Matias, E.F.F., Alves, E.F., Santos, B.S., 2013. Biological activities and chemical characteriza-
tion of Cordia verbenacea DC. as tool to validate the ethnobiological usage. Evidence-
based Complementary and Alternative Medicine 3, 3–7.
Morais, S.M., Lima, K.S.B., Siqueira, S.M.C., Cavalcanti, E.S.B., Souza, M.S.T., Menezes,
J.E.S.A., Trevisan, M.T.S., 2013. Correlação entre as atividades antiradical,
antiacetilcolinesterase e teor de fenóis totais de extratos de plantas medicinais de
farmácias vivas. Revista Brasileira de Plantas Medicinais 15, 575–582.
Navarro, V., Vilarreal, M.L., Rojas, G., Lozoya, X., 1996. Antimicrobial evaluation of some
plants used in Mexican traditional medicine for the treatment of infectious diseases.
Journal of Ethnopharmacology 53, 143–147.
Navarro, M.C., Montilla, M.P., Cabo, M.M., Galisteo, M., Cáceres, A., Morales, C., Berger, I.,
2003. Antibacterial, antiprotozoal and antioxidant activity of five plants used in Izabal
for infectious diseases. Phytotherapy Research 17, 325–329.
NCCLS, 1999. National Committee for Clinical Laboratory Standards. Methods for Deter-
mining Bactericidal Activity of Antimicrobial Agents: Approved Guide line M26-A
(Wayne, Pennsylvania, USA).
NCCLS, 2003. National Committee for Clinical Laboratory Standards. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacterium that Grow Aerobically: Approved
Standard, NCCLS document M7-A6, Wayne, Pennsylvania 19087–1898 USA.
Ramirez, V.R., Mostacero, L.J., Garcia, A.E., Mejia, C.F., Pelaez, P.F., Medina, C.D., Miranda,
C.H., 1988. Vegetales Empleados En Medicina Tradicional Norperuana. Banco Agrario
Del Peru & Nacional Universidad deTrujillo, Trujillo, Peru, p. 54 (June).
Rhee, I.K., Van de Meent, M., Ingkaninan, K., Verpoorte, R., 2011. Screening for acetylcho-
linesterase inhibitors from Amarillidaceae using silica gel thin-layer chromatography
in combination with bioactivity staining. Journal of Chromatography 915, 217–223.
Rutter, R.A., 1990. Catalogo de Plantas Utiles de la Amazonia Peruana. Instituto Linguistico
de Verano, Yarinacocha, Peru, p. 349.
Sousa, C.M.M., Silva, H.R., Vieira Júnior, G.M., 2007. Fenóis totais e atividade antioxidante
de cinco plantas medicinais. Quimica Nova 30, 351–355.
Sung, W.S., Lee, D.G., 2010. Antifungal action of chlorogenic acid against pathogenic fungi,
mediated by membrane disruption. Pure and Applied Chemistry 82, 219–226.
Syaefudin, W.T., Wahyuni, I.M., Artika, L., Sulistiyani, R., 2014. Antioxidant activity of fla-
vonoid from Guazuma ulmifolia Lam. leaves and apoptosis induction in yeast cells.
Journal of Biological Sciences 14, 305–310.
Urbina, J., 2009. A ergosterol biosynthesis and drug development for Chagas disease.
Memórias do Instituto Oswaldo Cruz 104, 311–318.
Veras, H.N.H., Santos, L.J.M., Santos, A.C.B., 2011. Comparative evaluation of antibiotic and
antibiotic modifying activity of quercetin and isoquercetin in vitro. Current Topics in
Nutraceutical Research 9, 25–30.
Wynn, R.L., Jabra-Rizk, M.A., Meiller, T.F., 2003. Fungal drug resistance, biofilms, and new
antifungals. General Dentistry 51, 94–98.
Yamaguchi, K.K.L., Alcântara, J.M., Veiga-Júnior, V.F., 2012. Investigação do potencial
antioxidante e anticolinesterásico de 20 espécies da família Lauraceae. Acta
Amazonica 42, 541–546.
Zhu, H., Li, Y.R., 2012. Oxidative stress and redox signalling mechanisms of inflammatory
bowel disease: updated experimental and clinical evidence. Experimental Biology
and Medicine (Maywood, N.J.) 237, 474–480.