SUPPLEMENTARY MATERIAL
Antioxidant and antifungal activities of Smilax campestris Griseb. (Smilacaceae)
Marcela Isis Moraisa, Maria Eduarda Amaral Pintoa, Sthéfane Guimarães Araújoa, Ana
Hortência Fonsêca Castroa, Joaquim Mauricio Duarte-Almeidaa, Luiz Henrique Rosab,
Carlos Augusto Rosab, Susana Johannb, Luciana Alves Rodrigues dos Santos Limaa
Abstract
Ethanol extract and fractions obtained from aerial parts of Smilax campestris
were examined in order to determine their phenolic composition, antioxidant capacity
and antifungal activities. High performance liquid chromatography coupled with DAD
analysis indicated that quercetin and rutin were the main phenolic compounds present in
butanol fraction and ethanol extract, respectively. The antioxidant activity assessed by
the scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was
significantly more pronounced for the ethanol extract and butanol fraction than that of
the commercial antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT). The antifungal
activity of extract and fractions was investigated by microdilution method using five
Candida and two Cryptococcus yeast strains. Ethanol extract and fractions showed
antifungal activities against C. albicans, C. glabrata, C. krusei, C. parapsilosis, C.
tropicalis and C. gattii. This work provided the knowledge of profile and content
flavonoids in the extract and fractions and their antioxidant and antifungal activities of
aerial parts of S. campestris.
Keywords: antifungal, antioxidant, flavonoids, Smilax campestris, yeast, DPPH.
Experimental
1. Plant material and extraction
Aerial parts of Smilax campestris Griseb. were collected in Lavras, South Minas
Gerais State, Brazil (21o13’46”S and 44o58’32”W, average altitude 908 m), in July
2011. The plant material was identified by Prof. Dr. Douglas Antônio de Carvalho, and
a voucher specimen (18.781) was deposited in the ESAL Herbarium of the Federal
University of Lavras, MG, Brazil.
The fresh plant material (105.2 g) was extracted by cold maceration in ethanol
P.A (Vetec, Brazil) for a period of 10 days at room temperature. After it was filtered
and concentrated in a rotary evaporator at 40°C under reduced pressure. Ethanol extract
(Et, 919.0 mg) was dissolved in EtOH/H2O (7:3), and then partitioned successively with
C6H14, CH2Cl2, AcOEt and n-BuOH (Vetec, Brazil; 15 mL, 3 times with each solvent),
resulting in 101.0, 79.6, 38.7, 451.2 and 236.2 mg of hexane (Hex), dichloromethane
(DCM), ethyl acetate (Ac), butanol (But) and hydroalcoholic (HE) fractions,
respectively (Araújo et al. 2013). The extract and fractions were screened for the
presence of different phytoconstituents such as saponins, tannins, alkaloids, steroids,
triterpenes, coumarins and flavonoids (Wagner et al. 2001).
2. Total flavonoid content
The total flavonoid contents were estimated according to the Dowd method
(Meda et al. 2005). Exactly 2 mL of 2% aluminum trichloride (AlCl3, Vetec, Brazil) in
methanol was mixed with the same volume of the extract or fraction solution (1.0
mg/mL). The absorbance was read at 415 nm using a Hitachi 2010 spectrophotometer
after 10 min, with a blank sample consisting of a 2-mL extract or fraction solution with
2 mL methanol without AlCl3. Quercetin (Sigma-Aldrich, St. Louis, MO) was used as
the reference compound to produce the standard curve, and total flavonoid contents
were expressed as g of quercetin equivalents/mg of extract or fraction. All assays were
performed in triplicate.
3. Phenolic profiles by HPLC-DAD
The phenolic substances in the butanol fraction and ethanol extract were carried
out using analytical reversed phase HPLC on an Agilent 1260 system with an
autosampler and quaternary pump coupled to a diode array detector. Compound
separation was performed by a Zorbax Eclipse Plus 5B RP-18 (5 μm, 250 x 4.6 mm,
Agilent, USA) column. The mobile phases were A - water/formic acid (99.9:0.1) - and
B - acetonitrile. The gradient consisted of 20% of B for 2 minutes, increased to 30% of
B after 10 minutes, 50% of B after another 10 minutes and 70% of B after an additional
10 minutes. For column cleaning, 90% of phase B was used (Duarte-Almeida et al.
2011). Each sample was injected in duplicate, ranging from 5–20 μL according to the
sample concentration. Determination was performed by comparing the retention times
and the UV spectra against those obtained from the standards [apigenin, luteolin,
quercetin and rutin, Sigma-Aldrich Chemical Co. (St. Louis, MO), and caffeic,
coumaric, ferulic, and chlorogenic acids, Apin Chemicals Ltd. (Abingdon, UK)]. The
determination was positive when the similarity between the chromatograms was equal
to or greater than 90%.
4. DPPH radical scavenging assay
The radical-scavenging abilities of extracts and fractions of S. campestris were
based on reactions with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) (Sigma, USA)
and compared to standards, 2,6-di-tert-butyl-4-methylphenol (BHT) and ascorbic acid
(AA) (Sigma, USA). Determination of antioxidant activity by the DPPH method was
adapted for use with microplates (Araújo et al. 2013). Briefly, a solution of DPPH
(0.002% w/v) was prepared in 80% methanol. Volumes of 75 L of samples or
standards (1, 10, 100, 250 e 500 g/mL) were added to wells in a 96-well flat-bottom
plate containing 150 L of DPPH solution. The plate was then covered and left in the
dark at room temperature (25°C). After 30 min, absorbance at 517 nm was measured in
a spectrophotometer (Biotek Power Wave XS2/US), and 80% methanol was used for
the baseline correction. Scavenging ability was expressed as the inhibition percentage
and was calculated by the following equation from Burda & Oleszek (2001):
Scavenging ability (%) = (Abscontrol − Abssample)/Abscontrol x 100, where Abscontrol =
absorbance of DPPH radical in 80% methanol and Abssample = absorbance of samples
and standards in 80% methanol + DPPH. The antioxidant activity of all samples was
expressed as IC50, which was defined as the concentration (in g/mL) of samples
required to inhibit the formation of DPPH radicals by 50%. IC50 values were calculated
by Probit analysis (Finney 1980).
5. Culture and maintenance of the yeast strains
Seven yeast strains, Candida albicans ATCC 18804, C. glabrata ATCC 2001, C.
krusei ATCC 200298, C. parapsilosis ATCC 22019, C. tropicalis ATCC 22019,
Cryptococcus gattii ATCC 32608 and Cryptococcus neoformans ATCC 2467, were
used in the biological assays. All the strains were stored at -80ºC.
Suspensions from the cultures of the Candida spp. and Cryptococcus spp. were
prepared according to the CLSI document M27-A3 (CLSI 2008) and modifications
suggested by Johann et al. (2010a) to obtain a final inoculum of 1.5 x 103 cells/mL
(CLSI 2008).
6. Determination of the minimal inhibitory concentration
The minimal inhibitory concentration (MIC) was obtained from the broth
microdilution tests performed in accordance with the guidelines in the CLSI document
M27-A3 (CLSI 2008). Amphotericin B (Sigma, USA), and fluconazole (Pfizer
Pharmaceutical, USA) were included as positive control; the stock solutions of
amphotericin were prepared in dimethylsulfoxide (DMSO) and fluconazole in water.
RPMI media (Sigma, USA) was used without samples or solvents as a control for
growth and sterility. The extract and fractions were dissolved in DMSO after the
addition of RPMI. Later, serial dilutions were made with RPMI, maintaining a constant
volume of 1000 μL in each tube. In this way, the samples were tested at eight
concentrations that varied from 15.6–2000 g/mL. From each dilution, brackets of 100
μL were transferred to the microplates. The solvent DMSO at the same volumes used in
the assay was used as a control for toxicity. After inoculation of Candida spp. and
Cryptococcus spp., plates were incubated at 37C for 48 h for Candida spp. and 72 h for
species of Cryptococcus. Amphotericin B was tested in the range of 0.008 to 1 g/mL,
and fluconazole of 0.125 to 16 g/mL. The endpoints were determined visually by
comparison with the drug-free growth control well. The MIC values were expressed in
g/ml and correspond to the lowest concentrations that did not allow for the detection of
any visual yeast growth. All assays were performed in triplicate and repeated at least
once.
7. Statistical Analysis
A Student’s t-test was utilized to evaluate the statistical difference between the
control group and the group exposed to ethanol extract and fractions of Smilax
campestris. The analyses were performed using the GraphPad Prism 5.0 software.
Values of p < 0.05 were considered statistically significant.
References
Araújo SG, Pinto MEA, Silva NL, Santos FJL, Castro AHF, Lima LARS. 2013.
Antioxidant and allelopathic activities of extract and fractions from Rosmarinus
officinalis. BBR – Biochem. Biotech. Rep. 2:35-43.
Burda S, Oleszek W. 2001. Antioxidant and antiradical activities of flavonoids. J.
Agric. Food Chem. 49:2774-2779.
[CLSI] Clinical and Laboratory Standards Institute. 2008. Reference method for broth
dilution antifungal susceptibility testing of yeast. Approved Standard M27-A3,
Wayne, PA, USA.
Duarte-Almeida JM, Salatino A, Genovese MI, Lajolo FM. 2011. Phenolic composition
and antioxidant activity of culms and sugarcane (Saccharum officinarum L.)
products. Food Chem. 125:660-664.
Finney DJ. 1980. Probit analysis, a statistical treatment of the sigmoid response curve.
Cambridge: University Press.
Johann S, Cisalpino PS, Watanabe GA, Cota BB, de Siqueira EP, Pizzolatti MG, Zani
CL, de Resende MA. 2010a. Antifungal activity of extracts of some plants used in
the Brazilian traditional medicine against the pathogenic fungus Paracoccidioides
brasiliensis. Pharm. Biol. 48:388-396.
Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG. 2005. Determination of the
total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as
their radical scavenging activity. Food Chem. 91:571-577.
Wagner H, Bladt S, Zgainski EM. 2001. Plant drug analysis a thin layer
chromatography atlas. Berlin Heidelberg: Springer-Verlag. 384p.
Table legends:
Table S1. Total flavonoids content, DPPH-scavenging activity and IC50 values of the
ethanol extract and fractions of Smilax campestris.
Table S2. Minimal inhibitory activity (MIC) of ethanol extract and fractions of Smilax
campestris against seven clinically important yeast species.
Figure legends:
Figure S1. HPLC chromatograms of Smilax campestris of ethanol extract (Et) and
fraction butanol (But) monitored at 350 nm. Peaks (1) no identified; (2 and 4) quercetin
derivative; (3) rutin derivative.
Table S1. Total flavonoids content, DPPH-scavenging activity and IC50 values of the ethanol extract and fractions of Smilax campestris.
Samples
DPPH-scavenging activity
Total
flavonoids
IC50 (g/mL)
1 g/mL
10 g/mL
100 g/mL
250 g/mL
500 g/mL
Et
13.00 ± 1.65
29.17 ± 0.29ab
42.76 ± 0.14ab
87.32 ± 0.38ab
90.20 ± 0.14ab
93.00 ± 0.51ab
13.61 ± 2.04ab
Hex
1.57 ± 0.14
38.40 ± 0.18ab
41.56 ± 0.17ab
44.30 ± 0.00ab
47.68 ± 0.66ab
54.54 ± 0.18ab
405.49 ± 48.67ab
DCM
5.42 ± 0.85
40.82 ± 0.32ab
42.51 ± 0.48ab
45.78 ± 0.66ab
49.26 ± 0.73ab
53.90 ± 0.66ab
298.91 ± 33.92ab
Ac
6.64 ± 0.87
40.51 ± 0.55ab
43.88 ± 0.18ab
50.42 ± 0.73ab
60.44 ± 0.55ab
77.85 ± 0.32ab
108.85 ± 5.71ab
But
13.79 ± 1.01
48.63 ± 0.37ab
53.90 ± 1.20ab
92.30 ± 0.48ab
95.36 ± 0.18a
96.94 ± 0.80ab
2.06 ± 0.14a
HE
7.42 ± 0.93
29.03 ± 0.39ab
30.21 ± 0.60ab
60.21 ± 0.45ab
91.39 ± 0.15b
94.23 ± 0.50b
34.46 ± 1.91ab
BHT
-
18.50 ± 0.24
25.90 ± 0.64
86.00 ± 0.56
91.40 ± 0.28
94.02 ± 0.64
16.36 ± 1.63
AA
-
39.10 ± 0.34
82.60 ± 0.26
90.80 ± 0.32
95.08 ± 0.43
99.80 ± 0.58
1.62 ± 0.25
Ethanol extract (Et), hexane (Hex), dichloromethane (DCM), ethyl acetate (Ac), butanol (But) and hydroalcoholic (HE) fractions, 2,6-di-tertbutyl-4-methylphenol (BHT) and ascorbic acid (AA).
1
Total flavonoids content: results expressed as g of quercetin equivalents/mg of extract or fraction.
2
IC50: concentration (in g/mL) of samples required to inhibit the formation of DPPH radicals by 50%.
Each value in the table is the mean ± standard deviation (n = 3).
a
p < 0.05 compared with BHT, b p < 0.05 compared with AA.
Table S2. Minimal inhibitory activity (MIC) of ethanol extract and fractions of Smilax campestris against seven clinically important yeast
species.
MIC (g/mL)
Fungi
Et
Hex
DCM
Ac
But
HE
Amphotericin B
Fluconazole
Candida albicans
2000*
≥ 2000
≥ 2000
≥ 2000
≥ 2000
≥ 2000
0.5
2
Candida glabrata
2000*
125*
≥ 2000
≥ 2000
≥ 2000
≥ 2000
0.125
4
*
*
65.2
≥ 2000
≥ 2000
0.5
32
*
*
Candida krusei
1000
500
2000
Candida parapsilosis
1000*
≥ 2000
≥ 2000
≥ 2000
≥ 2000
≥ 2000
0.5
1
Candida tropicalis
2000*
≥ 2000
2000*
≥ 2000
≥ 2000
≥ 2000
1.0
2
Cryptococcus gattii
1000*
≥ 2000
≥ 2000
1000*
≥ 2000
≥ 2000
1.0
nt
Cryptococcus neoformans
≥ 2000
≥ 2000
≥ 2000
≥ 2000
≥ 2000
≥ 2000
1.0
nt
Ethanol extract (Et), hexane (Hex), dichloromethane (DCM), ethyl acetate (Ac), butanol (But) and hydroalcoholic (HE) fractions.
nt = not tested
*
p < 0.05 compared with fluconazole and amphotericin B.
Figure S1. HPLC chromatograms of Smilax campestris of ethanol extract (Et) and fraction butanol (But) monitored at 350 nm. Peaks (1) no
identified; (2 and 4) quercetin derivative; (3) rutin derivative.