SUPPLEMENTARY MATERIAL
Molecular and cellular approach in the study of antioxidant/prooxidant
properties of Micromeria croatica (Pers.) Schott
Dunja Šameca, Jiri Gruzbc, Ksenija Durgod, Dario Kremere, Ivan Kosalece, Lidija Valek Žuljf,
Sanja Martinezf, Branka Salopek-Sondia, Jasenka Piljac-Žegaraca,*
Department of Molecular Biology, Institute ‘Ruđer Bošković, Bijenička c. 54, P.O. Box 180,
10000 Zagreb, Croatia; bLaboratory of Growth Regulators, Palacky´ University and Institute
of Experimental Botany ASCR, Šlechtitelu 11, CZ-783 71 Olomouc, Czech Republic; cCentre
of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science,
Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic; d Faculty of Food
Technology and Biotechnology, University of Zagreb, Zagreb, Croatia; e Faculty of
Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia;
f
Department of Electrochemistry, Faculty of Chemical Engineering and Technology, Savska
c.16/I, P.O. Box 177, 10000 Zagreb, Croatia
a
Corresponding author: Jasenka Piljac-Žegarac
The phytochemical composition and antioxidant activity of three wild populations of
endemic Illyric-Balcanic species Micromeria croatica (Pers.) Schott have been
evaluated with respect to plant organ and growing location. Multivariate analysis (PCA)
was performed to visualize (dis-)similarity among samples and identify correlations
between phytochemical variables that explain the most variability. The tested leaf
extract from locality Bačić kuk exhibited protective effects against ROS-induced
damage of DNA and inhibition of lipid peroxidation, while it caused oxidative
degradation of protein in the BSA assay at higher concentrations. This extract also
exhibited cytotoxic activity and facilitated the formation of ROS in the HEp2 cell line,
in a dose-dependent manner.
Keywords: antioxidant, cytotoxicity, Micromeria croatica (Pers.) Schott., polyphenols,
prooxidant
Experimental
Herbal material and extraction
Randomly selected samples of wild growing M. croatica plants were collected during the
blooming period in August of 2009, at three localities in Croatia: Bačić kuk (44.5781 N;
15.1012' E; 1260 m a.s.l.), Stupačinovo (44.5392 N; 15.1642 E; 970 m a.s.l.) and Bojinac
(44.3428° N; 15.4083° E; 1060 m a.s.l.). Voucher specimens (No. 1311 – 1341) were
deposited in the Herbarium of the Department of Pharmaceutical Botany, at ”Fran Kušan”
Pharmaceutical Botanical Garden, Faculty of Pharmacy and Biochemistry, University of
Zagreb in Croatia. Leaves were separated from flowers, air-dried in the dark and stored four
months until analysis. Triplicate extractions were performed with 60 mg of dried material in 2
mL of 80% methanol, at room temperature. (Šamec et al. 2014).
Phytochemical variability and antioxidant activity using biochemical assays
The total phenol (TP) content was determined according to the Folin-Ciocalteu assay
(Singleton & Rossi 1965) while the total flavonoid (TF) content was quantified according to
the AlCl3 colorimetric assay (Zhishen et al. 1999), adapted to small reaction volumes. Total
hydroxycinnamic acids and flavanols were determined according to Horward et al. (2003).
Proanthocyanidins were quantified using the vanilin-HCl method (Sun et al. 1998). UPLCMS/MS analysis of phenolic acids was performed as previously described (Gruz et al. 2008).
The Ferric Reducing/Antioxidant Power (FRAP) assay (Benzie & Strain 1999) was used to
estimate the antioxidant-reducing potential of extracts. The method reported by Prior et al.
(2003) with slight modification (Šamec et al. 2012) was used to quantify the Oxygen Radical
Absorbance Capacity (ORAC). Radical scavenging capacity was determined according to the
DPPH• radical scavenging assay (Brand-Williams et al. 1995) and the ABTS•+ radical cation
decolorization assay (Re et al. 1999). Cyclic voltammetry (CV) was used for electrochemical
characterization of antioxidants in methanol extracts (Piljac-Žegarac et al. 2010) with addition
of 0.1M LiClO4 to facilitate conductivity. In order to summarize the antioxidant assay data,
the ACI score was calculated (Seeram et al. 2008). The average of index scores obtained for
all four tests for a specific extract was defined as its ACI. The samples with the best ACI
index were used for biological activity testing.
Macromolecule-based assays
The extract of M. croatica exhibiting the best ACI score was freeze-dried and the pellets were
dissolved in methanol to the concentration of 10 mg/mL. The extraction yield was 12.8±2.3
%. The same extract was used in bioassays to determine the potential protective effects
against ROS – induced damage of DNA, lipids and protein. The DNA assay was based on the
conversion of supercoiled фX174 RF1 double-stranded DNA to open circular and linear
forms by hydroxyl radicals as damaging agents (Keum et al. 2000). A slightly modified
method reported by Wang et al. (2006) was used to assess the effect of M. croatica extract on
oxidative degradation of albumin. The data were expressed as oxidation percentage, with
reaction mixture without the test sample serving as the control. The inhibition of lipid
peroxidation was evaluated using the thiocyanate method (Yen & Hsieh 1998) and linoleic
acid as the model lipid. The reaction mixture was incubated at 37 °C for 5 h. Incubation
mixture without the plant extract was used as the negative control, while positive controls
contained Trolox and BHT in 1 mg/mL concentrations.
Cell-based assays
The human laryngeal carcinoma (HEp2) cell line was used in cell assays. The potential
cytotoxic effect of M. croatica leaf extract was examined by the Neutral red assay (Babich et
al. 1991) as we described earlier (Šamec et al., 2014). The percentage of survival was
calculated in comparison with the negative control. Free radicals were determined using the
dichlorofluorescein assay (Yang et al. 1998) with slight modification (Šamec et al., 2014).
The results were expressed as a ratio of fluorescence intensity and the number of viable cells.
All experiments were performed in triplicate.
Statistical analyses
One-way ANOVA and Post-hoc multiple mean comparisons (Tukey’s HSD test) were
performed in XLStat. All presented numeric values are means of three extractions ± standard
deviation (SD). The differences between measurements were considered to be significant at
p<0.05. PCA was performed on phytochemical and antioxidant activity data after
standardization, in order to visualize: 1) the distribution and the level of polyphenolic
compounds in different plant organs, and 2) the correlation between the polyphenol contents
of extracts and plant location.
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Glossary
3HBA, 3-hydroxybenzoic acid; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid); ACI, antioxidant composite index; BHT, butylated hydroxytoluene; CaA, caffeic acid;
ChA, chlorogenic acid; CV, cyclic voltammetry; DCF, dichlorofluorescein; DPPH, 2,2diphenyl-1-picrylhydrazyl; FA, ferulic acid; FRAP, Ferric Reducing/Antioxidant Power;
GaA, gallic acid; ORAC, Oxygen Radical Absorbance Capacity; PA, protocatechuic ; PAN,
proanthocyanidins; PCA, principal component analysis; pCoA, 4-coumaric acid; pHBA, 4hydroxybenzoic acid; ROS, reactive oxygen species; SA, salicylic acid; SiA, sinapic acid;
SyA, syringic acid; TF, total flavonoid; TFL, total flavanols; THA, total hydroxycinnamic
acids; TP, total phenol; VA, vanillic acid.
Table S1. Polyphenolic compounds: total phenol (TP), total flavonoid (TF), total
hydroxycinnamic acids (THA), total flavanols (TFL) and proanthocyanidins (PA) content of
flower, stem and leaf extracts of M. croatica collected at three different localities.
Stupačinovo
Bojinac
Bačić kuk
Average
Leaf
23.37±0.39c
26.31±1.47b
29.74±1.05a
26.49±3.19
TP
Flower
26.18±0.75b
23.07±0.97c
24.14±1.44bc
24.47±1.58
mg GAE/g dw
Stem
18.07±0.73d
12.81±0.75e
15.01±0.29e
15.30±2.64
per plant
67.62
62.25
68.89
Leaf
26.70±0.50ab
27.98±3.83a
29.57±0.04a
28.08±1.44
TF
Flower
27.65±0.66a
21.09±2.72bc
31.24±2.54a
26.66±5.15
mg CE/ g dw
Stem
18.26±0.61cd
11.05±0.31e
12.91±1.86de
14.07±3.74
per plant
72.61
60.13
73.73
Leaf
15.95±0.74cde 17.95±0.08bcd 19.26±0.16abc 17.72±1.67
THA
Flower
19.64±2.70ab
14.82±1.26def
22.44±1.83a
mg CAE/g dw
Stem
11.44±0.92fg
10.00±0.45g
12.75±0.62efg 11.40±1.38
per plant
47.03
42.77
54.45
Leaf
5,49±0,43bcd
6,41±0,33bc
6,03±0,10bc
5.97±0.46
TFL
Flower
9,31±1,22a
6,46±0,58b
9,71±1,16a
8.49±1.78
mg QE/g dw
Stem
4,52±0,61cd
4,05±0,16d
5,50±0,33bcd
4.69±0.74
per plant
19.31
16.91
21.24
Leaf
1,06±0,05b
1,01±0,12b
0,90±0,12bc
0.99±0.08
PA
Flower
1,74±0,10a
1,27±0,07b
1,77±0,07a
1.59±0.28
mg CE/g dw
Stem
0,56±0,08cd
0,49±0,06d
0,45±0,03d
0.50±0.06
per plant
3.36
2.77
3.12
18.96±3.85
Figure S1. Antioxidant capacities of M. croatica extracts measured by a) FRAP, b) ORAC, c)
DPPH, d) ABTS, and e) CV methods.
Table S2. Free phenolic acid content (μg/g dw) in M. croatica flower, steam and leaf extracts from three different localities measured by UPLCMS/MS.
Plant
part
Leaf
GaA
PA
pHBA
3HBA
SA
nd
5.4±0.1
10.1±0.9
nd
9.4±1.2
nd
nd
nd
14.5±0.5
nd
10.7±0.6 14.3±1.1
8.4±0.5 10.2±0.1
nd
Leaf
nd
4.8±0.7
10.2±0.1
Flower
nd
8.9±0.4
Stem
nd
Leaf
Flower
Stupačinovo Flower
Stem
Bojinac
Bačić kuk
ChA VA
CaA
SyA
pCoA
FA
SiA Total
nd
nd
nd
3.9±0.1
nd
nd
28.8
nd
nd
nd
nd
6.9±0.3
nd
nd
45.4
21.5±2.9
nd
nd
5.1±0.8
nd
87.0
nd
2.2±0.3
nd
nd
nd
nd
19.4
13.0±0.2
nd
5.8±0.3
nd
nd
nd
nd
nd
33.5
7.4±0.7
15.9±0.8
nd
14.2±0.5
nd
nd
nd
nd
65.4
nd
4.9±0.1
9.7±0.2
nd
7.6±0.5
nd
nd
nd
13.2±1.2 14.2±0.5 4.5±0.6
3.5±0.3
nd
nd
nd
25.7
nd
5.6±0.2
12.3±0.9
nd
9.7±0.2
nd
nd
nd
5.6±0.6
nd
33.2
18.6±3.1 16.6±0.8 6.6±1.0
2.2±0.3
nd
nd
nd
nd
5.8±0.3
nd
18.5±1.1 nd
nd 8.2±0.0 10.1±0.4
nd
nd
nd
15.9±1.4 6.6±0.9
nd
nd 59.3
PA, protocatechuic; pHBA, 4-hydroxybenzoic; 3HBA, 3-hydroxybenzoic; GaA, gallic; SA, salicylic; ChA, chlorogenic; VA, vanillic; CaA,
caffeic; SyA, syringic; pCoA, 4-coumaric; FA, ferulic; SiA, sinapic.
Stem
Table S3. Antioxidant potency composite index (ACI) for leaf, flower and stem extracts of M.
croatica from three different localities.
DPPH FRAP ABTS CV ORAC ACI
leaf
67
71
79
70
80
73
64
86
73
76
81
Stupačinovo flower
76
stem
40
53
49
51
52
49
leaf
76
79
85
63
100
81
flower
63
66
68
99
60
Bojinac
71
stem
29
38
44
68
14
39
leaf
100
91
100 100
77
94
92
100
78
88
74
Bačić Kuk flower
86
stem
29
40
46
44
44
41