PHYTOTHERAPY RESEARCH

Phytother. Res. 29: 120124 (2015)

Published online 30 September 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5236

Extracts from Calendula officinalis Offer in Vitro

Protection Against H2O2 Induced Oxidative Stress
Cell Killing of Human Skin Cells
Abdullah M. Alnuqaydan,1 Claire E. Lenehan,2 Rachel R. Hughes2 and Barbara J. Sanderson1*
1

Medical Biotechnology, School of Medicine, Faculty of Medicine, Nursing and Health Sciences, Flinders University, Australia
School of Chemical and Physical Sciences, Faculty of Science and Engineering, Flinders University, Australia

The in vitro safety and antioxidant potential of Calendula officinalis flower head extracts was investigated. The
effect of different concentrations (0.125, 0.5, 1.0, 2.0 and 5.0% (v/v)) of Calendula extracts on human skin cells
HaCaT in vitro was explored. Doses of 1.0% (v/v) (0.88 mg dry weight/mL) or less showed no toxicity. Cells were
also exposed to the Calendula extracts for either 4, 24 or 48 h before being exposed to an oxidative insult (hydrogen
peroxide H2O2) for 1 h. Using the MTT cytotoxicity assay, it was observed that two independent extracts of
C. officinalis gave time-dependent and concentration-dependent H2O2 protection against induced oxidative stress
in vitro using human skin cells. Pre-incubation with the Calendula extracts for 24 and 48 h increased survival relative
to the population without extract by 20% and 40% respectively following oxidative challenge. The antioxidant
potential of the Calendula extracts was confirmed using a complimentary chemical technique, the DPPH assay.
Calendula extracts exhibited free radical scavenging abilities. This study demonstrates that Calendula flower
extracts contain bioactive and free radical scavenging compounds that significantly protect against oxidative stress
in a human skin cell culture model. Copyright 2014 John Wiley & Sons, Ltd.
Keywords: Calendula officinalis; flower extracts; cytotoxicity; oxidative stress; antioxidants; human cells.

INTRODUCTION
There is a long history of using flowers and herbal infusions in folk medicine, and they have been studied for
their ability to protect against oxidative stress (Atoui
et al., 2005; Day et al., 1997). Calendula officinalis
(marigold or maravilla) is an herb that is native to the
Mediterranean region. The dried flower heads of
C. officinalis have been used in folk medicine. In some
countries they are also used as a spice or cultivated for
ornamental and medicinal properties (Fonseca et al.,
2010; Ozkol et al., 2012; Re et al., 2009). Calendula
extracts have been shown to exhibit antioxidant, antitumoral, anti-inflammatory, anti-viral and anti-HIV
properties (Jimnez-Medina et al., 2006; Kalvatchev
et al., 1997). The chemical compounds identified in methanol extracts of C. officinalis include polar compounds
phenolic acids (e.g. vanillic, protocatechuic, syringic,
p-coumaric, caffeic and chlorogenic acid) and flavonoid
glycosides (e.g. rutin, narcissin, 3-glucoside of isohamnetin,
isoquercitrin) (Matysik et al., 2005). Other constituents
include sterols, steriods, terpenoids, free and esterified
triterpenic alcohols (Re et al., 2009). Thus, it is clear that
C. officinalis contains a number of compounds with antioxidant and free radical scavenging potential.
Hydroxyl radicals (OH) are generated in biological
systems from hydrogen peroxide (H2O2) by the Fenton
reaction (Chen and Schopfer, 1999). One example of this

* Correspondence to: Barbara J Sanderson, Department of Medical

Biotechnology, School of Medicine, Flinders University of South Australia,
GPO Box 2100, Adelaide, SA 5001, Australia.
E-mail: Barbara.sanderson@flinders.edu.au

Copyright 2014 John Wiley & Sons, Ltd.

is when free radical processing is inadequate in melanin

synthesis and H2O2 is created, leading to the production
of OH and other reactive oxygen species (ROS) (Wang
et al., 2006). Although OH and other ROS are typically
short lived, they do interact with, and damage, cellular
components; including DNA, membrane lipids and
proteins (Chen and Schopfer, 1999). Antioxidants in
cells are required to stop these potentially destructive
reactions. Plant derived antioxidants have the potential
to perform this function. However, measuring the levels
of plant antioxidants in a cell system only partly correlates
with the measured levels of antioxidants in the cells
(Slatnar et al., 2012). Therefore non-cell-based assays
and cell-based bioassays are both necessary to determine the efficacy of plant derived extracts or individual
compounds. The current study examined the antioxidant
potential of two Calendula extracts using an in vitro
human skin cell model with hydrogen peroxide as an
oxidative challenge. This was complemented by use of
the DPPH chemical antioxidant assay.

MATERIALS AND METHODS

Materials. Materials used were 1640 medium (RPMI 1640,
Gibco, Invitrogen, Barcelona, Spain), foetal bovine
serum (FBS) (Gibco, Invitrogen, Barcelona, Spain),
penicillin/streptomycin (Gibco, Invitrogen, Barcelona,
Spain), phosphate buffered saline (PBS) (Sigma),
hydrogen peroxide (H2O2) (Sigma3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma),
Quercetin (Sigma) and 2,2-di(4-tert-octylphenyl)-1picrylhydrazyl (DPPH) (Sigma). All other chemicals
Received 31 January 2014
Revised 09 August 2014
Accepted 04 September 2014

OXIDATIVE STRESS PROTECTION BY CALENDULA OFFICINALIS EXTRACT

were reagent grade from Sigma. Media for growth

and treatment was RPMI with 10% FBS.
Plant extracts. Dried C. officinalis flower heads were supplied by an industry partner from Biodynamically grown
plants. Two extracts were prepared independently (C5 and
C6) using a proprietary process involving distillation and
percolation with water/ethanol-based solvents (Jurlique International, Mount Barker, Australia) and used as supplied.
When appropriate, extracts were diluted in media to prepare the concentration required of the treatment solution.
Plant extracts were characterised by DPPH assay; total
phenolics (see Folin-Ciocalteu Assay); dry weight and
density determination. Density determination was carried
out at 20 C by the average of 10 independent measurements. Data is reported (Table 1) as mean SD, with errors
calculated using the propagation of uncertainty equation when division is being utilised (since density =
mass/volume). Total dry weights (%) of C5 and C6 extracts were determined after samples were freeze dried at

56 C at 3.4 Pa for 24 h. Once all volatiles were removed
from samples, the total dry solids remaining were weighed.
Cell cultures. HaCaT, a human non-cancer keratinocytes
cell line, was obtained from ATCC (The Global Bioresource Center).The cell line was grown in RPMI-1640
medium (supplemented with L-glutamine 2 mM, HEPESNa 25 mM, penicillin 100 U/mL, streptomycin 100 g/mL
and 10% heat-inactivated FBS) at 37 C in a humidified
atmosphere containing 5% CO2. HaCaT cells were seeded
at 1 106 cells/mL and subcultured every 23 days after
6080% confluence was reached.
Cell treatment. Cells were treated to allow extract
expose prior to protection assay (see below). The 96 flat
bottom wells were seeded with 104 cells/well and incubated for 19 h to allow the adherence of cells at 37 C
in 5% CO2. The media were aspirated and replaced
with 200 L of the treatment solution per well and were
treated for 4, 24 and 48 h prior to MTT assay analysis.
Treatment solutions included C5 or C6 extracts (0,
0.125, 0.5,1, 2 and 5% (v/v)). Quercetin (16 g/mL final
concentration) was used as a positive control compound
with known antioxidant capacity. The negative or zero
control was media.

Table 1. The characterisation of the Calendula extracts C5 and C6.

Shown are the results for density determination; measurement of
total dry weight after freeze drying; and GAE results from the
Folin-Ciocalteau assay.
Characteristic
Density at 20 C
(mean SD)
Total dry weight
Gallic acid equivalents
(GAE)(mg GAE/g
dry weight)

Extract C5

Extract C6

1.003 g/mL 0.019 1.002 g/mL 0.019

8.8%
28

8.4%
26

The density of C5 was 1.003 g/mL and C6 was 1.002 g/mL.

Copyright 2014 John Wiley & Sons, Ltd.

121

Induction of oxidative stress to determine protection

activity. Following cell treatment, cells were exposed to
300-M H2O2 for 1 h to induce oxidative stress (Fenech
et al., 1999). Then MTT assay was performed.
MTT cell survival assay. To measure cell viability, the
MTT colorimetric assay was performed as published
(Young et al., 2005; Mosmann, 1983). Briefly, the treatment solution was aspirated, and wells were washed
twice with PBS; cells were then incubated with 200-L
MTT (0.5 mg/mL)/well for 4 h at 37 C. Formazan crystals
were dissolved with sodium dodecyl sulphate (20% SDS
in 0.02 M HCL). The absorbance was measured on an
ELISA plate reader using a reference wavelength of
630 nm and a test wavelength of 570 nm.
DPPH assay. The antioxidant activities of Calendula
extracts were measured in terms of radical scavenging
ability using the stable radical DPPH. A methanolic
stock solution of quercetin (1.6 mg/mL) was prepared as
the positive control with further dilutions of the stock in
methanol (5, 4, 3, 2 and 1% (v/v)) used to determine the linear range of the DPPH assay. Calendula extracts (C5 and
C6) were diluted in methanol (5, 4, 3, 2 and 1% (v/v))
before analysis. The sample (50 L) was put into a cuvette
with 2 mL of a methanolic DPPH solution (6 10
5 mol/l)
and analysed immediately following the method reported
by Politeo et al. (2006). The decrease in absorbance at
517 nm was determined with a Thermo Scientific Evolution
Array UVVisible spectrophotometer after 15 min for all
samples. Methanol was used to zero the spectrophotometer.
Percent inhibition of the DPPH radical by the samples
was calculated according to the formula:
%Inhibition AA0
AAt=AA0100
where AA(0) is the absorbance of the antioxidant solution
at t = 0 min and AA(t) is the absorbance of the antioxidant
solution at t = 15 min.
FolinCiocalteu assay. The FolinCiocalteu assay was
based on the method of Atanassova and Georgieva
(2010). Extracts C5 and C6 were prepared by diluting
200 L of extract up to 10 mL with water (50-fold dilution). Gallic acid aqueous standards were prepared by
adding 5.8, 12.8, 16.4, 22.8 and 27.8 mg in 250 mL of water.
For the FolinCiocalteu Assay, 0.4 mL of diluted extract
or gallic acid standard was added to 3.6 mL of water and
0.4 mL of FolinCiocalteu reagent. The mixture was
shaken and left for 5 min at room temperature. Four
millilitres of 7% sodium carbonate solution was then
added with 1.6-mL water. The mixture was left for 2 h at
room temperature. The absorbance of each solution at
765 nm and 750 nm was measured using a UVVis spectrophotometer. A calibration curve of gallic acid concentration versus absorbance was constructed and from this,
the gallic acid equivalents of the C5 and C6 extracts
were determined. Each gallic acid standard and Calendula
extract was prepared and analysed in triplicate.
Statistical analysis. The experiment was performed in
triplicate, and data is presented as the mean S.E.M.
Phytother. Res. 29: 120124 (2015)

122

A. M. ALNUQAYDAN ET AL.

Table 2. Relative survival at each time point (4, 24 and 48 h) for extracts C5 and C6. HaCaT cells were treated for the time indicated then
assayed by MTT assay (see Materials and Methods)
Dose
Extract
C5

C6

Time
4h
24 h
48 h
4h
24 h
48 h

0% (v/v)
100 28.0
100 0.4
100 0.1
100 30.5
100 0.1
100 0.1

0.125% (v/v)
70.2 15.4
114.6 7.3
108.6 1.9
91.6 0.5
88.9 4.7
107.9 2.6

0.5% (v/v)
85.3 0.9
101.0 6.0
107.5 3.1
94.0 1.2
96.2 5.5
114.1 3.7

1% (v/v)
85.8 1.6
72.3 2.7
97.1 4.7
92.6 5.9
83.7 10.4
108.1 7.2
*

2% (v/v)
90.11 0.9
29.86 2.6
*
60.90 4.7
103.0 5.2
*
52.86 5.4
*
49.97 7.0
*

5% (v/v)
*

62.1 5.3
*
7.1 3.6
*
4.2 2.1
*
74.8 1.0
*
10.9 3.7
*
6.1 2.7

*Significant difference at P < 0.05. Data shown as mean survival relative to the untreated control SEM; n = 3.

One way ANOVA with Turkeys post-hoc test was carried

out using SPSS software (version 19.0). Differences were
considered statistically significant when the P-value was
less than 0.05. The survival in each experiment following
pre-treatment with quercetin, C5 or C6 was calculated relative to the population of cells not treated with H2O2
(zero dose of extract and zero dose of H2O2) which are
represented as 100% survival.

RESULTS AND DISCUSSION

Characterisations of Calendula extracts
The extracts were characterised using multiple methods
to gain an appreciation of the physico-chemical characteristics of the C5 and C6. The average densities of C5
and C6 were 1.003 g/mL and 1.002 g/mL, respectively
(Table 1). The total dry weight, shown in Table 1, was
8.8% for C5 and 8.4% for C6. The FolinCiocalteu assay
was carried out to estimate polyphenol composition
which was expressed using per dry weight determinations
and gallic acid equivalents (GAE, Table 1). The values for
C5 and C6, 28 mg GAE/g dry weight and 26 mg GAE/g
dry weight, respectively, were two orders of magnitude
higher than those published by the authors outlining the
assay method (Atanassova and Georgieva, 2010). They
found values ranging between 0.27 and 0.49 mg GAE/g
dry weight for sage (Salvia officianalis), mint and lemon
balm. Sage is often used as a reference plant in such
circumstances because of the well recorded data on its
antioxidant activity. Clearly the Calendula extracts C5
and C6 contain more compounds with phenolic activity
than the extracts prepared from sage and these other plant
sources. Also, C5 and C6 extracts had higher GAE activity than that of a Calendula extract from another source
which had only 6.6-mg GAE/g extract (Miliauskas et al.,
2004). Presumably this would be even lower if expressed
per dry weight of extract, as is the case for our data.
Toxicity of Calendula plant extracts on HaCaT human
skin cells
The toxicity and protective effects of C5 and C6 Calendula
plant extracts on HaCaT cells in vitro was explored by
incubating cells with extract for 4, 24 and 48 h. Re et al.
(2009) state the need for toxicity studies to appreciate the
safety of plant extracts. In the current study, the two extracts of Calendula (extracts C5 and C6) showed only a
Copyright 2014 John Wiley & Sons, Ltd.

limited toxicity and this was at high doses. The C5 and

C6 extracts at the lowest doses, (0.125, 0.5 and 1.0%
(v/v)) were not toxic on HaCaT skin cells for any of
the treatment periods. However, the highest dose
(5%; C5 = 4.4 mg dry weight/mL and C6 = 4.2 mg dry
weight/mL) of extracts was not toxic for the 4 h treatment
only (Table 2). Hence, C5 and C6 extracts at the highest
doses (2% and 5% (v/v)) showed significant toxicity only
for the 48-h treatment (Table 2). Doses less than 1%
(v/v) (for C5 = 0.88 mg dry weight/mL; C6 = 0.84 mg dry
weight/mL) of the two extracts were not significantly toxic
at 24 h. These results suggest that C5 and C6 extracts
could have toxic substances in low concentration and with
low diffusivity through the cell membrane. Thus, only
high concentrations of extracts which are above those currently used in commercial products would have toxic
components in sufficient quantities to have an effect. This
effect could involve a period of contact between these
components and the cells, such that these toxic components would be taken up by cells, thus reducing the cellular viability. Therefore, in contrast, lower concentrations
of such extracts would not be toxic if incorporated into
products at the safe doses, identified by current findings.
The toxicity at the higher doses could be due to the
concentration of some bio actives becoming toxic above
a given dose and time exposure. Alternately, the reduction
in survival at higher doses could be a result of pro-oxidant
activity acting as a source of free radicals. The diffusion
through the cell membrane at long treatment times may
be such that high, but not low, doses have a pro-oxidant
effect. Consistent with this, Calendula extracts, infusions
and decoctions exhibit in vitro cytotoxic effects and growth
inhibition on human cancer cells in a dose-dependent
fashion (Matic et al., 2013; Jimnez-Medina et al., 2006).
Following C5 and C6 extract treatment at low doses,
there was an increase in cell number, reflected in relative
survival greater than 100%. Similar observations have been
made with Calendula-treated mouse fibroblasts (Fonseca
et al., 2010) and human fibroblasts (Matysik et al., 2005).
Protection by extracts against H2O2 induced oxidative
stress on human skin cells
Oxidative stress induced cell killing by 300-M H2O2
(with no pre-treatment) was significantly different from
the untreated control in every individual experiment.
The relative survival values for each experimental
series ranged from 5% to 35%, indicating that the
oxidative stress challenge induced cell killing (untreated
control + H2O2; Figs. 13). The level of cell killing by
Phytother. Res. 29: 120124 (2015)

123

140

140

120

120

Relative Survival (%)

Relative Survival (%)

OXIDATIVE STRESS PROTECTION BY CALENDULA OFFICINALIS EXTRACT

100
80
60
40
20

80
60
40
20
0

0
0

C5

C6

0.125

0.5

140
120
100
80
60
40
20
0
0

C5

C6

0.125

0.5

Concentration (% (v/v))
Untreated control+H2O2
Quercetin+H2O2

Figure 2. Survival relative to the untreated control, determined by

the MTT assay. Cells were plated in microplates at 10,000 cells/well
then treated for 24 h with C5 or C6, then for 1 h with 300 M H2O2,
except the 0% v/v dose C5 and C6 (untreated controls) that
had no H2O2 added. Q, populations of cells treated with the control
antioxidant quercetin; H2O2 = hydrogen peroxide = oxidative
stress; +, challenged with H2O2; , signficantly different from
untreated control + H2O2, p < 0.05; *, significantly different from
Q + H2O2, p < 0.05. Data shown as mean SEM; n = 3. Quercetin =
0.016 mg/ml; 1% C5 = 0.88 mg dry weight equiv/ml; 1%
C6 = 0.84 mg dry weight equiv/ml.

H2O2 varies however, due to the instability of ROS

(Fenech et al., 1999). Pre-treatment with extracts C5
and C6 of Calendula resulted in protection against
oxidative stress (free radical induced (H2O2) cell killing).
Quercetin, the positive control, offered protection at all
time-points for the dose tested (16 g/mL). For the 4-h
pre-treatment with extracts C5 and C6, there was dosedependent protection against oxidative stress, with 2%
(v/v) and 5% (v/v) offering significant protection against
H2O2 induced cell killing (Fig. 1).This is evident as greater
relative survival at these doses post-H2O2 challenge
(Unfilled bars; Fig. 1) compared to H2O2 without any
extract pre-treatment (Bars with horizontal stripes;
Fig. 1). Significant protection against the oxidative
stress was also observed for a 24-h pre-treatment for
1% (v/v) of C5 and C6 extracts (Fig. 2). Protection for
Copyright 2014 John Wiley & Sons, Ltd.

0.125

0.5

Concentration (% (v/v))

Concentration (% (v/v))
Untreated control+H2O2
Quercetin+H2O2

Figure 1. Survival relative to the untreated control, determined by

the MTT assay. Cells were plated in microplates at 10,000 cells/well
then treated for 4 h with C5 or C6, then for 1 h with 300 M H2O2,
except the 0% v/v dose C5 and C6 (untreated controls) that had no
H2O2 added. Q, populations of cells treated with the control antioxidant
quercetin; H2O2, hydrogen peroxide = oxidative stress; +, challenged
with H2O2; , significantly different from untreated 0 control + H2O2,
p < 0.05; *, significantly different from Q + H2O2, p < 0.05. Data shown
as mean SEM; n = 3. Quercetin = 0.016 mg/ml; 1% C5 = 0.88 mg
dry weight equiv/ml; 1% C6 = 0.84 mg dry weight equiv/ml.

Relative Survival (%)

100

C5

C6

Untreated control+H2O2

Quercetin+H2O2

Figure 3. Survival relative to the untreated control, determined by

the MTT assay. Cells were plated in microplates at 10,000 cells/well
then treated for 48 h with C5 or C6, then for 1 h with 300 M H2O2,
except the 0% v/v dose C5 and C6 (untreated controls) that had
no H2O2 added. Q, populations of cells treated with the control
antioxidant quercetin; H2O2, hydrogen peroxide = oxidative stress; +,
challenged with H2O2; , significantly different from untreated control +
H2O2, p < 0.05; *, significantly different from Q + H2O2, p < 0.05.
Data shown as mean SEM; n = 3. Quercetin = 0.016 mg/ml;
1% C5 = 0.88 mg dry weight equiv/ml; 1% C6 = 0.84 mg dry
weight equiv/ml.

48-h pre-treatment with the extracts was also observed.

At the lower doses (0.1251% (v/v)), relative cell
survival following pre-treatment and H2O2 challenge
was significantly above relative cell survival following
H2O2 treatment without any extract pre-treatment
(Fig. 3). The protection by extracts was time-dependent.
The 48 h pre-treatments offered more protection against
oxidative stress induced cell killing for HaCaT skin cells
than either the 4 or 24-h treatments. This could be due
to the longer incubation time offering more time for
cellular uptake of bio-actives in the extracts. This
time-course could reflect low diffusivity across the cell
membrane such that long times are required for the
antioxidants to enter and protect the cells. This shows
that not only is it safe to expose cells to low doses for
a longer time, but that these low doses for a longer
time offer the best protection against oxidative stress
induced cell killing. In vitro antioxidant protection by
Calendula extracts has also been demonstrated by
Ahmad et al. (2012) by screening for nitric oxide free
radical scavenging. They propose that such activity in
extracts could have the potential to slow down the

Figure 4. The percentage free radical inhibition of quercetin, C6

and C5 solutions, including the linear line of best fit between antioxidant concentration and free radical scavenging ability. 1% quercetin =
0.016 mg/ml; 1% C5 = 0.88 mg dry weight equiv/ml; 1%
C6 = 0.84 mg dry weight equiv/ml. The level of antioxidant activity
was determined by DPPH assay. Each value represents the mean
SEM; n = 3.
Phytother. Res. 29: 120124 (2015)

124

A. M. ALNUQAYDAN ET AL.

Table 3. Free radical inhibitory concentrations (ICs) determined by

the DPPH assay for Quercetin (Q) and extracts C5 and C6. Shown
is the dose inhibiting 20% of free radicals (IC20); the protective dose
for these agents based on performance in the MTT oxidative
challenge bioassay; and the IC level (predicted from the DPPH
assay) equivalent to the protective dose at 24 h (Fig. 1)
24-h extract pre-treatment

Sample
Q
C5
C6

IC20
52 g/mL
4.3% (v/v)
3.7% (v/v)

Dose significantly
protecting against
H2O2

Predicted IC
of protective dose

16 g/mL
1% (v/v)
1% (v/v)

IC8
IC7
IC11

ageing process. The current study is consistent with this

theory, in particular for cellular protection at the lower
non-toxic doses for a 48-h pre-treatment.
The DPPH assay revealed that C5, C6 and the positive control quercetin, all exhibited free radical inhibition
in a linear dose-dependent manner for a 10-min reaction
time (Fig. 4).
The IC20 values calculated from the graph are summarised in Table 3, and show that 4.3% (v/v) of C5 and
3.7% (v/v) of C6 had the same antioxidant activity (IC20)
as 52 g/mL of quercetin. Also shown in Table 3 are the
predicted IC values for the concentrations of quercetin,
C5 and C6 that showed protective effects in the 24-h
cellular challenge bioassay. The IC values are similar.
Extrapolating from the DPPH assay IC values, it would

be predicted that protection would occur at a level similar

to quercetin for the 1% (v/v) concentrations of the
extracts. However, for the 48 h pre-treatment, cellular
antioxidant protection at 0.1% (v/v) is equally as effective
as 16 g/mL quercetin (Fig. 3). The lower concentrations
would not be predicted from the DPPH assay results
to have such strong activity. Thus, both assays are
needed to gain an understanding of the antioxidant
potential of extracts.
In conclusion, Calendula flower extracts protect
HaCaT skin cells against an oxidative stress challenge
in the form of H2O2. Such oxidative stress can result
from normal metabolism giving rise to charged particles,
including OH and ROS. This can lead to oxidation of
cellular components, which in turn can lead to inflammation, skin ageing or cancer. C. officinalis dried flower
extracts could function as antioxidants to protect against
such damage. Such extracts have potential usefulness as
product additives that offer protection against oxidative
stress damage. However, further work is needed to fully
characterise the protective action of these extracts,
including studies on human skin fibroblasts.
Acknowledgements
Authors would like to thank the Ministry of Higher Education, Saudi
Arabia, for partial support of this project.

Conflict of Interest
The authors have declared that there is no conflict of interest.

REFERENCES
Ahmad H, Khan I, Wahid A. 2012. Antiglycation and antioxidation
properties of juglans regia and calendula officinalis: possible
role in reducing diabetic complications and slowing down ageing. J Tradit Chin Med 32(3): 411414.
Atoui AK, Mansouri A, Boskou G, Kefalas P. 2005. Tea and herbal
infusions: Their antioxidant activity and phenolic profile. Food
Chem 89: 2736.
Atanassova M, Georgieva S. 2010. Comparative polyphenol
compostion and antioxidant capactiy of the Bulgarian plants
(dry herbs). Elec J Environ Ag Food Chem 9(9): 15141523.
Chen SX, Schopfer P. 1999. Hydroxyl-radical production in physiological reactions. A novel function of peroxidase. Eur J
Biochem 260: 726735.
Day AP, Kemp HJ, Bolton C, Hartog M, Stansbie D. 1997. Effect of
Concentrated Red Grape Juice Consumption on Serum Antioxidant Capacity and Low-Density Lipoprotein Oxidation. Ann
Nutr Metabol 41: 353357.
Fenech M, Crott J, Turner J, Brown S. 1999. Necrosis, apoptosis,
cytostasis and DNA damage in human lymphocytes measured
simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide. Mutagenesis 14: 605612.
Fonseca YM, Catini CD, Vicentini FT, Nomizo A, Gerlach RF,
Fonseca MJ. 2010. Protective effect of Calendula officinalis
extract against UVB-induced oxidative stress in skin: evaluation of reduced glutathione levels and matrix metalloproteinase secretion. J Ethnopharmacol 127: 596601.
Jimnez-Medina E, Garcia-Lora A, Paco L, Algarra I, Collado A,
Garrido F. 2006. A new extract of the plant Calendula officinalis
produces a dual in vitro effect: cytotoxic anti-tumor activity and
lymphocyte activation. BMC Cancer 6: 119127.
Kalvatchev Z, Walder R, Garzaro D. 1997. Anti-HIV activity of extracts from Calendula officinalis flowers. Biomed Pharmacother
51: 176180.

Copyright 2014 John Wiley & Sons, Ltd.

Matysik G, Wojciak-Kosior M, Paduch R. 2005. The influence of

Calendulae officinalis flos extracts on cell cultures, and the
chromatographic analysis of extracts. J Pharm Biomed Anal
38: 285292.
Matic IZ, Juranic Z, Savikin K, Zdunic G, Nadvinski N, Godevac D.
2013. Chamomile and marigold tea: chemical characterization
and evaluation of anticancer activity. Phytother Res 27: 852858.
Miliauskas G, Venskutonis PR, van Beek TA. 2004. Screening of
radical scavenging activity of some medicinal and aromatic
plant extracts. Food Chem 85: 231237.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 5563.
Ozkol H, Tuluce Y, Koyuncu I. 2012. Subacute effect of cigarette
smoke exposure in rats: protection by pot marigold (Calendula
officinalis L.) extract. Toxicol Ind Health 28: 39.
Politeo O, Juki M, Milo M. 2006. Chemical composition and antioxidant activity of essential oils of twelve spice plants.
Croatica chemica acta 79: 545552.
Re TA, Mooney D, Antignac E, et al. 2009. Application of the
threshold of toxicological concern approach for the safety
evaluation of calendula flower (Calendula officinalis) petals
and extracts used in cosmetic and personal care products.
Food Chem Toxicol 47: 12461254.
Slatnar A, Jakopic J, Stampar F, Veberic R, Jamnik P. 2012. The
Effect of Bioactive Compounds on In Vitro and In Vivo Antioxidant Activity of Different Berry Juices. PLoS One 7: e47880.
Wang K-H, Lin R-D, Hsu F-L, et al. 2006. Cosmetic applications of selected traditional Chinese herbal medicines. J Ethnopharmacol
106: 353359.
Young FM, Phungtamdet W, Sanderson BJ. 2005. Modification of
MTT assay conditions to examine the cytotoxic effects of
amitraz on the human lymphoblastoid cell line, WIL2NS.
Toxicol Vitro 19: 10511059.

Phytother. Res. 29: 120124 (2015)