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HERMED 161 No. of Pages 9

Journal of Herbal Medicine xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Journal of Herbal Medicine

journal homepage: www.elsevier.com/locate/hermed

Research paper

In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant

activities and phenolic content in wild-grown flowers of common
daisy—A medicinal plant
Fatma Pehlivan Karakasa,b,* , Arzu Ucar Turkerb , Alper Karakasb , Vakhtang Mshvildadzec,
Andre Pichettec , Jean Legaultc
a
Abant Izzet Baysal University, Department of Field Crops, Faculty of Agriculture and Natural Sciences, Bolu, Turkey
b
Abant Izzet Baysal University, Department of Biology, Faculty of Science and Art, Bolu, Turkey
c
Laboratoire d’Analyse et de Séparation des Essences Végétales (LASEVE), Département des Sciences Fondamentales, Université du Québec à Chicoutimi
(UQAC), 555 boul. de l’Université, Chicoutimi, Québec, Canada

A R T I C L E I N F O A B S T R A C T

Article history:
Received 18 November 2015 Bellis perennis L. is a medicinal plant in the family Compositae. It has been used as a remedy for wounds,
Received in revised form 27 June 2016 rheumatism, eczema, eye diseases, inflammation and tonsillitis in folk medicine. In the present study, 19
Accepted 21 November 2016 different extracts and two fractions were obtained from wild-grown flowers, leaves and/or in vitro-grown
Available online xxx leaves of common daisy by using different solvents and extraction methods. Biological activities of these
extracts and fractions were assessed using selected bioassays: cytotoxic activity, disc diffusion assay,
Keywords: radical scavenging activity (DPPH), total phenolic content, oxygen radical absorbing capacity (ORAC) and
Bellis perennis 20 ,70 -dichlorofluorescin-diacetate (DCFH-DA) cell-based assays. The cytotoxic activity of extracts and
Cytotoxic
fractions was investigated against human lung carcinoma (A-549) and colon adenocarcinoma (DLD-1)
Antibacterial
cells. In vitro-grown leaf extracts showed the highest cytotoxic activity against selected cell lines.
Antioxidant
Anti-inflammatory Moreover, n-butanol (n-BuOH) and ethyl acetate (EtOAc) fractions of flowers exerted high levels of
Extraction cytotoxic activity. The MeOH extract and the EtOAc fraction of flowers exhibited broad-spectrum
antibacterial activity against Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis,
and Enterobacter cloacea. The strongest antioxidant activity was found in the EtOAc fraction of flowers
with the highest amount of phenolic content and ORAC value. The MeOH extract of flowers showed
strong anti-inflammatory activity on RAW 264.7 macrophages. The amount of the chosen 22 phenolic
compounds in dichloromethane (DCM), MeOH extracts, n-BuOH and EtOAc fractions of field-grown
flowers was detected using LC–ESI–MS/MS. The results of these studies support the potential use of B.
perennis for wounds, rheumatism, inflammation, cancer and eye diseases.
© 2016 Elsevier GmbH. All rights reserved.

1. Introduction bioassays is beneficial for the standardization and quality control

Many plant materials have been used as herbal teas or other
homemade remedies for many years in traditional medicine
(Dzhambazov et al., 2002). They have been generous sources of
medicines because they generate a host of secondary bioactive
compounds; most of which presumably evolved as chemical
defenses against bacterial infections (Cox and Balick, 1994).
Determination of the biological activities of medicinal plants with
* Corresponding author at: Abant Izzet Baysal University, Faculty of Agriculture
and Natural Sciences, Department of Field Crops, 14280 Bolu, Turkey.
E-mail addresses: fatmapehlivankarakas@gmail.com, pehlivan_f@ibu.edu.tr
(F.P. Karakas).
of heterogeneous plant secondary metabolites (McLaughlin et al.,
1998). Screening studies for medicinal plants are also important
because folkloric usage of these plants gains some scientific
justification.
Bellis perennis L. (common daisy) is a medicinal plant species in
the Compositae family (Panda, 2004; Davis, 1975), which has been
known as a popular wound-healing plant in Europe since ancient
times (Karakas et al., 2012a). The aerial parts of B. perennis have
been used for the treatment of rheumatism, (Morikawa et al.,
2011), common cold (Cakılcıoglu et al., 2010) and headache (Uzun
et al., 2004). They are utilized for their expectorant, sedative and
anti-inflammatory activities (Siatka and Kasparova, 2010) in
traditional medicine. Some traditional usages of B. perennis, such

http://dx.doi.org/10.1016/j.hermed.2016.11.003
2210-8033/© 2016 Elsevier GmbH. All rights reserved.

Please cite this article in press as: F.P. Karakas, et al., In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant activities and phenolic
content in wild-grown flowers of common daisy—A medicinal plant, J. Herbal Med. (2016), http://dx.doi.org/10.1016/j.hermed.2016.11.003
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HERMED 161 No. of Pages 9
2 F.P. Karakas et al. / Journal of Herbal Medicine xxx (2016) xxx–xxx
as for their wound healing (Karakas et al., 2012a) and sedative
activities (Karakas et al., 2011), have been verified with controlled
experimental animal studies. The aerial parts (especially flowers)
of B. perennis have been also used as an herbal tea against breast
and uterus cancer (Duke et al., 2002). Li et al. (2005) indicated the
cytotoxic activities of six triterpenoid saponins from the roots of B.
perennis against HL-60 human promyelocytic leukemia cells.
Furthermore, Karakas et al. (2015) showed the moderate
antiproliferative activity of MeOH extract obtained from B. perennis
flowers on MCF-7 human breast cancer and HepG2/C3A human
hepatocellular carcinoma cells.
Although common daisy has many areas of usage in traditional
medicine, there are no reports of some of the important biological
activities for field-grown flowers, leaves and in vitro-derived leaves
in the literature. In the present study, the cytotoxic activity of wild-
grown leaf, in vitro-grown derived leaf, and flower extracts was
investigated against human lung cancer (A-549) and human colon
cancer (DLD-1) cell lines with the resazurine reduction test. The
antibacterial activity of B. perennis extracts and fractions was
evaluated against ten different bacterial strains. The antioxidant
activity of 12 different B. perennis extracts [with hexane, dichloro-
methane (DCM), MeOH and water] of wild-grown flowers, leaves
and in vitro-derived leaves was examined using four different
antioxidant methods; free radical scavenging activity (DPPH), total
phenolic content (Folin-Ciocalteau), oxygen radical absorbing
capacity (ORAC) and direct antioxidant assays [cell-based assay
using 20 .70 -dichlorofluorescin-diacetate (DCFH-DA)]. Additionally,
the phenolic content of wild-grown flowers of B. perennis was
detected by LC–ESI–MS/MS analysis. The current research indi-
cates, to our knowledge for the first time, the antiproliferative,
antibacterial and antioxidant activities of B. perennis using
different extracts obtained from different extraction methods,
different plant parts and sources, and bioassays.
2. Materials and methods
2.1. Plant material and extract preparation
Aerial parts (flowers and leaves) of wild-grown B. perennis were
collected from Gölköy, Bolu/Turkey in May 2011. Identification of
the species was made by Prof. Dr. Arzu Ucar Turker (collection
number AUT-1909) and the specimen was deposited at Abant Izzet
Baysal University (AIBU) Herbarium, Bolu, Turkey. In vitro-derived
leaves of B. perennis were collected from plantlets that were
propagated with established protocol in plant tissue culture
laboratory (Karakas and Turker, 2013). Three different sources of
plant (wild-grown flowers, wild-grown leaves and in vitro-derived
leaves) were dried in an incubator at 37  C for three days. Five
different types of extraction methods (soxhlet, water bath,
decoction, infusion and liquid-liquid extraction) were performed
for flowers, and the Soxhlet extraction method was used for wild-
grown leaves and in vitro-derived leaves.
2.1.1. Soxhlet extraction
Three different plant materials [wild-grown flowers (70 g),
wild-grown leaves (70 g) and in vitro-derived leaves (55 g)] were
weighed for Soxhlet extraction. The dried powdered plant parts
from B. perennis were alternately extracted with hexane (at 65–
70  C), DCM (at 55–60  C), MeOH (at 60  C) and water (at 80  C)
using a Soxhlet apparatus (700 ml of each solvent for 24 h). The
extracts were filtered and pooled.
2.1.2. Water bath extraction
The powdered flowers of B. perennis (20 g) were extracted with
hot water (250 ml), cold water (250 ml), ethanol (EtOH; 200 ml),
Please cite this article in press as: F.P. Karakas, et al., In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant activities and phenolic
content in wild-grown flowers of common daisy—A medicinal plant, J. Herbal Med. (2016), http://dx.doi.org/10.1016/j.hermed.2016.11.003
methanol (200 ml) or acetone (200 ml) in a water bath at 45  C for
12 h and then filtered.
2.1.3. Decoction
The powdered flowers of B. perennis (10 g) were boiled in 100 ml
of distilled water for 1 h and the obtained decoction was filtered.
The same procedure was repeated three times and the results of
the three successive extractions were collected and combined.
2.1.4. Infusion
Pour 100 ml boiling distilled water over 10 g of powdered
flowers of B. perennis, mix and leave for 1 h at room temperature;
the resulting infusion was filtered and the procedure was repeated
three times as described above. Crude aqueous extracts were
lyophilized.
2.1.5. Liquid-liquid extraction
The powdered flowers of B. perennis (400 g) were comprehen-
sively extracted with MeOH (3 l, 60  C, three times, 1.5 h each time)
followed by MeOH-H2O 80:20 with heating. The extracts were
filtered and pooled. After evaporation of MeOH in vacuum, the
aqueous phase was extracted successively with DCM (3  500 ml),
EtOAc (5  500 ml) and saturated n-butanol (n-BuOH) with H2O
(60:40; 5  500 ml) using liquid-liquid extraction. The n-BuOH and
EtOAc phases were separated and decanted.
Aqueous extracts were evaporated using a lyophilizator at
55  C. All other solvents were evaporated under low pressure at
40  C using a rotary evaporator. The designation of the extracts,
name of the extracts, part used, and name of the extraction method
for each extract are given in Table 1.
2.2. Human cancer cell lines and culture conditions
The human lung carcinoma (A-549), colon adenocarcinoma
(DLD-1) and normal skin fibroblast (WS-1) cell lines were provided
from the American Type Culture Collection (ATCC, Manassas, VA,
USA). All cell lines were cultured in Dulbecco’s minimum essential
medium (DMEM) with Earle’s salts. A 10% fetal bovine serum, a
solution of vitamins, sodium pyruvate and non-essential amino
acids (all at a 1:100 v/v dilution of supplied solutions), penicillin
(100 IU/ml) and streptomycin (100 mg/ml) were added to the
culture medium. Cells were maintained at 37  C in a humidified
environment containing 5% CO2 (Legault and Pichette, 2007).
2.3. Cytotoxicity by resazurin assay
The cytotoxic activity of extracts obtained from wild-grown
flowers, wild-grown leaves and in vitro-grown leaves was analyzed
using resazurin on an automated 96-well Fluoroskan Ascent F1TM
plate reader (Labsystems) as described by O’Brien et al. (2000).
Etoposide was used as a positive control. Cytotoxic activities of
tested extracts were showed as means  standard deviation and
indicate the concentration inhibiting 50% of cell growth (IC50).
Each study was carried out three times in triplicate.
2.4. Antibacterial activity assay
The disc diffusion assay (Kirby-Bauer Method) was used to
evaluate the antibacterial activities of 19 different extracts
obtained from B. perennis, with some modifications (Karakaş
et al., 2012b). Ten bacterial strains were used in the bioassay: the
Gram-negative bacteria Escherichia coli (ATCC 25922), Pseudomo-
nas aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC
14028), Serratia marcescens (ATCC 8100), Proteus vulgaris (ATCC
13315), Enterobacter cloacae (ATCC 23355) and Klebsiella pneumo-
niae (ATTC 13883), and the Gram-positive bacteria Streptococcus
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F.P. Karakas et al. / Journal of Herbal Medicine xxx (2016) xxx–xxx
Table 1
Designation of the extracts, name of the extracts, part used and name of the extraction method for each extract.
Designation of the extracts
BFD
BFD
BFM
BFW
BFHW
BFCW
BFEt
BFMe
BFA
BF-dec.
BF-inf.
BFN
BFE
BLH
BLD
BLM
BLW
BIH
BID
BIM
BIW
pyogenes (ATTC 19615), Staphylococcus aureus (ATTC 25923) and
Staphylococcus epidermidis (ATCC 12228). Positive controls includ-
ed five different antimicrobial susceptibility test discs (Bio-
analyse1): erythromycin (15 mg) (E-15), ampicillin (10 mg) (AM-
10), carbenicillin (100 mg) (CB-100), tetracycline (30 mg) (TE-30)
and chloramphenicol (30 mg) (C-30). A negative control consisted
of water and DMSO. Inoculated plates with discs were placed in a
37  C incubator. After 16–18 h of incubation, the diameter (mm) of
the bacteria inhibition zone was measured. All experiments were
repeated three times.
2.5. Anti-inflammatory activity assay
To investigate the anti-inflammatory activity of wild-grown
flower, leaf and in vitro-grown leaf extracts obtained from B.
perennis, nitric oxide (NO) production in LPS-stimulated RAW
264.7 murine monocyte-macrophage (ATCC #TIB-71) cells was
examined as described by Green et al. (1990). Macrophage survival
was assessed using the resazurin reduction test.
2.6. Antioxidant activities of wild-grown leaves, flowers and in vitro-
grown leaves
2.6.1. DPPH radical scavenging assay
Antioxidant activities such as radical scavenging activity of
methanolic extracts from B. perennis against DPPH (2,2-diphenyl-
1-picrylhydrazyl; Sigma-Aldrich Chemie, Steinheim, Germany)
was performed by the method of Brand-Williams et al. (1995) with
some modifications. All methanolic extract stock solutions were
made by dissolving 0.003 g of dry extract in 3 ml of methanol. The
extract solutions were diluted with methanol (5, 10, 25, 50, 100 and
200 mg/ml) from the stock extract solutions. The solution of DPPH
in methanol (1.5  105 M) was prepared daily. 0.5 ml of this
solution was mixed with 1.5 ml of extract solution in a 10 cm path
length glass tube (the final mass ratio of extracts with DPPH was
3:1). The samples were maintained in the dark for 30 min at room
temperature and then the decrease in absorption was detected at
517 nm. The absorption of the blank sample (positive control)
Please cite this article in press as: F.P. Karakas, et al., In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant activities and phenolic
content in wild-grown flowers of common daisy—A medicinal plant, J. Herbal Med. (2016), http://dx.doi.org/10.1016/j.hermed.2016.11.003
3
Name of the extracts and used extraction methods
Hexane extract of B. perennis – flowers (soxhlet)
DCM extract of B. perennis – flowers (soxhlet)
MeOH extract of B. perennis – flowers (soxhlet)
Water extract of B. perennis – flowers (soxhlet)
Hot water extract of B. perennis – flowers (water-bath)
Cold water extract of B. perennis – flowers (water-bath)
EtOH extract of B. perennis – flowers (water-bath)
MeOH extract of B. perennis – flowers (water-bath)
Acetone extract of B. perennis – flowers (water-bath)
Decoction extract of B. perennis – flowers (decoction)
Infusion extract of B. perennis – flowers (infusion)
n-BuOH fraction of B. perennis – flowers (liquid-liquid extraction)
Ethyl acetate fraction of B. perennis – flowers (liquid-liquid extraction)
Hexane extract of B. perennis – leaves (soxhlet)
DCM extract of B. perennis – leaves (soxhlet)
MeOH extract of B. perennis – leaves (soxhlet)
Water extract of B. perennis – leaves (soxhlet)
Hexane extract of B. perennis – in vitro (soxhlet)
DCM extract of B. perennis – in vitro (soxhlet)
MeOH extract of B. perennis – in vitro (soxhlet)
Water extract of B. perennis – in vitro (soxhlet)
including the same amount of methanol and DPPH solution was
prepared daily and measured at the same time as the extract
samples. The experiment was carried out in triplicate. The radical
scavenging activity of B. perennis methanolic extracts was
calculated by the following formula (Gülçin et al., 2003):
% Inhibition = [(AB  AA)/AB]  100
AB = absorption of blank sample (control)
AA = absorption of tested extract solution.
2.6.2. Total phenolic assay (Folin-Ciocalteau method)
In this experiment, the total phenolic content of extracts was
detected using the Folin- Ciocalteu (FC) reagent according to the
method demonstrated by Singleton and Rossi (1965) with some
modifications. 50 ml, including increasing levels of extracts and
25 ml of FC reagent were added to 96-well plates. After 2 min,
125 ml of sodium bicarbonate (NaHCO3) solution was put into each
well. The tested 96-well plates were kept in the dark for 2 h at
22  2  C. Their absorbances were measured at 758 nm using an
automated 96-well Varioskan Ascent plate reader. The total
phenolic content was measured as tannic acid equivalents (TAE).
2.6.3. ORAC assay (oxygen radical absorbing capacity)
The ORAC values of wild-grown flower, leaf and in vitro-grown
leaf extracts were measured as described by Ou et al. (2001). ORAC
values were illustrated in mmoles of Trolox equivalents (TE) per
gram (mmol TE/g).
2.6.4. Antioxidant cell-based assay using 20 .70 -dichlorofluorescin-
diacetate (DCFH-DA)
The cellular antioxidant activity of wild-grown flower, leaf and
in vitro-grown leaf extracts was demonstrated using the DCFH-DA
assay as designated by Legault et al. (2003). Quercetin and trolox
were used as reference compounds. The IC50 was calculated using
the logarithmic regression of the dose-response curve. In all tests,
the coefficients of detection of the regression (R2) was higher than
0.95. The IC50 value of the tested extracts was calculated as the
mean  standard deviations of three different tests.
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2.7. Determination of phenolic compounds by LC–ESI–MS/MS analysis Table 2

In vitro cytotoxic results of B. perennis extracts and fractions on human lung
carcinoma (A-549) and colon adenocarcinoma (DLD-1) cell lines.
Determination of the selected 22 phenolics (apigenin, caffeic
acid, kaempferol 3-b-D-glucopyranoside, rutin hydrate, vanillic IC50 (mg ml1)a
acid, luteolin 7-O-b-D-glucoside, p-coumaric acid, coumarin, gallic A-549 DLD-1 WS-1
acid monohydrate, epicatechin, epigallocatechin, quercetin, pyro- BFH >200 >200 >200
catechol, procyanidin B1, genistein, procyanidin B2, daidzein, BFD 117  9 164  8 >200
procyanidin C1, resveratrol, taxifolin hydrate, myricetin and BFM 78  7 75  2 59  21
catechin) in the DCM and MeOH extracts, and the n-BuOH and BFW >200 >200 >200

EtOAc fractions of wild-grown flowers was performed using liquid

BF-inf. >200 >200 >200
chromatography-electrospray ionization-multi stage/mass spec- BF-inf. >200 >200 >200
trometry (LC–ESI–MS/MS) method. Analysis was performed by BFN 16  3 10  2 16  3
“METU Central Laboratory, Molecular Biology-Biotechnology BFE 25  8 20  4 54  7
Research and Development Center, Mass Spectroscopy Laboratory,
BLH >200 >200 >200
Ankara, Turkey”. Details of the method can be found in the study by BLD >200 >200 >200
Karakas and Turker (2013). BLM 88  8 93  12 67  6
BLW >200 >200 >200
3. Results and discussion
BIH 128  14 178  7 >200
BID 15.6  0.5 69  14 24  4
3.1. Cytotoxicity BIM 61  6 62  7 46  5
BIW >200 >200 >200
There are no reports about the cytotoxic activity of aerial parts
Etoposideb 8.9  0.9 mM 6.7  0.2 mM 34  3 mM
of B. perennis extracts on human lung carcinoma (A-549) and
human colon adenocarcinoma (DLD-1) cell lines up to now. Six Note: Mean values (standart deviation) for triplicate assays.
a
saponins isolated from the roots of B. perennis showed cytotoxic Concentration of extract that cause 50% inhibition of cell proliferation.
b
Positive control.
activity against HL-60 cells (Li et al., 2005). In another study,
moderate antiproliferative activity of a MeOH extract of B. perennis
on MCF-7 and HepG2/C3A cell lines was shown by the 3-(4,5- flowers, leaves and in vitro-derived leaves may be attributed to
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) their high saponin content. Extracts of different parts (field-grown
test (Karakas et al., 2015). In the present study, the cytotoxicity flowers, field-grown leaves and in vitro-derived leaves) of B.
of hexane, DCM, MeOH and water extracts of wild-grown flowers, perennis have different levels of cytotoxic activity against the A-
wild-grown leaves and in vitro-derived leaves, and two fractions 549 and DLD-1 human cell lines. Altogether, these data are
(n-BuOH and EtOAc) of B. perennis was evaluated against to human consistent with the traditional use of B. perennis in the treatment of
lung carcinoma (A-549) and human colon adenocarcinoma (DLD- some cancer types.
1) cell lines with the resazurin test. Inhibition of cell proliferation
was detected using the resazurin test as the vital strain. A tested 3.2. Antibacterial activity
medicinal plant extract is usually valued as significant for in vitro
cytotoxic activity when IC50 value is <100 mg/ml (Boyd, 1997). Antibacterial activity of 19 extracts of different plant parts
In the resazurin test, among the tested extracts, the in vitro- (field-grown flower, leaf and in vitro-derived leaf) obtained from B.
derived leaf extracts showed the best cytotoxic activity against the perennis was tested with the disc diffusion assay (Kirby-Bauer
chosen cell lines (Table 2). The DCM extract of in vitro-derived Method; Prescott et al., 1990; Karakaş et al., 2012b). Generally, the
leaves showed a strong cytotoxic activity against human lung Gram-positive bacteria (S. aureus, S. pyogenes and S. epidermidis)
carcinoma (A-549) cells, with IC50 values of 15.6  0.5 mg/ml and exhibited higher sensitivity to the flower extracts of B. perennis
showed moderate cytotoxic activity against human colon adeno- than the Gram-negative bacteria (Table 3). The sensitivity of Gram-
carcinoma (DLD-1) cells, with IC50 values of 69  14 mg/ml positive bacteria may be dependent on their cell wall structure
(Table 2). The MeOH extracts of in vitro-derived leaves showed consisting of one layer, while the cell wall of Gram-negative
moderate cytotoxic activity against A-549 and DLD-1 cell lines, bacteria is a multi-layered formation (Essawi and Srour, 2000).
with IC50 values of 61  6 and 62  7 mg/ml, respectively (Table 2). Usually, Gram-negative bacteria are more resistant than Gram-
However, hexane and water extracts were inactive against A-549 positive bacteria (Duraipandian and Ignacimuthu, 2007). Refer-
and DLD-1 with an IC50 value of >100 mg/ml (Table 2). In addition, ence antibiotics used as positive controls exhibited strong
the n-BuOH fraction exerted the most potent cytotoxic activity antibacterial activity to tested organisms. All tested extracts were
(with IC50 values of 16  3 mg/ml and 10  2 mg/ml), showing diluted with distilled water or DMSO, so they were used as a
stronger activity than the EtOAc fraction (with IC50 values of negative control and no inhibition zone was observed with them
25  8 mg/ml and 20  4 mg/ml) towards the A-549 and DLD-1 (Table 3).
human cell lines (Table 2). MeOH extracts of field-grown flowers In the present study, field-grown plant extracts indicated better
and leaves showed moderate cytotoxic activity against A-549 and antibacterial activities than in vitro-derived plant extracts. Among
DLD-1 cell lines, with IC50 values of 78  7; 88  8 and 75  2; all plant extracts, methanol extracts were better than hexane, DCM
93  12 mg/ml, respectively (Table 2). However, hexane, DCM and and water extracts. The best antibacterial activity was obtained
water extracts of field-grown flowers and leaves were inactive with the methanolic extract of field-grown flowers (water bath
against A-549 and DLD-1 with an IC50 value of >100 mg/ml extraction) (BFMe) against S. aureus (16 mm) and S. epidermidis
(Table 2). (23.2 mm), and the EtOAc fraction of field-grown flowers (BFE)
Saponins are well known compounds in B. perennis (Karakas against S. pyogenes (12.4 mm) and E. cloacae (15.9 mm) (Table 3).
et al., 2014). They have cytotoxic activity against HL-60 cells (Li BFMe (23.2 mm), acetone extract of field-grown flowers (BFA)
et al., 2005) and antitumor activities (Karakas and Turker, 2013). (20.3 mm), n-BuOH fraction of field-grown flowers (BFN)
Thus, in our present study, the high anticancer activity of the DCM (19.9 mm), cold water extract of field-grown flowers (BFCW)
extract of in vitro-derived leaves and MeOH extracts of field-grown (19.6 mm), ethanolic extract of field-grown flowers (BFEt)

Please cite this article in press as: F.P. Karakas, et al., In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant activities and phenolic
content in wild-grown flowers of common daisy—A medicinal plant, J. Herbal Med. (2016), http://dx.doi.org/10.1016/j.hermed.2016.11.003
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F.P. Karakas et al. / Journal of Herbal Medicine xxx (2016) xxx–xxx 5

Table 3
The antibacterial activity of B. perennis extracts and fractions. Data presented as zone of inhibition of bacterial growth in mm.

Mean diameter of inhibitory zones (mm  SE)

Gram (+) bacteria Gram () bacteria

Treatments S. pyogenes S. auerus S. epidermidis S. marcescens S. typhimurium P. aeruginosa P. vulgaris K. pneumonia E. cloacae E. coli
BFCW – 12.3  0.1g 19.6  0.3f – – – – – 8.1  0.9g –
BFHW – 10.4  0.1h,i 11.1  0.2i – – – – – – –
BFEt 10.9  0.2g 13.3  0.3f 13.9  0.3g – – – – – – –
BFMe – 16.0  0.3e 23.2  0.4e – – – – – 11.6  0.3e –
BFA 11.1  0.2g 9.4  0.2j 20.3  0.5f – – – – – – –

BFH 8.5  0.3h – – – – – – – – –

BFD – – – – – – – – – –
BFM 8.2  0.1h – 12.3  0.4h – – – – – – –
BFW – – 8.5  0.2j – – – – – – –

BFN – 9.7  0.4i,j 19.9  0.4f – – – – – – –

BFE 12.4  0.4f 10.7  0.3h 12.9  0.6h – – – – – 15.9  0.4d –

BLH 8.7  0.4h – – – – – – – – –

BLD – – – – – – – – – –
BLM – – 8.8  0.2j – – – – – – –
BLW – – – – – – – – – –

BIH – – – – – – – – – –
BID – – – – – – – – – –
BIM – – 8.6  0.1j – – – – – – –
BIW – – – – – – – – – –

Chloramphenicol (30 mg) 32.2  0.6e 26  0.3d 33.2  0.4c 25.0  0.5b 27.5  0.2a 8.6  0.2c 25.0  1c 28.6  0.3a 30.0  0.7b 29.0  0.8a
Tetracycline (30 mg) 35.9  0.5d 31.1  0.5c 8.1  0.1j 22.2  0.2c 23.5  0.2c 14.2  0.5b 31.0  0b 26.6  0.4b 29.3  0.4b 26.8  0.5b
Ampicillin (10 mg) 43.2  1.0b 39.6  0.7b 30.5  0.2d 14.2  0.1d 27.0  0.3b – 25.0  0c 10.0  0.2d 26.4  0.3c 22.5  0.5d
Carbenicillin (100 mg) 44.8  0.9a 42.6  0.4a 37.2  0.6b 28.4  0.3a 22.0  0.6d 20.0  0.9a 34.0  1a 8.0  0.4e 34.0  0.9a 25.0  0.3c
Erythromycin (15 mg) 38.9  1.5c 30.9  0.9c 39.0  0.4a 11.6  0.4e 12.4  0.7e – 14.6  1d 13.0  0.2c 9.3  0.1f 12.9  0.4e
DMSO – – – – – – – – – –
Water – – – – – – – – – –
a,b,c,d,e,f,g,h,i,j
Mean-values (standard error) with the same letters within vertical columns not being significantly different (P > 0.05).

(13.9 mm), (BFE) (12.9 mm), methanolic extract of field-grown
flowers (soxhlet extraction) BFM (12.3 mm), hot water extract of
field-grown flowers (BFHW) (11.1 mm), methanolic extract of field-
grown leaves (BLM) (8.8 mm), BIM (8.6 mm) and water extract of
field-grown flowers (BFW) (8.5 mm) extracts showed greater
antibacterial activity than the reference antibiotic tetracycline
(8 mm) against S. epidermidis (Table 3). The BFE (15.9 mm) and
BFMe (11.6 mm) extracts showed higher antibacterial activity than
the reference antibiotic erythromycin (9.3 mm) against E. cloacae
(Table 3).
In this study, the MeOH extract and EtOAc fraction of field-
grown flowers exhibited a broad-spectrum of activity against both
Gram-positive (S. pyogenes, S. aureus and S. epidermidis) and Gram-
negative bacteria (E. cloacea). However, S. marcences, S. typhimu-
rium, P. aeruginosa, P. vulgaris, K. pneumonia and E. coli did not show
any sensitivity to the extracts used (Table 3).
Kavalcioglu et al. (2010) showed the antimicrobial activity of
the methanolic extract and volatiles of B. perennis against six
bacteria (E. coli, S. aureus, P. aeruginosa, Enterobacter aerogenes, P.
vulgaris and S. typhimurium) using the Broth microdilution
method. The MIC (minimum inhibitory concentration) of the
methanol extract of B. perennis was found to be >2.0 mg/ml, with
moderate to low antimicrobial activity (Kavalcioglu et al., 2010).
Avato et al. (1997) also demonstrated the antimicrobial activity of
essential oils such as deca-4,6-diynoic acid and deca-4,6-diyne-
1,10-dioic acid and they were mainly effective against Gram-
positive (Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis
and S. aureus) and Gram-negative (Acinetobacter calcoaceticus and
E. coli) bacteria, respectively.
The antibacterial activities of field-grown flowers were better
than both the field-grown and in vitro-derived leaves. There is
evidence that the level of detected secondary metabolites in shoot
cultures is lower than donor plants (Stafford, 1991). For example,
the production of steroids obtained from shoot tip cultures of
Digitalis species was much lower than those found in the donor
plant (Seidel and Reinhard, 1987). The antibacterial activity of B.
perennis may be attributed to the phenolic constituents. Liquid–
liquid extraction of plant extracts with EtOAc is a general method
for extracting a flavonoid-rich fraction (Samsam-Shariat, 1992).
The good inhibition of E. cloacae bacterial growth by the EtOAc
fraction of B. perennis may be due to its high flavonoid content
(Table 6). The phenolic compounds that are the predominant active
chemicals in many medicinal plants have the greatest antibacterial
activity against Gram-positive bacteria (Rios and Recio, 2005). The
effectiveness of the methanol extracts of B. perennis may be
explained by the inhibitory effect of these compounds on bacteria.
The strong antibacterial activity of the flower extracts may be
attributed to their high flavonoid content (Table 6). Consistent with
our results, Nazaruk and Gudej (2001) demonstrated higher
flavonoid contents of flowers than leaves of B. perennis. The strong
antibacterial activity of B. perennis extracts against S. epidermidis
and E. cloacea may explain why common daisy is used in traditional
medicine as a vulnerary and anti-inflammatory herbal medicine.
3.3. Anti-inflammatory
The anti-inflammatory activities of 12 different B. perennis
extracts (hexane, DCM, MeOH and water) of field-grown flowers,
leaves and in vitro-derived leaves were determined by measuring
their ability to prevent cellular NO production. NO is an
endogenous free radical species that is synthesized from L-arginine
by nitric oxide synthase (NOS) in many living tissues. L-NAME

Please cite this article in press as: F.P. Karakas, et al., In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant activities and phenolic
content in wild-grown flowers of common daisy—A medicinal plant, J. Herbal Med. (2016), http://dx.doi.org/10.1016/j.hermed.2016.11.003
G Model
HERMED 161 No. of Pages 9
6 F.P. Karakas et al. / Journal of Herbal Medicine xxx (2016) xxx–xxx
(known as a NO synthase inhibitor) was used as a positive control;
it inhibits the production of NO in LPS-stimulated RAW 264.7
macrophages (Paul et al., 1997). It is a valuable promoter of physical
homeostasis, however high rates have been closely connected with
the pathophysiology of many inflammation-based diseases
(Marletta, 1993). Herewith, the prevention of NO generation can
be a valuable procedure for the therapy of different diseases that
involved inflammation (Bourgou et al., 2010).
As shown in Fig. 1, the MeOH extract of field-grown flowers
from B. perennis showed a stronger inhibitory effect on LPS-
induced NO secretion (96.3% inhibition) than DCM (86.4%
inhibition), hexane (25.3% inhibition) and water (14.2% inhibition)
extracts at a concentration of 20 mg/ml (Fig. 1). The DCM extract of
field-grown leaves from B. perennis showed a stronger inhibitory
effect on LPS-induced NO secretion (31.9% inhibition) than hexane
(15.7% inhibition), MeOH (19% inhibition) and water (7.1%
inhibition) extracts at 20 mg/ml (Fig. 1). The DCM extract of in
vitro-derived leaves from B. perennis showed a stronger inhibitory
effect on LPS-induced NO secretion (87.5% inhibition) than MeOH
(43.7% inhibition), hexane (26.9% inhibition) and water (7.2%
inhibition) extracts observed at 20 mg/ml (Fig. 1). Comparatively, L-
NAME prevented NO secretion by 65.3% at 250.0 mM (67.4 mg/ml).
The strong anti-inflammatory activity of the MeOH extract of
field-grown flowers may be attributed to the reported phenolic,
flavonoid and terpenoid constituents of the plants. These results
support the traditional use of B. perennis in remedies for
inflammatory diseases such as rheumatism, eczema, eye diseases
and tonsillitis.
3.4. Evaluation of antioxidant activity
Flower extracts of B. perennis have demonstrated some indirect
antioxidant activity, with DPPH radical scavenging activity,
reducing power, total antioxidant capacity (thiocyanate method),
total phenolic and total flavonoid contents (Siatka and Kasparova,
2010; Kavalcioglu et al., 2010). However, there are no data about
the antioxidant activity of field-grown leaf and in vitro-derived leaf
extracts and the direct antioxidant activity of B. perennis extracts.
In the present study, the antioxidant activity of hexane, DCM,
methanol and water extracts of field-grown flowers, leaves and in
120
100
80
NO Inhibition (%)
60
40
20
0
BFH
BLH
BFW
BFD
BLD
BFM
Treatments
Fig. 1. Effects of B. perennis extracts (20.0 mg/ml) and L-NAME (250 mM) on NO production in LPS-stimulated RAW-264.7 macrophages. Values are mean  S.D of three
replications.
Please cite this article in press as: F.P. Karakas, et al., In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant activities and phenolic
content in wild-grown flowers of common daisy—A medicinal plant, J. Herbal Med. (2016), http://dx.doi.org/10.1016/j.hermed.2016.11.003
vitro-derived leaves was assessed. Free radical scavenging activity
(DPPH), total phenolic content (Folin-Ciocalteau), ORAC and direct
antioxidant assays (cell-based assay using 20 .70 -dichlorofluorescin-
diacetate) were used in this assessment.
In the DPPH assay, methanol extracts of B. perennis exhibited
concentration-dependent free radical scavenging activity (%) by
scavenging the DPPH radical. Free radical scavenging activity was
elevated when concentrations increased from 5 to 200 mg/ml.
Methanol extracts of leaves (95.85% at 200 mg/ml) and flowers
(95.52% at 200 mg/ml) showed higher DPPH radical scavenging
activity than the methanol extract of in vitro-derived leaves
(55.61% at 200 mg/ml). The field-grown leaf and flower extracts
had active radical scavenging capability as high as ascorbic acid
(96.59% at 200 mg/ml), which was used as control antioxidant
(Table 4). Similarly, DPPH radical scavenging activity of aqueous
and methanol extracts of aerial parts of B. perennis was shown by
Kavalcioglu et al. (2010). The antioxidant mechanisms and their
phenols or flavonoids may be attributed to strong hydrogen
donating ability, and their effectiveness as scavengers of free
radicals (Gülçin et al., 2003).
The total phenolic content of common daisy extracts was
evaluated using the Folin-Ciocalteu reagent (Singleton and Rossi,
1965). The results were expressed as g of tannic acid equivalents
(TAE)/100 g dried extract (Table 5). The results shown in Table 5
indicated that the methanolic extracts of field-grown flowers and
leaves, and DCM extract of in vitro-derived leaves contained high
levels of phenolics with respectively, 5.20, 10.60 and 4.20 g TAE/
100 g of extract. The contents of total phenolics varied from 0.40 to
10.60 g TAE/100 g dried mass. There was a significant correlation
between the total phenolic content and the radical scavenging
activity. Our results were consistent with the results of Siatka and
Kasparova (2010) that total phenolics ranged from 2.81 to 3.57 mg
GAE/100 mg dry weight. Our study was also consistent with
Nazaruk and Gudej (2001) that the phenolic contents in the
flowers were higher than in the leaves of B. perennis. The amount
and type of phenolic content of extracts depends on the part of
plant material, type of extraction solvent and type of extraction
method used in extraction. Some non-phenolic reducing mole-
cules, such as ascorbic acid, sugars (Singleton and Rossi, 1965),
organic acids and amino acids inhibit the detection of total
BIH
BLW
BIW
BID
BLM
BIM
L-NAME
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F.P. Karakas et al. / Journal of Herbal Medicine xxx (2016) xxx–xxx 7

Table 4
The free-radical (DPPH) scavenging activity (%) of MeOH extracts of field-grown flowers (BFM), leaves (BLM) and in vitro-derived leaves (BIM) of B. perennis at different
concentrations and ascorbic acid (positive control).

Treatments % Inhibition of DPPH ( SD)

Concentrations

5 mg/ml 10 mg/ml 25 mg/ml 50 mg/ml 100 mg/ml 200 mg/ml

Ascorbic acid 25.33  0.03 66.44  0.02 96.01  0.03 96.27  0.04 96.57  0.03 96.59  0.04
BFM 14.20  0.02 17.66  0.05 19.28  0.01 36.47  0.09 63.79  0.01 95.52  0.05
BLM 24.66  0.04 31.58  0.03 49.14  0.01 75.85  0.05 95.63  0.01 95.85  0.03
BIM 15.25  0.02 16.09  0.02 17.60  0.05 22.88  0.03 33.63  0.05 55.61  0.01

Table 5
Antioxidant activities of B. perennis extracts and fractions of field-grown flowers, leaves and in vitro-grown leaves and positive controls (quercetine, trolox and tannic acid)
with using ORAC, Folin-Ciocalteu and cell-based assays.

Samples ORAC value Total phenolic compounds Cell-based assay

(mmol Trolox/mg)a (g/100 g)b WS1 IC50 (mg/ml)c
BFH 0.07  0.00 0.40  0.1 11  2
BFD 0.73  0.03 4.30  0.6 55  2
BFM 0.61  0.05 5.20  1.4 1.8  0.2
BFW 1.23  0.03 1.10  0.2 119  14
BFN 4.88  0.12 nd 1.00  6
BFE 9.05  0.38 nd 3.5  0.3

BLH 0.17  0.02 1.30  0.3 122  36

BLD 0.30  0.04 3.00  0.9 47  14
BLM 2.71  0.17 10.60  0.3 3  0.6
BLW 0.73  0.05 3.50  0.8 7.10  0.6

BIH 0.52  0.05 1.30  0.3 101  30.00

BID 1.21  0.08 4.20  1.2 13  1
BIM 0.86  0.08 3.10  0.4 19  2
BIW 0.51  0.04 1.60  0.6 43  4

Quercetine 20.65  2.01 nd 0.25  0.02

Trolox nd nd 0.15  0.05
Tannic Acid nd 93.10  10.6 nd

nd: not determined.

a
ORAC: oxygen radical antioxidant capacity.
b
g total phenolic compounds/100 g extract: tannic acid equivalents.
c
Concentration inhibiting 50% of cell growth (mean  SD; n = 3).

phenolic compounds in the Folin-Ciocalteu assay and lead to an
underestimation in the phenolic content of plant extracts (Siatka
and Kasparova, 2010).
The antioxidant potential of B. perennis extracts was also
assessed using the ORACFL assay. The EtOAc fraction of the flowers
was a strong antioxidant, with an ORAC value of 9.05  0.38 mmol
of TE/mg of dry mass (Table 5). In comparison, ORAC values of the
standard phenolic compound quercetin were 20.65  2.01 mmol
TE/mg. In the present study, there was a correlation between the
total phenolic contents and ORAC values. The methanol extracts of
flower and leaf, and DCM extract of in vitro-derived leaves were
moderately antioxidant, with ORAC values, respectively, of
1.61  0.05, 2.71  0.17 and 1.21  0.08 mmol of TE/mg of dry mass
(Table 5).
The antioxidant activity of flower, leaf and in vitro-derived leaf
extracts was also evaluated ex vivo using a cellular based-assay
(using DCFH-DA) (Girard-Lalancette et al., 2009). DCFH-DA is a
valuable indicator of reactive oxygen species (ROS) and oxidative
stress. Results presented in Table 5 showed that the n-BuOH
fraction (1  6 mg/ml), MeOH extract (1.8  0.2 mg/ml) and EtOAc
fraction of field-grown flowers (3.5  0.3 mg/ml), MeOH extract of
field-grown leaves (3  0,6 mg/ml) and DCM extract of in vitro-
grown leaves (13  1 mg/ml) inhibited the tBH-induced oxidation
of DCFH by 50%. In comparison, Trolox (0.15 mg/ml) and quercetin
(0.25 mg/ml) inhibited DCFH oxidation by 50%. These results
showed that flower extracts of B. perennis possessed a high value ex
vivo antioxidant activity (Table 5).
On the basis of the results of this study, it was mainly illustrated
that extracts of common daisy had strong antioxidant activity on in
vitro and ex vivo oxidative systems and contained high amounts of
phenolics. They can be utilized and produced as a natural source of
ROS scavenger and as a practical nutritional supplement, or in the
pharmaceutical industry.
3.5. Determination of phenolic compounds by LC–ESI–MS/MS analysis
The flower extracts and fractions were rich in phenolic
compounds. Considerable amounts of kaempferol, rutin hydrate,
caffeic acid, apigenin, quercetin, p-coumaric acid, luteolin-7-O-b-D
glucoside, gallic acid and myricetin were found in the methanol
extract and the EtOAc fraction of flowers. Those found in the
greatest quantities were kaempferol (0.4–14605.2 mg/g of dw) and
rutin hydrate (2.7–6252.9 mg/g of dw) in all tested extracts
(Table 6). In accordance with previous studies of B. perennis
(Nazaruk and Gudej, 2001), our study also detected the presence of
quercetin, apigenin and kaempferol in field-grown flower and leaf
extracts. Our research was focused on phenolic compounds.
Analysis of the phenolic profile indicates that B. perennis mainly
contains flavonoids (Nazaruk and Gudej, 2001). Consequently, the
most dominant flavonoids in both methanol extracts of field-

Please cite this article in press as: F.P. Karakas, et al., In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant activities and phenolic
content in wild-grown flowers of common daisy—A medicinal plant, J. Herbal Med. (2016), http://dx.doi.org/10.1016/j.hermed.2016.11.003
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8 F.P. Karakas et al. / Journal of Herbal Medicine xxx (2016) xxx–xxx

Table 6
Amount of 22 phenolic compounds in wild-grown flower extracts of B. perennis by using LC–ESI–MS/MS.

Retention time (min) Extracts (mg/g of dry extract)

Phenolic compounds BFD BFM BFN BFE

1 Gallic acid monohydrate 0.920 Nd 17.0  0.007 2.6  0.000 148.5  0.02
2a Pyrocatechol 1902 Nd Nd Nd Nd
Procyanidin B1 2225 Nd Nd Nd Nd
3a () Epigallocatechin 2497 Nd Nd Nd Nd
(+) Catechin 2539 Nd Nd Nd Nd
4 Procyanidin B2 2886 Nd Nd Nd Nd
5a Vanillic acid 3063 87.6  0.3 0.7  0.005 15.2 0.04 29.4  0.09
Caffeic acid 3092 10.2  0.003 373.3  0.30 8.2  0.006 1341.3  0.02
6a Procyanidin C1 3216 Nd Nd Nd Nd
() Epicatechin 3320 Nd Nd Nd Nd
7 p- Coumaric acid 3884 161.9  0.07 75.1  0.07 2.6  0.006 378.0  0.01
8 () Taxifolin hydrate 4070 Nd 0.1  0.000 Nd 5.1  0.005
9a Coumarin 4716 0.4  0.000 0.3  0.001 Nd 4.7  0.009
Luteolin-7-O-b-D glucoside 4809 0.4  0.000 70.1  0.07 32.1  0.05 232.4  0.01
Rutin hydrate 4898 38.9  0.06 6252.9  0.17 3073.7  0.3 3029.1  0.1
Resveratrol 4956 Nd Nd Nd Nd
10 Myricetin 5270 Nd 2.4  0.02 Nd 7.5  0.01
11 Kaempferol 3-b-D-glucopyranoside 5426 9.2  0.009 5207.4  0.5 426.4  0.03 14605.2  0.1
12 Daidzein 5829 Nd Nd Nd Nd
13 Quercetin 6073 19.5  0.10 174.6  0.1 0.4  0.001 775.2  0.09
14 Genistein 6425 26.1  0.06 Nd Nd Nd
15 Apigenin 6945 572.3  0.5 270.7  0.01 Nd 737.9  0.3

Total Phenolics 930  0.60 12440  0.60 3560  0.32 21295  0.31

Nd; not detected.

a
Indicates the same-close retention times. Values are means  SD of three measurements.

grown flower and leaf were rutin hydrate and kaempferol. Results
obtained for kaempferol in B. perennis were in agreement with the
data previously reported using the HPLC method by Nazaruk and
Gudej (2001). However, the presence of rutin in B. perennis was not
detected in their study (Nazaruk and Gudej, 2001).
In the present study, when we compared the fractions of EtOAc
and n-BuOH of flowers, the EtOAc (21295  0.31 mg/g) fraction had
a higher phenolic content than the n-BuOH (3560  0.32 mg/g)
fraction (Table 6). The higher amount of total phenolic content was
detected in the methanol extract (12440  0.6 mg/g) than dichloro-
methane extract (930  0.60 mg/g) of wild-grown flowers by LC–
MS/MS analysis (Table 6). The flowers of wild-grown B. perennis
included rutin hydrate, kaempferol, caffeic acid, apigenin, p-
coumaric acid, coumarin, gallic acid, genistein, luteolin-7-O-b-D
glucoside, myricetin, procyanidin C1, quercetin, taxifolin hydrate
and vanillic acid (Table 6). It has been previously shown that
phenolic constituents of B. perennis included flavonoids such as
kaempferol, quercetin, isorhamnetin, apigenin, (Nazaruk and
Gudej, 2001; Karakas and Turker, 2013) and some phenolic acids
(e.g., caffeic, p-coumaric, and salicylic acids) (Grabias et al., 1995).
Coumarin, genistein, luteolin-7-O-b-D glucoside, rutin hydrate,
myricetin, vanillic acid, procyanidin C1, and taxifolin hydrate were
determined in the flowers of B. perennis for the first time. All tested
extracts included rutin hydrate, kaempferol, apigenin, luteolin-7-
O-b-D glucoside, caffeic acid, p-coumaric acid, coumarin and
vanillic acid as the main phenolics (Table 6). In accordwith Nazaruk
and Gudej (2001), the flavonoid contents were higher in the
flowers than leaves (field-grown or in vitro-grown) in our study
(Karakas and Turker, 2013).
4. Conclusion
The bioassay results provide some basis to justify the common
use of B. perennis extracts in folk medicine as treatments for
respiratory disorders, rheumatism, cancer, certain skin diseases
and inflammatory conditions. These results provide some evidence
for the medicinal value of B. perennis. Definitive clinical studies are
needed to fully understand the many medicinal uses of common
daisy. Future work should include research into which secondary
metabolites can be isolated and described from the different
fractions of B. perennis so as to better understand the underlying
mechanism of their role on the cytotoxicity, antibacterial, anti-
inflammatory and antioxidant activity.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
This study was supported by the Abant Izzet Baysal University
Research Foundation (Project No: 2012.03.01.503). Great thanks
are expressed to Karl Girard-Lalancette, Catherine Dussault
(LASEVE Laboratuvary, Chicoutimi, Québec, Canada) for experi-
mental supports.
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