Industrial Crops & Products 124 (2018) 389–396

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Industrial Crops & Products

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

Bioactivity-guided isolation of cytotoxic secondary metabolites from the T

roots of Glycyrrhiza glabra and elucidation of their mechanisms of action
Dicle Çevika, Ş. Burçin Yılmazgözb, Yüksel Kanc, Ece Akhan Güzelcand, Irem Durmazd,
⁎
Rengül Çetin-Atalayd, Hasan Kırmızıbekmeza,
a
Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, TR-34755, Kayışdağı, İstanbul, Turkey
b
Faculty of Pharmacy, Yeditepe University, TR-34755, Kayışdağı, İstanbul, Turkey
c
Department of Medicinal Plants, Faculty of Agriculture, Selçuk University, TR-42070, Konya, Turkey
d
Cancer Systems Biology Laboratory, Graduate School of Informatics, METU, TR-06800, Ankara, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: Licorice (Glycyrrhiza glabra L.) is one of the most widely used plants worldwide for its various pharmacological
Glycyrrhiza glabra activities. The aim of this study was to isolate the potential cytotoxic secondary metabolites from the MeOH
Secondary metabolites extract prepared from the roots of Glycyrrhiza glabra through bioactivity-guided isolation procedure and to
Cytotoxic activity elucidate their mechanisms of action. The crude MeOH extract as well as CHCl3 and EtOAc subextracts sig-
Apoptosis
nificantly inhibited cell proliferation on hepatocelullar (Huh7), breast (MCF7) and colorectal (HCT116) cancer
Liver cancer
cell lines with IC50 values in the range of 5.6 to 33.6 μg/mL. Chromatographic seperations of the CHCl3 and
Huh7
EtOAc subextracts yielded 13 secondary metabolites. Structures were characterized based on NMR and MS data.
Amongst isolates, glabridin (1), 4′-O-methylglabridin (2), β-amyrin (3), kanzonol U (4), glabrene (7) and tet-
rahydroxymethoxychalcone (10) were established to be responsible for in vitro cytotoxicity of G. glabra, exerting
the best activity particularly against Huh7 cells. Further mechanistic studies demonstrated that 2 and 7 induced
caspase-dependent apoptosis by increasing cytochrome C release and subsequently cleaved caspase-9 level in
Huh7 cells. Moreover, both compounds decreased pRb and p21 levels and thus induced the accumulation of
Huh-7 cells in subG1 and G2/M phases. Compound 10 which displayed the most potent activity in Hoechst
staining and cell cycle assays through G2/M arrest, caused cell death by apoptosis in Huh7 cells.

1. Introduction
Cancer is the second leading cause of morbidity and mortality after
cardiovascular diseases in industrialized countries. It had been 14.1
million new cancer cases whereas 8.2 million cancer deaths in 2012.
Breast cancer has the highest incidence rate among women followed by
colorectal cancer. Among men colorectal cancer is the third whereas,
liver cancer which has a relatively poor survival and hence mortality is
the fifth common sites of cancer (WHO Cancer Report, 2014). Cancer
arises from the loss of balance between cell division and cell death.
Apoptosis is regulated destruction of cells, plays an important role and
is the main target in cancer treatment (Wong, 2011). This is a very
complicated process including both intrinsic and extrinsic pathways.
Release of cytochrom C from mitochondria into cytosol is occured via
intrinsic pathway which subsequently activates caspase-9 and thus
caspase-3 (Hengartner, 2000). Activation of these caspases eventually
leads to apotosis through caspase cascade by cleaving of poly (ADP-
ribose) polymerase (PARP) levels (Mohan et al., 2016; Shyu et al.,
2010).
Most of the drugs used in cancer treatment today are not effective
enough or have some serious side effects, thus new anticancer drugs are
needed to combat cancer. Natural resources particularly medicinal
plants play a very significant role for the discovery and development of
new drug leads. Clinically used anticancer drugs such as vincristine,
vinblastine, vindesine, paclitaxel, etoposide, teniposide, irinotecan and
topotecan are natural products or their derivatives which are produced
by semi-synthesis from a natural molecule (Saklani and Kutty, 2008).
The genus Glycyrrhiza (Fabaceae) commonly known as licorice,
contains approximately 30 species distributed worldwide including, G.
glabra, G. aspera and G. uralensis (Nomura et al., 2002). In Flora of
Turkey, it is represented by six species being G. glabra, the most pre-
valent one (Chamberlain, 1969). Glycyrrhiza glabra L. has a long history
of use in different folk medicines for the treatment of peptic ulcers,
against inflammatory diseases as well as an expectorant agent. In

⁎
Corresponding author.
E-mail address: hasankbekmez@yahoo.com (H. Kırmızıbekmez).

https://doi.org/10.1016/j.indcrop.2018.08.014
Received 7 April 2018; Received in revised form 5 August 2018; Accepted 6 August 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
D. Çevik et al. Industrial Crops & Products 124 (2018) 389–396

Traditional Chinese Medicine licorice which is known as Gancao, is Table 1

used for its nourishing, analgesic, spleen and stomach tonifying, ex- Cytotoxic activity results of crude MeOH extract, subextracts and the main
pectorant and relieving cough effects (Yang et al., 2015). It is one of the fractions of the active subextracts of G. glabra against Huh7, MCF7 and HCT116
most widely used plants in phytotherapy and food industry due to its cancer cell lines.
miscellaneous pharmacological activities and sweet taste. The sec- Extracts/Fractions Huh7 MCF7 HCT116
ondary metabolites, including triterpene saponins, flavonoids and iso-
a 2 a 2
flavonoids have been isolated from licorice as the major chemical IC50(μg/ml) R IC50(μg/ml) R IC50 (μg/ml)a R2

classes (Hosseinzadeh and Nassiri-Asl, 2015). Previous bioactivity stu- MeOH 18.9 0.6 28.8 0.6 33.6 0.7
dies on licorice and some of its secondary metabolites revealed anti- CHCl3 13.3 0.9 29.3 0.6 8.2 0.9
ulcer (Wittschier et al., 2009), antimicrobial (Kırmızıbekmez et al., Fr. B 14 0.8 14.6 0.8 18.5 0.8
2015), hepatoprotective (Ji et al., 2016), anti-inflammatory Fr. C 9.2 0.7 8.9 0.6 14.9 0.6
EtOAc 5.6 0.9 9.1 0.9 7.4 0.9
(Thiyagarajan et al., 2011), immunomodulatory, memory enhancing,
Fr. 2 NI – NI – NI –
antiprotozoal, cytotoxic and antitumor activities (Yang et al., 2015). Fr. 3 NI – NI – NI –
In recent years, biological activities particularly anticancer and Fr. 4 4.9 0.9 2.5 0.9 1.3 0.7
cytotoxic effects of Glycyrrhiza extracts and isolated secondary meta- n-buOH NI NI NI
H2O NI NI NI
bolites have attracted much interest. Previous in vitro studies demon-
strated that Glycyrrhiza extracts have cytotoxic activites against several
NI: No Inhibition.
cancer cells in different degrees (Aydemir et al., 2011; Park et al., 2014; a
IC50 values were calculated from the cell growth inhibition curves obtained
Vlaisavljević et al., 2018). Moreover, the secondary metabolites ob- from the treatments with increasing concentrations of extracts or fractions (30,
tained from several Glycyrrhiza species were shown to possess sig- 15, 7.5, 3.25 and 1.875 μM) for 72 h. Experiments were done in triplicate.
nificant cytotoxic and anticancer activities in recent years (Tang et al.,
2015; Ji et al., 2016; Li et al., 2017; Lin et al., 2017). Considering the specimen (YEF 15005) has been deposited at the Herbarium of the
wide utilization of G. glabra in folk medicines and phytotherapy as well Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe
as the recently documented bioactivities of several Glycyrrhiza species, University, İstanbul, Turkey.
we aimed to isolate the cytotoxic secondary metabolites from roots of
G. glabra through in vitro cytotoxicity-guided isolation techniques.
Furthermore, the underlying mechanisms behind this cytotoxic activites 2.3. Extraction and isolation
of the bioactive isolates were also elucidated.
The dried and powdered roots of G. glabra (370 g) were extracted
2. Materials and methods with MeOH (2 × 2.5 L, 4 h). The combined extracts were concentrated
under reduced pressure to afford crude MeOH extract (87.2 g) which
2.1. General procedures was suspended in 200 mL of H2O and partitioned three times with equal
volumes (each 200 mL) of CHCl3, EtOAc and n-buOH, respectively to
Chromatography: Column chromatography (CC) was performed on yield subextracts. The crude MeOH extract and subextracts were eval-
silica gel 60 (0.063-0.200 mm; Merck, Darmstadt, Germany), Sephadex uated for their cytotoxic activities by Sulphorodamine B (SRB) assay
LH-20 (25–100 μm; Sigma-Aldrich, St. Louis, MO, USA) and Polyamide (Table 1). The cytotoxically active subextracts, CHCl3 and EtOAc were
(50–160 μm; Fluka Analytical, Sigma-Aldrich, St. Louis, MO, USA). purified by successive chromatographic methods. CHCl3 subextract
Medium-pressure liquid chromatography (MPLC) was performed by (3.92 g) fractionated over Sephadex LH-20 (90 g) CC eluted with
Sepacore® Flash Systems X10/X50 (Buchi Labortechnik AG, Flawil, CH2Cl2:MeOH (1:1, 400 mL) and MeOH (200 mL), respectively to yield
Switzerland) on Redi sep columns LiChroprep C18 and SiO2 (Teledyne three main fractions (fr. A–C). EtOAc subextract (5.4 g) was loaded onto
Isco, Lincoln, Nebraska, USA). Thin layer chromatography (TLC) ana- poliamide column (40 g) eluting with H2O and stepwise gradient of
lyses were carried out on silica gel 60 F254 plates (9.5–11.5 μm; Merck, MeOH in H2O (25–100% in steps of 25%, each 100 mL) to obtain frs.1-
Darmstadt, Germany) on aluminum, visiualization was established by 4. These main fractions were also evaluated in the same cell panel
spraying with 1% vanillin/sulphuric acid solution followed by heating (Table 1). Active fractions (frs. B and C from CHCl3, fr. 4 from EtOAc)
at 105 °C for 2–3 min, detected with a UV at 254 and 365 nm. HPLC were subjected to chromatographic seperations. Fr. B (2.6 g) was se-
system, Agilents Technologies HP1100 (Agilent Technologies, parated by SiO2-MPLC (120 g) eluting with stepwise n-hexane:EtOAc
Waldbronn, Germany) equipped with a Vacuum degasser G1379 A, gradient (0–100% EtOAc) to obtain B1-18 subfractions. Subfraction B8
quaternary pump G1311 A, an auto-sampler G1313 A, a thermo-stated (34 mg) was further purified by SiO2-MPLC (12 g), eluted with the same
column compartment G1316 A and a diode array detector G1315B. The mobile phase (0–20% EtOAc) to isolate 2 (9 mg). Subfraction B11
separation was performed on an Agilent Zorbax Eclipse Plus C18 ODS (147 mg) was applied to Sephadex LH-20 (10 g) column with
column (5 μm, 250 mm × 9.4 mm, i.d.). UV Spectra: HP Agilent 8453 CH2Cl2:MeOH (1:1) to give 3 (3 mg). Subfraction fr. B13 (409 mg) was
spectrophotometer (Agilent Technologies, Santa Clara, CA, USA); λmax submitted to MPLC (SiO2, 40 g) eluting with stepwise gradient of n-
in nm. IR Spectra (KBr): PerkinElmer 2000 FT-IR spectrometer hexane:EtOAc (0–20% EtOAc) to yield 1 (13 mg) and fr. B13-b (100 mg)
(PerkinElmer, Waltham, Massachusetts, USA); υ in cm−1. NMR Spectra: which was purified by SiO2-MPLC (40 g) with n-hexane:EtOAc mixture
1
H (400 MHz), 13C (100 MHz), COSY, HSQC, HMBC and NOESY NMR (0–60%, EtOAc) to obtain 4 (7 mg). Fractionation of subfraction B14
spectra were recorded on a Varian Mercury FT spectrometer (Palo Alto, (199 mg) by MPLC (SiO2, 12 g) eluting with stepwise of n-hexane:EtOAc
CA, USA) in CD3OD or CDCl3; δ in ppm rel. Me4Si as internal standard, J gradient (20–100% EtOAc) yielded fr. B14-a (101 mg) which was further
in Hz. Mass Spectroscopy: Agilent G6530B TOF/Q-TOF mass spectro- applied to Sephadex LH-20 (8 g) with MeOH to obtain fr. B14-a-1
meter (Agilent Technologies, Santa Clara, CA, USA) in MeOH; positive- (20 mg). Compound 11 (4 mg) was purified from fr. B14-a-1 by semi-
ion mode; in m/z. preparative HPLC on Zorbax Eclipse XDB-C18 column (9.4 × 250 mm)
eluted with H2O:MeCN mixture (10–50%, MeCN). Fr. C (100 mg) was
2.2. Plant material fractionated over SiO2-MPLC (24 g) eluting with n-hexane:EtOAc
(10–50% EtOAc) to afford subfractions, C1-4. Compound 2 (2 mg) was
The roots of Glycyrrhiza glabra L. were collected from Medicinal isolated from subfraction C1 (20 mg) by Sephadex LH-20 CC (35 g)
Plant Experimental Garden, Faculty of Agriculture, Selçuk University, eluted with MeOH. Fr. C2 (20 mg) was separated by Sephadex LH-20
Konya, Turkey, where the plant species was cultivated. Voucher (6 g, MeOH) followed by MPLC (SiO2, 24 g, n-hexane-EtOAc, 90:10 and

390
D. Çevik et al.
80:20) to give 1 (3 mg). Fr. 4 (2 g) of EtOAc subextract was fractionated
over LiChroprep C18-MPLC (130 g) using stepwise H2O:MeOH gradient
(85:15–0:100) to yield subfractions 4a-j. Subfraction 4b (87 mg) was
applied to open cloumn chromatography (OCC, silica gel, 10 g) eluted
with CH2Cl2:MeOH:H2O (95:5:0, 93:7:0, 90:10:1, 80:20:2, respectively)
to purify 10 (3 mg). Subfraction 4f (141 mg) was applied to silica gel
column chromatography (OCC) using CH2Cl2: MeOH (95:5 and 90:10)
to afford a mixture of 8 and 12 (9 mg). Compound 7 (3 mg) was isolated
as consequence of submitting subfraction 4g (155 mg) to SiO2 column
with CH2Cl2:MeOH (97.5:2.5, 95:5, 90:10). Subfraction 4h (103 mg)
was seperated by SiO2 (15 g) column chromatography using n-hex-
ane:EtOAc (20–50% EtOAc) as mobile phase to yield fr. 4h-1 (30 mg)
which was subsequently applied to SiO2 (6 g) column again eluted with
same mobile phase (15–25% EtOAc) to give 1 (4 mg). The major
compounds of the inactive fractions (frs. 2 and 3) of the EtOAc sub-
extract were also isolated in order to make a phytochemical contribu-
tion to the plant species. Thus, fr. 2 (357 mg) was subjected to C18-
MPLC (50 g) eluting with H2O:MeOH (0–70% MeOH) to obtain sub-
fractions. Subfraction 2a (130 mg) was applied to SiO2 CC (20 g), eluted
with CH2Cl2:MeOH (5–40% MeOH) to isolate compound 13 (6 mg). Fr.
3 (950 mg) of EtOAc was subjected to C18-MPLC (130 g) eluting with
H2O:MeOH (20–50% MeOH) to give 5 and 6 (18 mg) as an inseperable
mixture along with subfraction 3e (350 mg). Compound 9 (5 mg) was
purified from subfr. 3e by SiO2-MPLC (40 g, CH2Cl2: MeOH; 5–40%
MeOH) and C18-MPLC (13 g, H2O:MeOH, 15–50% MeOH) methods.
2.4. Structure elucidation methods
The structures were elucidated based on spectroscopic methods in-
cluding UV, IR, 1D (1H and 13C) and 2D (COSY, HSQC, HMBC and
NOESY) NMR as well as MS.
2.5. Cell lines and cell culture
Human liver (Huh7), colon (HCT116) and breast (MCF7) cancer cell
lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM;
Gibco #31885) supplemented with 10% fetal bovine serum (FBS;
Invitrogen GIBCO #10270) and 1% penicillin (Gibco #15140-122). The
cell lines were maintained in an incubator at 37 °C under 5% CO2
(Hawash et al., 2017).
2.6. Sulforhodamine B assay
The sulforhodamine B (SRB) assay was performed to evaluate the
antiproliferative effects of crude extract, subextracts, main fractions as
well as purified compounds. SRB assay had been developed by US
National Cancer Institute (NCI) which is a rapid, sensitive and in-
expensive method and used for cell density determination, based on the
measurement of cellular protein content (Vichai and Kirtikara, 2006).
Human cancer cell lines were plated in 96-well plates (MCF7/Huh7:
2000 cell/well, HCT116: 3000 cell/well in 150 μl/well DMEM) and
grown for 24 h at 37 °C. After 24 h, cells were treated with five in-
creasing concentrations of the extracts (1.875–30 μg/mL), main frac-
tions or compounds (2.5–40 μM) dissolved in DMSO and applied
through serial dilution. After treatment process, the cells were in-
cubated at 37 under 5% CO2 for 72 h which was followed by washing
the plates with 1xPBS (CaCl2-, MgCl2-free). Fixation was performed
using 100 μL 10% (v/v) ice-cold trichloroacetic acid (TCA; Merck) and
incubated in dark +4 °C for one hour. Then TCA was washed away with
ddH2O 2 times and plates were air-dried. Finally, 100 μL of 0.4% (m/v)
of sulforhodamine (SRB) in 1% acetic acid solution was added to each
well for staining process. The unbound SRB was washed with 1% acetic
acid solution for 4 times to remove non-specific dye binding staining
and left for air drying. The bound sulforhodamine B was then solubi-
lized using 100 μL 10 mM Tris-base prior to absorbance measurement.
The absorbance was read at 515 nm. The experiments were done in
391
Industrial Crops & Products 124 (2018) 389–396
triplicates and IC50 values were calculated as μg/mL for extracts and
fractions and as μM for isolated compounds. The cell growth inhibition
ratios were calculated by formula of [1− (Absdrug × 100)/(AbsDMSO)].
2.7. Real time bioactivity assay (RT-CES xCELLigence)
The growth inhibitory effect of the most active compounds in SRB
assay on Huh7 cells were monitored by the xCELLigence System (Roche
Applied Sciences) which has a microelectronic biosensor technology to
do dynamic, real-time, label-free and non-invasive analysis of cellular
events, including cell number change, cell adhesion, cell viability, cell
morphology, and cell motility (Ke et al., 2011). Huh7 cancer cell lines
(2000 cells/well) were seeded into 96-well E-plates. Cells were treated
with the compounds with their IC50 concentrations. The proliferation of
cells were monitored every 30 min for 24 h after seeding, then treat-
ments with the compounds were done in log growth phase of cells. The
cell index (CI) values were recorded every 10 min for the first 24 h and
then every 30 min for the remaining 48 h. The cell growth ratios were
calculated by CIdrug/CIDMSO.
2.8. Apoptotic cell death assay (Hoechst staining)
Apoptotic cells were identified by Hoechst staining. Huh7 cells were
seeded onto coverslips in 6-well plates. After 24 h culture, cells were
treated with the compounds with their IC50 concentrations by RT-CES
assay. After 48 h, coverslips were washed twice with PBS, fixed in 1 mL
of ice-cold 100% methanol for 10 min and then incubated with Hoechst
33258 (Sigma-Aldrich) for 5 min in darkness. Cells were analyzed and
images of them were taken by using the fluorescence microscope
(Nikon Eclipse 50i).
2.9. Cell cycle assay (Fluorescence-activated cell sorting (FACS))
This method is used to investigate the effects of the cytotoxic mo-
lecules’ on cell cycle phases such as SubG1, G1, S and M. Propidium
Iodide (PI) dye binds to DNA in the presence of cytotoxic agent. Thus
this method determines the cell cycle phase by different type of DNA of
cells after staining with PI (Riccardi and Nicoletti, 2006). Huh7 cells
were seeded onto 100 mm culture dishes. After 24 h, cells were treated
with the compounds with their IC50 concentrations based on the RT-
CES results after 48 h. IC50 concentration of compound 10 distrupted
the cell cycle distribution of Huh7 cells, hence, we used IC25 con-
centration of compound 10 after 48 h. Cells were fixed with ice-cold
70% ethanol for 3 h at −20 °C after 48 h of incubation. Cell cycle
analysis was carried out by PI staining using Fluorescence-activated cell
sorting (FACS).
2.10. Cell protein assay (Western Blot)
Immunoblotting (western blotting) is a technique that has been used
to for the identification of specific antigens recognized by polyclonal or
monoclonal antibodies. It gives information about the identity of the
specific proteins, the change of their amounts and phosphorylation
character of them (Gallagher et al., 2008). Huh7 cancer cell lines were
inoculated into 150 mm culture dishes and after 24 h cells were treated
with the compounds (IC50 concentrations based on the RT-CES results
after 48 h) or DMSO control for 48 h. Novex® NuPAGE® Bis-Tris Elec-
trophoresis system was used according to the manufacturer's protocol
for all the western blotting analysis. For gel electrophoresis (NuPAGE),
20–50 μg of protein was used per well. Then the proteins were trans-
ferred to nitrocellulose membrane via XCell IITM Blot Module. p53
(#05-224, Millipore), phospho-Rb (Ser807/811) (#9308S Cell Sig-
naling), cytochrom C (sc7159, Santa Cruz), Mdm2 (OP46, Calbiochem),
p21 (OP64, Calbiochem), cleaved caspase-9 (sc-22182, Santa Cruz) and
cleaved PARP (#9532, Cell Signaling) antibodies were used in
1:200–1:500 5% BSA-TBS-T. β-actin (#A5441, Sigma) antibody was
D. Çevik et al.
used for equal loading (Hawash et al., 2017).
3. Results and discussion
3.1. Bioactivity-guided fractionation and isolation
The CHCl3 and EtOAc subextracts obtained from the crude MeOH
extract of G. glabra roots showed cytotoxic activity against hepatocel-
lular (Huh7), breast (MCF7) and colorectal (HCT116) cancer cell lines
with IC50 values in the range of 5.6–29.3 μg/mL whereas n-buOH and
remaing H2O subextracts were found to be inactive (Table 1). There-
fore, CHCl3 and EtOAc subextracts were fractionated by Sephadex LH-
20 and polyamide column respectively and the obtained main fractions
were submitted to SRB assay. The active fractions (frs. B and C of CHCl3
and fr. 4 of EtOAc) were selected for the isolation of the potential
bioactive metabolites (Table 1).
As a result of bioassay-guided fractionation and isolation studies,
compounds 1-4 and 11 were isolated from active fractions of CHCl3
while, 1, 7, 8, 10 and 12 were obtained from active fraction of EtOAc.
Moreover, the major compounds (5, 6, 9 and 13) of the inactive frac-
tions of the EtOAc subextract were also isolated in order to make a
phytochemical contribution to the plant species.
3.2. Structure elucidation of isolated molecules
As a result of sequential chromatographic techniques, totally 13
compounds were isolated from G.glabra roots. Among these compounds
5–6 and 8–12 were obtained as mixtures. To determine the exact
structure of the isolates spectroscopic techniques such as UV, IR, 1D and
2D NMR and MS were applied. The comparasion of obtained spectro-
scopic data with those published in the literatures led to the char-
acterization of the structures as glabridin (1), 4′-O-methylgrabridin (2)
(Vaya et al., 1997), β-amyrin (3) (Martins and Takahashi, 2010), kan-
zonol U (4) (Fukai et al., 1996), licuroside (5) and neolicuroside (6)
(Miething and Speicher-Brinker, 1989), glabrene (7) (Kinoshita et al.,
1997), isoliquiritigenin (8), isoliquiritigenin 4′-O-β-glucopyranoside (9)
(Sato et al., 2007), tetrahydroxymethoxychalcone (10) (Hatano et al.,
1997), abyssinone II (11) (Kamat et al., 1981), (2R,3R)-3,4′,7-trihy-
droxy-3′prenyl-flavanone (12) (Kuroda et al., 2010), liquiritin apioside
(13) (Montoro et al., 2011). The structures of the isolates are depicted
in Fig. 1.
3.3. Cytotoxic activity results of isolated molecules
The cytotoxic effects of the purified compounds were tested on the
epithelial cancer cell lines by SRB assay. In vitro cytotoxicity results of
isolated compounds with their calculated IC50 (μM) and R2 values are
given in Table 2. Since compounds 8 and 12 were obtained as a mixture
with different molecular weights, they were not tested. Among the
tested metabolites, 1-4, 7 and 10 showed cytotoxic effects while 5 + 6,
9 and 11 did not exert any activity. The active compounds (1-4, 7 and
10) exerted their best activity against Huh7 cancer cell lines in the
range of 1.8–16.7 μM, being the most potent ones were kanzonol U (4)
(IC50 = 1.8 μM) and glabrene (7) (IC50 = 2.3 μM). The same com-
pounds (1-4, 7 and 10) exerted cytotoxicity also against MCF7 cells in
different degrees (IC50 = 5.0–16.8 μM) being glabrene (7)
(IC50 = 5.0 μM) and β-amyrin (3) (IC50 = 6.8 μM) were the strongests.
Against HCT116 cells, 1-4, 7 and 10 were found to be cytotoxically
active (IC50 = 4.9–17.6 μM) and glabrene (7) was the most active one.
Hepatocellular carcinoma is fifth most common cancer in men
whereas eighth in women which is also the third leading cause of
cancer-related mortality causes 700.000 deaths per year across globe
(Montazeri et al., 2016). Among the cancer cell lines used in this study,
especially liver cancer cells were the most sensitive to cytotoxic effects
of the tested compounds except for 1 and 3 (Table 2). Based on the
results, glabridin (1), 4′-O-methylglabridin (2), β-amyrin (3), kanzonol
392
Industrial Crops & Products 124 (2018) 389–396
U (4), glabrene (7) and tetrahydroxymethoxychalcone (10) were iden-
tified as the bioactive secondary metabolites that are responsible for in
vitro cytotoxicity of G. glabra. The active compounds possessed iso-
flavane (1 and 2), triterpene (3), benzofuran (4), isoflavene (7) and
chalcone (10) skeletons. The inactive ones were flavanone derivative
(11) and chalcone glycosides (5 + 6 and 9). According to a study by
Ohno et al. (2013), chalcones were found to be cytotoxically more ac-
tive and to have more tumor specificity than flavanones. Moreover, it
was shown that number of phenolic hydroxyl groups had positively-
correlated with in vitro cytotoxicity. Conversely, addition of sugar units
in the molecule caused a decrease in the activity (Ji et al., 2016; Ohno
et al., 2013). The results of present study demonstrated that tetra-
hydroxymethoxychalcone (10) (IC50 = 8.3–13.9 μM) which had a
chalcone nucleus with four −OH groups displayed significant cytotoxic
effects againt all cancer cell lines tested. However, the flavanone deri-
vative, abyssinone II (11) was found to be inactive. Furthermore, none
of tested glycosides that were isolated (5 + 6 and 9) exerted any in vitro
cytotoxicity. Although glycosides do not usually show any significant
activity in in vitro screening models, after oral administration they may
be hydrolized in the gut to yield bioactive free aglycones.
3.4. Characterization of cell death mechanisms of the most active
compounds
The most active compounds, 2, 4, 7 and 10 (with IC50
values < 10 μM) were chosen for further mechanistic studies in Huh7
cells. First, time dependent cytotoxicity assay for 2, 4, 7 and 10 was
conducted by Real Time cell electronic sensing (RT-CES) in Huh7 cells.
Regarding to RT-CES results (Table 3), compounds with IC50 values <
10 μM after 48 h (2, 7 and 10) were accepted as the most active ones
that were justified to be mechanistically evaluated in Hoechst staining,
Fluorescence-activated cell sorting (FACS) and Western blot assays.
One of the most outstanding changes in apoptotic cells is the in-
crease in the concentration of the chromosomes. Apoptotic cells become
brighter blue color, while normal cells become milder after staining
with Hoechst dye which can be more invasive to cell nucleus and bound
to DNA in apoptotic cells (Kasibhatla, 2006). To gain a better under-
standing of the cell death of the most active compounds, Huh7 cells
were treated with different concentrations of the molecules based on
the results of RT-CES assay after 48 h (2: 10 μM, 7: 6 μM and 10: 5 μM).
Hoeschst staining was applied and the cellular changes were observed.
As shown in Fig. 2A compared to DMSO control, cells treated with
compound 10 were detached from the surface and showed condensed
nuclei which were positive for Hoechst staining, indicating apoptotic
cell death.
To elucidate the underlying molecular mechanisms of cell death
caused by 2, 7 and 10, the effect of these molecules on the cell cycle
distribution (SubG1, G1, S, G2 ve M) of Huh7 cells was evaluated by PI
staining using cell cycle assay (FACS). Huh7 cells were incubated with
compounds at different concentrations (2: 10 μM, 7: 6 μM, 10: 2,5 μM).
According to cell cycle results, there were significant increases in the
percentages of subG1 cells of Huh7 treated with 2, 7 and 10 indicating
increased number of apoptotic cells. Furthermore, accumulation of the
cells in G2/M phase was observed for each compound. Especially, the
G2/M arrest had become very dramatical for the cells treated with
compound 10.
To gain better understanding of cytotoxicity of the molecules 2, 7
and 10, next we examined the changes in protein expression levels
which are involved in apoptosis and cell cycle signaling pathways by
western blotting. For this purpose, cytochrome C, caspase-9, poly (ADP-
ribose) polymerase (PARP), phospho-Rb, p53, Mdm2 and p21 proteins
were examined for western blot technique in terms of apoptotic signal
networks. Cytochrome C is a pro-apoptotic protein released from mi-
tochondria into cytosol upon induction of apoptosis which plays an
important role for the activation of caspase-9. Caspases are key factors
of programmed cell death. The caspase cascade is triggered by
D. Çevik et al. Industrial Crops & Products 124 (2018) 389–396

Fig. 1. Chemical structures of compounds 1-13 isolated from Glycyrrhiza glabra.

Table 2 cleavage of PARP (Mohan et al., 2016; Shyu et al., 2010). As shown in
Cytotoxic activity results of purified and structurally determined compounds Fig. 3A, cytochrom C levels were significantly raised compared to
against Huh7, MCF7 and HCT116 cancer cell lines by SRB assay. DMSO control in Huh7 cells incubated with tested compounds (2, 7 and
Compounds Huh7 MCF7 HCT116 10). Besides, the levels of cleaved caspase-9 were increased with in-
cubation of the compounds, especially for 7 (Fig. 3A). On the other
2 2
IC50 R IC50 R IC50 (μM)a R2 hand, the increase in cleaved PARP levels were observed in cells treated
(μM)a (μM)a
with 7 and 10 (Fig. 3A). These findings suggest that the tested com-
Glabridin (1) 16.7 0.9 15.1 0.9 10.5 0.9 pounds, particularly 7 might reveal apoptosis through caspase cascade.
4′-O-methylgrabridin (2) 9.6 1 16.5 0.9 10.8 0.9 Phospho-Rb (pRb) is a retinoblastoma protein which is neccesary for
β-amyrin (3) 9.3 0.9 6.8 0.8 17.6 0.9 the continuation of cell cycle and decrease in phosphorylation of this
Kanzonol U (4) 1.8 0.8 16.8 0.9 14.0 0.8
protein is an indicator of the suppression in cell cycle. Besides being a
Glabrene (7) 2.3 0.99 5.0 0.7 4.9 0.92
Tetrahydroxymethoxychalcone 8.3 0.97 13.9 0.99 13.3 0.98
negative regulator of cell growth, it is also an anti-apoptotic factor and
(10) clevead during apoptotic pathways. This fragmentation impairs the
Abyssinone II (11) NI NI NI interaction of pRb with Mdm2 which is also an apoptosis inhibitor
Licuroside + Neolicuroside NI NI NI function together with pRb (Pucci et al., 2000). pRb proteins function in
(5 + 6)
G1 phase for the phosphorylation of transcriptional genes. As shown in
Isoliquiritigenin 4′-O-β- NI NI NI
glucopyranoside (9) Fig. 3B, pRb expression was decreased especially in cells incubated with
Camptothecin <1 <1 <1 2 and 7 which were important findings in terms of similarity with the
results of cell cycle analysis (Fig. 2B).
NI: No Inhibition. p53 gene is DNA-binding phosphoprotein, which plays an important
a
IC50 values were calculated from the cell growth inhibition curves obtained role in several aspects of cell cycle arrest, apoptosis, DNA repair etc.
from the treatments with increasing concentrations of compounds (40, 20, 10, 5
and regulates the cell mechanisms by transactivating various genes
and 2.5 μM) for 72 h. If IC50 value was beyond 2.5 μM the assays were repeated
including p21 and Mdm2 (Pucci et al., 2000). The dual activity of p21
with lower concentrations. Experiments were done in triplicate.
in apoptosis was shown that its downregulation or overexpression had
supressed the cellular proliferation, hence promoted apoptotic pro-
activation of caspases by proteolysis of inactive procaspases and con-
cesses and increased the G2 phase arrest (Chen et al., 2015). Based on
tinues the cleavage of downstream caspases and substrates such as
these findings, the significant decrease in p21 level in cells treated wih
PARP (Hengartner, 2000). Thus, activation of caspase-9 leads to
the compunds 2, 7, and 10 indicated induction of apoptotic response

Table 3
Real-time bioactivity assay (RT-CES xCELLigence) results of active compounds against Huh7 cells.
Compounds 24 h 48 h 72 h

2 2
IC50 (μM) R IC50 (μM) R IC50 (μM) R2

4′-O-methylgrabridin (2) 7.31 0.9 9.8 0.9 11.4 0.9

Kanzonol U (4) 10 0.9 13 0.8 14.7 0.9
Glabrene (7) 9 1 5.8 1 5.9 1
Tetrahydroxymethoxychalcone (10) 18 0.6 4.4 0.8 6.6 0.7

393
D. Çevik et al. Industrial Crops & Products 124 (2018) 389–396

Fig. 2. (A) Fluorescent microscopy images of Huh7 cells treated with DMSO or compounds for 48 h (2: 10 μM, 7: 6 μM, 10: 5 μM) stained with Hoechst 33258 nuclear
dye (B) Cell cycle analysis of Huh7 cells upon treatment with 2,7 and 10 (10 μM, 6 μM, and 2,5 μM respectively) and DMSO following 48 h of treatment.

Fig. 3. Effects of compounds 2, 7 and 10 on various cell protein levels (A, B) in Huh7 cells. (A) Apoptotic protein levels: cleaved caspase-9, cytochrom C and cleaved
PARP, (B) Cell cycle regulatory proteins: Mdm2, phosphoRb, p53, phospho-p53 and p21 proteins. Densities of the bands obtained by Western blot were compared
using ImageJ normalized to DMSO. Beta-actin was blotted as an equal loading control.

394
D. Çevik et al.
(Fig. 3B). Moreover, along with decreased p21 levels, the accumulation
of cells in G2 phase was also observed with these compounds particu-
larly with 10 (Fig. 2B). In addition, it was reported that p53 expression
usually mimics the p21 expression (Chen et al., 2015). The results of
this study supported this assumption, since the expression profiles of
p53 and p21 were found to be parallel in all tested samples especially
with the 7 and 10 (Fig. 3B). On the other hand, Mdm2 which is a ne-
gative regulator of p53 by binding to and promoting the degradation of
this protein (Pucci et al., 2000), were significantly decreased in cells
treated with the compounds. Hence, as shown in Fig. 3B, phosphor-
ylation of p53 was increased in cells incubated with compound 2.
The cytotoxic effects of glabrene (7), tetrahydroxymethoxychalcone
(10), neolicuroside and licuroside (5 + 6) were evaluated for the first
time against all three tested cell lines. Furthermore, these compounds
had never been studied before regarding their cytotoxic activities
against any cancer cells. In a very recent study, glabridin, a key bio-
logical and chemical marker of G. glabra, was reported to induce Huh7
cell death by inducing apoptosis through caspase cascade (Hsieh et al.,
2016). Cytotoxicity of isoliquiritigenin 4′-O-β-glucopyranoside (9), 4′-
O-methylglabridin (2), kanzonol U (4) and abyssinone II (11) against
HCT116 and Huh7, and β-amyrin (3) against Huh7 cells are also re-
ported with this study for the first time.
4. Conclusion
In conclusion, nine secondary metabolites were isolated and iden-
tified from G. glabra through bioactivity-guided fractionation. Amongst
isolates, glabridin (1), 4′-O-methylglabridin (2), β-amyrin (3), kanzonol
U (4), glabrene (7) and tetrahydroxymethoxychalcone (10) which ex-
erted the best potent activity against Huh7 cancer cell lines, were es-
tablished to be responsible for the in vitro cytotoxic effect of G. glabra.
The structure and cytotoxic activity relationships of the tested com-
pounds were also discussed. Furthermore, the mechanisms behind the
cytotoxic activities of the most active compounds were also elucidated.
4′-O-methylglabridin (2), glabrene (7) and tetra-
hydroxymethoxychalcone (10) contributed significantly to the in vitro
cytotoxic activity of G. glabra against Huh7 cancer cell lines by inducing
apoptosis through different mechanisms in liver cancer cells. Based on
these data, particularly 7 and 10 could be potential anticancer drug
candidates which deserve further in vivo and preclinical studies for the
development of new anti-cancer drug leads especially against hepato-
cellular carcinoma. The isolated bioactive compounds deserve to be
tested for their potential cytotoxic activities against other human
cancer cell lines. Based on these findings and the other results obtained
from previous studies on different Glycyrrhiza species put forward the
potential of licorice species to afford new promising anticancer drug
candidates.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgement
This study was supported by The Scientific and Technological
Research Council of Turkey (TÜBİTAK, Project No: 115S433).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2018.08.014.
References
Aydemir, E.A., Oz, E.S., Göktürk, R.S., Ozkan, G., Fiskin, K., 2011. Glycyrrhiza flavescens
subsp. antalyensis exerts antiproliferative effects on melanoma cells via altering TNF-
395
Industrial Crops & Products 124 (2018) 389–396
α and IFN-α levels. Food Chem. Toxicol. 49, 820–828. https://doi.org/10.1016/j.fct.
2010.12.003.
Chamberlain, D.F., 1969. Glycyrrhiza L. In: Davis, P.H. (Ed.), Flora of Turkey and East
Aegean Islands 3. Edinburgh University Press, Edinburgh, pp. 260–263.
Chen, A., Huang, X., Xue, Z., Cao, D., Huang, K., Chen, J., Pan, Y., Gao, Y., 2015. The role
of p21 in apoptosis, proliferation, cell cycle arrest, and antioxidant activity in UVB-
irradiated human HaCaT keratinocytes. Med. Sci. Monit. Basic Res. 21, 86–95.
https://doi.org/10.12659/MSMBR.893608.
Fukai, T., Sheng, C.B., Horikoshi, T., Nomura, T., 1996. Isoprenylated flavonoids from
underground parts of Glycyrrhiza glabra. Phytochemistry 43, 1119–1124. https://doi.
org/10.1016/S0031-9422(96)00391-3.
Gallagher, S., Winston, S.E., Fuller, S.A., Hurrel, J.G., 2008. Immunoblotting and im-
munodetection. Curr. Protoc. Mol. Biol. 83https://doi.org/10.1002/0471142727.
mb1008s83. 10.8.
Hatano, T., Tagagi, M., Ito, H., Yoshida, T., 1997. Phenolic constituents of liquorice. VII. A
new chalcone with a potent radical scavenging activity and accompanying phenolics
from liquorice. Chem. Pharm. Bull. 45, 1485–1492. https://doi.org/10.1248/cpb.37.
3229.
Hawash, M.M.A., Kahraman, D.C., Eren, F., Atalay, R.C., Baytas, S.N., 2017. Synthesis and
biological evaluation of novel pyrazolic chalcone derivatives as novel hepatocellular
carcinoma therapeutics. Eur. J. Med. Chem. 129, 12–26. https://doi.org/10.1016/j.
ejmech.2017.02.002.
Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407, 770–776. https://
doi.org/10.1038/35037710.
Hosseinzadeh, H., Nassiri-Asl, M., 2015. Pharmacological effects of Glycyrrhiza spp. and
its bioactive constituents: update and review. Phytother. Res. 29, 1868–1886.
https://doi.org/10.1002/ptr.5487.
Hsieh, M.-J., Chen, M.-K., Chen, C.-J., Hsieh, M.-C., Lo, Y.-S., Chuang, Y.-C., Chiou, H.-L.,
Yang, S.-F., 2016. Glabridin induces apoptosis and autophagy through JNK1/2
pathway in human hepatoma cells. Phytomedicine 23, 359–366. https://doi.org/10.
1111/bph.12626.
Ji, S., Li, Z., Song, W., Wang, Y., Liang, W., Li, K., Ye, M., 2016. Bioactive constituents of
Glycyrrhiza uralensis (Licorice): discovery of the effective components of a traditional
herbal medicine. J. Nat. Prod. 79, 281–292. https://doi.org/10.1021/acs.jnatprod.
5b00877.
Kamat, V.S., Chuo, F., Kubo, I., Nakanishi, K., 1981. Antimicrobial agents from an East
African medicinal plant Erythrina abyssinica. Heterocycles 15, 1163–1169. https://
doi.org/10.3987/S-1981-02-1163.
Kasibhatla, S., 2006. Staining of suspension cells with hoechst 33258 to detect apoptosis.
Cold Spring Harb. Protoc. 2006, 4492. https://doi.org/10.1101/pdb.prot4492.
Ke, N., Wang, X., Xu, X., Abassi, Y.A., 2011. The Xcelligence system for real-time and
label-free monitoring of cell viability. Methods Mol. Biol. 740, 33–43. https://doi.
org/10.1007/978-1-61779-108-6_6.
Kinoshita, T., Tamura, Y., Mizutani, K., 1997. Isolation and synthesis of two new 3-ar-
ylcoumarin derivatives from the root of Glycyrrhiza glabra (Licorice), and structure
revision of an antioxidant isoflavonoid glabrene. Nat. Prod. Lett. 9, 289–296. https://
doi.org/10.1080/10575639708043642.
Kırmızıbekmez, H., Uysal, G.B., Masullo, M., Demirci, F., Bağcı, Y., Kan, Y., Piacente, S.,
2015. Prenylated polyphenolic compounds from Glycyrrhiza iconica and their anti-
microbial and antioxidant activities. Fitoterapia 103, 289–293. https://doi.org/10.
1016/j.fitote.2015.05.003.
Kuroda, M., Mimaki, Y., Honda, S., Tanaka, H., Yokota, S., Mae, T., 2010. Phenolics from
Glycyrrhiza glabra roots and their PPAR-γ ligand-binding activity. Bioorg. Med. Chem.
18, 962–970. https://doi.org/10.1016/j.bmc.2009.11.027.
Li, K., Ji, S., Song, W., Kuang, Y., Lin, Y., Tang, S., Cui, Z., Qiao, X., Yu, S., Ye, M., 2017.
Glycybridins A-K, bioactive phenolic compounds from Glycyrrhiza glabra. J. Nat.
Prod. 80, 334–346. https://doi.org/10.1021/acs.jnatprod.6b00783.
Lin, Y., Kuang, Y., Li, K., Wang, S., Song, W., Qiao, X., Sabir, G., Ye, M., 2017. Screening
for bioactive natural products from a 67-compound library of Glycyrrhiza inflata.
Bioorg. Med. Chem. 25, 3706–3713. https://doi.org/10.1016/j.bmc.2017.05.009.
Martins, L.R., Takahashi, J.A., 2010. Rearrangement and oxidation of β-amyrin promoted
by growing cells of Lecanicillium muscarinium. Nat. Prod. Res. 24, 767–774. https://
doi.org/10.1080/14786410903262865.
Miething, H., Speicher-Brinker, A., 1989. Neolicurosid - ein neues Chalkonglykosid aus
der Suβholzwurzel. Arch. Pharm. 322, 141–143. https://doi.org/10.1002/ardp.
19893220305.
Mohan, V., Agarwal, R., Singh, R.P., 2016. A novel alkaloid, evodiamine causes nuclear
localization of cytochrome-c and induces apoptosis independent of p53 in human
lung cancer cells. Biochem. Biophys. Res. Commun. 477, 1065–1071. https://doi.
org/10.1016/j.bbrc.2016.07.037.
Montazeri, M., Sadeghizadeh, M., Pilehvar-Soltanahmadi, Y., Zarghami, F., Khodi, S.,
Mohaghegh, M., Sadeghzadeh, H., Zarghami, N., 2016. Dendrosomal curcumin na-
noformulation modulate apoptosis-related genes and protein expression in hepato-
carcinoma cell lines. Int. J. Pharm. 509, 244–254. https://doi.org/10.1016/j.
ijpharm.2016.05.039.
Montoro, P., Maldini, M., Russo, M., Postorino, S., Piacente, S., Pizza, C., 2011. Metabolic
profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by
ESI/ MS/ MS and determination of major metabolites by LC-ESI/ MS and LC-ESI/
MS/MS. J. Pharm. Biomed. 54, 535–544. https://doi.org/10.1016/j.jpba.2010.10.
004.
Nomura, T., Fukai, T., Akiyama, T., 2002. Chemistry of phenolic compounds of licorice
(Glycyrrhiza species) and their estrogenic and cytotoxic activities. Pure Appl. Chem.
74, 1199–1206. https://doi.org/10.1351/pac200274071199.
Ohno, H., Araho, D., Uesawa, Y., Kagaya, H., Ishihara, M., Sakagami, H., Yamamoto, M.,
2013. Evaluation of cytotoxiciy and tumor-specificity of licorice flavonoids based on
chemical structure. Anticancer Res. 33, 3061–3068.
D. Çevik et al.
Park, S.Y., Kim, E.J., Choi, H.J., Seon, M.R., Lim, S.S., Kang, Y.-H., Choi, M.-S., Lee, K.W.,
Park, J.H.Y., 2014. Anti-carcinogenic effects of non-polar components containing
licochalcone A in roasted licorice root. Nutr. Res. Pract. 8, 257–266. https://doi.org/
10.4162/nrp.2014.8.3.257.
Pucci, B., Kasten, M., Giordano, A., 2000. Cell cycle and apoptosis. Neoplasia 2, 291–299.
Riccardi, C., Nicoletti, I., 2006. Analysis of apoptosis by propidium iodide staining and
flow cytometry. Nat. Protoc. 1, 1458–1461. https://doi.org/10.1038/nprot.2006.
238.
Saklani, A., Kutty, S.K., 2008. Plant-derived compounds in clinical trials. Drug Discov.
Today 13, 161–171. https://doi.org/10.1016/j.drudis.2007.10.010.
Sato, Y., He, J.-X., Nagai, H., Tani, T., Akao, T., 2007. Isoliquiritigenin, one of the anti-
spasmodic principles of Glycyrrhiza ularensis roots, acts in the lower part of intestine.
Biol. Pharm. Bull. 30, 145–149. https://doi.org/10.1248/bpb.30.145.
Shyu, M.H., Kao, T.C., Yen, G.C., 2010. Oleanolic acid and ursolic acid induce apoptosis in
HuH7 human hepatocellular carcinoma cells through a mitochondrial-dependent
pathway and downregulation of XIAP. J. Agric. Food Chem. 58, 6110–6118. https://
doi.org/10.1021/jf100574j.
Tang, Z.-H., Li, T., Tong, Y.-G., Chen, X.-J., Chen, X.-P., Wang, Y.-T., Lu, J.-J., 2015. A
systematic review of the anticancer properties of compounds isolated from licorice
(Gancao). Planta Med. 81, 1670–1687. https://doi.org/10.1055/s-0035-1558227.
Thiyagarajan, P., Chandrasekaran, C.V., Deepak, H.B., Agarwal, A., 2011. Modulation of
lipopolysaccharide-induced pro-inflammatory mediators by an extract of Glycyrrhiza
396
Industrial Crops & Products 124 (2018) 389–396
glabra and its phytoconstituents. Inflammopharmacology 19, 235–241. https://doi.
org/10.1007/s10787-011-0080-x.
Vaya, J., Belinky, P.A., Aviram, M., 1997. Antioxidant constituents from licorice roots:
isolation, structure elucidation and antioxidative capacity toward LDL oxidation. Free
Radic. Biol. Med. 23, 302–313. https://doi.org/10.1016/S0891-5849(97)00089-0.
Vichai, V., Kirtikara, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity
screening. Nat. Protoc. 1, 1112–1116. https://doi.org/10.1038/nprot.2006.179.
Vlaisavljević, S., Šibul, F., Sinka, I., Zupko, I., Ocsovszki, I., Jovanović-Šanta, S., 2018.
Chemical composition, antioxidant and anticancer activity of licorice from Fruska
Gora locality. Ind. Crops Prod. 112, 217–224. https://doi.org/10.1016/j.indcrop.
2017.11.050.
WHO, 2014. World Cancer Report 2014. . http://www.who.int/cancer/publications/
WRC_2014/en/.
Wittschier, N., Faller, G., Hensel, A., 2009. Aqueous extracts and polysaccharides from
Liquorice roots (Glycyrrhiza glabra L.) inhibit adhesion of Helicobacter pylori to human
gastric mucosa. J. Ethnopharmacol. 125, 218–223. https://doi.org/10.1016/j.jep.
2009.07.009.
Wong, R.S., 2011. Apoptosis in cancer: from pathogenesis to treatment. J. Exp. Clin.
Cancer Res. 30, 87. https://doi.org/10.1186/1756-9966-30-87.
Yang, R., Wang, L.Q., Yuan, B.C., Liu, Y., 2015. The pharmacological activities of licorice.
Planta Med. 81, 1654–1669. https://doi.org/10.1055/s-0035-1557893.