Вы находитесь на странице: 1из 8

Industrial Crops & Products 124 (2018) 389–396

Contents lists available at ScienceDirect

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 ribose) polymerase (PARP) levels (Mohan et al., 2016; Shyu et al.,
2010).
Cancer is the second leading cause of morbidity and mortality after Most of the drugs used in cancer treatment today are not effective
cardiovascular diseases in industrialized countries. It had been 14.1 enough or have some serious side effects, thus new anticancer drugs are
million new cancer cases whereas 8.2 million cancer deaths in 2012. needed to combat cancer. Natural resources particularly medicinal
Breast cancer has the highest incidence rate among women followed by plants play a very significant role for the discovery and development of
colorectal cancer. Among men colorectal cancer is the third whereas, new drug leads. Clinically used anticancer drugs such as vincristine,
liver cancer which has a relatively poor survival and hence mortality is vinblastine, vindesine, paclitaxel, etoposide, teniposide, irinotecan and
the fifth common sites of cancer (WHO Cancer Report, 2014). Cancer topotecan are natural products or their derivatives which are produced
arises from the loss of balance between cell division and cell death. by semi-synthesis from a natural molecule (Saklani and Kutty, 2008).
Apoptosis is regulated destruction of cells, plays an important role and The genus Glycyrrhiza (Fabaceae) commonly known as licorice,
is the main target in cancer treatment (Wong, 2011). This is a very contains approximately 30 species distributed worldwide including, G.
complicated process including both intrinsic and extrinsic pathways. glabra, G. aspera and G. uralensis (Nomura et al., 2002). In Flora of
Release of cytochrom C from mitochondria into cytosol is occured via Turkey, it is represented by six species being G. glabra, the most pre-
intrinsic pathway which subsequently activates caspase-9 and thus valent one (Chamberlain, 1969). Glycyrrhiza glabra L. has a long history
caspase-3 (Hengartner, 2000). Activation of these caspases eventually of use in different folk medicines for the treatment of peptic ulcers,
leads to apotosis through caspase cascade by cleaving of poly (ADP- 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. Industrial Crops & Products 124 (2018) 389–396

80:20) to give 1 (3 mg). Fr. 4 (2 g) of EtOAc subextract was fractionated triplicates and IC50 values were calculated as μg/mL for extracts and
over LiChroprep C18-MPLC (130 g) using stepwise H2O:MeOH gradient fractions and as μM for isolated compounds. The cell growth inhibition
(85:15–0:100) to yield subfractions 4a-j. Subfraction 4b (87 mg) was ratios were calculated by formula of [1− (Absdrug × 100)/(AbsDMSO)].
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) 2.7. Real time bioactivity assay (RT-CES xCELLigence)
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) The growth inhibitory effect of the most active compounds in SRB
to afford a mixture of 8 and 12 (9 mg). Compound 7 (3 mg) was isolated assay on Huh7 cells were monitored by the xCELLigence System (Roche
as consequence of submitting subfraction 4g (155 mg) to SiO2 column Applied Sciences) which has a microelectronic biosensor technology to
with CH2Cl2:MeOH (97.5:2.5, 95:5, 90:10). Subfraction 4h (103 mg) do dynamic, real-time, label-free and non-invasive analysis of cellular
was seperated by SiO2 (15 g) column chromatography using n-hex- events, including cell number change, cell adhesion, cell viability, cell
ane:EtOAc (20–50% EtOAc) as mobile phase to yield fr. 4h-1 (30 mg) morphology, and cell motility (Ke et al., 2011). Huh7 cancer cell lines
which was subsequently applied to SiO2 (6 g) column again eluted with (2000 cells/well) were seeded into 96-well E-plates. Cells were treated
same mobile phase (15–25% EtOAc) to give 1 (4 mg). The major with the compounds with their IC50 concentrations. The proliferation of
compounds of the inactive fractions (frs. 2 and 3) of the EtOAc sub- cells were monitored every 30 min for 24 h after seeding, then treat-
extract were also isolated in order to make a phytochemical contribu- ments with the compounds were done in log growth phase of cells. The
tion to the plant species. Thus, fr. 2 (357 mg) was subjected to C18- cell index (CI) values were recorded every 10 min for the first 24 h and
MPLC (50 g) eluting with H2O:MeOH (0–70% MeOH) to obtain sub- then every 30 min for the remaining 48 h. The cell growth ratios were
fractions. Subfraction 2a (130 mg) was applied to SiO2 CC (20 g), eluted calculated by CIdrug/CIDMSO.
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 2.8. Apoptotic cell death assay (Hoechst staining)
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 Apoptotic cells were identified by Hoechst staining. Huh7 cells were
purified from subfr. 3e by SiO2-MPLC (40 g, CH2Cl2: MeOH; 5–40% seeded onto coverslips in 6-well plates. After 24 h culture, cells were
MeOH) and C18-MPLC (13 g, H2O:MeOH, 15–50% MeOH) methods. 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
2.4. Structure elucidation methods 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
The structures were elucidated based on spectroscopic methods in- images of them were taken by using the fluorescence microscope
cluding UV, IR, 1D (1H and 13C) and 2D (COSY, HSQC, HMBC and (Nikon Eclipse 50i).
NOESY) NMR as well as MS.
2.9. Cell cycle assay (Fluorescence-activated cell sorting (FACS))
2.5. Cell lines and cell culture
This method is used to investigate the effects of the cytotoxic mo-
Human liver (Huh7), colon (HCT116) and breast (MCF7) cancer cell lecules’ on cell cycle phases such as SubG1, G1, S and M. Propidium
lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Iodide (PI) dye binds to DNA in the presence of cytotoxic agent. Thus
Gibco #31885) supplemented with 10% fetal bovine serum (FBS; this method determines the cell cycle phase by different type of DNA of
Invitrogen GIBCO #10270) and 1% penicillin (Gibco #15140-122). The cells after staining with PI (Riccardi and Nicoletti, 2006). Huh7 cells
cell lines were maintained in an incubator at 37 °C under 5% CO2 were seeded onto 100 mm culture dishes. After 24 h, cells were treated
(Hawash et al., 2017). with the compounds with their IC50 concentrations based on the RT-
CES results after 48 h. IC50 concentration of compound 10 distrupted
2.6. Sulforhodamine B assay 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
The sulforhodamine B (SRB) assay was performed to evaluate the 70% ethanol for 3 h at −20 °C after 48 h of incubation. Cell cycle
antiproliferative effects of crude extract, subextracts, main fractions as analysis was carried out by PI staining using Fluorescence-activated cell
well as purified compounds. SRB assay had been developed by US sorting (FACS).
National Cancer Institute (NCI) which is a rapid, sensitive and in-
expensive method and used for cell density determination, based on the 2.10. Cell protein assay (Western Blot)
measurement of cellular protein content (Vichai and Kirtikara, 2006).
Human cancer cell lines were plated in 96-well plates (MCF7/Huh7: Immunoblotting (western blotting) is a technique that has been used
2000 cell/well, HCT116: 3000 cell/well in 150 μl/well DMEM) and to for the identification of specific antigens recognized by polyclonal or
grown for 24 h at 37 °C. After 24 h, cells were treated with five in- monoclonal antibodies. It gives information about the identity of the
creasing concentrations of the extracts (1.875–30 μg/mL), main frac- specific proteins, the change of their amounts and phosphorylation
tions or compounds (2.5–40 μM) dissolved in DMSO and applied character of them (Gallagher et al., 2008). Huh7 cancer cell lines were
through serial dilution. After treatment process, the cells were in- inoculated into 150 mm culture dishes and after 24 h cells were treated
cubated at 37 under 5% CO2 for 72 h which was followed by washing with the compounds (IC50 concentrations based on the RT-CES results
the plates with 1xPBS (CaCl2-, MgCl2-free). Fixation was performed after 48 h) or DMSO control for 48 h. Novex® NuPAGE® Bis-Tris Elec-
using 100 μL 10% (v/v) ice-cold trichloroacetic acid (TCA; Merck) and trophoresis system was used according to the manufacturer's protocol
incubated in dark +4 °C for one hour. Then TCA was washed away with for all the western blotting analysis. For gel electrophoresis (NuPAGE),
ddH2O 2 times and plates were air-dried. Finally, 100 μL of 0.4% (m/v) 20–50 μg of protein was used per well. Then the proteins were trans-
of sulforhodamine (SRB) in 1% acetic acid solution was added to each ferred to nitrocellulose membrane via XCell IITM Blot Module. p53
well for staining process. The unbound SRB was washed with 1% acetic (#05-224, Millipore), phospho-Rb (Ser807/811) (#9308S Cell Sig-
acid solution for 4 times to remove non-specific dye binding staining naling), cytochrom C (sc7159, Santa Cruz), Mdm2 (OP46, Calbiochem),
and left for air drying. The bound sulforhodamine B was then solubi- p21 (OP64, Calbiochem), cleaved caspase-9 (sc-22182, Santa Cruz) and
lized using 100 μL 10 mM Tris-base prior to absorbance measurement. cleaved PARP (#9532, Cell Signaling) antibodies were used in
The absorbance was read at 515 nm. The experiments were done in 1:200–1:500 5% BSA-TBS-T. β-actin (#A5441, Sigma) antibody was

391
D. Çevik et al. Industrial Crops & Products 124 (2018) 389–396

used for equal loading (Hawash et al., 2017). U (4), glabrene (7) and tetrahydroxymethoxychalcone (10) were iden-
tified as the bioactive secondary metabolites that are responsible for in
3. Results and discussion vitro cytotoxicity of G. glabra. The active compounds possessed iso-
flavane (1 and 2), triterpene (3), benzofuran (4), isoflavene (7) and
3.1. Bioactivity-guided fractionation and isolation chalcone (10) skeletons. The inactive ones were flavanone derivative
(11) and chalcone glycosides (5 + 6 and 9). According to a study by
The CHCl3 and EtOAc subextracts obtained from the crude MeOH Ohno et al. (2013), chalcones were found to be cytotoxically more ac-
extract of G. glabra roots showed cytotoxic activity against hepatocel- tive and to have more tumor specificity than flavanones. Moreover, it
lular (Huh7), breast (MCF7) and colorectal (HCT116) cancer cell lines was shown that number of phenolic hydroxyl groups had positively-
with IC50 values in the range of 5.6–29.3 μg/mL whereas n-buOH and correlated with in vitro cytotoxicity. Conversely, addition of sugar units
remaing H2O subextracts were found to be inactive (Table 1). There- in the molecule caused a decrease in the activity (Ji et al., 2016; Ohno
fore, CHCl3 and EtOAc subextracts were fractionated by Sephadex LH- et al., 2013). The results of present study demonstrated that tetra-
20 and polyamide column respectively and the obtained main fractions hydroxymethoxychalcone (10) (IC50 = 8.3–13.9 μM) which had a
were submitted to SRB assay. The active fractions (frs. B and C of CHCl3 chalcone nucleus with four −OH groups displayed significant cytotoxic
and fr. 4 of EtOAc) were selected for the isolation of the potential effects againt all cancer cell lines tested. However, the flavanone deri-
bioactive metabolites (Table 1). vative, abyssinone II (11) was found to be inactive. Furthermore, none
As a result of bioassay-guided fractionation and isolation studies, of tested glycosides that were isolated (5 + 6 and 9) exerted any in vitro
compounds 1-4 and 11 were isolated from active fractions of CHCl3 cytotoxicity. Although glycosides do not usually show any significant
while, 1, 7, 8, 10 and 12 were obtained from active fraction of EtOAc. activity in in vitro screening models, after oral administration they may
Moreover, the major compounds (5, 6, 9 and 13) of the inactive frac- be hydrolized in the gut to yield bioactive free aglycones.
tions of the EtOAc subextract were also isolated in order to make a
phytochemical contribution to the plant species. 3.4. Characterization of cell death mechanisms of the most active
compounds
3.2. Structure elucidation of isolated molecules
The most active compounds, 2, 4, 7 and 10 (with IC50
As a result of sequential chromatographic techniques, totally 13 values < 10 μM) were chosen for further mechanistic studies in Huh7
compounds were isolated from G.glabra roots. Among these compounds cells. First, time dependent cytotoxicity assay for 2, 4, 7 and 10 was
5–6 and 8–12 were obtained as mixtures. To determine the exact conducted by Real Time cell electronic sensing (RT-CES) in Huh7 cells.
structure of the isolates spectroscopic techniques such as UV, IR, 1D and Regarding to RT-CES results (Table 3), compounds with IC50 values <
2D NMR and MS were applied. The comparasion of obtained spectro- 10 μM after 48 h (2, 7 and 10) were accepted as the most active ones
scopic data with those published in the literatures led to the char- that were justified to be mechanistically evaluated in Hoechst staining,
acterization of the structures as glabridin (1), 4′-O-methylgrabridin (2) Fluorescence-activated cell sorting (FACS) and Western blot assays.
(Vaya et al., 1997), β-amyrin (3) (Martins and Takahashi, 2010), kan- One of the most outstanding changes in apoptotic cells is the in-
zonol U (4) (Fukai et al., 1996), licuroside (5) and neolicuroside (6) crease in the concentration of the chromosomes. Apoptotic cells become
(Miething and Speicher-Brinker, 1989), glabrene (7) (Kinoshita et al., brighter blue color, while normal cells become milder after staining
1997), isoliquiritigenin (8), isoliquiritigenin 4′-O-β-glucopyranoside (9) with Hoechst dye which can be more invasive to cell nucleus and bound
(Sato et al., 2007), tetrahydroxymethoxychalcone (10) (Hatano et al., to DNA in apoptotic cells (Kasibhatla, 2006). To gain a better under-
1997), abyssinone II (11) (Kamat et al., 1981), (2R,3R)-3,4′,7-trihy- standing of the cell death of the most active compounds, Huh7 cells
droxy-3′prenyl-flavanone (12) (Kuroda et al., 2010), liquiritin apioside were treated with different concentrations of the molecules based on
(13) (Montoro et al., 2011). The structures of the isolates are depicted the results of RT-CES assay after 48 h (2: 10 μM, 7: 6 μM and 10: 5 μM).
in Fig. 1. Hoeschst staining was applied and the cellular changes were observed.
As shown in Fig. 2A compared to DMSO control, cells treated with
3.3. Cytotoxic activity results of isolated molecules compound 10 were detached from the surface and showed condensed
nuclei which were positive for Hoechst staining, indicating apoptotic
The cytotoxic effects of the purified compounds were tested on the cell death.
epithelial cancer cell lines by SRB assay. In vitro cytotoxicity results of To elucidate the underlying molecular mechanisms of cell death
isolated compounds with their calculated IC50 (μM) and R2 values are caused by 2, 7 and 10, the effect of these molecules on the cell cycle
given in Table 2. Since compounds 8 and 12 were obtained as a mixture distribution (SubG1, G1, S, G2 ve M) of Huh7 cells was evaluated by PI
with different molecular weights, they were not tested. Among the staining using cell cycle assay (FACS). Huh7 cells were incubated with
tested metabolites, 1-4, 7 and 10 showed cytotoxic effects while 5 + 6, compounds at different concentrations (2: 10 μM, 7: 6 μM, 10: 2,5 μM).
9 and 11 did not exert any activity. The active compounds (1-4, 7 and According to cell cycle results, there were significant increases in the
10) exerted their best activity against Huh7 cancer cell lines in the percentages of subG1 cells of Huh7 treated with 2, 7 and 10 indicating
range of 1.8–16.7 μM, being the most potent ones were kanzonol U (4) increased number of apoptotic cells. Furthermore, accumulation of the
(IC50 = 1.8 μM) and glabrene (7) (IC50 = 2.3 μM). The same com- cells in G2/M phase was observed for each compound. Especially, the
pounds (1-4, 7 and 10) exerted cytotoxicity also against MCF7 cells in G2/M arrest had become very dramatical for the cells treated with
different degrees (IC50 = 5.0–16.8 μM) being glabrene (7) compound 10.
(IC50 = 5.0 μM) and β-amyrin (3) (IC50 = 6.8 μM) were the strongests. To gain better understanding of cytotoxicity of the molecules 2, 7
Against HCT116 cells, 1-4, 7 and 10 were found to be cytotoxically and 10, next we examined the changes in protein expression levels
active (IC50 = 4.9–17.6 μM) and glabrene (7) was the most active one. which are involved in apoptosis and cell cycle signaling pathways by
Hepatocellular carcinoma is fifth most common cancer in men western blotting. For this purpose, cytochrome C, caspase-9, poly (ADP-
whereas eighth in women which is also the third leading cause of ribose) polymerase (PARP), phospho-Rb, p53, Mdm2 and p21 proteins
cancer-related mortality causes 700.000 deaths per year across globe were examined for western blot technique in terms of apoptotic signal
(Montazeri et al., 2016). Among the cancer cell lines used in this study, networks. Cytochrome C is a pro-apoptotic protein released from mi-
especially liver cancer cells were the most sensitive to cytotoxic effects tochondria into cytosol upon induction of apoptosis which plays an
of the tested compounds except for 1 and 3 (Table 2). Based on the important role for the activation of caspase-9. Caspases are key factors
results, glabridin (1), 4′-O-methylglabridin (2), β-amyrin (3), kanzonol of programmed cell death. The caspase cascade is triggered by

392
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. Industrial Crops & Products 124 (2018) 389–396

(Fig. 3B). Moreover, along with decreased p21 levels, the accumulation α and IFN-α levels. Food Chem. Toxicol. 49, 820–828. https://doi.org/10.1016/j.fct.
of cells in G2 phase was also observed with these compounds particu- 2010.12.003.
Chamberlain, D.F., 1969. Glycyrrhiza L. In: Davis, P.H. (Ed.), Flora of Turkey and East
larly with 10 (Fig. 2B). In addition, it was reported that p53 expression Aegean Islands 3. Edinburgh University Press, Edinburgh, pp. 260–263.
usually mimics the p21 expression (Chen et al., 2015). The results of Chen, A., Huang, X., Xue, Z., Cao, D., Huang, K., Chen, J., Pan, Y., Gao, Y., 2015. The role
this study supported this assumption, since the expression profiles of 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.
p53 and p21 were found to be parallel in all tested samples especially https://doi.org/10.12659/MSMBR.893608.
with the 7 and 10 (Fig. 3B). On the other hand, Mdm2 which is a ne- Fukai, T., Sheng, C.B., Horikoshi, T., Nomura, T., 1996. Isoprenylated flavonoids from
gative regulator of p53 by binding to and promoting the degradation of underground parts of Glycyrrhiza glabra. Phytochemistry 43, 1119–1124. https://doi.
org/10.1016/S0031-9422(96)00391-3.
this protein (Pucci et al., 2000), were significantly decreased in cells Gallagher, S., Winston, S.E., Fuller, S.A., Hurrel, J.G., 2008. Immunoblotting and im-
treated with the compounds. Hence, as shown in Fig. 3B, phosphor- munodetection. Curr. Protoc. Mol. Biol. 83https://doi.org/10.1002/0471142727.
ylation of p53 was increased in cells incubated with compound 2. mb1008s83. 10.8.
Hatano, T., Tagagi, M., Ito, H., Yoshida, T., 1997. Phenolic constituents of liquorice. VII. A
The cytotoxic effects of glabrene (7), tetrahydroxymethoxychalcone
new chalcone with a potent radical scavenging activity and accompanying phenolics
(10), neolicuroside and licuroside (5 + 6) were evaluated for the first from liquorice. Chem. Pharm. Bull. 45, 1485–1492. https://doi.org/10.1248/cpb.37.
time against all three tested cell lines. Furthermore, these compounds 3229.
had never been studied before regarding their cytotoxic activities 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
against any cancer cells. In a very recent study, glabridin, a key bio- carcinoma therapeutics. Eur. J. Med. Chem. 129, 12–26. https://doi.org/10.1016/j.
logical and chemical marker of G. glabra, was reported to induce Huh7 ejmech.2017.02.002.
cell death by inducing apoptosis through caspase cascade (Hsieh et al., Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407, 770–776. https://
doi.org/10.1038/35037710.
2016). Cytotoxicity of isoliquiritigenin 4′-O-β-glucopyranoside (9), 4′- Hosseinzadeh, H., Nassiri-Asl, M., 2015. Pharmacological effects of Glycyrrhiza spp. and
O-methylglabridin (2), kanzonol U (4) and abyssinone II (11) against its bioactive constituents: update and review. Phytother. Res. 29, 1868–1886.
HCT116 and Huh7, and β-amyrin (3) against Huh7 cells are also re- 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.,
ported with this study for the first time. 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.
4. Conclusion 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
In conclusion, nine secondary metabolites were isolated and iden- herbal medicine. J. Nat. Prod. 79, 281–292. https://doi.org/10.1021/acs.jnatprod.
tified from G. glabra through bioactivity-guided fractionation. Amongst 5b00877.
Kamat, V.S., Chuo, F., Kubo, I., Nakanishi, K., 1981. Antimicrobial agents from an East
isolates, glabridin (1), 4′-O-methylglabridin (2), β-amyrin (3), kanzonol
African medicinal plant Erythrina abyssinica. Heterocycles 15, 1163–1169. https://
U (4), glabrene (7) and tetrahydroxymethoxychalcone (10) which ex- doi.org/10.3987/S-1981-02-1163.
erted the best potent activity against Huh7 cancer cell lines, were es- Kasibhatla, S., 2006. Staining of suspension cells with hoechst 33258 to detect apoptosis.
tablished to be responsible for the in vitro cytotoxic effect of G. glabra. 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
The structure and cytotoxic activity relationships of the tested com- label-free monitoring of cell viability. Methods Mol. Biol. 740, 33–43. https://doi.
pounds were also discussed. Furthermore, the mechanisms behind the org/10.1007/978-1-61779-108-6_6.
cytotoxic activities of the most active compounds were also elucidated. 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
4′-O-methylglabridin (2), glabrene (7) and tetra- revision of an antioxidant isoflavonoid glabrene. Nat. Prod. Lett. 9, 289–296. https://
hydroxymethoxychalcone (10) contributed significantly to the in vitro doi.org/10.1080/10575639708043642.
cytotoxic activity of G. glabra against Huh7 cancer cell lines by inducing 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-
apoptosis through different mechanisms in liver cancer cells. Based on microbial and antioxidant activities. Fitoterapia 103, 289–293. https://doi.org/10.
these data, particularly 7 and 10 could be potential anticancer drug 1016/j.fitote.2015.05.003.
candidates which deserve further in vivo and preclinical studies for the 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.
development of new anti-cancer drug leads especially against hepato-
18, 962–970. https://doi.org/10.1016/j.bmc.2009.11.027.
cellular carcinoma. The isolated bioactive compounds deserve to be Li, K., Ji, S., Song, W., Kuang, Y., Lin, Y., Tang, S., Cui, Z., Qiao, X., Yu, S., Ye, M., 2017.
tested for their potential cytotoxic activities against other human Glycybridins A-K, bioactive phenolic compounds from Glycyrrhiza glabra. J. Nat.
Prod. 80, 334–346. https://doi.org/10.1021/acs.jnatprod.6b00783.
cancer cell lines. Based on these findings and the other results obtained
Lin, Y., Kuang, Y., Li, K., Wang, S., Song, W., Qiao, X., Sabir, G., Ye, M., 2017. Screening
from previous studies on different Glycyrrhiza species put forward the for bioactive natural products from a 67-compound library of Glycyrrhiza inflata.
potential of licorice species to afford new promising anticancer drug Bioorg. Med. Chem. 25, 3706–3713. https://doi.org/10.1016/j.bmc.2017.05.009.
candidates. 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.
Conflict of interest 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.
The authors have no conflict of interest to declare. 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
Acknowledgement 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.,
This study was supported by The Scientific and Technological Mohaghegh, M., Sadeghzadeh, H., Zarghami, N., 2016. Dendrosomal curcumin na-
Research Council of Turkey (TÜBİTAK, Project No: 115S433). 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.
Appendix A. Supplementary data 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/
Supplementary material related to this article can be found, in the
MS/MS. J. Pharm. Biomed. 54, 535–544. https://doi.org/10.1016/j.jpba.2010.10.
online version, at doi:https://doi.org/10.1016/j.indcrop.2018.08.014. 004.
Nomura, T., Fukai, T., Akiyama, T., 2002. Chemistry of phenolic compounds of licorice
References (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.,
Aydemir, E.A., Oz, E.S., Göktürk, R.S., Ozkan, G., Fiskin, K., 2011. Glycyrrhiza flavescens 2013. Evaluation of cytotoxiciy and tumor-specificity of licorice flavonoids based on
subsp. antalyensis exerts antiproliferative effects on melanoma cells via altering TNF- chemical structure. Anticancer Res. 33, 3061–3068.

395
D. Çevik et al. Industrial Crops & Products 124 (2018) 389–396

Park, S.Y., Kim, E.J., Choi, H.J., Seon, M.R., Lim, S.S., Kang, Y.-H., Choi, M.-S., Lee, K.W., glabra and its phytoconstituents. Inflammopharmacology 19, 235–241. https://doi.
Park, J.H.Y., 2014. Anti-carcinogenic effects of non-polar components containing org/10.1007/s10787-011-0080-x.
licochalcone A in roasted licorice root. Nutr. Res. Pract. 8, 257–266. https://doi.org/ Vaya, J., Belinky, P.A., Aviram, M., 1997. Antioxidant constituents from licorice roots:
10.4162/nrp.2014.8.3.257. isolation, structure elucidation and antioxidative capacity toward LDL oxidation. Free
Pucci, B., Kasten, M., Giordano, A., 2000. Cell cycle and apoptosis. Neoplasia 2, 291–299. Radic. Biol. Med. 23, 302–313. https://doi.org/10.1016/S0891-5849(97)00089-0.
Riccardi, C., Nicoletti, I., 2006. Analysis of apoptosis by propidium iodide staining and Vichai, V., Kirtikara, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity
flow cytometry. Nat. Protoc. 1, 1458–1461. https://doi.org/10.1038/nprot.2006. screening. Nat. Protoc. 1, 1112–1116. https://doi.org/10.1038/nprot.2006.179.
238. Vlaisavljević, S., Šibul, F., Sinka, I., Zupko, I., Ocsovszki, I., Jovanović-Šanta, S., 2018.
Saklani, A., Kutty, S.K., 2008. Plant-derived compounds in clinical trials. Drug Discov. Chemical composition, antioxidant and anticancer activity of licorice from Fruska
Today 13, 161–171. https://doi.org/10.1016/j.drudis.2007.10.010. Gora locality. Ind. Crops Prod. 112, 217–224. https://doi.org/10.1016/j.indcrop.
Sato, Y., He, J.-X., Nagai, H., Tani, T., Akao, T., 2007. Isoliquiritigenin, one of the anti- 2017.11.050.
spasmodic principles of Glycyrrhiza ularensis roots, acts in the lower part of intestine. WHO, 2014. World Cancer Report 2014. . http://www.who.int/cancer/publications/
Biol. Pharm. Bull. 30, 145–149. https://doi.org/10.1248/bpb.30.145. WRC_2014/en/.
Shyu, M.H., Kao, T.C., Yen, G.C., 2010. Oleanolic acid and ursolic acid induce apoptosis in Wittschier, N., Faller, G., Hensel, A., 2009. Aqueous extracts and polysaccharides from
HuH7 human hepatocellular carcinoma cells through a mitochondrial-dependent Liquorice roots (Glycyrrhiza glabra L.) inhibit adhesion of Helicobacter pylori to human
pathway and downregulation of XIAP. J. Agric. Food Chem. 58, 6110–6118. https:// gastric mucosa. J. Ethnopharmacol. 125, 218–223. https://doi.org/10.1016/j.jep.
doi.org/10.1021/jf100574j. 2009.07.009.
Tang, Z.-H., Li, T., Tong, Y.-G., Chen, X.-J., Chen, X.-P., Wang, Y.-T., Lu, J.-J., 2015. A Wong, R.S., 2011. Apoptosis in cancer: from pathogenesis to treatment. J. Exp. Clin.
systematic review of the anticancer properties of compounds isolated from licorice Cancer Res. 30, 87. https://doi.org/10.1186/1756-9966-30-87.
(Gancao). Planta Med. 81, 1670–1687. https://doi.org/10.1055/s-0035-1558227. Yang, R., Wang, L.Q., Yuan, B.C., Liu, Y., 2015. The pharmacological activities of licorice.
Thiyagarajan, P., Chandrasekaran, C.V., Deepak, H.B., Agarwal, A., 2011. Modulation of Planta Med. 81, 1654–1669. https://doi.org/10.1055/s-0035-1557893.
lipopolysaccharide-induced pro-inflammatory mediators by an extract of Glycyrrhiza

396