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Phytomedicine
journal homepage:www.elsevier.com/locate/phymed
Original Article
Mechanisms of action of cytotoxic phenolic compounds from Glycyrrhiza
iconica roots
Dicle Çevik
a, Yüksel Kan
b, Hasan K
ırmızıbekmez
a,⁎aDepartment of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, Kayışdağı, İstanbul TR-34755, Turkey bDepartment of Medicinal Plants, Faculty of Agriculture, Selçuk University, Konya TR-42070, Turkey
A R T I C L E I N F O Keywords: Glycyrrhiza iconica Secondary metabolites Cytotoxic activity Apoptosis Liver cancer Huh7 A B S T R A C T
Background: Glycyrrhiza (licorice) species are rich in bioactive secondary metabolites and their roots are used traditionally for the treatment of several diseases. In recent years, secondary metabolites of licorice are gaining popularity, especially due to their significant cytotoxic and antitumor effects. However, Glycyrrhiza iconica, an endemic species to Turkey, was not investigated in terms of its anticancer secondary metabolites previously. Purpose: This study aimed to isolate the cytotoxic compounds from G. iconica through bioactivity-guided frac-tionation and to elucidate mechanisms of action of the most potent compounds.
Methods: Total MeOH extract and CHCl3, EtOAc, n-buOH and rH2O subextracts were prepared from G. iconica
roots. Sequential chromatographic techniques were conducted for the isolation studies. The chemical structures of the isolates were established based on NMR and HR-MS analysis. Sulforhodamine B assay was used to evaluate the cytotoxic activity of extracts, main fractions as well as isolates against hepatocellular (Huh7), breast (MCF7) and colorectal (HCT116) cancer cell lines. The mechanisms underlying the cytotoxicity of the most active compounds in Huh7 cells were elucidated by using Hoechst staining, Fluorescence-activated cell sorting and Western blot assays.
Results: A new dihydrochalcone, iconichalcone (1) along with 15 known phenolic compounds were isolated from the active CHCl3, EtOAc and n-buOH subextracts. Compounds2–5, 7–16 were found to be responsible for
the in vitro cytotoxic activity of G. iconica against all tested cancer cell lines with IC50values ranging from 2.4 to
33 µM. Amongst these compounds, licoricidin (10), dehydroglyasperin C (12), iconisoflaven (13) and 1-meth-oxyficifolinol (15) were found to be the most active compounds according to SRB and real time bioactivity assays and submitted to further mechanistic investigations in Huh7 cells. Compounds10, 12, 13 and 15 caused ac-cumulation of cells in different phases of cell cycle. Moreover, 10, 12, 13 and 15 induced apoptosis through caspase activation. Besides,12 showed activation of p53 expression and thus G2/M arrest as well as a condensed
nuclei, established very promising results.
Conclusion: The results demonstrated that the aforementioned compounds, particularly12 could be potential lead molecules for anticancer drug development that deserve further in vivo and clinical investigations.
Introduction
Cancer is a major public health problem and one of the leading cause of mortality and morbidity worldwide. It is characterized by uncontrolled growth and multiplication of abnormal cells, related to inadequate amount of apoptosis. According to GLOBOCAN (2018)
statics, there are estimated 18.1 million new cancer cases, 9.6 million cancer-related deaths and 43.8 million prevalence of cancer in 2018 around the world (WHO, 2018). Up until now, there have been some commonly used strategies in cancer treatment. However, these thera-pies have been associated with severe adverse effects as well as multi-drug resistance. More effective new anticancer multi-drugs with less adverse
https://doi.org/10.1016/j.phymed.2019.152872
Received 18 October 2018; Received in revised form 1 January 2019; Accepted 19 February 2019
Abbreviations: 1D and 2D NMR, one and two dimensional nuclear magnetic resonance spectroscopy; CC, column chromatograph; CD3OD, deuterated methanol;
CHCl3, chloroform; CH2Cl2, dichloromethane; COSY, correlation spectroscopy; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; FACS,fluorescence activated cell
sorting; fr., fraction; frs., fractions; HMBC, heteronuclear multi-bond correlation; HR-ESI-MS, high resolution-electrospray ionisation-mass spectrometry; HSQC, heteronuclear single-quantum coherence; IC50, the half maximal inhibitory concentration; IR, infrared spectroscopy; MeCN, acetonitrile; MeOH, methanol; Me4Si,
tetramethylsilane; MPLC, medium pressure liquid chromatography
⁎Corresponding author.
E-mail address:hasankbekmez@yahoo.com(H. Kırmızıbekmez).
0944-7113/ © 2019 Elsevier GmbH. All rights reserved.
effects are needed to combat cancer. Natural resources particularly medicinal plants play a crucial role for the discovery of lead drug molecules. From 1940s to date, 48.6% of the total number of small anticancer drugs have been obtained by either natural resources or semi-synthesis of them (Newman and Cragg, 2012). Today, numerous natural products of plant origin are in clinical use as anticancer agents such as vinblastine, vindesine, vincristine, paclitaxel, etoposide, teni-poside, irinotecan and topotecan which are obtained from plants by isolation or produced by the semi-synthesis of the isolates (Balunas and Kinghorn, 2005). New cancer drugs are targeting several aspects of apoptosis due to its important role in both carcinogenesis and cancer treatment. Apoptosis is an ordered, programmed cell death. Under-standing its underlying mechanism is one of the most studied topics among cell biologists due to its important role in pathogenesis of many diseases (Wong, 2011).
Some species of the genus Glycyrrhiza (Fabaceae), commonly known as licorice, have been widely used traditionally in different folk medi-cines for many years as antiulcer, antiinflammatory and expectorant agent (Nassiri-Asl and Hosseinzadeh, 2008). Besides being one of the most commonly used remedy in phytotherapy, licorice is also used as flavoring and sweetening agent in food industry. From Glycyrrhiza species, over 400 compounds have been isolated belonging to triterpene saponins, flavonoids and isoflavonoids chemical classes (Zhang and Ye, 2009). Different extracts of the genus Glycyrrhiza as well as the isolated secondary metabolites were shown to possess antiulcer, anti-microbial, antiprotozoal, hepatoprotective, anti-inflammatory, anti-diabetic, antioxidative, memory enhancing, immunomodulatory, cyto-toxic and antitumor activities (Hosseinzadeh and Nassiri-Asl, 2015; Yang et al., 2015). In recent years, secondary metabolites isolated from licorice most interestingly flavonoids are receiving attention particu-larly for their significant cytotoxic and antitumor effects. For instance, some phenolic compounds including isoflavones, pterocarpans and isoflavans obtained from G. pallidiflora were shown to have significant cytotoxicity against cancer cells (Shults et al., 2017). Moreover, a number of prenylatedflavonoids purified from G. uralensis and G. in-flata remarkably inhibited cancer cell viability (Ji et al., 2016; Lin et al., 2017).
In our very recent study, we reported the cytotoxic secondary me-tabolites from G. glabra and explained their possible cellular mechan-isms (Çevik et al., 2018). As a continuation of our search for new cy-totoxic compounds from the genus Glycyrrhiza, we investigated G. iconica, which is an endemic species to Konya province of Turkey (Chamberlain, 1969). This species was previously studied in terms of its antimicrobial and antioxidant constituents by us (Kırmızıbekmez et al., 2015). Herein, reported are the isolation and the structure elucidation of the secondary metabolites that are responsible for the cytotoxic effect of G. iconica extract against hepatocellular (Huh7), breast (MCF7) and colorectal (HCT116) cancer cell lines. Moreover, the mechanisms un-derlying the cytotoxicity of the most active compounds in Huh7 cells were also elucidated.
Materials and methods General experimental procedures
The reagents and instrumentation utilized for extraction, isolation and structure characterization throughout this study were described previously in details (Çevik et al., 2018).
Plant material
The Glycyrrhiza iconica Hub.-Mor. roots were collected from Medicinal Plant Experimental Garden, Faculty of Agriculture, Selcuk University, Konya, Turkey, where the plant species was cultivated. A herbarium specimen (YEF 15006) was deposited in the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe
University,İstanbul, Turkey.
Extraction and isolation
The air-dried and powdered G. iconica roots (210 g) were macerated with 2.3 l of MeOH at room temperature forfive days and extracted twice at 45 °C for 4 h to afford 43.3 g of MeOH extract (yield, 20.6%). The crude MeOH extract was dispersed in 150 ml of H2O and
parti-tioned against CHCl3 (3 × 150 ml), EtOAc (3 × 150 ml) and n-buOH
(3 × 150 ml), respectively to yield CHCl3, (9.3 g), EtOAc (9.7 g),
n-buOH (8.21 g) and H2O (13.3 g) subextracts. The CHCl3, EtOAc and
n-buOH subextracts were found to possess pronounced cytotoxic activity in Sulforhodamine B (SRB) assay (Table 1) and thus submitted to chromatographic seperations.
CHCl3subextract (3.9 g) was loaded onto Sephadex LH-20 (100 g)
column chromatography eluted with CHCl3:MeOH (1:1, 400 ml) and
MeOH (350 ml), respectively to yield three main fractions (frs. A-C). EtOAc subextract (9.7 g) was fractionated over poliamide column (75 g) eluting with H2O and stepwise gradient of MeOH in H2O (25–100% in
steps of 25%, each 150 ml) to afford four main fractions (frs.1–4). In order to fractionate n-buOH subextract into four main frs. D-GD, 7.8 g of this extract was submitted to polyamide (65 g) CC eluted with H2O
and then stepwise gradient of MeOH in H2O (25–100% in steps of 25%,
each 150 ml). The obtained main fractions were also assesed for their cytotoxicity in the same cancer cell panels. The active fractions (frs. A-C from CHCl3, frs. 1–4 from EtOAc and frs. E-G from n-buOH) (Table 1)
were subjected to further purification procedures. Fr. B (1.54 g) was separated by SiO2−MPLC (120 g) eluting with stepwise
n-hex-ane:EtOAc gradient (10 to 100% EtOAc) to obtain subfractions B1-15.
Subfraction B1 (147 mg) was further loaded on SiO2−MPLC (24 g),
eluted with CH2Cl2:MeOH (0–10% MeOH) to isolate compound 11
(6.5 mg). Subfraction B2(80 mg) was applied to Sephadex LH-20 (30 g)
column (MeOH) to obtain B2-b(31 mg) which was further purified by
SiO2−MPLC (4 g) to afford compound 10 (7 mg) by eluting with
n-hexane:EtOAc (0 to 10% EtOAc). Compound5 (11 mg) was purified by Sephadex LH-20 CC (30 g, MeOH) from subfraction B6(113 mg). Fr. C
(1.4 g) was fractionated over LiChroprep C18−MPLC (150 g) eluting
with H2O:MeOH gradient (40–100% MeOH) to afford subfractions, C 1-16. Isolation of14 (12 mg) and 16 (3 mg) was achieved by successive
Sephadex LH-20 (35 g, MeOH) and SiO2−MPLC (12 g; CHCl3:MeOH,
Table 1
Cytotoxic activity results of crude MeOH extract, subextracts and the main fractions from G. iconica against Huh7, MCF7 and HCT116 cancer cell lines.
Huh7 MCF7 HCT116 Extracts/ Fractions: IC50(µg/ ml)a R2 IC 50(µg/ ml)a R2 IC 50(µg/ ml)a R2 MeOH 12.7 0.9 7.4 0.9 4.3 1 CHCl3 7 0.9 0.9 0.8 2.4 0.8 Fr. A 10.1 0.9 < 0.4 0.9 11.7 0.9 Fr. B 4.4 0.9 0.4 0.9 4.1 0.8 Fr. C 5.3 0.9 < 0.4 0.9 6.0 0.9 EtOAc: 6.4 0.9 1.1 0.9 <0.4 0.9 Fr. 1 4.3 0.8 6.7 0.7 4.8 0.9 Fr. 2 4.0 0.9 < 0.4 0.8 3.0 0.9 Fr. 3 12.8 0.9 10.4 0.9 5.8 0.7 Fr. 4 11.6 0.9 9.0 0.9 3.0 0.7 n-buOH: 7.7 0.9 0.6 0.9 2.1 0.9 Fr. D NI – NI – NI – Fr. E 16.4 0.7 23.7 0.7 NI – Fr. F 8.5 0.9 13.1 0.9 20.3 0.8 Fr. G 7.9 0.8 12.1 0.8 5.9 0.7 r H2O NI 0.8 2.6 0.8 NI 0.8 a IC
50values were calculated from the cell growth inhibition curves obtained
from the treatments with increasing concentrations of extracts or fractions for 72 h. Experiments were done in triplicate.
100:0, 95:5) methods from C7(86 mg). Fractionation of subfraction C9
(72 mg) by MPLC (SiO2, 12 g) eluting with CH2Cl2:MeOH (0 to 5%
MeOH) yielded fr. C9-a(45 mg) which was further applied to Sephadex
LH-20 (25 g) column with MeOH to obtain13 (7 mg). Similarly, sub-fraction C10(135 mg) was separated by SiO2−MPLC (12 g) eluting with
CHCl3:MeOH (0 to 5%, MeOH) to give fr. C10-b (66 mg) which was
further loaded onto Sephadex LH-20 (35 g; MeOH) to purify compound 10 (23 mg). Subfraction C14 (50 mg) was subjected to MPLC (SiO2,
12 g), eluted with CH2Cl2:MeOH mixture (0 to 5% MeOH) to give15
(6 mg).
Fraction 2 (1.46 g) of EtOAc subextract was seperated by C18−MPLC (150 g) using H2O:MeOH mixture (10–100% MeOH) to
afford subfractions (subfrs. 2a-2 s) in addition to compound1 (20 mg).
Subfraction 2d (50 mg) was applied to Sephadex LH-20 (30 g) with
MeOH to isolate 2 (23 mg). Compound 4 (8.5 mg) was purified by Sephadex LH-20 (30 g) CC of subfr. 2i(27 mg). Subfraction 2k(78 mg)
was subjected to MPLC (SiO2,12 g) eluting with n-hexane:EtOAc (5 to
50% EtOAc) to obtain compound9 (3 mg). Similarly, purification of 12 (10 mg) was achieved through SiO2−MPLC (12 g) of subfr. 2l(101 mg)
eluted with same mobile phase system. Subfr. 2n(80 mg) was applied to
SiO2−MPLC (12 g; CHCl3:MeOH, 100:0 to 90:10) to isolate compound
14 (30 mg). An aliquot of fr. 3 (1.2 g) was separated by C18−MPLC
(130 g) eluted with H2O:MeCN (20 to 70% MeCN) to yield subfractions
3a-3 h. Compound8 (5 mg) was purified from fr. 3f(120 mg) by using
SiO2CC (18 g, n-hexane: EtOAc). The remaining amount fr. 3 (1.1 g)
was subjected to SiO2−MPLC (120 g), eluted with n-hexane:EtOAc (20
to 100% EtOAc) in order to obtain subfractions (subfrs. 3i-v). Subfr. 3j
(38 mg) was separated by Sephadex LH-20 CC (30 g) eluted with MeOH to purify5 (4 mg) and 7 (4 mg). Compound 3 (4 mg) was purified by Sephadex LH-20 (30 g, MeOH) CC of fr. 3o(50 mg). Fraction 4 (1.2 g)
was submitted to C18−MPLC (150 g) eluting with H2O:MeOH (20 to
100% MeOH) to give compounds2 (7 mg) and 14 (10 mg) as well as subfractions (subfrs. 4a-l). Fr. G (160.2 mg) was subjected to C18−MPLC
(30 g) eluted with H2O:MeOH (30 to 100% MeOH) to isolate4 (5 mg).
Fr. F (470 mg) was fractionated over by C18−MPLC (100 g) eluting
with H2O:MeOH (5 to 80% MeOH) to afford subfractions F1-13.
Subfractions F10 (21 mg) was separated by SiO2−MPLC (4 g,
CHCl3:MeOH; 0 to 50% MeOH) to give compound 14 (3 mg). Fr. E
(1.3 g) was separated by C18−MPLC (150 g) to obtain subfractions (E1
-E9) using H2O:MeOH (10 to 100% MeOH). Compound 6 (6 mg) was
purified from subfr. E3 (135 mg) by sequential C18−MPLC (30 g,
10–30% MeOH in H2O) and Sephadex LH-20 (35 g, MeOH) CCs.
Iconichalcone (1). Amorphous powder; UV (MeOH): λmax= 231,
283 nm; IR (KBr): υ = 3433, 1648, 1597, 1512, 1472 cm−1; HR-ESI-MS: [M+Na]+m/z 359.0790 (calcd for C
16H16O8Na, 359.0743), [(M
+Na)–H2O]+ m/z 341.0648 (calcd for C16H14O7Na, 341.0632) and
[(M + H)–H2O]+ m/z 319.0820 (calcd for C16H15O7,319.0812); 1H NMR (400 MHz, CD3OD):δ 3.65 (3H, s, OMe), 4.84 (1H, d, J = 8.6 Hz, H-α), 5.58 (1H, d, J = 8.6 Hz, H-β), 6.57 (1H, d, J = 8.4 Hz, H-5′), 6.61 (1H, d, J = 8.5 Hz, H-5), 6.93 (1H, d, J = 8.5 Hz, H-6), 7.05 (1H, dd, J = 8.4, 2.1 Hz, H-6′), 7.21 (d, J = 2.1 Hz, H-2′);13C NMR (100 MHz, CD3OD):δ 59.6 (d, C-α), 61.5 (q, OCH3), 82.5 (d, C-β), 112.5 (d, C-5), 115.9 (d, C-5′), 116.4 (d, C-2′), 118.9 (d, C-6), 124.3 (d, C-6′), 125.7 (s, C-1), 130.5 (s, C-1′), 139.4 (s, C-3), 146.2 (s, C-3′), 147.8 (s, C-4), 148.1 (s, C-2), 152.7 (s, C-4′), 199.9 (s, C = O).
Cytotoxicity and mechanistic assays
The cytotoxic activities of the crude extract, subextracts, main fractions as well as the isolates were evaluated by sulforhodamine B (SRB) assay in human liver (Huh7), colon (HCT116) and breast (MCF7) cancer cell lines as previously described (Çevik et al., 2018). Real time bioactivity, Hoechst staining, Fluorescence-activated cell sorting (FACS) and Western blot assays were conducted in Huh7 cells also with regard to the published procedures (Çevik et al., 2018).
Results and discussion
Bioactivity guided fractionation and isolation
The crude MeOH extract and the subextracts of G. iconica roots were tested for their in vitro cytotoxic activities against Huh7, MCF7 and HCT116 cancer cell lines by SRB assay. As presented inTable 1, CHCl3,
EtOAc and n-buOH subextracts showed significant cytotoxic effects against all tested cell lines with IC50values in the range of < 0.4–
7.7 µg/ml, while remaining H2O subextract was found to be active only
against MCF7 cells. CHCl3subextract was fractionated over Sephadex
LH-20 while EtOAc and n-buOH subextracts were fractionated by polyamide CC to yield main fractions which were also submitted to cytotoxicity assay. Based on the results, frs. B and C from CHCl3, frs.
2–4 from EtOAc and frs. E-G from n-buOH subextracts were used for isolation procedures to yield cytotoxic compounds (Table 1).
Structure elucidation of isolates
Totally 16 secondary metabolites including a new one (1) were isolated from cytotoxically active main fractions of G. iconica roots as a result of chromatographic seperations. The structures were elucidated by using 1D and 2D NMR experiments as well as HR-ESI-MS. The chemical structures of the isolated compounds are shown inFig. 1.
Compound1 was obtained as yellowish amorphous powder. The HR-ESI-MS of1 exhibited [M + Na]+ion at m/z 359.0790 (calcd for
359.0743 C16H16O8Na) and [(M+Na)-H2O]+ ion at m/z 341.0648
(calcd for 341.0632 C16H14O7Na) and [(M + H)-H2O]+ion at m/z
319.0820 (calcd for 319.0812 C16H15O7), supporting a molecular
for-mula of C16H16O8. The UV spectrum of1 showed absorption maxima at
231 and 283 nm, while its IR spectrum displayed absorption bands at 3433 (hydroxyl groups), 1648 (ketone C = O), 1597, 1512 and 1472 (aromatic rings) cm−1. The 1H NMR spectrum of1 displayed three
aromatic signals ascribable to an ABX system at 7.21 (d, J = 2.1 Hz), 7.05 (dd, J = 8.4, 2.1 Hz) and 6.57 (d, J = 8.4 Hz). Moreover, two aromatic proton signals as an AB system were detected at 6.93 (d, J = 8.5 Hz) and 6.61 (d, J = 8.5 Hz). In addition, signals for two oxy-methines at 5.58 (d, J = 8.6 Hz) and 4.84 (d, J = 8.6 Hz) as well as one methoxy signal at 3.65 (s, 3H) were observed. The13C NMR spectrum
contained sixteen resonances including one ketone (δC 199.8), two
oxymethine (δC82.5 and 59.6), one methoxy (δC 61.5) and twelve
aromatic ring signals. Detailed analysis of COSY, HSQC and HMBC spectra, revealed that 1 is a dihydrochalcone derivative. Two oxy-methine signals were corresponded to the hydroxylation of C-α and C-β positions. Long-range heteronuclear correlations of C = O signal (δC
199.8) with H-2′ (δH7.21) and H-6′ (δH7.05) supported this foreseen
structure. The location of methoxy unit was determined to be C-2 by the cross-peak betweenδC148.1 (C-2) and methoxy atδH3.65 in the HMBC
experiment. The relative configuration of the hydroxyl groups at C-α and C-β positions was tentatively identified by NOESY spectrum. The NOE correlations between H-α/H-β, H-α/H-2′, H-α/H-6 and H-β/H-6 in the NOESY spectrum suggested α configuration for both hydroxyl groups at C-α/β. The large coupling constants, Jαβ= 8.6 Hz, also
sup-ported the threo configuration (∼3.0 Hz for erythro and ∼ 8.0 for threo) of these hydroxyl groups (Zhang et al., 2017). On the basis of the above evidence, the structure of1 was established as 3,4,3′,4′-tetrahydroxy-2‑methoxy‑α,β-dihydroxychalcone and named as iconichalcone. The structure of1 was very similar to compound 2 however the only dif-ference between them was dihydroxylation of theα,β-conjugated bonds in1. In previous studies,α-hydoxylated chalcone derivatives were re-ported from the genus Glycyrrhiza (Li et al., 2017). However, this is the first report of the occurrence of α,β-dihydroxychalcone in the genus Glycrrhiza.
The known compounds were identified as tetra-hydroxymethoxychalcone (2) (Çevik et al., 2018), 2 ′-O-methylisoli-quiritigenin (3), 3-O-methylkaempferol (4) (Hatano et al., 1989),
topazolin (5) (Kırmızıbekmez et al., 2015), violanthin (6), glycyr-rhisoflavone (7) (Hatano et al., 1988), licocoumarone (8), glyasperin C (9) (Zeng et al., 1992), licoricidin (10) (Fukai et al., 1998), licoriso-flavan A (11) (Shih et al., 1987), dehydroglyasperin C (12) (Shibano et al., 1997), iconisoflaven (13) (Kırmızıbekmez et al., 2015), glycycoumarin (14) (Hatano et al., 1988), 1-methoxyficifolinol (15) (Ahn et al., 2015) and edudiol (16) (Guchu et al., 2007) by compara-sion of obtained spectroscopic data with those published in literatures. Among the isolates, compounds 2–4, 6–9, 12, 15 and 16 are being reported from G. iconica for thefirst time while 3 is new for the genus Glycyrrhiza.
Cytotoxic activity results of isolates by SRB assay
The cytotoxic effects of the isolates were evaluated against Huh7, MCF7 and HCT116 cancer cell lines by SRB assay and their IC50(µM)
values were determined (Table 2). Amongst tested compounds, tetra-hydroxymethoxychalcone (2) was also obtained from G. glabra as one of the most potent compounds against the same cancer cell lines in our previous study (Çevik et al., 2018). The compounds having IC50values
below 40 µM were accepted as active. All of the isolates except for iconichalcone (1) and violanthin (6) displayed cytotoxicity against three tested cancer cell lines with IC50values ranging from 2.4 to 33 µM
(Table 2). The active compounds (2–5, and 7–16) exerted their best cytotoxicity against Huh7 cancer cells (IC50= 2.4–17.2 µM) except for
compound9. Besides, the same compounds exhibited cytotoxic activity also against MCF7 and HCT116 cells with IC50values in the range of
4.6–20 µM and 5.0–33 µM, respectively. Based on the SRB results, 2–5, 7–16 were identified to be responsible for the in vitro cytotoxic activity of G. iconica. It is noteworthy to mention that 1-methoxyficifolinol (15) was determined to be the most cytotoxic compound against Huh7, MCF7 and HCT116 with IC50values of 2.4, 4.6 and 5.0 µM,
respec-tively. Furthermore, the results also demonstrated that Huh7 was the most sensitive cell line to the cytotoxic effects of the tested isolates
(Table 2).
In terms of structure activity relationships, the active compounds possessed chalcone (2,3),flavonol (4,5), isoflavone (7), benzofuran (8), isoflavan (9–11), isoflaven (12,13), 3-arylcoumarin (14) and pter-ocarpan (15,16) skeletons. The presence of isoprenyl chain in flavo-noids might enhance cytotoxic activity via increasing their lipophilicity and thus the affinity to biological membranes (Botta et al., 2005). Tang et al. (2016)showed that the number of isoprenyl groups in A and B rings, the hydroxyl groups at ortho position of isoprenyl on A ring and conjugated plane of C ring were positively-correlated with anticancer activity of flavonoids (Tang et al., 2016). According to our results (Table 2), licoricidin (10) (IC50= 4.7- 9.3 µM) which bears two
iso-prenyl units in the rings A and B, was found to be more active than glyasperin C (9) (IC50= 16.8–20 µM) which lacks one of the isoprenyl
units. Concerningflavonol derivatives (4 and 5), the presence of iso-prenyl moiety in5 caused a significant increase in cytotoxic activity. Similarly, the occurence of isoprenyl moiety in the pterocarpan deri-vatives (15 and 16) enhanced the cytotoxic activity significantly. Fur-thermore, dehydrogenation between C-3/C-4 position of C ring in de-hydroglyasperin C (12) (IC50= 5.8–9.6 µM) increased the cytotoxic
activity when compared to glyasperin C (9) (IC50= 16.8–20 µM).
Be-sides, 12 was found to be more active than glycycoumarin (14) (IC50= 10.7–17.5 µM). This phenomenon could be explained by the
introduction of ketone function at C-2 on isoflavenes caused around 2-fold decrease in the activity. Violanthin (6), aflavonoid glycoside did not exert any cytotoxicity against any tested cell lines. Flavonoid gly-cosides only displayed weak to moderate activities for most of the in vitro screening models (Çevik et al., 2018; Lin et al., 2017; Shults et al., 2017). However, they might be metabolized to their bioactive free forms in gastrointestinal system after oral administration, and thus may play an important role in therapeutic effects of licorice.
The in vitro cytotoxic effects of 2′-O-methylisoliquiritigenin (3), 3-O-methylkaemferol (4), violanthin (6), licorisoflavan A (11), iconiso-flaven (13), 1-methoxyficifolinol (15) and edudiol (16) were studied for Fig. 1. Chemical structures of isolates (1–16).
the first time against Huh7, MCF7 and HCT116 cancer cell lines. Moreover,3, 6, 11, 13 and 16 were evaluated for thefirst time for their cytotoxic properties. In vitro cytotoxicity of topazolin (5), glycyrrhiso-flavone (7), licocoumarone (8), glyasperin C (9) and dehydroglyasperin C (12) against MCF7 cells were demonstrated in previous studies (Lin et al., 2017) whereas their cytotoxic effects against Huh7 and HCT116 cells are being reported for thefirst time within this study. In addition, it is thefirst report demonstrating the cytotoxic activity of licoricidin (10) against Huh7 cells.
Mechanistic studies of most potent isolates
The compounds with IC50values less than 10μM (2, 5, 7, 10, 12,
13, 15 and 16) were chosen for further mechanistic studies in Huh7 cells. First, these compounds were conducted to real time cell electronic sensing (RT-CES) assay. According to RT-CES results (Table 3), com-pounds exerting their activities with IC50values less than or equal to10
μM at 48th
h in Huh7 cells were established as the most active ones (10, 12, 13 and 15) and hence, determined to be evaluated regarding their mechanism of cytotoxic actions by Hoechst staining, fluorescence-ac-tivated cell sorting (FACS) and western blot assays. In our previous study, tetrahydroxymethoxychalcone (2), which was also isolated from G. glabra, was already investigated for its mechanism of cytotoxic action in Huh7 cells and it was demonstrated that 2 induced apoptosis by increasing cytochrome C, cleaved caspase-9 and PARP levels as well as decreasing p21 level, caused increase in apoptotic subG1cell population
and most notably G2/M arrest and showed nuclei with condensed
chromatin as the morphological indicator of apoptosis in Huh7 cells (Çevik et al., 2018).
To examine the nuclear changes and apoptotic body formation, which are characteristic indicators for apoptosis, fluorescent micro-scopy using Hoechst 33,258, which stains the condensed chromatin of apoptotic cells more brightly than the chromatin of normal cells was employed for the selected compounds. Huh7 cells treated with com-pounds10 and 12 showed condensed nuclei, comparing to DMSO, in-dicated the existence of apoptotic cells (Fig. 2A). Effects of 10, 12, 13 and15 on cell cycle were further characterized by FACS analysis, using PI stain. Dysregulation of cell cycle is a key feature of cancer cells and a significant target in cancer therapies. As shown inFig. 2B, all of the tested compounds particularly 13 caused accumulation of cells in SubG1phase which is the indicator of apoptotic cell death. Besides,10,
12 and 13 induced apoptosis also by the cell cycle arrest in G2/M phase.
Moreover,10 and 15 induced the accumulation of Huh7 cells at G1
phase.
In order to clarify the cellular cytoxicity mechanisms of these compounds in Huh7 cells more specifically, their effects against the expressions of apoptotic (cytochrome C, cleaved caspase-3/−9 and PARP) and cell cycle regulatory proteins (Mdm2, pRb, p21 and p53) were investigated by western blot analysis. Caspases are cysteine pro-tease enzymes, accepted to be central mechanisms of apoptosis, acti-vation of them consists of both intrinsic and extrinsic pathways. The intrinsic pathway is initiated within the cell as a result of increased mitochondrial permeability and release of pro-apoptotic molecules such as cytochrome C from mitochondria into cytosol by induction of apoptosis. Released cytochrome C in cytosol activates caspase-9 by forming a complex called apoptosome early during the apoptotic pro-cess (Wong, 2011). The activated caspase-9 subsequently stimulates proteolytic activity of other downstream caspases including executioner caspase-3 which leads to cleavage of various proteins such as PARP and results in cell death (Pfeffer and Singh, 2018). According to our find-ings, treatment with all of the tested compounds (10, 12, 13 and 15) increased cytochrome C levels in Huh7 cells which might be con-sequence of increased mitochondrial permeability and cytochrome C release into cytosol (Fig. 3A). Among the tested compounds,10, 12 and 15 caused an increase in cleaved caspase-9 levels, while increased cleaved caspase-3 levels were observed in Huh-7 cells treatments with 12 and 13. In accordance with these results, elevated cleaved PARP protein levels were detected most importantly with compound12 as well as10 (Fig. 3A). Taken together, thesefindings suggested that all tested compounds particularly 12 might exert pro-apoptotic effects through intrinsic mitochondrial pathway of caspase cascade.
Table 2
Cytotoxic activity of isolates against Huh7, MCF7 and HCT116 cancer cell lines by SRB assay.
Huh7 MCF7 HCT116 Compounds IC50(µM)a R2 IC50(µM)a R2 IC50(µM)a R2 Iconichalcone (1) > 40 0.78 > 40 0.88 > 40 0.65 Tetrahydroxymethoxychalcone (2)b 8.3 0.97 13.9 0.99 13.3 0.98 2′-O-Methylisoliquiritigenin (3) 12.1 0.83 13.8 0.98 18.4 0.93 3-O-methylkaempferol (4) 14.7 0.98 19.6 0.97 33 0.99 Topazolin (5) 7.2 0.96 10.5 0.99 11.8 0.9 Violanthin (6) > 40 0.64 > 40 – > 40 0.95 Glycyrrhisoflavone (7) 8.7 0.89 11 0.91 11.1 0.98 Licocoumarone (8) 10 0.87 10.5 0.95 18.3 0.93 Glyasperin C (9) 17.2 0.77 16.8 0.86 20 0.74 Licoricidin (10) 4.7 0.95 9.3 0.97 9.2 0.99 Licorisoflavan A (11) 11.5 0.99 12.8 0.99 13 0.97 Dehydroglyasperin C (12) 5.8 0.97 6.4 0.99 9.6 0.99 Iconisoflaven (13) 7.1 0.98 17.1 0.91 18 0.8 Glycycoumarin (14) 10.7 0.87 15 0.85 17.5 0.9 1-Methoxyficifolinol (15) 2.4 0.98 4.6 0.98 5.0 0.96 Edudiol (16) 7.5 0.86 20 0.91 20 0.96 Camptothecin <1 <1 <1 a
IC50values were calculated from the cell growth inhibition curves obtained from the treatments with increasing concentrations of compounds (40, 20, 10, 5 and
2.5μM) for 72 h. If IC50value was beyond 2.5 µM the assays were repeated with lower concentrations. Experiments were done in triplicate. NI: No Inhibition. b (Results are fromÇevik et al., 2018).
Table 3
Real-time bioactivity assay (RT-CES xCELLigence) results of active compounds against Huh7 cells.
24 h 48 h 72 h Compound IC50(µM) R2 IC50(µM) R2 IC50(µM) R2 Topazolin (5) 6.9 0.5 19 0.7 16 0.9 Glycyrrhisoflavone (7) > 10 – > 10 – > 10 – Licoricidin (10) 6.9 0.7 10 0.7 11.8 0.9 Dehydroglyasperin C (12) 4.2 0.7 3.5 0.7 4 0.8 Iconisoflaven (13) 4.8 1 6.5 1 6.3 1 1-Methoxyficifolinol (15) 3.8 0.7 4.3 0.8 5.7 0.9 Edudiol (16) > 10 – > 10 – > 10 –
Decrease in the phosporylation of retinoblastoma protein (pRb) is an indicator of cell cycle arrest in G1 phase (Pucci et al., 2000).
Phospho-Rb levels were decreased in cells treated with13 and 15 as shown inFig. 3B. These results were in accordance with cell cycle assay results in which accumulation of cells in subG1and/or G1phases were
observed also with the same compounds. Moreover, silencing of p21 significantly promoted apoptosis and increased G2 phase arrest
(Chen et al., 2015). p21 levels were observed to be significantly de-creased in all samples compared to DMSO, indicating apoptotic process by treating with all tested compounds and also supporting the G2/M
arrest in cell cycle assay with compounds10, 12 and 13 (Fig. 3B). p53 is a tumour suppressor gene which is mutated in approximately more than 50% of human cancers, functions to eliminate and inhibit the proliferation of abnormal cells (Pucci et al., 2000). In normal cells, p53 remains in“standby” mode, whereas its activation causes increasing levels of p53 protein and induces variety of cellular responses, most notably in cell cycle and apoptosis pathways (Bai and Zhu, 2006). Ac-tivity of p53 can be regulated by multiple cellular proteins particularly by mouse double minute 2 (Mdm2) which is not only a physiological antagonist of p53 but also a direct target for the transcriptional acti-vation by p53 thus establishes a negative autoregulatory feedback loop where p53 activates its own inhibitor (Oren and Rotter, 1999). In our present study, treatment of Huh7 cells with compound12 caused an increase in the levels of both p53 and Mdm2 indicating that12 sti-mulated the anti-tumour gene p53 (Fig. 3B). It is known that p53 plays an important role in G1and G2phases of cell cycle (Sagar et al., 2014).
Overexpression of p53 inhibits the cells entrying to mitosis by causing cell cycle arrest in G1and G2/M (Bai and Zhu, 2006; Pucci et al., 2000).
Upregulation of p53 in Huh7 cells treated with compound12 might be another possible explanation for the higher percentage of cells in subG1
and G2/M phases. In addition, p53 regulates expression of pro- and
anti-apoptotic proteins of Bcl-2 family, the relative levels of these proteins are crucial for apoptosis (Sagar et al., 2014). P53 transcriptionally
activates Bax, a pro-apoptotic member of Bcl-2 family and down reg-ulates anti-apoptotic Bcl-2 protein which leads to decreased ratio be-tween Bcl-2/Bax and therefore causes increase in the permeability of mitochondrial membrane causing cytochrome C release and finally leads to p53 mediated and caspase dependent apoptosis (Bai and Zhu, 2006; Sagar et al., 2014). Treatment with compound12 revealed cas-pase dependent apoptosis as mentioned above, might be mediated by p53 activation.
Conclusion
In vitro cytotoxic activity guided fractionation of G. iconica roots resulted in the isolation of 16 secondary metabolites including a new one, iconichalcone (1). Compounds 2–5, 7–16 were found to be re-sponsible for the in vitro cytotoxic activity of G. iconica against Huh7, MCF7 and HCT116 cell lines. The mechanisms behind the cytotoxic activity of the most active compounds were also investigaed by Hoechst staining, cell cycle assay and western blot analyses. The results in-dicated that induced apoptosis of Huh7 cells by10, 12, 13 and 15 were mediated via caspase activation. Furthermore, 10, 12, 13 and 15 caused accumulation of cells in different phases of cell cycle including subG1, G1and/or G2/M. The underlying reasons of these cell cycle
ar-rests might be due to decrease in phospho-Rb levels (13 and 15), p21 level (10, 12 and 13) or increase in p53 expression (12). Amongst the tested compounds, 12 that showed activation of p53 expression and thus G2/M arrest as well as a condensed nuclei, established very
pro-mising results, might cause cytotoxicity by inducing Huh7 cells to apoptosis through p53 mediated caspase cascade. Taken together, tet-rahydroxymethoxychalcone (2), licoricidin (10), dehydroglyasperin C (12), iconisoflaven (13) and 1-methoxyficifolinol (15) were found to be cytotoxic phenolic compounds of G. iconica against liver cancer by enhancing apoptosis and displaying anticancer functions via promoting pro-apoptotic factors such as cytochrome C and caspases. The results Fig. 2. (A): Fluorescent microscopy images of Huh7 cells treated with DMSO or compounds for 48 h (10: 10μM, 12: 4 μM, 13: 7 μM, 15: 5 μM) stained with Hoechst 33,258 nuclear dye. (B): Cell cycle analysis of Huh7 cells upon treatment with 10, 12, 13 and 15 (10μM, 4 μM, 7 μM and 5 μM respectively) and DMSO following 48 h of treatment.
demonstrated that the aforementioned compounds, particularly 12 could be potential lead molecules for anticancer drug development that deserve further in vivo and clinical investigations. Based on our pro-mising results on two Glycyrrhiza species to date, the other Glycyrrhiza species also deserve attention in terms of their potential cytotoxic me-tabolites on the way to discover new anticancer drug candidates from natural sources.
Acknowledgments
This study was financially supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK, Project No: 115S433). The authors thank Dr. Ece Akhan Güzelcan and Prof. Dr. Rengül Çetin Atalay (Cancer Systems Biology Laboratory, Graduate School of Informatics, METU, TR-06800, Ankara, Turkey) for per-forming bioactivity assays.
Conflict of interest
The authors have no conflict of interest to declare.
Supplementary materials
Supplementary material associated with this article can be found, in the online version, atdoi:10.1016/j.phymed.2019.152872.
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