In vitro cytotoxicity of compounds isolated from Desbordesia glaucescens against human carcinoma cell lines

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In vitro cytotoxicity of compounds isolated from Desbordesia glaucescens

against human carcinoma cell lines

V. Kuete

a,b,

⁎

, F.L. Dongmo Mafodong

c

, I. Celik

d

, S.A.T. Fobofou

c,e

, B.L. Ndontsa

c

, O. Karaosmano

_ğlu

b,f

,

L.A. Weissjohann

e

, P. Tane

c

, A.T. Koparal

b

, H. Sivas

b

a

Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon b_{Department of Biology, Science Faculty, Anadolu University, Eskişehir, Turkey}

c

Department of Organic Chemistry, Faculty of Science, University of Dschang, Dschang, Cameroon d_{Department of Chemistry, Faculty of Science, Anadolu University, Eskişehir, Turkey}

e_{Department of Biorganic Chemistry, Liebniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany} f

Department of Biology, KamilÖzdağ Science Faculty, Karamanoğlu Mehmetbey University, Karaman, Turkey

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 14 October 2016

Received in revised form 21 December 2016 Accepted 9 March 2017

Available online 20 March 2017 Edited by L Verschaeve

Malignancies constitute a global health concern and chemotherapy remains the main mode of treatment. The present study was designed to evaluate the cytotoxicity of 8 compounds from Desbordesia glaucescens namely lanosta-7,24-dien-3-one (1), friedelanone (2), friedelanol (3), 3,3′-di-O-methylellagic acid (4), 3,3′,4′-tri-O-methylellagic acid (5), ellagic acid (6), 3′,4′-di-O-3,3′,4′-tri-O-methylellagic acid 4-O-β-D-glucopyranoside (7) and

3,3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside (8) against 4 human carcinoma cell lines and normal CRL2120 ﬁ-broblasts. The neutral red uptake (NRU) assay was used for cytotoxicity testing. Caspase-Glo assay, cell cycle anal-ysis, measurements of mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were used to evaluate apoptosis induction. Compounds 4 and 6 as well as doxorubicin had IC50values below

45μM in the four tested cancer cell lines meanwhile other compounds displayed selective activity. The IC50values

ranged from 11.23μM (towards breast adenocarcinoma MCF-7 cells) to 44.65 μM (colon carcinoma Caco-2 cells) for 4, from 14.07μM (towards MCF-7 cells) to 77.73 μM (Caco-2 cells) for 6 and from 0.07 μM (towards SPC212 cells) to 1.01μM (A549 cells) for doxorubicin. Compound 4 induced apoptosis in MCF-7 cells mediated by MMP loss. The constituents of Desbordesia glaucescens and especially ellagic acid (6) and its derivative 4 are potential cytotoxic compounds that deserve more investigations towards developing novel antiproliferative drugs against human carcinoma.

Keywords: Apoptosis Carcinoma Cytotoxicity Ellagic acid Terpenoids Mode of action 1. Introduction

Cancer is one of theﬁve leading causes of death globally and accord-ing to the International Agency for Research on Cancer (IARC) for 2012, 14.1 million new cancer cases and 8.2 million deaths occurred in 2012 worldwide (Bray et al., 2013; Ferlay et al., 2013). The most occurring cancers in both developed and less developed countries include lung, breast, colon prostate, liver, stomach and cervix cancers (Torre et al., 2015). Chemotherapy remains the major mode of the treatment of var-ious neoplastic diseases. Although there have been vast discoveries of potent cytotoxic agents from plants, there is a growing need of novel cy-totoxic drugs to diversify the armory of anticancer agents and to offer

new therapeutic alternatives (Zeytinoglu et al., 2003; Ipek et al., 2005;

Zeytinoglu et al., 2008; Unlu et al., 2010). Drug discovery from African

plants is of relevant interest because Africa hosts 57,704 species of the world'sﬂora (Fadeyi et al., 2013). In the past, evidence of the anticancer activity of several plants and compounds from Africanﬂora was docu-mented (Kuete and Efferth, 2015). In our continuous search of cytotoxic molecules to combat malignant diseases, the present study was de-signed to investigate the antiproliferative potential of compounds iso-lated from Desbordesiaglaucescens (Engl.) Tiegh. (Irvingiaceae). Desbordesiaglaucescens is found around forest zones in South Nigeria, West Cameroon, Gabon, Congo, and Angola (Letouzey, 1982). In Congo, the seeds are used as a food additive while bark-decoctions are taken to attenuate stomach-ache, and as an aphrodisiac. Ointments made with barks and palm-oil are used to treat chicken-pox and head-ache (DongmoMafodong et al., 2015). Previous phytochemical studies on D. glaucescens reported the isolation of ellagic acid derivatives and triterpenes (DongmoMafodong et al., 2015).

⁎ Corresponding author at: Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon.

E-mail address:Kuetevictor@yahoo.fr(V. Kuete).

http://dx.doi.org/10.1016/j.sajb.2017.03.031

Contents lists available atScienceDirect

South African Journal of Botany

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s a j b

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2. Materials and methods 2.1. General procedure

Mass spectral data [Electrospray ionization mass spectrometry (ESI-MS)] were measured on a Waters Synapt HDMS spectrometer. NMR Spectra were recorded with an Agillent DD2 NMR (400 MHz) spectrom-eter. Column chromatography was performed on silica gel Merck 60 F254[(0.2–0.5 mm) and (0.2–0.063 mm)] 70–230 and 230–400

mesh (Darmstadt, Germany). Pre-coated silica gel 60 F254thin layer

chromatography (TLC) plates (Merck, Germany) were used for moni-toring fractions and spots were detected with UV light (254 and 365 nm) and then sprayed with 50% sulphuric acid (H2SO4) followed

by heating to 100 °C. 2.2. Plant material

The stem of Desbordesia glaucescens (Irvingiaceae) were collected in Suza (Littoral Region-Cameroon) in March 2012. The plant material was authenticated (voucher number 53939/HNC) at the National Herbarium in Yaoundé by a botanist (Mr. Mezili Paul).

2.3. Extraction and isolation

Air-dried and powdered leaves (2.6 kg) of D. glaucescens were ex-tracted with EtOH (10 L) at room temperature for 48 h andﬁltered. Theﬁltrate was concentrated to dryness under vacuum to give 158.7 g of crude extract (CE). The obtained extract was suspended in distilled water and subjected to a liquid–liquid extraction using EtOAc and n-butanol to give ethyl acetate (EAF), and n-n-butanol (n-ButF) fractions, respectively.

Part of crude extract (151 g) was suspended in distilled water and subjected to a liquid–liquid extraction using EtOAc to give 50 g of ethyl acetate (EAF) and 82 g n-butanol fractions (n-ButF), respectively. Ethyl acetate (EAF) fraction (47 g) was subjected to CC over silica gel and eluted with a gradient of n-Hex-EtOAc and EtOAc-MeOH of increas-ing polarity. 59 fractions of 300 mL each were collected as follows: n-Hex (frs1–6); n-Hex-EtOAc (90:10) (frs7–23); n-Hex-EtOAc (85:15) (frs24–28); n-Hex-EtOAc (80:20) (frs29–38); n-Hex-EtOAc (60:40) (frs39–41); n-Hex-EtOAc (30:70) (frs42–44); EtOAc (frs45–51); EtOAc-MeOH (50:50) (frs52_{–57); MeOH (frs58–59). They were then} combined into four sub-fractions based on their thin layer chromatogra-phy (TLC) proﬁle: EAFA (09 g; frs1–8); EAFB (08 g; frs9–32); EAFC (11 g; frs33_{–44) and EAFD (12 g; frs45–59).}

EAFA (07.5 g) was subjected to CC over silica gel and eluted with n-Hex-CH2Cl2mixture of increasing polarity. 53 new sub-frs of 75 mL each

were collected. n-Hex (sub-frs1–12); n-Hex-CH2Cl2(95:05)

(sub-frs13–19); n-Hex-CH2Cl2 (90:10) (sub-frs20–25); n-Hex-CH2Cl2

(85:15) (sub-frs26–33); n-Hex-CH2Cl2 (80:20) (sub-frs34–37);

n-Hex-CH2Cl2 (60:40) (sub-frs38–41); n-Hex-CH2Cl2 (50:50)

(sub-frs42–45); CH2Cl2−MeOH (50:50) (sub-frs46–51); MeOH

(sub-frs52–53). They were then combined into ﬁve sub-fractions based on their thin layer chromatography (TLC) proﬁle: EAFA1 (0.7 g; sub-frs1– 14); EAFA2 (1.3 g; sub-frs15–21); EAFA3 (2.3 g; sub-frs22–31); EAFA4 (1.7 g; sub-frs32–44) and EAFA5 (0.7 g; sub-frs45–51). sub-fr EAFA1 (1–14) and sub-fr EAFA2 (15–21) was further subjected to silica gel CC eluting with n-Hex-CH2Cl2mixture of increasing polarity to afford

1 (230 mg). The ordinaryﬁltration of sub-fr EAFA3 (22–31) afforded 2 (60 mg). The sub-fr EAFA4 (32–44) was further subjected to another sil-ica gel CC eluting with n-Hex-CH2Cl2(90:10) isocratic system to afford

(3; 16 mg).

EAFC (10 g) was subjected to CC over silica gel and eluted with n-Hex-AcOEt mixture of increasing polarity. 69 new sub-frs of 100 mL each were collected. The ordinaryﬁltration of sub-frs 49–55 afforded white powder identiﬁed 3,3′-di-O-methylellagic acid (4; 26 mg).

n-Butanol fraction (n-ButF) (77 g) was subjected to CC over silica gel and eluting with mixture solvent of EtOAc-MeOH of increasing polarity. 44 fractions of 300 mL each were collected as follows: EtOAc (frs1_–11); EtOAc-MeOH (90:10) (frs12–20); EtOAc-MeOH (80:20) (frs21–32); EtOAc-MeOH (70:30) (frs33–36); EtOAc-MeOH (50:50) (frs37–39); MeOH (frs40_{–44). They were then combined into ﬁve sub-fractions} based on their thin layer chromatography (TLC) proﬁle: n-But A (11 g; frs1–11); ButFB (18 g; frs12–27); ButFC (20 g; frs28–36) and n-ButFD (20 g; frs37–44).

Fraction n-ButFA (11 g) was subjected to CC over silica gel and elut-ed with CH2Cl2-MeOH mixture of increasing polarity. 72 new sub-frs of

75 mL each were collected as follows: CH2Cl2(sub-frs1–7); CH2Cl2

-MeOH (95:05) (sub-frs8–20); CH2Cl2-MeOH (90:10) (sub-frs21–35);

CH2Cl2-MeOH (80:20) (sub-frs36–55); CH2Cl2-MeOH (50:50)

(sub-frs56–70); MeOH (sub-frs71–72). They were further pooled into 4 other sub-frs according to their TLC pro_{ﬁles: n-ButFA1 (0.8 g;} sub-frs1–14); n-ButFA2 (1.8 g; sub-frs15–31); n-ButFA3 (2.4 g; sub-frs32– 53); and n-ButFA4 (2.1 g; sub-frs54–72). sub-fr n-ButFA3 (23–38) was further subjected to CC over silica gel and eluted with an isocratic sol-vent system [CH2Cl2-acetone (90:10)] to give 8 (12 mg). sub-fr n-ButF

A4 (54–72) was also subjected to CC over silica gel and eluted with an isocratic solvent system [CH2Cl2-MeOH (80:20)] afforded 6 (21 mg).

Fraction n-ButFB (18 g) was subjected to CC over silica gel and eluted with CH2Cl2-MeOH mixture of increasing polarity. 38 new sub-frs of

100 mL each were collected as follows: CH2Cl2-MeOH (90:10)

(sub-frs1–7); CH2Cl2-MeOH (80:20) (sub-frs8–14); CH2Cl2-MeOH (70:30)

(sub-frs15–24); CH2Cl2-MeOH (60:40) (sub-frs25–28); CH2Cl2-MeOH

(50:50) (sub-frs29–36); MeOH (sub-frs37–38). The simple ﬁltration of sub-frs1–3 yielded 5 (9 mg).

Fraction n-ButFC (20 g) was subjected to CC over silica gel and eluted with CH2Cl2-MeOH mixture of increasing polarity. 28 new sub-frs of

100 mL each were collected as follows: CH2Cl2-MeOH (80:20)

(sub-frs1–5); CH2Cl2-MeOH (70:30) (sub-frs6–13); CH2Cl2-MeOH (60:40)

(sub-frs14–19); CH2Cl2-MeOH (50:50) (sub-frs20–26); MeOH

(sub-frs27–28). They were further pooled into 4 other sub-frs according to their TLC proﬁles: n-ButFC1 (2.1 g; frs1–6); n-ButFC2 (1.8 g; sub-frs7–17); n-ButFC3 (2.6 g; frs18–24) and n-ButFC4 (4.2 g; sub-frs25–28). sub-fr n-ButFA2 (7–17) was subjected to further sephadex LH20 puriﬁcation with CH2Cl2-MeOH (50:50) to give 7 (12 mg).

2.4. Chemicals

The natural compounds 1–8 (Fig. 1) used in this study were isolated from stem of D. glaucescens as described above. Doxorubicin 98.0% was purchased from Sigma-Aldrich (Munich, Germany) and used as refer-ence drug.

2.5. Cell lines and culture

Four human cancer cell lines and one normal cell line were used in this study. They included A549 human non-small cell lung cancer (NSCLC) cell line, obtained from the Institute for Fermentation, Osaka (IFO, Japan), Caco2 colorectal adenocarcinoma cells obtained from the ŞAP Institute of Turkey (Ankara), HepG2 hepatocarcinoma cells, MCF-7 breast adenocarcinoma cells and the normal CRL2120 human skin ﬁ-broblasts was obtained from ATCC. The cells were maintained as a monolayer in DMEM medium (Sigma-Aldrich, Munich, Germany) me-dium supplemented with 10% fetal calf serum and 1% penicillin (100 U/mL)-streptomycin (100μg/mL) in a humidiﬁed 5% CO2atmosphere

at 37 °C.

2.6. Neutral red uptake (NRU) assay

The cytotoxicity of samples was performed by NRU uptake assay as previously described (Borenfreund et al., 1988). This method is based on the ability of viable cells to incorporate and bind the supravital dye

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NRU in the lysosomes. The procedure is cheaper and more sensitive than other cytotoxicity tests (Repetto et al., 2008). Samples were added in the culture medium so that dimethylsufoxide (DMSO) used prior for dilution did not exceed 0.1%ﬁnal concentration. Brieﬂy, cells were detached by treatment with 0.25% trypsin/EDTA (Invitrogen) and an aliquot of 1 × 104_{cells was placed in each well of a 96-well}

cell culture plate (Thermo Scientiﬁc, Germany) in a total volume of 200μL. The cells were allowed to attach overnight and subsequently treated with different concentrations of the 12 compounds and doxoru-bicin. Each of the studied samples were immediately added in varying concentrations in additional 100μL of culture medium to obtain a total volume of 200μL/well. After 72 h incubation in humidiﬁed 5% CO2atmosphere at 37 °C, the medium was removed and 200μL fresh

medium containing 50μg/mL NRU was added to each well and incuba-tion continued for an addiincuba-tional 3 h at 37 °C in 5% CO2atmosphere. The

dye medium was then removed and each well was then washed rapidly with 200_{μL phosphate buffer saline (PBS) followed by addition of} 200μL of acetic acid–water–ethanol in water (1:49:50). The plates were kept for 15 min at room temperature to extract the dye and then shaken for a few minutes on a GFL 3012 shaker (Gesellschaft für Labortechnik mbH, Burgwedel, Germany). Absorbance was measured on ELx 808 Ultra Microplate Reader (Biotek) equipped with a 540 nm ﬁlter. Each assay was done at least three times, with three replicates each. The viability was evaluated based on a comparison with untreated cells. The IC50values represent the sample's concentrations required to

inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel (Kuete et al., 2011). 2.7. Flow cytometry for cell cycle analysis and detection of apoptotic cells

The cell-cycle analysis was performed byﬂow cytometry using BD cycletest™ Plus DNA Kit Assay (BD Biosciences, San Jose, USA). The BD Cycletest™ Plus DNA kit provides a set of reagents for isolating and staining cell nuclei. Flow cytometric analysis of differentially stained cells is used to estimate the DNA index (DI) and cell-cycle phase distri-butions. Brieﬂy, MCF-7 cells (3 mL, 1 × 105_{cells/mL) were seeded into}

each well of 6-well plates and allowed to attach for 24 h. The cells which were treated with 1/4 × IC50, 1/2 × IC50and IC50concentrations

of compound 4 and the standard drug, doxorubicin were then grown in 6-well plates for 72 h. The untreated cells (control) were also includ-ed in the assay. They were further trypsinizinclud-ed and suspendinclud-ed in 1 mL PBS, then centrifuged at 400g for 5 min at room temperature (RT). The cells were further processed according to the manufacturer protocol: addition of 250μL of solution A (trypsin buffer), 10 min incubation at RT followed by the addition of 200μL of solution B (trypsin inhibitor and RNase buffer), 10 min incubation at RT followed by the addition of 200μL of solution C (2–8 °C) (propidium iodide stain solution), 10 min on ice. The cells were further measured on a BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dickinson, Germany). For each sample 104_{cells were counted. For PI excitation, an argon-ion laser} Fig. 1. Chemical structures of the tested compounds. Lanosta-7,24-dien-3-one (1); friedelanone (2); friedelanol (3); 3,3′-di-O-methylellagic acid (4); 3,3′,4′-tri-O-methylellagic acid (5); ellagic acid (6); 3′,4′-di-O-methylellagic acid 4-O-β-D-glucopyranoside (7); 3,3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside (8).

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emitting at 488 nm was used. Cytographs were analyzed using BD FACSDiva_{™ Flow Cytometry Software Version 6.1.2 (Becton-Dickinson).} 2.8. Caspase-Glo 3/7 and caspase-Glo 9 assay

Caspase activity in MCF-7 cells was detected using Caspase-Glo 3/7 and Caspase-Glo 9 Assay kits (Promega, Mannheim, Germany) as previ-ously reported (Kuete et al., 2013a, 2013b, 2014). Cells were treated with compound 4 at their 1/2 × IC50and IC50values with DMSO as

sol-vent control for 6 h. Luminescence was measured using an BioTek Synergy™ HT multi-detection microplate reader. Caspase activity was expressed as percentage of the untreated control.

2.9. Analysis of mitochondrial membrane potential (MMP)

The MMP was analyzed in MCF-7 cells by 5,5 ′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) (JC-1; Biomol, Hamburg, Germany) staining as previously reported (Kuete et al.,

2013a, 2013b, 2014). Cells (3 mL, 1 × 105_{cells/mL) treated for 72 h}

with different concentrations (1/4 × IC50, 1/2 × IC50and IC50) of

com-pound 4 and doxorubicin (drug control) or DMSO (solvent control) were incubated with JC-1 staining solution for 30 min according to the manufacturer's protocol as reported earlier. Subsequently, cells were measured in a BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dick-inson, Germany). The JC-1 signal was measured at an excitation of 561 nm (150 mW) and detected using a 586/15 nm band-passﬁlter. The signal was analyzed at 640 nm excitation (40 mW) and detected using a 730/45 nm bandpass_{ﬁlter. Cytographs were analyzed using} BD FACSDiva™ Flow Cytometry Software Version 6.1.2 (Becton-Dickin-son). All experiments were performed at least in triplicate.

2.10. Measurement of reactive oxygen species (ROS)

The 2′,7′-dichlorodihydroﬂuorescein diacetate (H2DCFH-DA)

(Sigma-Aldrich) was used for the detection of ROS in MCF-7 cells treat-ed with compound 4 and doxorubicin (drug control) or DMSO (solvent control) using OxiSelect™ Intracellular ROS Assay Kit (Green

Fluorescence) as recommended by the manufacturer, Cell Biolabs Inc. (San Diego, USA). This is a cell-based assay for measuring hydroxyl, peroxyl, or other reactive oxygen species activity within a cell. The assay employs the cell-permeable fluorogenic probe 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA). DCFH-DA is diffused into cells and is deacetylated by cellular esterases to non-fluorescent 2′,7′-dichlorodihydrofluorescin (DCFH), which is rapidly oxidized to highly fluorescent 2′,7′-dichlorofluorescein (DCF) by ROS. Cells (1 × 104_{cells) were treated with samples at 1/4 × IC}

50, 1/2 × IC50and

IC50for 24 h. After addition of 100μL 1X DCFH-DA/DMEM solution to

cells and incubation at 37 °C for 30–60 min, the ﬂuorescence was mea-sured using SpectraMax® M5 Microplate Reader (Molecular Devices, Biberach, Germany) at 480/530 nm. All experiments were performed at least in triplicate.

3. Results and discussion

Compounds isolated from the stem of Desbordesia glaucescens were elucidated using physical and NMR data and comparison with literature (Data available in supporting information). They included three triterpenoids namely lanosta-7,24-dien-3-one C30H48O (1; m/z: 424)

(Nana et al., 2012), friedelanone C30H50O (2; m/z: 426; m.p.

264–265 °C) (Mahato and Kundu, 1994) and friedelanol C30H52O (3;

m/z: 428; m.p. 288–289 °C) (Kamperdick et al., 1997) andﬁve ellagic acid derivatives: 3,3_{′-di-O-methylellagic acid C}16H10O8(4; m/z = 330)

(Ye et al., 2007), 3,3′,4′-tri-O-methylellagic acid C17H12O8(5; m/z:

344; m.p. 287–289 °C) (Ye et al., 2007), ellagic acid C14H6O8(6; m/z:

330) (Li et al., 1999), 3′,4′-di-O-methylellagic acid 4-O-β-D -glucopyranoside C22H20O13 (7; m/z: 492; m.p. 245 et 247 °C)

(DongmoMafodong et al., 2015) and 3,3′-di-O-methylellagic acid

4′-O-β-D-xylopyranoside C21H18O12(8; m/z: 462) (Liu et al., 2014).

Com-pound 7 as well as a novel ellagic acid derivative, desglauside, and oleanolic acid andβ-sitosterol-3-O-β-D-glucopyranoside were previ-ously isolated from the leaves of D. glaucescens (DongmoMafodong et al., 2015).

The cytotoxicity of the 8 isolated compounds and doxorubicin was determined by the NRU uptake assay and the recorded IC50values are

3.1 62.8 15.2 12.8 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Control G row th (pe rc e nt a g e of c o nt ro l) G2/M S G0/G1 Sub-G0 27.6 47.8 60 27.8 16.7 11.5 12.9 10.1 7.6 12.5 11.8 12.8 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

1/4xIC50 1/2xIC50 IC50

G row th (pe rc e nt a g e of c o nt ro l) Doxorubicin G2/M S G0/G1 Sub-G0 23.5 28.6 31.8 38.3 37 34.3 15.6 13.7 12.4 11.6 9.8 9 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

1/4xIC50 1/2xIC50 IC50

G row th (pe rc e nt a g e of c o nt ro l) Compound 4 G2/M S G0/G1 Sub-G0

Fig. 2. Effects 3,3′-di-O-methylellagic acid (4) and doxorubicin on cell cycle distribution in MCF-7 cells. IC50values were 11.23μM (4) and 0.35 μM (doxorubicin). Table 1

Cytotoxicity of tested compounds and doxorubicin towards cancer cell lines and normal cells as determined by the neutral red assay. Compounds Cell lines, IC50values (inμM) and selectivity index* (in bracket)

A549 Caco-2 MCF-7 HepG2 CRL2120

1 82.43 ± 6.42 (N1.15) N94.39 N94.39 43.63 ± 3.17 (N2.16) N94.39 2 N93.90 82.39 ± 7.02 (N1.14) 60.61 ± 4.39 (N1.54) N93.90 N93.90 3 80.79 ± 5.69 (N1.16) 35.37 ± 2.18 (N2.64) N93.46 22.24 ± 2.01 (N4.20) N93.46 4 31.70 ± 1.76 (N3.84) 44.65 ± 4.35 (N2.72) 11.23 ± 0.17 (N10.83) 13.47 ± 0.55 (N9.02) N121.58 5 N116.28 N116.28 N116.28 55.44 ± 3.91 (N2.10) N116.28 6 77.73 ± 5.96 (N1.72) 74.83 ± 6.22 (N1.78) 14.07 ± 0.88 (N9.48) 22.17 ± 1.37 (N6.01) N133.33 7 N81.30 N81.30 N81.30 57.95 ± 3.16 (N1.40) N81.30 8 N86.58 N86.58 N86.58 N86.58 N86.58 Doxorubicin 1.01 ± 0.19 0.72 ± 0.13 0.35 ± 0.05 0.18 ± 0.03 0.59 ± 0.01

(*): The selectivity index was determined as the ratio of IC50value in the CRL2120 normalﬁbroblasts divided by the IC50in the cancer cell lines. In bold: signiﬁcant activity (Boik, 2001;

Brahemi et al., 2010; Kuete and Efferth, 2010, 2015); lanosta-7,24-dien-3-one (1); friedelanone (2); friedelanol (3), 3,3′-di-O-methylellagic acid (4), 3,3′,4′-tri-O-methylellagic acid (5),

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summarized inTable 1. The selectivity index was determined as the ratio of IC50value in the CRL2120 normalﬁbroblast divided by the IC50

in the cancer cell line. Compounds 4 and 6 as well as doxorubicin had IC50values below 45μM in the four tested cancer cell lines meanwhile

other compounds displayed selective activity. The obtained IC50values

ranged from 11.23μM (towards breast adenocarcinoma MCF-7 cells) to 44.65μM (colon carcinoma Caco-2 cells) for 4, from 14.07 μM (to-wards MCF-7 cells) to 77.73_{μM (Caco-2 cells) for 6 and from 0.07 μM} (towards SPC212 cells) to 1.01μM (A549 cells) for doxorubicin. The two most active compounds (4 and 6) were generally less toxic towards normal CRL2120_{ﬁbroblast than carcinoma cells, and the obtained} selec-tivity indexes were above 1.00 in the majority of the cases (Table 1). Compound 4 having the lowest IC50values than other compounds as

well as doxorubicin were further tested for the effects on cell cycle dis-tribution, caspases activity, MMP loss and ROS production in MCF-7 cells.

Compound 4 was analyzed for its ability to alter the distribution of the cell cycle of MCF-7 breast cancer cells (Fig. 2). It was observed that this phytochemical induced concentration-dependent cell cycle modi ﬁ-cations with progressive increase of sub-G0/G1 phase cells. It induced cell cycle arrest in S phases. MCF-7 cells treated with the compound 4 progressively underwent apoptosis, with increase of sub-G0/G1 cells from 27.6% (1/4 IC50) to 60% (IC50) whilst doxorubicin also caused up

to 60% sub-Go/G1 phase with IC50treatment in comparison to only

3.1% in non-treated cells. Upon treatment of MCF-7 cells with equiva-lent (eq.) to the 1/2 × IC50and IC50for 6 h, compound 4 did not activate

caspase 3/7 and caspase 9 contrary to doxorubicin (Fig. 3). The integrity of the MMP was also investigated upon treatment of MCF-7 cells with

Fig. 4. Effects of 3,3′-di-O-methylellagic acid (4) and doxorubicin on MMP in MCF-7 cells for 72 h. IC50values were 11.23μM (4) and 0.35 μM (doxorubicin). Cells were treated with 1/ 4 × IC50(C1), 1/2 × IC50(C2) and IC50(C3) of each compound.

0 0.5 1 1.5 2 2.5

½×IC(50) IC(50) ½×IC(50) IC(50) Compound 4 Doxorubicin Fol d i n c re a s e caspase 3/7 caspase 9

Fig. 3. Effects of 3,3′-di-O-methylellagic acid (4) and doxorubicin on the activation caspases 3/7 and 9 in MCF-7 cells. IC50values were 11.23μM (4) and 0.35 μM (doxorubicin).

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compound 4 and doxorubicin. Treatment with compound 4 at eq. to the 1/4 × IC50, 1/2 × IC50and IC50values for 72 h induced

concentration-dependent depletion of MMP (Fig. 4). The percentage loss of MMP ranged from 9.9% (1/4 × IC50) to 16.5% (IC50). In similar experimental

condition, doxorubicin caused 26% loss of MMP meanwhile only 4.3% was observed with non-treated control. After treatment of MCF-7 cells with compound 4 and doxorubicin at eq. to the 1/4 × IC50, 1/2 × IC50

and IC50values for 24 h, the production of ROS in cells was also analyzed

(Fig. 5). Compound 4 slightly induced increased ROS levels (less than

2-fold (at IC50) as compared with non-treated cells meanwhile the

in-crease was more than 2-fold after treatment with doxorubicin. In regards to the high incidence of cancer globally, there is a contin-ued need for new alternatives for treatment, and natural products play an important role in the discovery of cytotoxic compounds (George et al., 2010; Kuete and Efferth, 2011; Dzoyem et al., 2012; Kuete and

Efferth, 2015). In the present work, compounds isolated from

Desbordesia glaucescens were tested for their cytotoxic effects against various carcinoma cell lines, including breast, colon, lung and liver can-cers. These cancer types are amongst the leading cause of cancer death worldwide (Torre et al., 2015). According to the National Cancer Insti-tute (NCI), phytochemicals having IC50 values around or below

4μg/mL or 10 μM (Boik, 2001; Brahemi et al., 2010; Kuete and Efferth, 2015) can be considered as potential cytotoxic substances. In the pres-ent study IC50values obtained were all above 10μM; however, the

IC50values of 11.23μM and 13.47 μM obtained with compound 4 against

breast adenocarcinoma MCF-7 cells and hepatocarcinoma HepG2 re-spectively, were not far from this set bar. This suggests that this com-pound could be explored more to develop a possible cytotoxic drug. Possible modiﬁcation of the chemical structure of both compounds 4 and 6 could be envisaged to improve their activity. Importantly, the se-lectivity indexes obtained with the two compounds were above 1, indi-cating that they were more toxic towards carcinoma cells than towards normal CRL2120ﬁbroblast in all the cases. The cytotoxicity of the ellagic acid derivatives 4 and 6 are in accordance with previous studies; in fact, ellagic acid has been proposed as a promising candidate for therapeutic use in colon cancer (Yousef et al., 2016). In this study, this compound was active towards Caco-2 colon adenocarcinoma, though the effect was rather low (IC50of 74.83μM). An isomer of compound 4 namely

4,4′-di-O-methylellagic acid also efﬁciently inhibits colon cancer cell growth through a mechanism involving WNT16 (Ramirez de Molina

et al., 2015), conﬁrming the role of ellagic acid as potential cytotoxic

drugs. In this work we provided additional information on the cytotox-icity of this compound on breast, lung and liver cancers. Compound 4 in-duces apoptosis in MCF-7 cells, mediated by alteration on MMP. Ellagic acid was also found to induce apoptosis via depolarization of mitochon-dria in human neuroblastoma cells (Alfredsson et al., 2014). It has been reported that the breast cancer cell line MCF-7 lacks a functional caspase-3 gene product (Jänicke et al., 1998)). This could explained

the lack of induction of apoptosis by compound 4 in MCF-7 cells as ob-served in the present study.

Regarding the structure_{–activity relationship, it appears that both} terpenoids and phenolics isolated from Desbordesia glaucescens had cy-totoxic effects. The best activities were obtained with ellagic acid (6) and its derivative 4. Within terpenoids, friedelanol is more active than friedelanone on Caco-2, MCF-7 and HepG2 cells; the presence of hy-droxyl (−OH) group at C-3 inﬂuences for the anticancer activity. With-in phenolics, the 3,3′-di-O-méthylellagic acid is more active than the 3,3′,4′-tri-O-méthylellagic acid on A549, Caco-2, MCF-7 and HepG2 cells; hence, the–OH group at C-4 also seems to inﬂuence the cytotoxic activity ellagic acid derivatives.

4. Conclusions

In the present study, we demonstrated the cytotoxicity of com-pounds isolated from Desbordesia glaucescens against human carcinoma cell lines. Ellagic acid (6) and its derivative 4 displayed cytotoxic effects on all tested cancer cell lines. Compound 4 induced apoptosis in MCF-7 cells mediated by MMP loss. The studied compounds and especially 4 and 6 deserve more investigations to develop novel cytotoxic drugs against cancers. Abbreviations 1 lanosta-7,24-dien-3-one 2 friedelanone 3 friedelanol 4 3,3_{′-di-O-methylellagic acid} 5 3,3′,4′-tri-O-methylellagic acid 6 ellagic acid

7 3′,4′-di-O-methylellagic acid 4-O-β-D-glucopyranoside 8 3,3′-di-O-methylellagic acid 4′-O-β-D-xylopyranoside

DMSO dimethylsufoxide

H2DCF 2′,7′-Dichlorodihydroﬂuorescein

H2DCFH-DA 2′,7′-Dichlorodihydroﬂuorescein diacetate

MMP mitochondrial membrane potential NRU neutral red uptake

PBS phosphate buffer saline ROS reactive oxygen species

Acknowledgments

V.K. and H.S. are thankful to Scientific and Technological Research Counsel of Turkey (TÜBİTAK) for 6 months travel grant (to V.K.) and to Scienti_{fic Research Projects Commission of Anadolu University,} Eski-sehir, Turkey for the funding grant 1507F563 (to V.K. and H.S.). A grant for part of this work was also provided by International Science Pro-gramme, Uppsala University, Sweden (ISP)-KEN-02 project. IC would like to thank the Scientific Research Projects Commission of Anadolu University, Eskisehir, Turkey for the funding grant (1306F110). Authors are also thankful toŞennur Görgülü for FACS measurements.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.

doi.org/10.1016/j.sajb.2017.03.031.

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