Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/315799330

AntioxidantandselectiveanticanceractivitiesoftwoEuphorbiaspeciesinhumanacutemyeloidleukemia

ArticleinBiomedicine&Pharmacotherapy·April2017

DOI:10.1016/j.biopha.2017.03.072

CITATIONS

4

READS

219

7authors,including:

Someoftheauthorsofthispublicationarealsoworkingontheserelatedprojects:

BioactivecompoundsofhalophytesViewproject

HalophytecultureViewproject

NolwennHymery

UniversitédeBretagneOccidentale

28PUBLICATIONS256CITATIONS

SEEPROFILE

SoumayaBourgou

CentredeBiotechnologie,TechnopoleBorjC…

35PUBLICATIONS871CITATIONS

SEEPROFILE

AhmedJdey

UniversitédeBretagneOccidentale

7PUBLICATIONS38CITATIONS

SEEPROFILE

ChristianMagné

UniversitédeBretagneOccidentale

74PUBLICATIONS1,656CITATIONS

SEEPROFILE

AllcontentfollowingthispagewasuploadedbyChristianMagnéon25September2017.

Theuserhasrequestedenhancementofthedownloadedfile.

Biomedicine & Pharmacotherapy 90 (2017) 375–385

Original article

Antioxidant and selective anticancer activities of two Euphorbia speciesin human acute myeloid leukemia

Soumaya Ben Janneta,d,*, Nolwenn Hymeryc, Soumaya Bourgoua, Ahmed Jdeya,b,Mokhtar Lachaald, Christian Magnéb, Riadh Ksouria

a Laboratoire des Plantes Aromatiques et Médicinales, Centre de Biotechnologie de Borj-Cédria (CBBC), BP 901, 2050 Hammam-Lif, Tunisieb Laboratoire de Géoarchitecture (EA 2219), Université de Brest, 6 avenue V. Le Gorgeu, CS 93837, Brest Cedex 3, Francec Laboratoire de Biodiversité et d’Écologie Microbienne (EA 3882), Université de Brest, Parvis Blaise Pascal, 29280 Plouzané, FrancedUniversité de Tunis El Manar, Unité de Physiologie et Biochimie de la Réponse des Plantes aux Contraintes Abiotiques, Département de Biologie, 1068 Tunis,Tunisie

A R T I C L E I N F O

Article history:Received 10 January 2017Received in revised form 23 March 2017Accepted 24 March 2017

Keywords:E. terracinaE. paraliasIn vitro cytotoxicityLeukemiaApoptosisInflammatoryAntioxidants

A B S T R A C T

In this study, two Euphorbia species (i.e. terracina and paralias) were investigated for their cytotoxic andantioxidant activities. Cytotoxicity of plant methanol and chloroform fractions was examined towardshuman acute myeloid leukemia (THP1) and human colon epithelial (Caco2) cancer cell lines, as well as CD14 and IEC-6 normal cells by targeting various modulators of apoptosis or inflammation. Moreover,secondary metabolite pools (phenolic classes, alkaloids, terpenes, saponins) and antioxidant activities(DPPH, ABTS and O2

�� scavenging, as well as FRAP tests) were assessed in plant extracts.Both Euphorbia species appeared to be rich in phenolic compounds and terpenoids, Moreover, E.

terracina polar and apolar fractions and E. paralias polar fraction were highly active against THP1 cells,with IC50 values of 2.08, 14.43 and 54.58 mg/mL, respectively. However, no cytotoxicity was found againstnormal cells (CD14+ monocytes). The results indicate that the three fractions induce apoptosis in THP1cell line after 6 h of exposure. Furthermore, apoptosis caused by apolar fraction was related to a caspase-dependent process, whereas other death pathways seemed to be involved with the polar fractions. Anenhanced production of reactive oxygen species was detected upon cell treatment with plant extracts.Interestingly, they have no effect on cytokine TNF-a secretion in THP1 and normal cells compared tountreated cells, indicating that the three fractions caused no inflammation.Euphorbia terracina and E. paralias polar fractions showed strong antioxidant activity with potent

scavenging capacity against DPPH, ABTS and superoxide radicals. Moreover, these fractions displayed avery high ferric reducing power.These findings confirm the strong antioxidant capacity of Euphorbia plants and suggest a targeted anti-

cancer effect with a potent anti-proliferative property of E. terracina and E. paralias extracts, which induceprogrammed cell death in leukemia cell lines but not in normal monocytes cells.

© 2017 Elsevier Masson SAS. All rights reserved.

Available online at

ScienceDirectwww.sciencedirect.com

Abbreviations: ABTS, 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid);ACP, principal component analysis; Apol.F, apolar fraction; BHT, Butylatedhydroxytoluene; DMSO, Dimethyl sulfoxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl;MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-nyl)-2H-tetrazolium, inner salt; NADH, nicotinamide adenine dinucleotide hydrate;NBT, p-Nitrotetrazolium blue; PI, percentage of inhibition; PI, propidium iodide;PMS, 5-Methylphenazinium methyl sulfate; Pol.F, polar fraction; ROS, reactiveoxygen species.* Corresponding author at: Laboratoire des Plantes Aromatiques et Médicinales,

Centre de Biotechnologie de Borj-Cédria (CBBC), BP 901, 2050 Hammam-Lif, TunisieE-mail address: Soumaya.BJannet.Lachaal@gmail.com (S. Ben Jannet).

http://dx.doi.org/10.1016/j.biopha.2017.03.0720753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction

Cancer is one of the most common diseases around the worldwith the highest mortality rates. In 2012, the disease had anincident rate of more than 14.1 million cases and 8.2 million deaths.After lung and breast cancers, colorectal was the most diagnosedcancer (1.36 million), while hematological tumors represent onetenth of malignancy cases worldwide [1]. Projections are evenmore unsettling because deaths from cancer are expected to rise toover 13.1 million in 2030 [2]. Cancer’s treatment worldwide is verycostly, requiring expensive facilities and drugs. Moreover, it hasbeen well demonstrated that chemotherapy drugs kill rapidly

376 S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385

growing cancer cells but can also harm perfectly healthy cells,causing side effects throughout the body. Accordingly, newapproaches, finding effective anticancer agents with fewer effectsand less toxic in the clinic, are needed to provide valid therapeuticregimes that offer safe and reliable treatment.

Plants have long been considered in the therapeutic arsenal ofhumanity. Over the last few decades, they have become a prolificsource of structurally diverse bioactive molecules, yielding someof the most important products in the pharmaceutical industry[3,4]. In particular, antioxidant molecules have raised muchattention, these secondary metabolites having many pharmaco-logical properties [5]. Today, approximately 25% of all prescrip-tions contain one or more active ingredients from plants [6], andmost of the anticancer drugs have been discovered from plantsources [7].

The genus Jatropha (Euphorbiaceae) is widely used as atraditional medicinal herb since ancient times in all themediterranean countries [8]. In addition, five euphorbia species,E. pekinensis,E. kansui, E. lathyris, E. humifusa, and E. maculata havebeen recorded in Chinese pharmacopoeias for the treatment ofoedema, gonorrhea, migraine and wart cures. Moreover, severalphytochemical and pharmacological studies have reported a widespectrum of medicinal properties of this genus (antimicrobial,anti-cancer, antiviral and antidiabetic) revealing an importantbiological potential [8,9]. These plants represent a vast and largelyyet untapped source of bioactive molecules and powerful newpharmaceutical products [8–11]. Therefore, searching for alterna-tive medicines and natural therapeutics for cancer therapy,Euphorbia genus appears as among the most promising targets.

In folk medicine, E. terracina and E. paralias infusions were usedto treat fever and paralysis [12,13].To the best of our knowledge,the cytotoxic activity of Euphorbia paralias and Euphorbia terracinaagainst Human acute myeloid leukemia and human colonepithelial cancer have not been investigated previously, nor theantioxidant activities of E. terracina. Therefore, the present studywas addressed to investigate the phytochemical composition ofshoot extracts from Euphorbia paralias and Euphorbia terracina andto evaluate their antioxidant properties. Moreover, cytotoxicity ofboth Euphorbia species was assessed on human monocytesprimary cell culture, two human cancer cell lines (acute myeloidleukemia THP1, and colon epithelial cancer Caco2), and a ratintestinal cell lineage. To understand toxicity mechanism impliedin each case, several parameters were followed, including cellviability, apoptosis, intracellular reactive oxygen species (ROS)generation and the inflammatory response.

2. Experimental

2.1. Plant material and preparation of extracts

>E. paralias and E. terracina shoots were harvested at fullflowering stage from Kelibia (North-Eastern Tunisia). The collectedmaterial was identified at the Biotechnology Center of Borj-Cédriaby Pr. Abderrazak Smaoui, and a voucher specimen were depositedat the Herbarium of the laboratory (Laboratoire des PlantesMédicinales et Aromatiques, CBBC, Tunisia). Crude extract wasobtained by homogenizing 200 mg of powder with 20 mLmethanol/chloroform/water (12:5:3) under magnetic stirring for20 min. Three successive extractions were performed. After phaseseparation by adding deionized water and decantation, apolar(chloroformic) and polar (aqueous methanol) fractions wereconcentrated by rotary evaporation at 40 �C. Polar and apolarresidues were weighed and dissolved in 1% DMSO and methanol,respectively.

2.2. Phytochemical study of secondary metabolites

Total phenolic and flavonoid contents were measured using acolorimetric assay developed by Dewanto [14]. The proanthocya-nidins were determined according to the method of Sun andRichardo da Silvia [15]. Furthermore, plant extracts were screenedfor the presence of alkaloids, terpenoids, steroids and saponins,according to Tona [16].

2.3. Biological assays

2.3.1. Cell culture of the studied lineages� Human leukemia monocytes

THP-1 cells were purchased from the European Collection ofCell Cultures (ECACC; number 88081201, Salisbury, UK) andmaintained in 75 cm2

flasks in the Roswell Park Memorial Institutemedium (RPMI-1640, Sigma-Aldrich St. Louis, MO, USA) supple-mented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 2 mMl-glutamine, and 1% penicillin/streptomycin (Gibco-BRL, Paisley,Renfrewshire, Scotland). Cells were grown at 37 �C, 100% relativehumidity and under 5% CO2 to a density between 0.2 and1 �106 cells/mL as recommended by ECACC. Culture mediumwas replaced every 2 to 3 days before cytotoxic assay experiments.

� Colon adenocarcinoma cells

Caucasian colon adenocarcinoma cells (Caco-2) were obtainedfrom the European Collection of Cell Cultures (ECACC 86010202,Salisbury, UK). They were maintained in 75 cm2

flasks usingDulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich St.Louis, MO, USA) supplemented with 10% FBS, 2 mM of L-glutamine,1% penicillin/streptomycin (Gibco-BRL, Paisley, Renfrewshire,Scotland). The medium was changed every 2–3 days. When thecell monolayer reached 80% confluence, cells were detached with atrypsin-EDTA aqueous solution (0.5 mg/mL) and reseeded at adensity of 5 �104 cells/cm2.

� Rat intestinal cells

Normal rat small intestine cell line (IEC-6, ATCC CRL 1592), wasgrown in DMEM (BE12-604F, Biowhittaker) supplemented with 5%foetal bovine serum (FBS), 2 mM L-glutamine (Sigma) and 1%penicillin/streptomycin (Gibco-BRL), at 37 �C, in 5% CO2 and 100%humidity. Cells were detached with a trypsin-EDTA aqueoussolution (0.2 mg/mL) and reseeded at a density of 5 �104 cells/cm2.

2.3.2. Primary cell culture of human monocytesCD14+ monocytes were used as control cells to assess the

cytotoxicity of plant extracts against previous cell lines. Mononu-clear cells originating from human umbilical cord blood sampleswere collected in citrated tubes from placentas after normaldeliveries in Brest University Hospital (France), with the consentof donors. Low density cells were recovered by density gradientcentrifugation (d = 1.077 g/mL) at 400 � g for 30 min, according toHymery [17]. Monocytes were purified by a positive selectionenrichment method (Miltenyi Biotech, Bergisch Gladbach, Nordr-hein-Westfalen, Germany) using 5 �107 mononuclear cells resus-pended in 1 mL of phosphate buffered saline (PBS, Sigma-Aldrich)with 6% (v/v) FBS. The cell suspension was incubated for 15 min at4 �C with 100 mL of CD14 Microbeads (Miltenyi Biotech). Afterlabeling, monocytes were selected using positive selection columns(MS, Miltenyi Biotech) and cultured for each test. Cell viability ofeach culture was assessed prior to start every experiment, and onlythe batches showing more than 95% cell viability were used in thestudy.

S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385 377

2.3.3. Cell proliferation assayThe cytotoxic effect of polar and apolar extracts was studied on

cancerous cells and compared to non-cancerous cells. Thus,cytotoxicity was evaluated using the Cell Titer 96AQueous Onecell proliferation assay (Promega, Madison, Wisconsin, USA), asdescribed by Hymery [17]. This colorimetric method determinescell viability based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoli-um (MTS) to formazan by mitochondrial dehydrogenases in viablecells. After 24 and 48 h of incubation in the presence of samples,cells were washed with PBS, resuspended in 100 mL of the samebuffer, and seeded in 96-well plates at 37 �C, under 5% CO2

atmosphere and 100% humidity. Then, 20 mL of CellTiter 96AQue-ous One solution was added to each well and the cells were furtherincubated for 3 h at 37 �C, under 5% CO2 atmosphere and 100%humidity. The absorbance was measured at 490 nm. Cytotoxicity isexpressed as the concentration of samples inhibiting cell growthby 50% (IC50), compared with the control (cells treated with 1%DMSO). All tests and analyses were run in triplicate and averaged.Concerning CD14+ monocytes, three different umbilical cord bloodsamples were used.

2.3.4. Annexin V/propidium iodide stainingHuman cancer cells (4 105 cells/mL) were cultured in 96-well

plates and treated for 6 h with different concentrations of eachextract. Cells were washed with 1 mL PBS, resuspended in 100 mLstaining buffer (Annexin V-FITC staining Kit, Miltenyi Biotech,Germany) and analyzed immediately. The Quantification of cellpopulations (i.e. necrotic and apoptotic cells) was performed withBD accuri C6 flow cytometer (Becton Dickinson, New Jersey, USA).The 488 nm laser was used for excitation. Bright field (430–480 nm), annexin-V (505–560 nm) and propidium iodide (595–642 nm) channels were measured and at least 10,000 events ofsingle cells per sample were collected. Color compensation wasnecessary as annexin-V and PI have overlapping emission spectra.Data were analyzed with BD accuri C6 software.

2.3.5. Caspase activityTo investigate the mechanism underlying the apoptotic activity

of the plant fractions, the activation of caspase 3 was detectedusing the Caspase-Glo Assay (Promega, Madison, WI). THP1 cellswere plated in duplicate in 96-well plates and treated with plantfractions. Forty eight hours after exposure, caspase activities weremeasured according to the manufacturer’s instructions. Sampleswere read after 1 h of incubation with the caspase substrate. Therelative fold increase of caspase 3 was calculated by dividing theluminescence reading from the compound treatment to that ofcontrol.

2.3.6. Intracellular reactive oxygen species (ROS) generationROS generation in the THP1 cell treated with plant extracts was

measured by luminescent probe (luciferin) using ROS-Glo H2O2

Assay (Promega, France) according to the manufacturer’s instruc-tions. THP-1 cells were treated with 50 mg/mL of plant fractions for6, 24 and 48 h, and H2O2 (0.3%, v/v) was used as positive control.Intensity of yellow luminescence was measured with a GloMax1

luminometer. The average RLU and standard deviation of triplicatesamples were calculated.

2.3.7. Cytokine measurementsCells were incubated for 24 h in the presence of plant extracts.

The amount of Tumor Necrosis Factor alpha (TNF-a) in thesupernatant of different cell cultures was quantified using an ELISAkit following manufacturer’s instructions (Promocell, Heidelberg,Germany).

2.4. Antioxidant activities

2.4.1. DPPH radical assayDPPH-scavenging activity was measured according to the

method of Chen [18]. An aliquot of the reaction solution wasmixed with 100 mL of 100 mM DPPH in 90% methanol and 100 mL ofplant extract diluted in ethanol at different concentrations.Mixtures were vigorously shaken and left for 30 min in the dark.Absorbance was measured at 517 nm using ethanol as a blank. Thefollowing equation was used to calculate quenching of DPPHradical: PI (%) = 100*(A0� As)/A0, where A0 is the absorbance of thecontrol, and As is the absorbance of the tested sample. Theantiradical activity was expressed as IC50 (mg/mL), the concentra-tion of the sample that caused 50% quenching. All tests andanalyses were run in triplicate and mean values were recorded.Butylated hydroxytoluene (BHT) was used as a positive standard.

2.4.2. ABTS radical assayABTS radical-scavenging activity was measured according to

the method described by Re [19]. Briefly, ABTS was dissolved inwater to a 7 mM concentration and the ABTS+ radical was producedby adding potassium persulfate to a final concentration of2.45 mM. This solution was then diluted with ethanol to adjustits absorbance at 734 nm to 0.70 � 0.02. To determine thescavenging activity, 950 mL of diluted ABTS+ solution was addedto 50 mL of plant extract (or water for the control), and theabsorbance at 734 nm was measured 6 min after the initial mixing,using ethanol as the blank. IC50 was calculated the same way asdescribed in DPPH radical assay, and BHT served as a positivestandard.

2.4.3. Superoxide scavenging activityThe method described by Duh [20] was used to determine

scavenging activity of plant extracts toward superoxide anionradical. Superoxide was generated in a PMS-NADH system (byoxidation of NADH) and assayed by the reduction of NBT. In thereaction medium, samples at different concentrations were addedwith 0.2 mL PMS (60 mM), 0.2 mL NADH (677 mM), and 0.2 mL NBT(144 mM). Mixture was incubated at room temperature for 5 min,and the absorbance was read at 560 nm. All solutions wereprepared in phosphate buffer (0.1 M, pH 7.4). The scavengingactivity was calculated as described previously in DPPH radicalassay. Samples were analyzed in triplicate.

2.4.4. FRAP assayThe capacity of plant extracts to transform Fe3+ to Fe2+ was

determined according to the method of Oyaizu [21]. Samples atdifferent concentrations were mixed with 2.5 mL of 0.2 Mphosphate buffer (pH 6.6) and 2.5 mL of potassium ferricyanide(1% w/v). The tubes were incubated at 50 �C for 20 min. Then,2.5 mL of 10% TCA were added in each tube and the mixture wascentrifuged for 10 min at 1,000g. An aliquot of the supernatant(2.5 mL) was mixed with distilled water (2.5 mL) and 0.5 mL offerric chloride (0.1% w/v), and the absorbance was read at 700 nm.Ascorbic acid was used as authentic standard, and EC50 value(effective concentration of the extract which corresponds to 50% ofcontrol absorbance) was obtained from linear regression analysis.

2.5. Statistical analyses

Results are expressed as means � standard deviation of threereplicates. Different mean value groups were compared to controlvalues according to the least significant difference test ofmultifactor analysis of variance (ANOVA) using Statistica software.Three technical replicates were done for each biological assay (forcell culture analyses). Phenolic contents and biological activities

378 S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385

(antioxidant and cytotoxic datasets) were analyzed individuallyand in combination by principal component analysis (PCA) andrespective heat maps were generated.

3. Results

3.1. Phytochemical study of plant extracts

Estimation of total phenolic, flavonoid and condensed tannincontents of E. terracina and E. paralias fractions are reported inTable 1. Polar fractions of the two species were rich in phenoliccompounds, E. terracina exhibiting 3.5-fold higher total phenoliccontent than E. paralias. Accordingly, E. terracina was rich inflavonoids, whereas E. paralias flavonoid content was low (43.1 vs4.7 mg CE/g DW, respectively). Condensed tannin contents in thetwo polar fractions were statistically similar. No phenolics weredetected in the apolar extracts. Other secondary metabolite poolswere investigated in the different fractions (Table 2). Thus,preliminary characterisation showed that polar fractions of bothspecies are rich in terpenoids, steroids and saponins, as shown byan intense color obtained from each test and a high index of foam.Terpenoids and steroids were also detected in apolar fractions of E.terracina and E. paralias, but not saponins.

3.2. Cytotoxicity and anti-proliferative effect of plant extracts

Cytotoxicity of E. terracina and E. paralias fractions towards celllines (THP1, Caco-2, IEC-6) and monocytes (CD14+) was evaluatedwith different concentration (ranging from 1 to 100 mg/mL) at twodifferent times of exposure (24 and 48 h). As shown in Fig. 1, THP1viability varied depending on the extract concentration andtreatment time. After 24 h, no antiproliferative activity wasobserved whatever the extract concentration. Conversely, after48 h of incubation, a strong suppression of cell proliferation wasinduced by the polar fraction of E. paralias and the two fractions ofE. terracina. A significant decrease of cell viability was observed atthe high concentration (100 mg/mL), reaching 8.69 and 13.74% forpolar fractions of E. terracina and E. paralias, respectively.

These results revealed that E. terracina fractions exhibited apotent inhibitory activity on THP1cell proliferation, with an IC50

values ranged between 2.08 (polar fraction) and 14.43 mg/mL(apolar fraction), followed by polar fraction of E. paralias (IC50 =54.8 mg/mL). In order to evaluate their anticancer activity, the

Table 1Total phenolic, total flavonoid and condensed tannin contents in polar and apolar fractiofollowed by different letters differ statistically at P < 0.05.

Species Fraction Total phenolics (mg GAE/g DW)

E. paralias Polar 22.73 � 0.32 b

Apolar n.d

E. terracina Polar 78.01 � 8.83 a

Apolar n.d

n.d: not detected.

Table 2Phytochemical characterisation of secondary metabolite pools in Euphorbia paralias an

Species Fractions Alkaloids

Mayer test Dragendorff tes

E. paralias Polar � �

Apolar � �

E. terracina Polar � �

Apolar � �

+ Positive test, � negative test, Pol.F: polar fraction, Apol.F: apolar fraction.

previous action of Euphorbia fractions on THP1 cell line wascompared to that on non-cancerous cells. Thus, no cytotoxic effectwas noted on human mononuclear cells (CD14+) purified fromhuman umbilical cord blood at any concentrations tested. On theother hand, Caco2 line was found to be much more resistant thanTHP1, and Caco2 cells were not affected by any of the testedfractions (Table 3).

Interestingly, no cytotoxic effects were noted on humanmononuclear cells (CD14 + ), indicating that both species inhibitpreferentially THP1 cell line.

3.3. Apoptosis mechanism in leukemia cells

Based on the antiprolifative data, the most sensitive cell line(THP1) and cytotoxic fractions (both E. terracina extracts and polarfraction of E. paralias) were retained for complementary studies toinvestigate apoptosis mechanism via flow cytometry technique.Thus, annexin V and propidium iodide (PI) were used inconjunction to distinguish between apoptotic and necroticpathway. Annexin V is a binding protein with high affinity andselectivity for phosphatidylserine (PS). The translocation of PS tothe external cell surface is one of the reliable biochemical markersof apoptosis. Alternatively, annexin may enter cell and bind to PSupon cell necrosis. The difference between these two forms of celldeath is that during the initial stages of apoptosis the cellmembrane remains intact and impermeable to dyes such aspropidium iodide (PI). As shown in Fig. 2, after 6 h of treatmentwith 50 mg/mL of each selected fraction, apoptosis was inducedleading to the subsequent inhibition of cellular proliferation.Indeed, compared to control group, the percentage of apoptoticcells was significantly increased from 0.8% to 43, 34 and 33% by E.paralias polar fraction, E. terracina polar and apolar fractions,respectively. However, the proportion of necrotic cells did notexceed 6.7%.

To further explore pathways of apoptosis mechanism underly-ing the cytotoxicity activity of plant extracts, we tested theinduction of caspase 3 activity. THP1 cells were treated for 48 hwith 50 mg/mL plant extracts. Upon exposure to E. terracina apolarfraction, a significant (2.2 fold) increase in caspase 3 activity wasobserved, compared to the untreated cells (Fig. 3). Moreover, in thepresence of the pancaspase inhibitor Z-VAD.fmk, a decrease ofcaspase activity was found. Such changes were not recorded uponexposure to E. terracina polar fraction or E. paralias extract.

ns of E. paralias and E. terracina. Mean � SD of three replicates in the same column

Total flavonoids (mg CE/g DW) Condensed tannins (mg CE/g DW)

4.66 � 0.09 b 1.30 � 0.06 an.d n.d

43.08 � 3.04 a 1.65 � 0.10 an.d n.d

d Euphorbia terracina fractions.

Terpenoids Steroids Saponins

t Sulfuric acid test Salkowski test Foam test

+++ ++ +++++ + �+++ +++ ++++ + �

Fig.1. Effect of E. paralias and E. terracina fractions on the proliferation of THP-1cells after 24 and 48 h of exposure. The percentage of cell viability was measured by MTT assay.All experimental data are means � SD of triplicate determinations. * Statistically significant differences at P<0.05 when compared with solvent control.

Table 3Cytotoxicity of Euphorbia paralias and E. terracina fractions, expressed as IC50 (mg/mL), on two cancer cell lines (THP-1 and Caco-2) and normal cells (CD14+ and IEC6)after 48 h of exposure. Means � SD of three replicates in the same row followed bydifferent letters differ statistically at P < 0.05.

Cell line E. paralias E. terracina

Polar Apolar Polar Apolar

THP-1 54.58 � 4.43 c* >100 d* 2.08 � 0.26 a* 14.43 � 0.31 b*

Caco-2 NT NT NT NTCD14+ NT NT NT NTIEC-6 NT NT NT NT

NT: Non Toxic.* Statistically different from Caco-2 after the same time of exposure.

S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385 379

3.4. Effect of plant extracts on ROS production

A putative pro-oxidant effect of plant extract was investigatedon THP-1 cells treated with active fractions for 6, 24 and 48 h. Asshown in Fig. 4, a significant rise in ROS levels was observed inTHP1 cell line compared to untreated cells (P < 0.05). Regardless ofincubation time, each extract induced a 5 to 10-fold increase in ROSproduction.

3.5. Effect of plant extracts on inflammatory response

The inflammatory response was quantified by assayingcytokines released in the extracellular medium of cells treatedfor 48 h with 50 mg/mL plant extracts. None of the studied extractsinduced TNF-a production by THP1 and CD14+ cells, compared tountreated cells (Fig. 5). Cytokine secretion by leukemic cell line

Fig. 2. Apoptotic effects of E. terracina fractions and E. paralias polar fraction on THP1 cell line. THP1 cells were incubated with 50 mg/mL for 6 h and analyzed by flowcytometry after annexin V-FITC and propidium iodide staining. Untreated cells were used as negative controls. Means � SD of six replicates followed by at least one same letterwere not significantly different between fractions at P < 0.05. * Statistically significant differences when compared with untreated cells.

Fig. 3. Effects of E. terracina fractions and E. paralias polar fraction on caspase 3 activation. The result represents means values � SD of three replicates.* indicates significantlydifferent from untreated cells, P < 0.05.

Fig. 4. ROS production in THP-1 cells upon exposure to E. terracina and E. paralias fraction treatments. The result represents mean values � SD of three replicates.* indicatessignificant difference with untreated cells, P < 0.05.

380 S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385

Fig. 5. Effects of E. terracina fractions, and E. paralias polar fraction on TNF-a secretion. Each experiment was performed in triplicate. Three technical replicates for eachbiological replicate were done. Standard deviations were established from three biological replicates.

S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385 381

(THP1) and their homologue normal cells (CD14+) did not exceed15 and 18 pg/mL, respectively.

3.6. Antioxidant activities of Euphorbia extracts

Antiradical power of the different fractions of E. terracina and E.paralias was evaluated in vitro using DPPH, ABTS and superoxideradicals. Polar fractions of E. terracina and E. paralias showed strongantioxidative activity against peroxyl radicals, whereas little or noactivity was found in apolar fractions (Table 4). DPPH-scavengingactivity was similar in the two species, with an IC50 of about 5 mg/mL, and close to that of the positive control (BHT). E. terracinaexhibited the strongest ABTS-quenching capacity (1.1 mg/mL), bothspecies being even more powerful than BHT (10.02 mg/mL). Inaddition, polar Euphorbia extracts exhibited a high superoxide-scavenging power, E. Terracina being the most active (3 mg/mL).

The antioxidant activity of plant extracts was also assessedthrough the ferric reducing antioxidant power (FRAP) assay. Asfound with antiradical bioassays, polar fractions showed strongreducing capacity, with EC50 values below 2 mg/mL. Here again, E.terracina appeared to be more active than E. paralias, and far morethan ascorbic acid.

4. Discussion

For the first time, the apoptotic and proliferative effects ofEuphorbia terracina and E. paralias extracts were investigated onhuman acute myeloid leukemia (THP1) and colon epithelial cancer(Caco2) cell lines, as well as on CD14+ normal monocytes and (IEC6normal cells). Since some relationships between cytotoxicity and

Table 4DPPH, ABTS and superoxide radical-scavenging activities and ferric reducing power of Eupand EC50 (mg/mL). Means � SD of three replicates in the same column followed by at l

Species DPPH IC50 (mg/mL) ABT

E. paralias Polar 5.25 � 0.32 a 1.94Apolar 266.2 � 11.2 c >50

E. terracina Polar 4.90 � 0.10 a 1.16Apolar 244.8 � 17.9 c >50

BHT 9.65 � 0.79 b 10.0Ascorbic acid – –

n.d: not detected.

antioxidant activity have been substanciated [4–22], the antioxi-dant potential of Euphorbia extracts was investigated, throughDPPH, ABTS and superoxide radicals scavenging, and iron reducingtests. Moreover, as an attempt to identify putative chemical class ofcompound responsible for cytotoxic action, plant extracts werescreened for secondary metabolite classes, including phenolics,terpenoids, steroids and saponins.

Results first highlighted the richness in phenolic compounds,particularly in flavonoids, of polar fractions of E. terracina, and to alower extent E. paralia. Polyphenol levels have not been reportedhitherto in E. terracina. However, similar trends were observed onother Euphorbia species originating from Tunisia, including E.helioscopia and E. guyoniana [23,24]. Moreover, our results ofphytochemical characterisation are in agreement with previousinvestigations highlighting the abundance of terpenoids inEuphorbia plants [10–25]. Thus, a number of diterpenes have beenisolated from the shoots and latex of spurges [12–28]. According toour preliminary screening, alkaloids were not detected in the twospecies studied. These results did not support previous observa-tions indicating the presence of this class of molecules in severalother Euphorbiaceae species [10]. Of the two cancer linesinvestigated, THP1 was the most sensitive to E. terracina extractsand polar fraction of E. paralias. Moreover, cell proliferation uponexposure to plant extracts decreased in distinct dose and time-dependent manners. Interestingly, no effect on normal monocytes(CD14+) viability was recorded, suggesting that the three fractionsare not cytotoxic to non-cancerous cells. Moreover, same fractionsshowed no obvious cytotoxicity at 100 mg/mL on Caco2 cells. Acomparison of antiproliferative action revealed that the twostudied species have potent selective anticancer effects,

horbia terracina and Euphorbia paralias fractions. All activities were expressed as IC50

east one same letter are not significantly different at P < 0.05.

S IC50 (mg/mL O2�� IC50 (mg/mL) FRAP EC50 (mg/mL)

� 0.07 a 7.89 � 1.43 c 1.92 � 0.05 b0d >500d n.d

� 0.01 b 2.97 � 0.02 b 0.48 � 0.03 a0d >500d n.d

2 � 12.09 c 0.20 � 0.01 a –

– 28.33 � 1.01 c

382 S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385

particularly on leukemia cells. These results are in agreement witha recent study which indicated the antigrowth and cytotoxiceffects of methanol extract of E. terracina against humanhepatocellular carcinoma cell line (HepG2) [29]. Furthermore,new diterpenoid with anticancer effect have been isolated andcharacterized from active extract of Euphorbia paralias [30].

Although the MTS assay can be used to assess cytotoxicity, itdoes not differentiate between the different modes of cell death.Therefore, cytotoxicity mechanism was further examined by flowcytometry, using FITC-conjugated annexin V and propidium iodidedouble staining on the THP1 line. Cells exposed to selectedEuphorbia fractions showed a significant increase in apoptosis(more than 30%, P < 0.05) and a very low rate of necrosis (less than6%), suggesting that E. terracina fractions and polar E. paraliasextract induce THP1 cytotoxicity through apoptosis mechanism.Therefore, those active fractions should be considered of particularinterest since numerous studies identified pro-apoptosis andantiproliferation as the main therapeutic goals of anti-leukemiadrugs [31]. Indeed, leukemia is characterized by proliferation ofbone marrow and peripheral blood cells following the inhibition ofapoptosis, driving usually inexplosive cell growth in an uncon-trolled manner [32]. This makes this type of cancer a highlycomplex multi-targeted disorder [33], hardly curable.

On the basis of increased apoptosis in treated THP1 cells, theinvolvement of caspases, which play a major role in execution ofirreversible apoptotic events, was then adressed. Indeed, apoptosisis a complex process that includes caspase-dependent andcaspase-independent mechanisms [4]. Upon exposure to apolarfractions, the intracellular caspase cascade was activated, whereaspolar fractions seemed to induce more complex process withcaspase-independent factors. They may include oxidative stresscompounds and/or autophagic processes, apoptosis inducingfactor (AIF), endonuclease G [34]. It can be assumed that thepolarity or hydrophobicity of major compounds present in eachfraction explain their interaction with the sites responsible for thecellular regulation involved in cell death.

In the other hand, cytotoxic Euphorbia fractions caused amarked accumulation of reactive oxygen species (ROS) in THP1cells. Our data are in accordance with previous studies reportingthe pro-oxidant effect in cancer lines of plant secondarymetabolites like polyphenols [35–37] or terpenoids [38]. Indeed,these natural products have been shown to cause an acumulationof intracellular ROS beyond the cellular tolerance level, by (i) theformation of a labile aroxil radical, or a labile redox complex with ametal cation. This aroxil radical can react with oxygen, resulting inthe formation of O2�� (ii) activating the intracellular production ofROS by NOX (iii) the reduction of metal ions involved in redox-cycling, and promoting the generation of hydroxyl radicals throughFenton and Fenton-like reactions leading to subsequent apoptosis[38–40].

Since we found high levels of polyphenols and terpenoids inactive Euphorbia fractions, our results support the view that suchsecondary metabolites may be involved in the pro-apoptoticproperties of these fractions. Accordingly, terpenoids (especiallyditerpenes and sesquiterpenes) were reported as the mainanticancer substances in Euphorbia genus [10]. Furthermore,euphornin, euphoscopin and jatrophane (type diterpene), isolatedpreviously from other Euphorbia species, exhibited a strong growthinhibition effect on murine leukemia (P388), human cervicalcarcinoma (Hela), human breast adenocarcinoma (MCF-7), humanhepatocellular (HepG2), human colon carcinoma (HCT116), lungadenocarcinoma (A549) and ovarian cancer cell lines (Caov4,OVCAR3) [41,42–43–44,45]. Terpenoids constitute promisful com-pounds in drug discovery particularly for cancer chemotherapy.Their selective anticancer properties have been recently the focusof intense research [46–48]. They caused inhibition on cell

proliferation, tumor growth, angiogenesis and metastasis ofhuman cancers mostly by targeting apoptotic pathways [49,50].

Besides, our results showed that polar fractions of bothEuphorbia species contain significant contents of saponins. Yet,the cytotoxic activity of several saponins was recently reported byKoczurkiewicz [51]. Indeed, it is worth noting that saponins exhibitselectivity in their antitumoral action, possess high efficiency incarcinogenesis inhibition, and they can act in a comprehensivemanner. This may explain in part the powerful selectivecytotoxicity of Euphorbia fractions in our study.

Previous works reported the cytotoxicity of Euphorbia plantsagainst several cancer lines [44–52]. However, there is noinvestigation on inflammatory response of leukemia cancer linesand normal monocytes to Euphorbia. In this study, inflammatoryresponse to E. paralias was similar to that observed with E.terracina: none of polar or apolar fractions induced secretions ofpro-inflammatory cytokines in THP1 and CD14+ cells, indicatingthat those fractions do not cause inflammation.

Examination of the antioxidant properties of E. terracina and E.paralias polar fractions showed strong antiradical activity withpotent scavenging capacity. As far as we know, this is the firstreport on E. terracina antioxidant activities. In E. paralias, a singlestudy mentioned a modest antioxidant activity in aqueous andethanol extracts [13]. Our results appear more spectacular, theactivities measured being even stronger than those of authenticstandard. Such discrepancy might be due to the geographical originof the plant, or to the development stage [53,54]. The choice ofsolvents used and studied organs may also affect the secondarymetabolite pool extracted, therefore the antioxidant level [55]. Thestrong antiradical capacity of E. terracina and E. paralias found inour study corroborates results from another spurge, Euphorbiaguyoniana [24]. Kawashty [56] showed the abundance offlavonoids in E. paralias, and quercetin was found to be a majorphenolic constituent in Euphorbia hirta [57]. These classes are wellknown for their strong antioxidant activity, which may explain themarked antioxidant capacities of the two species studied here. It iswell known that phenolic compounds contributed significantly tothe antioxidant capacity of plant tissues, a positive correlationbeing found frequently between phenolic pool and antioxidantactivities [5]. Besides, the contribution of terpenoids to the strongantioxidant activity of Euphorbia species should not be excluded, asit has been substantiated by Graßmann [58].

Numerous studies have reported a strong relationship betweenantioxidant and anticancer activities. Indeed, most of the plant-derived anticancer drugs discovered hitherto have potent antioxi-dant activities. This is the case for some promising polyphenolicssuch as resveratrol, curcumin, and genistein [59–61]. Therefore,statistical analysis was conducted to describe the relationshipbetween phenolic levels and biological activities of Euphorbiaparalias and Euphorbia terracina extracts. PCA were carried out inorder to determine the relationship between the activities(antioxidant and cytotoxic activities) of fractions on the basis oftheir composition (PPT, FVT, CT). Matrix correlation showed adirect correlation between total polyphenols and different in vitroantioxidant activities (Fig. 6), indicating that phenolic compoundsplay a crucial role in the antioxidant activity of Euphorbia fractionsand corroborating previously published data [55]. However, a lowcorrelation between polyphenol pools and cytoxic activities wasobserved, although cytotoxicity was significantly correlated withantioxidant activities (r = 0.58). These results can also be deducedfrom the PCA (Fig. 7). Actually, different substances may beinvolved in anticancer action, including terpenoids and saponins aspreviously mentioned [4]. From our study, the accurately effect ofplants fractions on the anti-tumoral responses remains to beelucidated. However, is could be that treatment of THP-1 cells withE. terracina and E. paralias fractions activates some specific and

Fig. 6. Correlation coefficient between phenolic contents (PPT, FVT, TC) and biological activities determined by different assays. MTT, apoptosis percentage (obtained on THP1cell line). ***: significant correlation (P < 0.05).

S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385 383

rapid signal transduction pathway committed to the generation ofan apoptotic cascade, thanks to the antioxidant molecules presentin the extracts. Such mechanism was already found by Totta [62]for naringenin polyphenol.

5. Conclusion

The aim of our study was to investigate enthnopharmacologicalclaims of two Tunisian spurges in cancer treatment. Our resultsreveal, for the first time, a targeted anti-cancer effect of Euphorbiaparalias and Euphorbia terracina extracts, with no risk to normalhuman monocytes. Our data show the high anti-proliferative

effects of these species against human acute myeloid leukemiaTHP-1 through the induction of apoptosis. However, fractionstarget different death pathways. We demonstrated that caspaseactivation plays an important role in the antiproliferative effectinitiated by apolar fraction but not in cells treated with polarfraction. Moreover, studied fractions induced intracellular ROSproduction without causing inflammation under the same con-ditions. A high phenolic and terpenoid content, correlated topotent antioxidant activity, was also found.

Taken together, E. terracina and E. paralias fractions could have aselective action on leukemia. However, it is still unclear whichmolecules were involved in this antitumor response. Therefore,

Fig. 7. Bi-plot of E. paralias and E. terracina fractions obtained from principal component analysis of data matrix of the phenolic compounds (PPT, FVT, TC), antioxidant DPPH,ABTS, O2

��) and cytotoxic (MTT) activities with first two principal components.

384 S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385

fractionation of active fractions of E. paralias and E. terracina areunderway to isolate the individual active compound(s) and toelucicate their molecular mechanism.

Conflict of interest

There is no conflict of interest.

Acknowledgments

This work was supported by the Tunisian Ministry of HigherEducation and Scientific Research LR15CBBC02 and Campus Francein the framework of the PHC CMCU programme (no. 15G0812).

References

[1] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M.Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources,methods and major patterns in GLOBOCAN 2012, Int. J. Cancer 136 (2015)E359–386.

[2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015, CA. Cancer J. Clin. 65(2015) 5–29.

[3] I.F.F. Benzie, S. Wachtel-Galor, Herbal Medicine: Biomolecular and ClinicalAspects, 2nd ed., Boca Raton, 2011.

[4] H. Gali-Muhtasib, R. Hmadi, M. Kareh, R. Tohme, N. Darwiche, Cell deathmechanisms of plant-derived anticancer drugs: beyond apoptosis, Apoptosis20 (2015) 1531–1562.

[5] R. Ksouri, W.M. Ksouri, I. Jallali, A. Debez, C. Magné, I. Hiroko, C. Abdelly,Medicinal halophytes: potent source of health promoting biomolecules withmedical: nutraceutical and food applications, Crit. Rev. Biotechnol. 32 (2012)289–326.

[6] S.Y. Pan, S.F. Zhou, S.H. Gao, Z.L. Yu, S.F. Zhang, M.K. Tang, J.N. Sun, D.L. Ma, Y.F.Han, W.F. Fong, K.M. Ko, S.Y. Pan, S.F. Zhou, S.H. Gao, Z.L. Yu, S.F. Zhang, M.K.Tang, J.N. Sun, D.L. Ma, Y.F. Han, W.F. Fong, K.M. Ko, New perspectives on how todiscover drugs from herbal medicines: CAM’s outstanding contribution tomodern therapeutics, new perspectives on how to discover drugs from herbalmedicines: CAM’s outstanding contribution to modern therapeutics,evidence-Based complementary and alternative medicine, Evid. BasedComplement. Altern. Med. 2013 (2013) 2013.

[7] Encyclopedia of Cancer, in: M. Schwab (Ed.), Springer Berlin Heidelberg, Berlin,Heidelberg, 2012.

[8] C.W. Sabandar, N. Ahmat, F.M. Jaafar, I. Sahidin, Medicinal property,phytochemistry and pharmacology of several Jatropha species(Euphorbiaceae): A review, Phytochemistry 85 (2013) 7–29.

[9] H.A. Abdelgadir, J. Van Staden, Ethnobotany, ethnopharmacology and toxicityof Jatropha curcas L. (Euphorbiaceae): a review, S. Afr. J. Bot. 88 (2013) 204–218.

[10] T.J. Mwine, P. Van Damme, Why do Euphorbiaceae tick as medicinal plants?: areview of Euphorbiaceae family and its medicinal features, J. Med. Plants Res. 5(2011) 652–662.

[11] S. Tariq, M. Mussarat, Adnan Review on ethnomedicinal, phytochemical andpharmacological evidence of Himalayan anticancer plants, J. Ethnopharmacol.164 (2015) 96–119.

[12] S.A. Abdelgaleil, S.M. Kassem, M. Doe, M. Baba, M. Nakatani, Diterpenoids fromEuphorbia paralias, Phytochemistry 58 (2001) 1135–1139.

[13] A. Aboul-Enein, F. Abu El-Ela, A. Shalaby Emad, A. El-Shemy, Traditionalmedicinal plants research in Egypt: studies of antioxidant and anticanceractivities, J. Med. Plants Res. 5 (2012) 689–703.

[14] V. Dewanto, X. Wu, K.K. Adom, R.H. Liu, Thermal processing enhances thenutritional value of tomatoes by increasing total antioxidant activity, J. Agric.Food Chem. 50 (2002) 3010–3014.

[15] B. Sun, J.M. Ricardo-da-Silva, I. Spranger, Critical factors of vanillin assay forcatechins and proanthocyanidins, J. Agric. Food Chem. 46 (1998) 4267–4274.

[16] L. Tona, K. Kambu, N. Ngimbi, K. Cimanga, A.J. Vlietnick, Antiamoebic andphytochemical screening of some congolese medical plants, J.Ethnopharmacol. 61 (1998) 57–65.

[17] N. Hymery, F. Masson, G. Barbier, E. Coton, Cytotoxicity and immunotoxicity ofcyclopiazonic acid on human cells, Toxicol. In Vitro 28 (2014) 940–947.

S. Ben Jannet et al. / Biomedicine & Pharmacotherapy 90 (2017) 375–385 385

[18] Y. Chen, M. Wang, R.T. Rosen, C.T. Ho, 2,2-Diphenyl-1-picrylhydrazyl radical-scavenging active components from Polygonum multiflorum thunb, J. Agric.Food Chem. 47 (1999) 2226–2228.

[19] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice Evans,Antioxidant activity applying an improved ABTS radical cation decolorizationassay, Free Radic. Biol. Med. 26 (1999) 1231–1237.

[20] P.D. Duh, Y.Y. Tu, G.C. Yen, Antioxidant activity of water extract of harng jyur(Chrysanthemum morifolium Ramat), LWT-Food Sci. Technol. 32 (1999) 269–277.

[21] M. Oyaizu, Studies on products of browning reaction prepared from glucoseamine, Jap. J. Nut. 44 (1986) 307–315.

[22] P.K. Sahu, Design structure activity relationship, cytotoxicity and evaluation ofantioxidant activity of curcumin derivatives/analogues, Eur. J. Med. Chem. 121(2016) 510–516.

[23] M. Ben Mohamed, A. Jelassi, I. Hassen, S. Ould Mohamed, A. Ould Boukhari,Antioxidant proprieties of methanolic and ethanolic extracts of Euphorbiahelioscopia, (L.) aerial parts, Inter Food Res J. 19 (2012) 1125–1130.

[24] M. Bouaziz, A. Dhouib, S. Loukil, M. Boukhris, S. Sayadi, Polyphenols content:antioxidant and antimicrobial activities of extracts of some wild plantscollected from the south of Tunisia, Afr. J. Biotechnol. 24 (2009) 7017–7027.

[25] A. Vasas, D. Rédei, D. Csupor, J. Molnár, J. Hohmann, Diterpenes from EuropeanEuphorbia species serving as prototypes for natural product based drugdiscovery, Eur. J. Org. Chem. 2012 (2012) 5115–5130.

[27] I.S. Aljan9ci�c, M. Peši�c, S.M. Milosavljevi�c, N.M. Todorovi�c, M. Jadranin, G.Milosavljevi�c, D. Povrenovi�c, J. Bankovi�c, N. Tani�c, I.D. Markovi�c, S. Ruždiji�c, V.E.Vajs, V.V. Teševi�c, Isolation and biological evaluation of jatrophanediterpenoids from Euphorbia dendroides, J. Nat. Prod. 74 (2011) 1613–1620.

[28] S. Aichour, H. Haba, M. Benkhaled, D. Harakat, C. Lavaud, Terpenoids and otherconstituents from Euphorbia bupleuroides, Phytochem. Lett.10 (2014) 198–203.

[29] M. El Manawaty, W. Fayad, N.M. El-Fiky, G.M. Wassel, B.S. El Menshawi, High-throughput screening of 75 euphorbiaceae and myrtaceae plant extracts forin-vitro antitumor and pro-apoptotic activities on human tumor cell lines, andlethality to brine shrimp, Int. J. Pharm. Pharmaceut. Sci. 5 (2013) 178–183.

[30] G. Corea, A. Di Pietro, C. Dumontet, E. Fattorusso, V. Lanzotti, Jatrophanediterpenes from Euphorbia spp as modulators of multidrug resistance incancer therapy, Phytochem. Rev. 8 (2009) 431–447.

[31] E. Robles-Escajeda, D. Lerma, A.M. Nyakeriga, J.A. Ross, R.A. Kirken, R.J.Aguilera, A. Varela-Ramirez, Searching in mother nature for anti-canceractivity: anti-proliferative and pro-apoptotic effect elicited by green Barley onLeukemia/Lymphoma Cells, PLoS One 8 (2013) e73508.

[32] B.T. Gjertsen, H. Wiig, Investigation of therapy resistance mechanisms inmyeloid leukemia by protein profiling of bone marrow extracellular fluid,Expert Rev. Proteom. 9 (2012) 595–598.

[33] S. Ramadevi Mani, B.S. Lakshmi, G1 Arrest and Caspase-mediated apoptosis inHL-60Cells by dichloromethane extract of Centrosema pubescens, Am. J. Chin.Med. 38 (2010) 1143–1159.

[34] C. Candé, N. Vahsen, C. Garrido, G. Kroemer, Apoptosis-inducing factor (AIF):caspase-independent after all, Cell Death Differ. 11 (2004) 591–595.

[35] D.O. Moon, M.O. Kim, Y.H. Choi, J.W. Hyun, W.Y. Chang, G.Y. Kim, Buteininduces G2/M phase arrest and apoptosis in human hepatoma cancer cellsthrough ROS generation, Cancer Lett. 288 (2010) 204–213.

[36] V. Sosa, T. Moliné, R. Somoza, R. Paciucci, H. Kondoh, M.E.L. Leonart, Oxidativestress and cancer: an overview, Ageing Res. Rev. 12 (2013) 376–390.

[37] A.J. León-González, C. Auger, V.B. Schini-Kerth, Pro-oxidant activity ofpolyphenols and its implication on cancer chemoprevention andchemotherapy, Biochem. Pharmacol. 98 (2015) 371–380.

[38] D.Y. Eum, J.Y. Byun, C.H. Yoon, W.D. Seo, K.H. Park, J.H. Lee, H.Y. Chung, S. An, Y.Suh, M.J. Kim, S.J. Lee, Triterpenoid pristimerin synergizes with taxol to inducecervical cancer cell death through reactive oxygen species-mediatedmitochondrial dysfunction, Anticancer Drugs 22 (2011) 763–773.

[39] S. Yasuda, S. Yogosawa, Y. Izutani, Y. Nakamura, H. Watanabe, T. Sakai,Cucurbitacin B induces G2 arrest and apoptosis via a reactive oxygen species-dependent mechanism in human colon adenocarcinoma SW480 cells, Mol.Nutr. Food Res. 54 (2010) 559–565.

[40] E. Mayola, C. Gallerne, D.D. Esposti, C. Martel, S. Pervaiz, L. Larue, B. Debuire, A.Lemoine, C. Brenner, C. Lemaire, Withaferin-A induces apoptosis in humanmelanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2, Apoptosis 16 (2011) 1014–1027.

[41] Z.Q. Lu, S.H. Guan, X.N. Li, G.T. Chen, J.Q. Zhang, H.L. Huang, X. Liu, D.A. Guo,Cytotoxic diterpenoids from Euphorbia helioscopia, J. Nat. Prod. 71 (2008) 873–876.

View publication statsView publication stats

[42] H.W. Tao, X.J. Hao, P.P. Liu, W.M. Zhu, Cytotoxic macrocyclic diterpenoids fromEuphorbia helioscopia, Arch. Pharm. Res. 31 (2008) 1547–1551.

[43] Z.Y. Wang, H.P. Liu, Y.C. Zhang, L.Q. Guo, Z.X. Li, X.F. Shi, Anticancer potential ofEuphorbia helioscopia L extracts against human cancer cells, Anat. Rec. 295(2012) 223–233.

[44] D.S. Yang, W.B. Peng, Z.L. Li, X. Wang, J.G. Wei, Q.X. He, Y.P. Yang, K.C. Liu, X.L. Li,Chemical constituents from Euphorbia stracheyi and their biological activities,Fitoterapia 97 (2014) 211–218.

[45] M. Ghanadian, H. Saeidi, M. Aghaei, M.R. Rahiminejad, E. Ahmadi, S.M.Ayatollahi, M.I. Choudhary, B. Bahmani, New jatrophane diterpenes fromEuphorbia osyridea with proapoptotic effects on ovarian cancer cells,Phytochem. Lett. 12 (2015) 302–307.

[46] A. Ghantous, H. Gali-Muhtasib, H. Vuorela, N.A. Saliba, N. Darwiche, Whatmade sesquiterpene lactones reach cancer clinical trials? Drug Discov. Today15 (2010) 668–678.

[47] C.T. Yeh, W.C. Huang, Y.K. Rao, M. Ye, W.H. Lee, L.S. Wang, D.T.W. Tzeng, C.H.Wu, Y.S. Shieh, C.Y.F. Huang, Y.J. Chen, M. Hsiao, A.T.H. Wu, Z. Yang, Y.M. Tzeng,A sesquiterpene lactone antrocin from Antrodia camphorata negativelymodulates JAK2/STAT3 signaling via microRNA let-7c and induces apoptosis inlung cancer cells, Carcinogenesis 34 (2013) 2918–2928.

[48] C. Bosio, G. Tomasoni, R. Martínez, A.F. Olea, H. Carrasco, J. Villena, Cytotoxicand apoptotic effects of leptocarpin a plant-derived sesquiterpene lactone, onhuman cancer cell lines, Chem. Biol. Interact. 242 (2015) 415–421.

[49] K.Y. Chiu, C.C. Wu, C.H. Chia, S.L. Hsu, Y.M. Tzeng, Inhibition of growth,migration and invasion of human bladder cancer cells by antrocin, asesquiterpene lactone isolated from Antrodia cinnamomea, and its molecularmechanisms, Cancer Lett. 373 (2016) 174–184.

[50] K. Nakagawa-Goto, J.Y. Chen, Y.T. Cheng, W.L. Lee, M. Takeya, Y. Saito, K.H. Lee,L.F. Shyur, Novel sesquiterpene lactone analogues as potent anti-breast canceragents, Mol. Oncol. 10 (2016) 921–937.

[51] P. Koczurkiewicz, J. Czy _z, I. Podolak, K. Wójcik, A. Galanty, Z. Janeczko, M.Michalik, Multidirectional effects of triterpene saponins on cancer cells �mini-review of in vitro studies, Acta Biochim. Pol. 62 (2015) 383–393.

[52] A. Ashraf, R.A. Sarfraz, M.A. Rashid, M. Shahid, Antioxidant, antimicrobial,antitumor, and cytotoxic activities of an important medicinal plant (Euphorbiaroyleana) from Pakistan, J. Food Drug Anal. 23 (2015) 109–115.

[53] A. Ghasemi Pirbalouti, A. Siahpoosh, M. Setayesh, L. Craker, Antioxidantactivity, total phenolic and flavonoid contents of some medicinal and aromaticplants used as herbal teas and condiments in Iran, J. Med. Food 17 (2014) 1151–1157.

[54] I. Bajalan, M. Mohammadi, M. Alaei, A.G. Pirbalouti, Total phenolic andflavonoid contents and antioxidant activity of extracts from differentpopulations of lavandin, Ind. Crops Prod. 87 (2016) 255–260.

[55] N. Trabelsi, W. Megdiche, R. Ksouri, H. Falleh, S. Oueslati, S. Bourgou, H.Hajlaoui, C. Abdelly, Solvent effects on phenolic contents and biologicalactivities of the halophyte Limoniastrum monopetalum leaves, LWT-Food Sci.Technol. 43 (2010) 632–639.

[56] S.A. Kawashty, M.F. Abdalla, M.N. El Hadidit, N.A.M. Saleh, Thechemosystematics of egyptian Euphorbia species, Biochem. Syst. Ecol. 7(1990) 487–490.

[57] L. Tona, R.K. Cimanga, K. Mesia, C.T. Musuamba, T. de Bruyne, S. Apers, N.Hernans, S. van Miert, L. Pieters, J. Totte, In vitro antiplasmodial activity ofextracts and fractions from seven medicinal plants used in the DemocraticRepublic of Congo, J. Ethnopharmacol. 93 (2004) 27–32.

[58] J. Graßmann, Terpenoids as plant antioxidants, Vitam. Horm. 72 (2005) 505–535.

[59] R. Frazzi, M. Tigano, The multiple mechanisms of cell death triggered byResveratrol in Lymphoma and Leukemia, Int. J. Mol. Sci. 15 (2014) 4977–4993.

[60] S.H. Kim, C.W. Kim, S.Y. Jeon, R.E. Go, K.A. Hwang, K.C. Choi, Chemopreventiveand chemotherapeutic effects of genistein, a soy isoflavone, upon cancerdevelopment and progression in preclinical animal models, Lab. Anim. Res. 30(2014) 143.

[61] P.P. Sordillo, L. Helson, Curcumin and cancer stem cells: curcumin hasasymmetrical effects on cancer and normal stem cells, Anticancer Res. 35(2015) 599–614.

[62] P. Totta, F. Acconcia, S. Leone, I. Cardillo, M. Marino, Mechanisms of naringenin-induced apoptotic cascade in cancer cells: involvement of estrogen receptor aand ß signalling, IUBMB Life 56 (2004) 491–499.