Chemical characterization and cytotoxic activity evaluation of Lebanese propolis

Biomedicine & Pharmacotherapy 95 (2017) 298–307

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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Chemical characterization and cytotoxic activity evaluation of Lebanese propolis

MARK

Hiba Noureddinea,1, Rouba Hage-Sleimanb,1, Batoul Wehbic, A. Hussein Fayyad-Kazand, Salem Hayara, Mohamad Traboulssie, Osama A. Alyamanif, Wissam H. Faourg, Yolla ElMakhoura,⁎ a

Environmental Health Research Lab (EHRL), Faculty of Sciences V, Lebanese University, Nabatieh, Lebanon Department of Biology, Faculty of Sciences, Lebanese University, Hadath, Lebanon c Faculty of sciences, Lebanese University, Lebanon d Laboratory of Cancer Biology and Molecular Immunology, Faculty of Sciences I, Lebanese University, Hadath, Lebanon e British institute of homeopathy for Lebanon & Middle East, Lebanon f Toxicology Lab Research and Training Center, Faculty of Health Sciences, American University of Science & Technology (AUST), Beirut, Lebanon g School of Medicine, Lebanese American University (LAU), Byblos,, P.O. Box 36, Lebanon b

A R T I C L E I N F O

A B S T R A C T

Keywords: Lebanese propolis Apoptosis Cytotoxicity Plant extract

Chemical composition, anti-proliferative and proapoptotic activity as well as the effect of various fractions of Lebanese propolis on the cell cycle distribution were evaluated on Jurkat leukemic T-cells, glioblastoma U251 cells, and breast adenocarcinoma MDA-MB-231 cells using cytotoxic assays, flow cytometry as well as western blot analysis. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis revealed that ferulic acid, chrysin, pinocembrin, galangin are major constituents of the ethanolic crude extract of the Lebanese propolis, while the hexane fraction mostly contains chrysin, pinocembrin, galangin but at similar levels. Furthermore chemical analysis was performed using gas chromatography-mass spectrometry (GC–MS) to identify major compounds in the hexane fraction. Reduction of cell viability was observed in Jurkat cells exposed to the ethanolic crude extract and the hexane fraction, while viability of U251 and MDA-MB-231 cells was only affected upon exposure to the hexane fraction; the other fractions (aqueous phase, methylene chloride, and ethyl acetate) were without effect. Maximum toxic effect was obtained when Jurkat cells were cultivated with 90 μg/ml of both the crude extract and hexane faction. Toxicity started early after 24 h of incubation and remained till 72 h. Interestingly, the decrease in cell viability was accompanied by a significant increase in p53 protein expression levels and PARP cleavage. Cell cycle distribution showed an increase in the SubG0 fraction in Jurkat, U251 and MDA-MB-231 cells after 24 h incubation with the hexane fraction. This increase in SubG0 was further investigated in Jurkat cells by annexinV/PI and showed an increase in the percentage of cells in early and late apoptosis as well as necrosis. In conclusion, Lebanese propolis exhibited significant cytotoxicity and anti-proliferative activity promising enough that warrant further investigations on the molecular targets and mechanisms of action of Lebanese propolis.

1. Introduction Natural products exhibiting anticancer properties attracted considerable attention as potential adjunctive complementary interventions to conventional treatment methods [1–3]. First, natural products are widely available and accessible, not expensive, can be prepared as easy-to-administer types of dosage forms (e.g per os), and widely

consumed well-known popular natural products seems to be associated with low to moderate toxicity profile. However, the lack of strong clinical and experimental evidence is a major obstacle toward an established use of natural products as adjunct therapy to conventional regimens. Propolis is a glue-like resinous honey bee product collected by Apis mellifera from various plant sources [4]. A long time ago, propolis was used by human for embalming purposes to insulate and

Abbreviations: LC–MS/MS, Liquid chromatography-tandem mass spectrometry; GC–MS, Gas chromatography-mass spectrometry; PARP, Poly-ADP ribose polymerase; DAD, diode array detector; FACS, Fluorescence-activated cell sorting; EDTA, Ethylenediaminetetraacetic acid ⁎ Corresponding author at: Department of Biology, Faculty of Sciences Section V Lebanese University Nabatieh, Lebanon. E-mail address: [email protected] (Y. ElMakhour). 1 Authors contributed equally. http://dx.doi.org/10.1016/j.biopha.2017.08.067 Received 18 May 2017; Received in revised form 28 July 2017; Accepted 13 August 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.

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2.1.2. LC–MS/MS analysis LC–MS/MS analysis was performed on an API 4000™LC/MS/MS system. Two phenolic acids (caffeic acid and trans-ferulic acid) and nine flavonoids including (rutin, quercetin, daidzein, genistein, apigenin, kaempferol, chrysin, pinocembrin and galangin) were investigation. The chromatographic system consisted of a pump LC-20AD (Flow: 0.3 ml/min, Pressure range: 0–276 bar), an autosampler, a degasser and an automatic thermostatic column compartment. The injection volume was 25 ml. The LC was run on a reversed phase octadecylsilane C18column with standard mesures (250mḿ4 mm i.d., 5 mm particle diameter) and its temperature was maintained at 30 °C. The mobile phase was composed of solvent (A) 0.1% (v/v) formic acid in water, and solvent (B) acetonitrile, which were previously degassed and filtered. The solvent gradient started with 80% (A) and 20% (B), reaching 30% (B) at 10 min, 40% (B) at 40 min, 60% (B) at 60 min, 90% (B) at 80 min and followed by the return to the initial conditions. The freeze-dried extracts (10 mg of each) was dissolved in 1 ml of 80% of ethanol prior analysis. All samples were filtered through a 0.2 mm Nylon membrane (Whatman). The flow rate was 1 ml/min and split out 200 ml/min to MS. The MS used was an AB SCIEX triple quadrupole ion trap mass spectrometer MS equipped with an ESI source. Nitrogen above 99% purity was used and the gas pressure was 520 kPa (75 psi). The instrument was operated in negative and positive ion modes, with ESI needle voltage set at 5.00 kV and the ESI capillary temperature at 325 °C. Multiple Reaction Monitoring (MRM) were used for targeted quantification and screening. The MS data were simultaneously acquired for the selected precursor ion. The collision induced decomposition and MS experiments were performed using helium as the collision gas, with a collision energy of 25–40 eV. Compounds were characterized based on their mass spectra, using the precursor ion, fragment ions, and comparison of the fragmentation patterns with the eleven standards investigated and molecules described in the literature. Quantification was established based on spectra of the analytical standard solutions showing the precursor ion.

preserve cadavers and bodies as well as to prevent bacterial and fungal overgrowth and decomposition [5]. Additionally, propolis paste is included as an essential ingredient in various dosage forms such as tablets, capsules, toothpaste [6,7], solutions and mouthwash preparations [8,9], cream [10], and ointments [11]. More recently, a series of biological evaluations demonstrated that propolis exhibited several beneficial anti-inflammatory [12], antioxidant [13], cardioprotective [14], antimicrobial [15,16], antiviral [17,18], antiangiogenic [19], and importantly anticancer properties [20–22]. These various biological effects are mediated by a mixture of compounds found in the resin that can largely vary according to vegetation of the geographic areas [23–27]. To date, the major chemical compounds found in propolis are fatty acids, aliphatic and aromatic acids, flavonoids, alcohols, terpenes, sugars and various types of esters that include, but not limited to, Chrysin, apigenin, acacetin, galangin, kaempferol, kaempferid, quercetin, cinnanic acid, o-coumaric acid, m-coumaric acid, p-coumaric caffeic acid and caffeic acid phenylethyl ester (CAPE). However, the list of chemicals and/or the level of each constituent can largely differ between the types of propolis [28]. Therefore, identification and characterization of new chemical compounds with important biological properties e.g., anti-inflammatory and/or anticancer, still emerge due to the heterogeneity in propolis sources. Several studies documented the anticancer activity of propolis, collected from various geographic locations, in various cancer cell lines such as colon and prostate cancer cells [29,30], MCF-7 (human breast cancer cells) [22,31], HT-29 (human colon adenocarcinoma) [29,32], Caco-2 (human epithelial colorectal adenocarcinoma) [32], B16F1 (murine melanoma) [32–34], and human lung and cervical cancer cell lines [33,35,36]. Propolis induced cancer cell death is thought to be mediated through induction of apoptosis, mitochondrial dysfunction and cell cycle arrest [30–32,36]. In this study, we investigated whether Lebanese propolis extracts exhibit potential anti-proliferative activity. Our results indicated that Lebanese propolis exhibited important anti-proliferative activity, and the viability of Jurkat, U251, MDA-MB-231 cells was significantly reduced upon exposure to Lebanese propolis hexane fraction. Furthermore, the toxic effect of Lebanese propolis was mediated by apoptosis as shown by apoptotic PARP cleavage, p53 protein induction and Annexin V positive cells. As a result, the cytotoxic activity of Lebanese propolis in different cancer cell lines is promising to warrant further investigation of its cytotoxic activity.

2.1.3. Gas chromatography–mass spectrometry (GC–MS) analysis 2.1.3.1. Instruments. Gas chromatography-mass spectrometry was carried out on an Agilent GCMS, GC 7890 B MS in EI mode 5977 gas chromatograph with Auto-injector G4513A. The chromatographic column for the analysis was DB–5 ms Ultra Inert capillary column (30m × 250 μm i.d., 0.25 μm film thickness). The carrier gas used was high-purity helium (99.999%, UAE). The injection was performed in pulsed splitless mode (200 kPa until 0.5 min, Pressure 89.149 kPa), the injector temperature was maintained at 280 °C, the flow rate was of 1.2 ml/min for 16 min then 10 ml/min to 1.7 ml/min for 12 min, the purge flow was of 15 ml/min at 1 min. The injection volume was 1 μl. Samples were analyzed with the column held initially at 100 °C for 1 min and then increased to 300 °C with a 15 °C/min heating ramp for 14.3333 min. Run time was of 28.666 min. The interface temperature was 280 °C. Full-scan EI (Electron Impact) spectra were recorded from 30- 550 m/z (Mass/charge) with 2 scans per second. Peaks were identified by computer searches in reference libraries.

2. Materials and methods 2.1. Chemical evaluation 2.1.1. Preparation of the ethanolic crude extract and different fractions of Lebanese propolis 43 g of crude greenish powered propolis collected from the South of Lebanon were extracted with 50 ml of 70% ethanol by maceration for a week in a shaker regulated at a speed of 100 rpm and temperature of 30 °C. The insoluble portion was then separated by filtration and the filtrate was again extracted by maceration for a week in the same conditions. After the second filtration, the filtrate was kept in a freezer at −18 °C overnight and filtered again at this temperature to reduce the wax content of the extract. Part of the obtained extract was used as the crude extract. The other part of extract obtained was then used for a serial liquid–liquid fractionation process using 3 successive organic solvents from the less polar one to the more polar one: hexane, methylene chloride and ethyl acetate. Each fraction obtained was filtered, and concentrated at reduced pressure with a rotary evaporator at 40 °C. The crude extract and the aqueous phase were dried by lyophilization. Dry extracts of propolis were obtained and weighed: ethanolic crude extract (m = 18.5734 g), hexane (m = 1.5482 g); methylene Chloride (m = 0.5521 g), ethyl acetate (m = 0.4510 g) and aqueous phase (m = 5.761 g).

2.1.3.2. Sample preparation for injection. 3 mg of dry hexane propolis extract was diluted in 3 ml Ethyl Acetate then centrifuged for 10 min at 13k RPM. For underivatized free sample, 1 μl of the sample was directly injected into the GC–MS chromatograph. For derivatization, 100 μl of the diluted sample was evaporated under N2 Stream without heating, then reconstituted in 1000 μl of bis-(trimethyl-silyl) trifluoroacetamide (BSTFA) and incubated for 30 min at 80 °C. 1 μl of this derivatized sample was then injected into the GC–MS chromatograph. 2.2. Biological evaluation 2.2.1. Cell lines and culture conditions Human Jurkat leukemic T cells, human glioblastoma U251 cells, 299

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ab1101), and anti-GAPDH (Santa Cruz, 47724). Protein bands on the membrane were visualized by an enhanced chemiluminescence detection system (ECL) according to the manufacturer's instructions.

and breast adenocarcinoma MDA-MB-231 cells were grown in T75 cell culture flasks (Corning Canted Neck) containing RPMI 1640 medium for Jurkat cells and DMEM medium for MDA-MB-231 and U251 cell lines, both supplemented with 10% fetal calf serum (Gibco), 1% penicillin (100 IU/mL) and streptomycin (10 μg/ml) (Gibco). All cells were incubated in a humidified incubator at 37 °C and 5% CO2.

2.3. Statistics Results are expressed as mean ± standard error of mean (SEM). Statistical significance was determined using Student’s t-test (Erithacussoftware, UK). Differences were considered significant when P value < 0.05.

2.2.2. Cytotoxicity test The viability of Jurkat, MDA-MB-231 and U251 cells was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyl tetrazoliumbromide) assay. MTT is reduced intracellularly in a mitochondrion-dependent reaction to yield insoluble formazan cristals. The ability of cells to reduce MTT indicates mitochondrial activity and serves as a measure of cell viability. Briefly, 20 000 cells were seeded in 96-well plates and treated with 30, 60, or 90 μg/ml of each of the propolis fraction for 24, 48 or 72 h. Every 24 h, 10 μl of MTT solution (Sigma) was added to each well of each plate. After 3 h of incubation at 37 °C, formazan crystals were solubilized with 100 μl of acidified isobutanol. The absorbance was measured spectrophotometrically with an ELISA microplate reader (ELISA reader/Biotech) at 595 nm wavelengths. The number of viable cells was directly correlated to the amount of purple formazan crystals formed.

3. Results 3.1. Determination of Lebanese propolis chemical composition LC–MS/MS analysis of the crude ethanol extract revealed the presence of ferulic acid (1.184 mg/g), and low amount of caffeic acid (0.142 mg/g). While chrysin (5.699.463 mg/g), pinocembrin (2.688 mg/g) and galangin (4.064 mg/g) were the principal compound among the tested flavonoids. Of note, low but detectable levels were measured for the following flavonoids: rutin (0.206 mg/g), quercetin (0.474 mg/g), apigenin (0.176 mg/g), kaempferol (0.571 mg/g). However, the levels of daidzein and genistein were insignificant. The results of the hexane extract only revealed the presence of chrysin, pinocembrin and galangin but relatively in high amount (Table 1), while low levels of rutin (0.072 mg/g), quercetin (0.055 mg/g) and kaempferol (0.714 mg/g) were also detected. The levels caffeic acid, ferulic acid, daidzein, genistein and apigenin were insignificant. The hexane fraction was further studied by GC–MS in order to identify the different types of organic compounds. The dominant compounds determined from the peak percentage areas were as follow for the derivatized compounds (Supplemental Table 1): benzoic acid (2.10%); hydrocinnamic acid (1.48%); alpha curcumene (8.47%); cynnamic acid (10.67%); beta.H, 5.alpha-Eremophil-1(10)-ene, 11(trimethylsiloxy)-/8.427 (25.52%); cynnamic acid, P-methoxy-, trimethylsilyl ester/9.430 (2.25%); 3-methyl-3-butenyl isoferulate (3.04%); 3-methyl-2-butenyl isoferulate-TMS-derivative/12.443 (1.95%); pinostrobin chalcone (8.52%); styracin (15.09%); abietic acid, trimethylsilyl ester (2.68%); 3-hydroxyanthrannilic acid, methyl ester, di(trimethylsilyl)-/13.950 (6.94%); tectochrysin (6.49%). As for the non-derivatized compounds (supplemental Table 2 Table 2), the dominant compounds were cinnamic acid (1.6%); cinnamyl acetate (1.01), alpha-cucrcumene (10.68%); beta-bisabolene (1.48%); guaiol (8.95%); gamma-eudesmol (4.67%); alpha-eudesmol (13.73%); bulnesol (7.32%); alpha-bisabolol (6.18%); ferulic acid (3.28%); pinostrobin chalcone (25.57%); pinocembrin, galangin flavanone (2.96%); tectochrysin (7.43%); vitamin E (1.3%); 2-phenylethyl laurate (1.18%).

2.2.3. Analysis of cell cycle distribution by flow cytometry Cells were incubated with 30, 60 or 90 μg/ml of the hexane propolis fraction for 24 h. Treated and untreated cells were then collected and incubated 1 h at 4 °C with propidium iodide (0.05 mg/ml) solution containing Nonidet-P40 (0.05%). Cells were analyzed using BD Fluorescence-activated cell sorting (FACS) Aria SORP cell sorter (BD, Bioscience Europe) and cell cycle distribution was determined using FACS DIVA Software. 2.2.4. Apoptosis assay by AnnexinV/PI staining Jurkat cells (500, 000) were seeded in T25 flask. The next day, cells were treated with the vehicle, 30, 60 or 90 μg/ml of crude extract or hexane propolis fraction for 5, 24 or 48 h. At each time point cells were collected by centrifugation at 1500 rpm for 5 min. Then using AnnexinV-FLUOS Staining Kit from Roche, pellets were incubated with 400 μl of incubation buffer (HEPES buffer) and 2 μl of Annexin-V-FITC labeling agent and 2 μl propidium iodide solution (PI), and then kept for 15min at room temperature in the dark. FITC fluorescence (Fl-1) and PI (Fl-2) fluorescence were read by BD Fluorescence-activated cell sorting (FACS) Aria SORP cell sorter (BD, Bioscience Europe). The results of 10,000 events were analyzed and the percentages of populations were determined using FACS DIVA Software. Alive cells are negatively stained for both Annexin V and PI. Early apoptotic cells are Annexin V positive and PI negative. Late apoptotic are both Annexin V and PI positive. Necrotic cells are Annexin V negative and PI positive. 2.2.5. Western blot analysis Jurkat cells (2 × 105) were seeded in T25 flasks and incubated with 10, 30, 45, 60 or 90 μg/ml of the propolis fraction for 24, 48 or 72 h. Then, the cells were washed, collected and lysed in RIPA lysis buffer [1% nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate (pH 7.2), 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate and 100 units/ml aprotinin]. After incubation of the lysate for 30 min on ice, the cell lysates were centrifuged at 1200 rpm for 5 min and the supernatant was collected and used or immediately stored at −20 °C. Protein concentration of total cell lysate was measured by the Bradford assay and a total of 50 μg were subjected to 10% SDS-polyacrylamide gels electrophoresis and the proteins were then transferred onto nitrocellulose membrane. After blocking the transfer with a blocking buffer (1.5 mM Tris–HCl (pH 8), 5 mM NaCl, 0.1% Tween 20, 5% non-fat dry milk) for 1 h at room temperature, the blots were incubated overnight at 4° C with primary antibodies then with HRP conjugated secondary antibodies. The antibodies used: anti-PARP (Cell signaling, 9542), anti-p53 (Abcam,

Table 1 Content of phenolic compounds and flavonoids of CE and hexane fraction from propolis using their spectral characteristic in positive ion mode in LC–MS/MS. Compounds identified in the Propolis Extracts by negative mode LC–MS/MS

300

Crude Ethanol

Hexane

Compounds

RT (min)

Quantity (mg/g)

RT (min)

Quantity (mg/g)

Caffeic acid Rutin Ferulic acid Quercetin Daidzein Genistein Apigenin Keampferol Chrysin Pinocembrin Galangin

4.79 4.85 5.24 5.69 5.83 6.16 6.18 6.24 7.09 7.10 7.18

0.142 0.206 1.184 0.474 n.d. 0.015 0.176 0.571 5.696 2.688 4.064

4.76 4.81 5.16 5.82 5.68 6.16 6.18 6.23 7.09 7.11 7.18

n.d. 0.072 n.d. 0.055 n.d. n.d. n.d. 0.714 3.480 3.112 3.080

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percentage of cells in the SubG0 phase of the cell cycle. The percentage of Jurkat cells in SubG0 increased significantly after 24 h of incubation with 60 μg/ml of both the crude extract and hexane fraction (Fig. 3A). Significant increase in the percentage of cells in the SubG0 phase was also observed in both U251 (Fig. 3B) and MDA-MB-231 (Fig. 3C) after 24 h exposure to the hexane fraction.

Table 2 IC50 values for the crude ethanol extract and hexane fraction of Lebanese propolis in 3 different cell lines as measured by MTT Assay. Cell line

Disease

IC50- Hexane fraction (μg/ ml) at 48 h

IC50- Hexane fraction (μg/ ml) at 72 h

IC50- Ethanol crude extract (μg/ml) at 72 h

Jurkat

Acute T Cell Leukemia Human Glioblastoma Breast Cancer

51

44

72

77

63

–

75

61

–

U251 MDAMB231

3.3. PARP cleavage, apoptosis and p53 expression upon exposure to Lebanese propolis Since PARP cleavage is a common phenomenon that occurs during apoptosis, it was studied by western blot on Jurkat, U251 and MDA-MB231 cells upon exposure to different concentrations of Lebanese propolis. PARP cleavage was observed early at 24 h post-exposure to 90 μg/ml of both the crude and hexane fractions in Jurkat cells and 90 μg/ml of hexane fraction for U251 and MDA-MB-231 cells (Fig. 4). Interestingly, Annexin V/PI staining showed that in Jurkat cells treated with crude extract, apoptosis started early after 5 h post-exposure to 90 μg/ml of the crude extract. The proportions of early apoptotic, late apoptotic and necrotic cells significantly increased after 24 h of stimulation reaching highly significant values at 48 h (Fig. 5A). Similar results were obtained when Jurkat cells were incubated with the hexane fraction (Fig. 5B). Furthermore, incubation of Jurkat cells with the crude extract (90 μg/ml) and hexane fraction resulted in significant increase in the expression level of p53 protein that started after 24 h of stimulation (for 60 and 90 μg/ml) and continued after 48 h (for 30, 60, and 90 μg/ml) (Fig. 6). All these results show that the cytotoxic effect of Lebanese propolis on Jurkat cells acts by triggering an apoptotic response mediated by p53.

3.2. Cytotoxic effect of Lebanese propolis on different cancer cells The effects of various fractions of Lebanese propolis were evaluated on Jurkat leukemic T cells, glioblastoma U251 cells, and breast adenocarcinoma MDA-MB-231 cells using MTT cytotoxicity assay. The IC50 of hexane fractions in U251 (77 and 63 μg/ml) and MDA-MB-231 (75 and 61 μg/ml) cells were higher than in Jurkat (51 and 44 μg/ml) cells after 48 and 72 h, respectively (Table 2). In order to cover a range of values from less to more than the IC50, the three concentrations 30, 60, and 90 μg/ml were chosen for the subsequent experiments. Cell viability was reduced in Jurkat cells in a dose and time-dependent manner after exposure to 30, 60, and 90 μg/ml of ethanolic crude extract and hexane fraction of Lebanese Propolis (Fig. 1A and B). The decrease in cell viability was highly significant after 72 h of exposure to 90 μg/ml of the crude extract or hexane fraction but more prominent effect was observed for the hexane fraction with an IC50 of 44 μg/ml as compared to 72 μg/ml for the crude extract (Table 2). The viability of U251 and MDA-MB-231 cells was only affected upon exposure to the hexane fraction in a dose and time-dependent manner after 48 and 72 h (Fig. 1C and D). The aqueous phase, methylene chloride, and ethyl acetate fractions showed no significant effect on the Jurkat cells unlike the wax fraction that showed an effect but dose-independent (Fig. 2). None of the above fractions (aqueous phase, methylene chloride, ethyl acetate, and wax) was effective on U251 and MDA-MB-231 (data not shown). The anti-proliferative and cytotoxic effect exerted by the crude extract and hexane fraction of Lebanese propolis is by triggering cell death rather than cell cycle arrest as observed by the increase in the

4. Discussion The incidence of breast cancer in Lebanon is steadily increasing since 1998 reaching alarming rates and also found in high proportions in relatively young women patients [37]. Also, breast cancer projection in females 2020 reached 40.4% from all cancers and ranked first among all cancers [38]. Moreover, glioblastoma brain tumors are more common in males [39]. Finally, acute lymphoblastic leukemia is the most common malignancy diagnosed in children [40]. All these evidences empowered us to test the cytotoxic effect of Lebanese propolis first on these three cell lines. Natural remedies have been proven to be Fig. 1. Effect of crude extract and hexane fraction of Lebanese propolis on the viability of cancer cells. The percentage of viable cells was determined by measuring the optical density of solubilized formazan crystals in treated cells as compared to the control. (A) Jurkat cells incubated with the vehicle, 30, 60 and 90 μg/ml of ethanolic crude extract (CE) for 24 h, 48 h or 72 h (B) Jurkat cells incubated with the vehicle, 30, 60 and 90 μg/ml of hexane fraction (HF) for 24 h, 48 h or 72 h (C) U251 cells incubated with the vehicle, 30, 60 and 90 μg/ml of hexane fraction for 24 h, 48 h or 72 h (D) MDA-MB231 cells incubated with the vehicle, 30, 60 and 90 μg/ml of hexane fraction for 24 h, 48 h or 72 h. Results presented are mean ± SEM of three independent experiments, each performed in triplicate. *p < 0.05, **p < 0.005 and ***p < 0.0001 represent the significant differences from the control.

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Fig. 1. (continued)

three different types of cancer cells. In this current study on Lebanese propolis, our results were consistent with previous studies done with other propolis on several other cancer cell lines using both in vivo and in vitro cancer cell models [42–44]. Importantly, Benguedouar et al. showed that Algerian propolis significantly reduces invasiveness in in vivo melanoma mouse model and contains high amount of galangin. Galangin significantly reduces the number of melanoma cells in vitro [42]. Therefore, in the above-mentioned study the cytotoxic effect of propolis is significant in both in vitro and in vivo skin cancer models. Interestingly, galangin is abundantly found in Lebanese propolis which validates its cytotoxic activity on cancer cells and suggests a potential in vivo anti-cancer effect. Furthermore, the cytotoxic doses of our extract used in the current study are considered similar to the doses normally ingested by a person and thus documents their safety on normal cells. Given that the chemical mixture of propolis varies according to the geographic location, the chemical compositions of several fractions of the Lebanese propolis were determined. Noteworthy, the cytotoxic effect was only observed in the ethanolic crude extract and the hexane

an important source of chemicals that can serve as precursors or candidate anticancer agents [1,3]. Moreover, deleterious side effects associated with anticancer pharmacotherapy are major challenges facing today’s medicine [41]. For the time being, adjunct therapies that may reduce the adverse events of cancer chemotherapy are gaining importance. Ethanolic and aqueous propolis extracts have been used in folk medicine longtime ago. With the exception of few documented cases of dermatitis associated with propolis ointments, the safety profile of other propolis dosage forms is well established [24]. In this study, we demonstrated that Lebanese propolis inhibited cell proliferation and induced cell death in several cancer cell lines including Jurkat, U251, and MDA-MB-231 cells. Furthermore, the cell death observed was mostly due to apoptosis as shown in Jurkat cells. PARP cleavage, increased p53 protein expression and AnnexinV positive cells are features of apoptosis. Interestingly, the cytotoxic doses were relatively low and the anti-proliferative effect appeared at a concentration as low as 30 μg/ml of the extract that reached a prominent effect, at a concentration of 90 μg/ml. In addition, the cytotoxic effect was effective on

Fig. 2. Effect of aqueous, methylene chloride, ethyl acetate (EA) and wax fractions on the viability of Jurkat cells. The percentage of viable cells was determined by measuring the optical density of solubilized formazan crystals in treated cells as compared to the control. Jurkat cells were incubated with the vehicle, 30, 60 and 90 μg/ml of each fraction for 24 h, 48 h or 72 h (A, B). Results presented are mean ± SEM of three independent experiments, each performed in triplicate. *p < 0.05 represents the significant difference from the control.

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Fig. 3. Effect of Lebanese propolis on the cell cycle distribution of cancer cells. The percentage of cells was determined after incubation with propidium iodide. (A) Jurkat cells incubated with the vehicle, 30, 60 and 90 μg/ml of ethanolic crude extract and hexane fraction for 24 h (B) U251 cells incubated with the vehicle, 30, 60 and 90 μg/ml of hexane fraction for 24 h (C) MDA-MB-231 cells incubated with the vehicle, 30, 60 and 90 μg/ml of hexane fraction for 24 h. Results presented are mean ± SEM of three independent experiments. *p < 0.05, **p < 0.005 and ***p < 0.0001 represent the significant differences from the control.

Fig. 4. Induction of PARP cleavage in cancer cells. PARP cleavage was assessed by western blot using an antibody that can detect both the uncleaved PARP (116 kDa) and cleaved PARP (89 kDa). Jurkat cells were incubated with vehicle, 5, 10, 30, 60 and 90 μg/ml of hexane fraction, or with 90 μg/ml of crude extract for 24 h and 48 h. U251 and MDA-MB-231 cells were incubated with vehicle, 5, 10, 30, 60 and 90 μg/ml of hexane fraction for 24 h and 48 h. Expression levels were normalized to GAPDH protein content. This figure is representative of three independent experiments.

differentiation into osteoblasts [51]. Furthermore, galangin effect potentiated cancer cells cytotoxicity by activating adenosine monophosphate kinase (AMPK), an enzyme linked to energy homeostasis [52]. Finally, galangin was found to synergistically act with berberine to inhibit esophageal carcinoma cells by reducing the expression of WNT and β-catenin. The latter effect is significantly enhanced in the presence of ROS scavenger [53]. Interestingly, Li et al. recently showed that galangin suppressed the proliferation and induced autophagy in hepatocellular carcinoma cells [54]. Since Lebanese propolis contains significant amount of galangin, then, it can be assumed that the cytotoxic effect of Lebanese propolis can be mediated through at least one of the above mentioned signaling mechanisms in parallel to apoptotic pathway. Chrysin, another major constituent of Lebanese propolis, induced apoptosis and cytotoxic effect by targeting hexokinase-2 [55] or inducing reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress [56]. Furthermore, chrysin is shown to possess prominent anti-inflammatory and immunomodulatory effects which were recently reviewed by Zeinali et al. [57]. Finally, ferulic acid is shown to modulate several molecular pathways involved in a multitude of biological

fraction, while the other fractions including aqueous, methylene chloride and ethyl acetate didn’t show any significant effect. Similarly, the ethanolic extract fractions from Lebanese propolis and propolis from other geographic area (such as Thailand and Turkey) were shown to be associated with the most promising anticancer and cytotoxic effect [42–44]. Importantly, recent studies showed a wide range of biological activities of propolis ethanolic extracts, including anti-inflammatory activity of polyphenol rich fractions in Brazil and Argentina [45,46], antiparasitic and bacterial activities in Russia, Nigeria and Libya [47–49] as well as antioxidant properties in Greece [50]. Strong body of evidence suggests that bees’ hives contain antibacterial and antifungal activities; therefore we suggest that Lebanese propolis might also contain promising antibacterial compounds. In our study, the chemical analysis revealed that galangin, chrysin, pinocembrin and ferulic acid are abundantly found in Lebanese propolis. Galangin was found to mediate anticancer activity through multiple signaling pathways. Recently, Liu et al. showed that galangin inhibited osteosarcoma cells proliferation through induction of transforming growth factor-beta (TGF-β) release which led to cancer cells 303

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Fig. 5. Induction of apoptosis in response to crude extract and hexane fraction of Lebanese propolis. Annexin V-FITC/PI flow cytometry analysis was done on Jurkat cells following treatment with 30, 60 and 90 μg/ ml of (A) crude extract and (B) hexane fraction for 5, 24 and 48 h. Living cells are negative for both Annexin V and PI staining; Early apoptotic cells are positive for Annexin V and negative for PI staining; Late apoptotic cells are positive for both Annexin V and PI staining; Necrotic cells are negative for Annexin V and positive for PI staining. Results presented are mean ± SEM of two independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 represent the significant differences from the control.

biological effects through multiple intracellular signaling pathways. LC–MS/MS analysis using specific selected standards showed that kaemferol, pincembrin and galangin exist in both ethanolic crude extract and the hexane fraction but at different proportion but interestingly, ferulic and caffeic acid were only detected in the crude extract. Additionally, quercetin and its phenolic derivative rutin are found but at low levels, the latter compounds are known for their potential anticancer properties [61–63]. Whilst various biological activities were attributed to ferulic acid [64,65], its distinct anti-proliferative effect recently appeared in the literature [65,66], and most of the previously

functions. Accordingly, ferulic acid suppressed indoleamine 2, 3-dioxygenase (IDO) expression by inhibiting nuclear factor kappa B (NF-қB) translocation and p38 phosphorylation in LPS-activated microglial cells [58]. Also, by downregulating the expression of ATP-binding cassette transporter ABAC1, ferulic acid reversed the multiple drug resistance against paclitaxel [59]. Finally, ferulic acid induced-regulation of epithelial-to-mesenchymal transition inhibited metastasis in MDA-MB-231 breast cancer cells [60]. Therefore, the abundant presence of at least the above mentioned compounds makes Lebanese propolis a prominent source of anti-inflammatory and anticancer agents that exert their 304

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Fig. 6. Expression level of p53 in response to crude extract and hexane fraction of Lebanese propolis. Expression level was assessed by western blot on Jurkat cells incubated with vehicle, 5, 10, 30, 60 and 90 μg/ml of hexane fraction, or with 90 μg/ml of crude extract for 24 h and 48 h. Expression levels were normalized to GAPDH protein content. This figure is representative of three independent experiments.

the hexane faction. While other fractions seem to be devoid of any cytotoxic effect, it is also important to assess their chemical composition since they might contain chemical entities involved in protecting normal cells from the potential toxic effect of conventional chemotherapeutic agents.

published data showed that ferulic acid produced its anticancer effect by acting in a concerted manner to increase the sensitivity of cancer cells to the parent anticancer compounds [59,67–69]. Importantly, in these published studies the anti-proliferative effect of ferulic and rutin was only observed at extremely high doses that ranged between 100 μM to 1000 μM, while in the current study we exposed the cells to concentrations of ferulic acid and rutin in the nanomolar range. Although the concentration of ferulic acid in the hexane fraction is relatively low, it seems that the presence of kaempferol or galangin rendered very low doses of ferulic acid effective in inhibiting cancer cells proliferation. Similarly, galangin was shown to enhance cancer cells sensitivity to anticancer agents or distinctly induce cancer cell death [52,53,70]. However, the dose of galangin used in these studies was in the micromolar range, while in our study galangin amount was significantly lower within the nanomolar range. These data suggest that the Lebanese propolis in addition to having similar composition to propolis found in other geographic areas, the chemical composition and the proportion of each constituent is unique. Knowing that in the hexane fraction only 3 major constituents were found including it is of considerable importance to observe that cancer cells exposed to a combination of these compounds and at very low concentration showed significant reduction in cell survival and/or increased cell death. GC–MS analysis revealed the detailed chemical composition of the hexane fraction. In fact, the Lebanese propolis chemical composition is relatively distinct when compared to the chemical composition of Brazilian, Saudi and Algerian propolis [71–74]. Most of the compounds are commonly found in all propolis but at different concentrations. However, alpha-curcumene and eremophila derivatives were only found in the Lebanese propolis and at very high proportions. To date the potential anticancer and anti-proliferative activities of alpha-curcumene are still uncovered, but plant extracts containing curcumene at high levels were shown to be toxic on malaria, chikungunya and St. Louis encephalitis mosquito vectors [75]. Interestingly, eremophila compounds were shown to have promising anticancer properties [76]. Lebanese propolis-induced cell death seems to be mostly mediated through induction of early apoptosis. Although, the latter finding is not unique to Lebanese propolis and similar data was found with propolis collected from various geographic locations [22,28,31,32,53], anticancer compounds found in the Lebanese propolis can have modified chemical structures. Therefore, identification of these parent compounds and their specific anticancer synergistic effect at very low concentrations will provide a rational for the potential use of Lebanese propolis as an adjunctive anticancer therapy.

Funding This study was supported by grant from the Research Program of the Lebanese University, Grant number: 4/5740. Competing of interest The authors declare having no financial competing interests. References [1] R. Duffy, C. Wade, R. Chang, Discovery of anticancer drugs from antimalarial natural products: a MEDLINE literature review, Drug Discov. Today 17 (17–18) (2012) 942–953. [2] B. Orlikova, N. Legrand, J. Panning, M. Dicato, M. Diederich, Anti-inflammatory and anticancer drugs from nature, Cancer Treat. Res. 159 (2014) 123–143. [3] V. Srivastava, A.S. Negi, J.K. Kumar, M.M. Gupta, S.P. Khanuja, Plant-based anticancer molecules: a chemical and biological profile of some important leads, Bioorg. Med. Chem. 13 (21) (2005) 5892–5908. [4] M. Hanssen, The Healing Power of Pollen and Other Products from the Beehive Propolis Royal Jelly, Honey, Thorsons, Wellingborough, 1979. [5] S. Castaldo, F. Capasso, Propolis, an old remedy used in modern medicine, Fitoterapia 73 (Suppl. 1) (2002) S1–S6. [6] T. Morawiec, A. Dziedzic, I. Niedzielska, A. Mertas, M. Tanasiewicz, D. Skaba, J. Kasperski, A. Machorowska-Pieniazek, M. Kucharzewski, K. Szaniawska, W. Wieckiewicz, M. Wieckiewicz, The biological activity of propolis-containing toothpaste on oral health environment in patients who underwent implant-supported prosthodontic rehabilitation, Evid.-Based Complement. Altern. Med.: eCAM 2013 (2013) 704947. [7] D. Skaba, T. Morawiec, M. Tanasiewicz, A. Mertas, E. Bobela, E. Szliszka, M. Skucha-Nowak, M. Dawiec, R. Yamamoto, S. Ishiai, Y. Makita, M. Redzynia, B. Janoszka, I. Niedzielska, W. Krol, Influence of the toothpaste with brazilian ethanol extract propolis on the oral cavity health, Evid.-Based Complement. Altern. Med.: eCAM 2013 (2013) 215391. [8] H. Koo, J.A. Cury, P.L. Rosalen, G.M. Ambrosano, M. Ikegaki, Y.K. Park, Effect of a mouthrinse containing selected propolis on 3-day dental plaque accumulation and polysaccharide formation, Caries Res. 36 (6) (2002) 445–448. [9] N. Malhotra, S.P. Rao, S. Acharya, B. Vasudev, Comparative in vitro evaluation of efficacy of mouthrinses against Streptococcus mutans, Lactobacilli and Candida albicans, Oral Health Prev. Dent. 9 (3) (2011) 261–268. [10] S.R. Gregory, N. Piccolo, M.T. Piccolo, M.S. Piccolo, J.P. Heggers, Comparison of propolis skin cream to silver sulfadiazine: a naturopathic alternative to antibiotics in treatment of minor burns, J. Alter. Complement. Med. 8 (1) (2002) 77–83. [11] M. Kucharzewski, M. Kozka, T. Urbanek, Topical treatment of nonhealing venous leg ulcer with propolis ointment, Evid.-Based Complement. Altern. Med.: eCAM 2013 (2013) 254017. [12] N. Paulino, S.R. Abreu, Y. Uto, D. Koyama, H. Nagasawa, H. Hori, V.M. Dirsch, A.M. Vollmar, A. Scremin, W.A. Bretz, Anti-inflammatory effects of a bioavailable compound, Artepillin C, in Brazilian propolis, Eur. J. Pharmacol. 587 (1–3) (2008) 296–301. [13] A.A. Lopes, T.S. Ferreira, R.T. Nesi, M. Lanzetti, K.M. Pires, A.M. Silva, R.M. Borges, A.J. Silva, S.S. Valenca, L.C. Porto, Antioxidant action of propolis on mouse lungs

5. Conclusion In conclusion, Lebanese propolis expectedly showed promising cytotoxic activity that was restricted to the ethanolic crude extract and 305

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H. Noureddine et al.

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41] A. Carlotto, V.L. Hogsett, E.M. Maiorini, J.G. Razulis, S.T. Sonis, The economic burden of toxicities associated with cancer treatment: review of the literature and analysis of nausea and vomiting, diarrhoea, oral mucositis and fatigue, PharmacoEcon 31 (9) (2013) 753–766. [42] L. Benguedouar, M. Lahouel, S.C. Gangloff, A. Durlach, F. Grange, P. Bernard, F. Antonicelli, Ethanolic extract of algerian propolis and galangin decreased murine melanoma T, Anticancer Agents Med. Chem. 16 (9) (2016) 1172–1183. [43] S. Demir, Y. Aliyazicioglu, I. Turan, S. Misir, A. Mentese, S.O. Yaman, K. Akbulut, K. Kilinc, O. Deger, Antiproliferative and proapoptotic activity of Turkish propolis on human lung cancer cell line, Nutr. Cancer 68 (1) (2016) 165–172. [44] S. Khacha-Ananda, K. Tragoolpua, P. Chantawannakul, Y. Tragoolpua, Propolis extracts from the northern region of Thailand suppress cancer cell growth through induction of apoptosis pathways, Invest. New Drugs 34 (6) (2016) 707–722. [45] B. Bueno-Silva, M. Franchin, C.F. Alves, C. Denny, D.F. Colon, T.M. Cunha, S.M. Alencar, M.H. Napimoga, P.L. Rosalen, Main pathways of action of Brazilian red propolis on the modulation of neutrophils migration in the inflammatory process, Phytomedicine 23 (13) (2016) 1583–1590. [46] A.L. Salas, M.R. Alberto, I.C. Zampini, A.S. Cuello, L. Maldonado, J.L. Rios, G. Schmeda-Hirschmann, M.I. Isla, Biological activities of polyphenols-enriched propolis from Argentina arid regions, Phytomedicine 23 (1) (2016) 27–31. [47] A. Ambi, J. Bryan, K. Borbon, D. Centeno, T. Liu, T.P. Chen, T. Cattabiani, C. Traba, Are Russian propolis ethanol extracts the future for the prevention of medical and biomedical implant contaminations? Phytomedicine 30 (2017) 50–58. [48] R. Omar, J.O. Igoli, T. Zhang, A.I. Gray, G.U. Ebiloma, C.J. Clements, J. Fearnley, R. Edrada Ebel, T. Paget, H.P. de Koning, D.G. Watson, The chemical characterization of nigerian propolis samples and their activity against trypanosoma brucei, Sci. Rep. 7 (1) (2017) 923. [49] W. Siheri, T. Zhang, G.U. Ebiloma, M. Biddau, N. Woods, M.Y. Hussain, C.J. Clements, J. Fearnley, R.E. Ebel, T. Paget, S. Muller, K.C. Carter, V.A. Ferro, H.P. De Koning, D.G. Watson, Chemical and antimicrobial profiling of propolis from different regions within Libya, PLoS One 11 (5) (2016) e0155355. [50] K.M. Kasiotis, P. Anastasiadou, A. Papadopoulos, K. Machera, Revisiting greek propolis: chromatographic analysis and antioxidant activity study, PLoS One 12 (1) (2017) e0170077. [51] C. Liu, M. Ma, J. Zhang, S. Gui, X. Zhang, S. Xue, Galangin inhibits human osteosarcoma cells growth by inducing transforming growth factor-beta1-dependent osteogenic differentiation, Biomed. Pharmacother. 89 (2017) 1415–1421. [52] W. Song, C.Y. Yan, Q.Q. Zhou, L.L. Zhen, Galangin potentiates human breast cancer to apoptosis induced by TRAIL through activating AMPK, Biomed. Pharmacother. 89 (2017) 845–856. [53] K. Ren, W. Zhang, G. Wu, J. Ren, H. Lu, Z. Li, X. Han, Synergistic anti-cancer effects of galangin and berberine through apoptosis induction and proliferation inhibition in oesophageal carcinoma cells, Biomed. Pharmacother. 84 (2016) 1748–1759. [54] X. Li, Y. Wang, Y. Xiong, J. Wu, H. Ding, X. Chen, L. Lan, H. Zhang, Galangin induces autophagy via deacetylation of LC3 by SIRT1 in HepG2Cells, Sci. Rep. 6 (2016) 30496. [55] D. Xu, J. Jin, H. Yu, Z. Zhao, D. Ma, C. Zhang, H. Jiang, Chrysin inhibited tumor glycolysis and induced apoptosis in hepatocellular carcinoma by targeting hexokinase-2, J. Exp. Clin. Cancer Res. 36 (1) (2017) 44. [56] S. Ryu, W. Lim, F.W. Bazer, G. Song, Chrysin induces death of prostate cancer cells by inducing ROS and ER stress, J. Cell. Physiol. (2017). [57] M. Zeinali, S.A. Rezaee, H. Hosseinzadeh, An overview on immunoregulatory and anti-inflammatory properties of chrysin and flavonoids substances, Biomed. Pharmacother. 92 (2017) 998–1009. [58] M. Koshiguchi, H. Komazaki, S. Hirai, Y. Egashira, Ferulic acid suppresses expression of tryptophan metabolic key enzyme indoleamine 2, 3-dioxygenase via NFkappaB and p38 MAPK in lipopolysaccharide-stimulated microglial cells, Biosci. Biotechnol. Biochem. 81 (5) (2017) 966–971. [59] G. Muthusamy, A. Balupillai, K. Ramasamy, M. Shanmugam, S. Gunaseelan, B. Mary, N.R. Prasad, Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines, Eur. J. Pharmacol. 786 (2016) 194–203. [60] X. Zhang, D. Lin, R. Jiang, H. Li, J. Wan, H. Li, Ferulic acid exerts antitumor activity and inhibits metastasis in breast cancer cells by regulating epithelial to mesenchymal transition, Oncol. Rep. 36 (1) (2016) 271–278. [61] A.J. Alonso-Castro, F. Dominguez, A. Garcia-Carranca, Rutin exerts antitumor effects on nude mice bearing SW480 tumor, Arch. Med. Res. 44 (5) (2013) 346–351. [62] J.R. Araujo, P. Goncalves, F. Martel, Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines, Nutr. Res. 31 (2) (2011) 77–87. [63] J.P. Lin, J.S. Yang, J.J. Lin, K.C. Lai, H.F. Lu, C.Y. Ma, R. Sai-Chuen Wu, K.C. Wu, F.S. Chueh, W. Gibson Wood, J.G. Chung, Rutin inhibits human leukemia tumor growth in a murine xenograft model in vivo, Environ. Toxicol. 27 (8) (2012) 480–484. [64] J. Naowaboot, P. Piyabhan, N. Munkong, W. Parklak, P. Pannangpetch, Ferulic acid improves lipid and glucose homeostasis in high-fat diet-induced obese mice, Clin. Exp. Pharmacol. Physiol. 43 (2) (2016) 242–250. [65] C. Niu, Y. Sheng, E. Zhu, L. Ji, Z. Wang, Ferulic acid prevents liver injury induced by Diosbulbin B and its mechanism, Biosci. Trends 10 (5) (2016) 386–391. [66] U. Fahrioglu, Y. Dodurga, L. Elmas, M. Secme, Ferulic acid decreases cell viability and colony formation while inhibiting migration of MIA PaCa-2 human pancreatic cancer cells in vitro, Gene 576 (1 Pt. 3) (2016) 476–482. [67] T. Eitsuka, N. Tatewaki, H. Nishida, K. Nakagawa, T. Miyazawa, Synergistic anticancer effect of tocotrienol combined with chemotherapeutic agents or dietary components: a review, Int. J. Mol. Sci. 17 (10) (2016). [68] N. Nasr Bouzaiene, S. Kilani Jaziri, H. Kovacic, L. Chekir-Ghedira, K. Ghedira, J. Luis, The effects of caffeic coumaric and ferulic acids on proliferation, superoxide production, adhesion and migration of human tumor cells in vitro, Eur. J.

exposed to short-term cigarette smoke, Bioorg. Med.l Chem. 21 (24) (2013) 7570–7577. G.J. Chang, C.J. Chang, W.J. Chen, Y.H. Yeh, H.Y. Lee, Electrophysiological and mechanical effects of caffeic acid phenethyl ester, a novel cardioprotective agent with antiarrhythmic activity, in guinea-pig heart, Eur. J. Pharmacol. 702 (1–3) (2013) 194–207. S.I. Falcao, N. Vale, P. Cos, P. Gomes, C. Freire, L. Maes, M. Vilas-Boas, In vitro evaluation of portuguese propolis and floral sources for antiprotozoal, antibacterial and antifungal activity, Phytother. Res.: PTR 28 (3) (2014) 437–443. M. Popova, R. Dimitrova, H.T. Al-Lawati, I. Tsvetkova, H. Najdenski, V. Bankova, Omani propolis: chemical profiling, antibacterial activity and new propolis plant sources, Chem. Cent. J. 7 (1) (2013) 158. F.R. Chang, Y.C. Hsieh, Y.F. Chang, K.H. Lee, Y.C. Wu, L.K. Chang, Inhibition of the Epstein-Barr virus lytic cycle by moronic acid, Antiviral Res. 85 (3) (2010) 490–495. D. Diaz-Carballo, K. Ueberla, V. Kleff, S. Ergun, S. Malak, M. Freistuehler, S. Somogyi, C. Kucherer, W. Bardenheuer, D. Strumberg, Antiretroviral activity of two polyisoprenylated acylphloroglucinols, 7-epi-nemorosone and plukenetione A, isolated from Caribbean propolis, Int. J. Clin. Pharmacol. Ther. 48 (10) (2010) 670–677. J.B. Daleprane, T. Schmid, N. Dehne, M. Rudnicki, H. Menrad, T. Geis, M. Ikegaki, T.P. Ong, B. Brune, D.S. Abdalla, Suppression of hypoxia-inducible factor-1alpha contributes to the antiangiogenic activity of red propolis polyphenols in human endothelial cells, J. Nutr. 142 (3) (2012) 441–447. C.N. Chen, C.J. Hsiao, S.S. Lee, J.H. Guh, P.C. Chiang, C.C. Huang, W.J. Huang, Chemical modification and anticancer effect of prenylated flavanones from Taiwanese propolis, Nat. Prod. Res. 26 (2) (2012) 116–124. E. Szliszka, A. Sokol-Letowska, A.Z. Kucharska, D. Jaworska, Z.P. Czuba, W. Krol, Ethanolic extract of polish propolis: chemical composition and TRAIL-R2 death receptor targeting apoptotic activity against prostate cancer cells, Evid.-Based Complement. Altern. Med.: eCAM 2013 (2013) 757628 B. F. Yang, H. Jin, J. Pi, J.H. Jiang, L. Liu, H.H. Bai, P.H. Yang, J.Y. Cai, Anti-tumor activity evaluation of novel chrysin-organogermanium(IV) complex in MCF-7 cells, Bioorg. Med. Chem. Lett. 23 (20) (2013) 5544–5551. C.S. Daugsch, P. Moraes, Brazilian red propolis-chemical composition and botanical origin, Evid.-Based Complement. Altern. Med.: eCAM 2013 5 (4) (2008) 435–441. A.C. de Groot, Propolis: a review of properties, applications, chemical composition, contact allergy, and other adverse effects, Dermat.: Contact Atopic Occup. Drug 24 (6) (2013) 263–282. K. Midorikawa, A.H. Banskota, Y. Tezuka, T. Nagaoka, K. Matsushige, D. Message, A.A. Huertas, S. Kadota, Liquid chromatography–mass spectrometry analysis of propolis, Phytochem. Anal. 12 (6) (2001) 366–373. B.B. Silva, P.L. Rosalen, J.A. Cury, M. Ikegaki, V.C. Souza, A. Esteves, S.M. Alencar, Chemical composition and botanical origin of red propolis, a new type of brazilian propolis, Evid.-Based Complement. Altern. Med.: eCAM 5 (3) (2008) 313–316. V.C. Toreti, H.H. Sato, G.M. Pastore, Y.K. Park, Recent progress of propolis for its biological and chemical compositions and its botanical origin, Evid.-Based Complement. Altern. Med.: eCAM 2013 (2013) 697390. D. Sawicka, H. Car, M.H. Borawska, J. Niklinski, The anticancer activity of propolis, Folia histochemica et cytobiologica/Polish Academy of Sciences, Polish Histochem. Cytochem. Soc. 50 (1) (2012) 25–37. M. Ishihara, K. Naoi, M. Hashita, Y. Itoh, M. Suzui, Growth inhibitory activity of ethanol extracts of Chinese and Brazilian propolis in four human colon carcinoma cell lines, Oncol. Rep. 22 (2) (2009) 349–354. I. Valenca, F. Morais-Santos, V. Miranda-Goncalves, A.M. Ferreira, C. AlmeidaAguiar, F. Baltazar, Portuguese propolis disturbs glycolytic metabolism of human colorectal cancer in vitro, BMC Complement. Altern. Med. 13 (2013) 184. T. Kamiya, H. Nishihara, H. Hara, T. Adachi, Ethanol extract of Brazilian red propolis induces apoptosis in human breast cancer MCF-7 cells through endoplasmic reticulum stress, J. Agric. Food Chem. 60 (44) (2012) 11065–11070. M.K. Choudhari, R. Haghniaz, J.M. Rajwade, K.M. Paknikar, Anticancer activity of Indian stingless bee propolis: an in vitro study, Evid.-Based Complement. Altern. Med.: eCAM 2013 (2013) 928280. F. Li, S. Awale, Y. Tezuka, S. Kadota, Cytotoxicity of constituents from Mexican propolis against a panel of six different cancer cell lines, Nat. Prod. Commun. 5 (10) (2010) 1601–1606. E. Pichichero, R. Cicconi, M. Mattei, A. Canini, Chrysin-induced apoptosis is mediated through p38 and Bax activation in B16-F1 and A375 melanoma cells, Int. J. Oncol. 38 (2) (2011) 473–483. T.H. Hsu, C.C. Chu, M.W. Hung, H.J. Lee, H.J. Hsu, T.C. Chang, Caffeic acid phenethyl ester induces E2F-1-mediated growth inhibition and cell-cycle arrest in human cervical cancer cells, FEBS J. 280 (11) (2013) 2581–2593. G. Ozturk, Z. Ginis, S. Akyol, G. Erden, A. Gurel, O. Akyol, The anticancer mechanism of caffeic acid phenethyl ester (CAPE): review of melanomas, lung and prostate cancers, Eur. Rev. Med. Pharmacol. Sci. 16 (15) (2012) 2064–2068. A. Shamseddine, A. Saleh, M. Charafeddine, M. Seoud, D. Mukherji, S. Temraz, A.M. Sibai, Cancer trends in Lebanon: a review of incidence rates for the period of 2003–2008 and projectionsuntil 2018, Popul. Health Metr. 12 (1) (2014) 4. I. Soerjomataram, J. Lortet-Tieulent, D.M. Parkin, J. Ferlay, C. Mathers, D. Forman, F. Bray, Global burden of cancer in 2008: a systematic analysis of disability-adjusted life-years in 12 world regions, Lancet 380 (9856) (2012) 1840–1850. T. Sun, N.M. Warrington, J. Luo, M.D. Brooks, S. Dahiya, S.C. Snyder, R. Sengupta, J.B. Rubin, Sexually dimorphic RB inactivation underlies mesenchymal glioblastoma prevalence in males, J. Clin. Invest. 124 (9) (2014) 4123–4133. J.M. Ribera, A. Oriol, Acute lymphoblastic leukemia in adolescents and young adults, Hematol Oncol. Clin. North Am. 23 (5) (2009) 1033–1042 (vi).

306

Biomedicine & Pharmacotherapy 95 (2017) 298–307

H. Noureddine et al.

[73] C.O. Frozza, C.S. Garcia, G. Gambato, M.D. de Souza, M. Salvador, S. Moura, F.F. Padilha, F.K. Seixas, T. Collares, S. Borsuk, O.A. Dellagostin, J.A. Henriques, M. Roesch-Ely, Chemical characterization, antioxidant and cytotoxic activities of Brazilian red propolis, Food Chem. Toxicol. 52 (2013) 137–142. [74] E.K. Soltani, R. Cerezuela, N. Charef, S. Mezaache-Aichour, M.A. Esteban, M.M. Zerroug, Algerian propolis extracts: chemical composition, bactericidal activity and in vitro effects on gilthead seabream innate immune responses, Fish Shellfish Immunol. 62 (2017) 57–67. [75] M.M. AlShebly, F.S. AlQahtani, M. Govindarajan, K. Gopinath, P. Vijayan, G. Benelli, Toxicity of ar-curcumene and epi-beta-bisabolol from Hedychium larsenii (Zingiberaceae) essential oil on malaria, chikungunya and St. Louis encephalitis mosquito vectors, Ecotoxicol. Environ. Saf. 137 (2017) 149–157. [76] G. Xia, Y. Huang, M. Xia, L. Wang, N. Kang, L. Ding, L. Chen, F. Qiu, A new eremophilane glycoside from the fruits of Physalis pubescens and its cytotoxic activity, Nat. Prod. Res. (2017) 1–8.

Pharmacol 766 (2015) 99–105. [69] N. Roy, A. Narayanankutty, P.A. Nazeem, R. Valsalan, T.D. Babu, D. Mathew, Plant phenolics ferulic acid and P-coumaric acid inhibit colorectal cancer cell proliferation through EGFR down-regulation, Asian Pac. J. Cancer Prev. 17 (8) (2016) 4019–4023. [70] Z. Yang, X. Li, W. Han, X. Lu, S. Jin, W. Yang, J. Li, W. He, Y. Qian, Galangin suppresses human osteosarcoma cells: an exploration of its underlying mechanism, Oncol. Rep. 37 (1) (2017) 435–441. [71] S.M. Alencar, T.L. Oldoni, M.L. Castro, I.S. Cabral, C.M. Costa-Neto, J.A. Cury, P.L. Rosalen, M. Ikegaki, Chemical composition and biological activity of a new type of Brazilian propolis: red propolis, J. Ethnopharmacol. 113 (2) (2007) 278–283. [72] Y.A. Elnakady, A.I. Rushdi, R. Franke, N. Abutaha, H. Ebaid, M. Baabbad, M.O. Omar, A.A. Al Ghamdi, Characteristics, chemical compositions and biological activities of propolis from Al-Bahah, Saudi Arabia, Sci. Rep. 7 (2017) 41453.

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