Apoptotic induction by pinobanksin and some of its ester derivatives from Sonoran propolis in a B-cell lymphoma cell line

Accepted Manuscript Apoptotic induction by pinobanksin and some of its ester derivatives from Sonoran Propolis in a B-cell lymphoma cell line Efrain Alday, Dora Valencia, Ana Laura Carreño, Patrizia Picerno, Anna Lisa Piccinelli, Luca Rastrelli, Ramon Robles-Zepeda, Javier Hernandez, Carlos Velazquez PII:

S0009-2797(15)30066-1

DOI:

10.1016/j.cbi.2015.09.013

Reference:

CBI 7470

To appear in:

Chemico-Biological Interactions

Received Date: 8 June 2015 Revised Date:

21 August 2015

Accepted Date: 9 September 2015

Please cite this article as: E. Alday, D. Valencia, A.L. Carreño, P. Picerno, A.L. Piccinelli, L. Rastrelli, R. Robles-Zepeda, J. Hernandez, C. Velazquez, Apoptotic induction by pinobanksin and some of its ester derivatives from Sonoran Propolis in a B-cell lymphoma cell line, Chemico-Biological Interactions (2015), doi: 10.1016/j.cbi.2015.09.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Apoptotic induction by pinobanksin and some of its ester derivatives from Sonoran Propolis in a B-cell lymphoma cell line.

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Efrain Aldaya, Dora Valenciab, Ana Laura Carreñoa, Patrizia Picernoc, Anna Lisa Piccinellic, Luca Rastrellic, Ramon Robles-Zepedaa, Javier Hernandezd, Carlos

a

Department of Chemistry-Biology, University of Sonora. Blvd. Luis Encinas y Rosales

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s/n, 83000, Hermosillo (Son.), Mexico. b

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Velazqueza*

Department of Chemical Biological and Agropecuary Sciences, University of Sonora.

Av. Universidad e Irigoyen, 83600, Caborca (Son.), Mexico. c

Dipartimento di Farmacia, University of Salerno, Via Giovanni Paolo II, 132 84084

d

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Fisciano (SA), Italy.

Unidad de Servicios de Apoyo en Resolución Analítica, Universidad

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Veracruzana,575, Xalapa (Ver.), Mexico.

*Corresponding author. Adress: Department of Chemistry-Biology, University of

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Sonora. Blvd. Luis Encinas y Rosales s/n. Hermosillo, Sonora 83000, Mexico. Phone numbers: +52 (662) 259-21-63, +52 (662) 259-21-63. Fax number: +52 (662) 259-2163.

E-mail: [email protected].

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ACCEPTED MANUSCRIPT Abstract Propolis is a resinous substance produced by honeybees (Apis mellifera) from the selective collection of exudates and bud secretions from several plants. In previous

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works, we reported the antiproliferative activity of Sonoran propolis (SP) on cancer cells; in addition we suggested the induction of apoptosis after treatment with SP due to the presence of morphological changes and a characteristic DNA fragmentation pattern.

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Herein, in this study we demonstrated that the antiproliferative effect of SP is induced through apoptosis in a B-cell lymphoma cancer cell line, M12.C3.F6, by an annexin V-

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FITC/Propidium iodide double labeling. This apoptotic effect of SP resulted to be mediated by modulations in the loss of mitochondrial membrane potential (∆Ψm) and through activation of caspases signaling pathway (3, 8 and 9). Afterward, in order to characterize the chemical constituents of SP that induce apoptosis in cancer cells, an

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HPLC-PDA-ESI-MS/MS method followed by a preparative isolation procedure and NMR spectroscopy analysis have been used. Eighteen flavonoids, commonly described in propolis from temperate regions, were characterized. Chrysin, pinocembrin,

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pinobanksin and its ester derivatives are the main constituents of SP and some of them have never been reported in SP. In addition, two esters of pinobanksin (8 and 13) are

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described by first time in propolis samples in general. The antiproliferative activity on M12.C3.F6 cells through apoptosis induction was exhibited by pinobanksin (4), pinobanksin-3-O-propanoate (14), pinobanksin-3-O-butyrate (16), pinobanksin-3-Opentanoate (17), and the already reported galangin (11), chrysin (9) and CAPE. To our knowledge this is the first report of bioactivity of pinobanksin and some of its ester derivatives as apoptosis inducers. Further studies are needed to advance in the

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ACCEPTED MANUSCRIPT understanding of the molecular basis of apoptosis induction by SP and its constituents, as well as the structure-activity relationship of them.

Keywords: Sonoran propolis; induction of apoptosis; pinobanksin ester derivatives; chemical

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characterization.

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ACCEPTED MANUSCRIPT 1. Introduction Propolis is a resinous substance produced by honeybees (Apis mellifera) from the collection of exudates and bud secretions produced by several plants; due to its

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biological properties, this natural product has been used in traditional medicine dating back at least to 300 BC [1,2].

The chemical composition of propolis is qualitatively and quantitatively

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variable, depending on the vegetation at the site from which it was collected and the time of collection [3,4]. A diversity of chemical compounds has been identified in

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different propolis samples, including phenolic acids and esters, flavonoids, terpenes, aromatic aldehydes and alcohols, fatty acids, stilbenes and steroids [5,6]. The presence and abundance of chemical compounds in propolis provides multiple biological activities that characterize different geographical samples, such as

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anesthetic [7], antibacterial [8,9], anti-inflammatory [1,5,10], immunomodulatory [11], antioxidant [9,12], antiparasitic [13], antiproliferative [14,15], among other activities. One of the most investigated biological activities in different geographical

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samples of propolis is its cytotoxic effect and its ability to inhibit cancer cell proliferation. Although the mechanisms of action of propolis on cancer cells are not

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completely understood, apoptosis has been reported, and in this regard, many efforts are directed to specify the genesis of pro-apoptotic induction by propolis [16]. Apoptosis is a quietly complex cellular event, and it may be triggered by both

extrinsic and intrinsic factors. Since cellular proliferation is preferentially shifted than apoptosis in cancer cells, the identification of apoptosis inducers has a great importance for the development of novel, efficient and safer anticancer drugs [17,18].

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ACCEPTED MANUSCRIPT Several propolis constituents such as caffeic acid phenetyl ester (CAPE) [19], chrysin [20], propolin C [21], and artepillin C [22] have been identified as apoptotic inducers in cancer cells. In previous works, we reported that B-cell lymphoma cells

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(M12.C3.F6) treated with Sonoran propolis (SP) and one of its constituent, CAPE, exhibited morphological changes and a characteristic DNA fragmentation pattern related to apoptosis [14].

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In order to continue our studies on the antiproliferative activity of SP, herein we report the induction of apoptosis by SP on M12.C3.F6 cells, detected by annexin V-

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FITC/Propidium iodide double staining. Moreover we found that the loss of mitochondrial membrane potential and the activation of caspase-signaling pathways are involved in apoptosis induced by SP. Additionally with the aim to characterize the SP chemical constituents that induce apoptosis in cancer cells, an HPLC-PDA-ESI/MS/MS

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analysis followed by an isolation procedure and a NMR analysis have been done. Some of the chemical constituents have never been reported in SP and in propolis in general. Pinobanksin and some of its ester derivatives present in SP induced apoptosis in a

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similar manner than chrysin did. To our knowledge this is the first report of apoptosis

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induction by pinobanksin and its ester derivatives.

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. General Experimental Procedures. Optical rotations were determined on a model DIP-1000 polarimeter (Jasco,

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Easton, MD) equipped with a sodium lamp (589 nm) and a 10 cm microcell. A Bruker DRX-600 NMR spectrometer, operating at 599.19 MHz for 1H and 150.85 MHz for 13

C, using the WINXNMR software package, was used for NMR experiments in

CD3OD. Chemical shifts are expressed in δ (parts per million) referring to the solvent

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peaks δH 3.31 and δC 49.05 for CD3OD, with coupling constants, J, in Hertz. 1H-1H

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DQF-COSY, 1H-13C HSQC and HMBC experiments were obtained using conventional pulse sequences [23]. HPLC-PDA-ESI/MS/MS analyses were performed using a HPLC system (Thermo Finnigan, San Jose, CA) including a Surveyor LC pump, a Surveyor autosampler, a Surveyor PDA detector, and an LCQ Advantage ion trap mass

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spectrometer equipped with Xcalibur 3.1 software. Exact masses were measured by linear ion trap Orbitrap hybrid mass spectrometer (LTQ OrbiTrap XL, ThermoFisher Scientific), with electrospray ionization (ESI). Chromatography was performed over

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Sephadex LH-20 (Pharmacia, Uppsala, Sweden) employing MeOH as the solvent. Semipreparative HPLC, with isocratic elution, was performed with a Waters 590 series

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pumping system equipped with a Waters R401 refractive index detector, a Rheodyne injector (100 µL loop), and a Luna C-8 column (250 × 10.00 mm, particle size 10 µm, flow rate 3.0 mL/min) Phenomenex (Castel Maggiore (BO), Italy). Thin-layer chromatography (TLC) analysis was performed with Macherey−Nagel precoated silica gel 60 F254 plates (Delchimica, Napoli, Italy). All the solvents used were of analytical grade. HPLC-grade MeOH, and acetic acid (AcOH) were purchased from J. T. Baker (Baker Mallinckrodt, Phillipssburg, NJ). HPLC-grade water (18 mΩ) was prepared by a

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ACCEPTED MANUSCRIPT Milli-Q50 purification system (Millipore Corp., Bedford, MA). 3-(4,5-dimethylthiazol2-yl)-2,5-dimethyltetrazoliumbromide (MTT), dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle’s Medium (DMEM ), annexin V-FITC/propidium iodide, 5,5’,6,6’-

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tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and deuterated methanol (CD3OD) were purchased from Sigma Chemicals (St. Louis, MO, USA). FBS was purchased from Gibco®. CH3OH for chromatographic separation was purchased

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from Carlo Erba (Milan, Lombardia, It). Caffeic acid phenethyl ester (CAPE) was synthesized in our lab based on the procedure of Grunberger et al. [24].

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2.2. Propolis Sample.

Propolis samples used in this study were collected from 12 hives from March 2008 to March 2009. The hives were located in the area known as “El Coyote”, in Ures, Sonora, Mexico (N 29°27.181´, W 110°23.398). Propolis sample (10 g) was cut into

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small pieces and exhaustively extracted at room temperature with methanol (70 ml x 3 times) for several days (usually 3–4 days) with occasional stirring (2–3 times per day). Then, the pooled extract was filtered through Whatman grade No. 4 filter paper and

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concentrated under reduced pressure in a Yamato RE300 Rotary Evaporator. To remove waxes, a solution of 10% of the dried propolis extract was prepared in

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methanol, dissolving entirely and then stored at -20°C for 24 hours to allow insoluble material precipitate. After that, the filtering and concentration process of the extract was repeated. Propolis was finally stored in the dark at -20°C until analysis. Both the sample and the dried methanol extract were stored at 5 °C in the dark. 2.3. Cell Culture. The cancer cell line M12.C3.F6, derived from murine B-cell lymphoma, was provided by Dr. Emil R. Unanue (Department of Pathology and Immunology,

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ACCEPTED MANUSCRIPT Washington University in St. Louis, MO, USA). We evaluated the apoptotic effect of SP and some of its chemical constituents on this B-cell lymphoma line due to its high sensitivity to antiproliferative compounds and to propolis extracts [14].

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2.4. Cell viability Assays. To evaluate the effect of SP and its chemical constituents on the proliferation of M12.C3.F6 cells, we used the standard MTT assay [25] with some modifications

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[14,26]. In brief, 1x104 cells (50 µL) were placed in each well of a flat 96 well plate

(Costar, Corning, N.Y. USA), after 24 h of incubation at 37 °C in an atmosphere of 5 %

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CO2, aliquots (50 µL) of D5F (DMEM supplemented at 5 % with FBS) cell culture medium containing different concentrations of chemical constituents were added, and the cell cultures were incubated for 48 h. In the last 4 h of the cell culture, 10 µL of a MTT solution (5 mg/mL) were added to each well. The cell viability was assessed by

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the ability of metabolically active cells to reduce tetrazolium salt to colored formazan compounds. The formazan crystals formed were dissolved with acidic isopropyl alcohol (Sigma-Aldrich). The absorbance of the samples was measured with an ELISA plate

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reader (Multiskan EX, ThermoLabSystem), using a test wavelength of 570 nm and reference wavelength of 650 nm. The antiproliferative activity of compounds was

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reported as IC50 values (IC50 was defined as the required concentration of a pure compound to inhibit cell proliferation by 50 %). 2.5. Annexin V FITC/Iodide Propidium Staining. In order to distinguish apoptotic cells from necrotic cells, we used double

staining with annexin V-FITC/propidium iodide [17]. Cellular suspensions of 2 x105 cell/mL were prepared and incubated in 6 well, flat bottom cell culture plates (Costar, Corning, N.Y. USA) (6 x 105 cells per well, 3 mL). According to preliminary

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ACCEPTED MANUSCRIPT experiments, a SP concentration of 50 µg/mL and a 12 h incubation time were the optimal conditions to perform the apoptosis assay. We tested the SP extract (50 µg/mL) and its chemical compounds (50 µM) for 12 h. The cell cultures were maintained at 37

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°C, in an atmosphere of 5 % CO2 and 80-90 % relative humidity in an incubator (Fisher Scientific, USA). CAPE (3.75 µM) was used as a positive control for apoptosis

induction. Once the apoptosis induction time has passed, the cells were harvested, and

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then washed twice with cold PBS (543 x g, 7 min, 4°C). The cell bottom was suspended in 50 µL of binding solution (annexin V at 1 µg/mL concentration and a high

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percentage of calcium ions that mediates the binding of annexin V to

phosphatidylserine; Sigma-Aldrich). The cells were incubated 10 minutes at room temperature in the dark. Subsequently, propidium iodide was added (0.5 µg/mL final concentration; Sigma-Aldrich). Then, cells were incubated (10 min) at room

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temperature in the dark. Finally, cells were washed twice with PBS (543 x g, 7 min, 4 °C) and resuspended in 200 µL of D5F cell culture medium and analyzed immediately by flow cytometry (Canto II FACS, Becton Dickinson, CA, USA).

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2.6. JC-1 staining of Mitochondria.

To evaluate the loss of mitochondrial membrane potential (∆Ψm) the cationic

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lipophilic dye JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine iodide) was used, which is an autofluorescence dual molecule. In non apoptotic cells, the intact ∆Ψm allows the lipophilic dye JC-1 to accumulate as aggregates in the mitochondrial matrix which stain red, whereas, in apoptotic and necrotic cells, JC-1 exists in monomeric form and stains the cytosol green. The displacement in its fluorescence is directly associated to the loss of mitochondrial membrane potential [27]. Cellular suspensions of 2 x 105 cells/mL were

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ACCEPTED MANUSCRIPT prepared and placed in 6 well, flat bottom cell culture plates (Costar, Corning, N.Y. USA) (4.5 x 106 cells per well, 2 mL) and incubated with the propolis extract at different periods of time (0.25, 1, 3, 6, 12, and 24 h). Cell cultures were maintained at

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37 °C, in an atmosphere of 5 % CO2 and 80-90 % relative humidity in an incubator (Fisher Scientific, USA). CAPE was used as a positive control for apoptosis induction (7.5 µM). After incubation period, the cells were harvested, and washed twice with cold

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PBS (543 x g, 7 min, 4 °C). The cell bottom was suspended in a dyeing solution (JC-1 10 µg/mL in cell culture medium), and settled in an incubator for 15 minutes. Finally,

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two washes with cell culture medium were performed, and then cells were resuspended in 400 µL of D5F and analyzed immediately by flow cytometry (Canto II FACS, Becton Dickinson, CA, USA). 2.7. Activity of Caspases.

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The activation of caspases (3, 8 and 9) was performed using commercial kits purchased by Abcam Company (www.abcam.com/technical). Cellular suspensions (2 x 105 cells/mL) were prepared and placed in 6-well, flat bottom cell culture plates

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(Costar, Corning, N.Y. USA) (6 x 105 cells per well, 3 mL) and incubated in presence of propolis extract (50 µg/mL) for different times (0.25, 1, 3, 6, and 12 h). CAPE was

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used as positive control of induction of apoptosis (7.5 µM). Subsequently, cells were harvested and washed twice with cold D5F, the cell bottom was resuspended in 300 µL of cell culture medium, and 1 µL (using 1:20 reagent dilution) of corresponding caspase inhibitor marked with FITC (FITC-DEVD-FMK, FITC-IETD-FMK and FITC-LEHDFMK for 3, 8 and 9 caspases, respectively) was added. Cells were incubated 1 h at 37 °C, in an atmosphere of 5 % CO2 and 80-90 % relative humidity. Afterward, cells were washed twice with 300 µL of wash buffer (543 x g, 7 min, 4 °C), then resuspended (300

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ACCEPTED MANUSCRIPT µL) in wash buffer and maintained in ice during the flow cytometry analysis (Canto II FACS, Becton Dickinson, CA, USA) was performed analyzed. 2.8. HPLC-PDA-ESI-MS Analysis.

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HPLC separations were accomplished using a Luna C18 column (150 mm × 2.0 mm i.d., 5 µm, Phenomenex) protected by its guard cartridge (4 mm × 2.0 mm i.d.) and a binary gradient composed of water (solvent A) and MeOH (solvent B), both

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containing 0.1% (v/v) formic acid (FA). The following gradient was adopted: a linear

gradient of B from 30 to 50 for 20 min, from 50 to 65% B for 10 min, from 65 to 100%

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B for 15 min, followed by washing and re-equilibrating of the column. Elution was performed at flow rate of 0.2 mL min-1, and the volume of the injection was 5 µL. Detection by diode array was performed at two different wavelengths: 280 and 330 nm. The UV spectra were recorded with a 200–600 nm range. The mass analyses were

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performed in the negative ion mode. The data were acquired in the full scan (range of m/z 100-1000) and MS/MS scanning mode, the maximum injection time was 50 ms, with a sole microscan, and for the MS/MS scanning mode the percentage of collision

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energy were 40 and 50%. The optimized instrumental parameters were as follows: capillary temperature, 250 °C; capillary voltage, -28 V; spray voltage, 4.90 kV; sheath

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and auxiliary gas flow rates, 30 and 30 (nitrogen, arbitrary units), respectively. 2.9. Isolation Procedure of Main Constituents. A portion of dry methanol extract sample (2.5 g) was fractionated on a

Sephadex LH 20 column (100 cm × 5.0 cm) using methanol as solvent. After TLC analysis, fractions with similar Rf values were combined in five major fractions (I-V) and analyzed by HPLC-DAD-MS to guide the isolation of main compounds. Fractions II, III, and V, were purified by semipreparative RP-HPLC. Fraction II, containing

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ACCEPTED MANUSCRIPT pinobanksin derivatives, was purified using MeOH-H2O (7:3), v/v, as mobile phase to yield the compounds 1, 3, 12, 14, 16, 17, and 18. Fractions III, and V, separated using MeOH-H2O (6:4), v/v, afforded compounds 2, 4, and 10, (Fr. III), as well as

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compounds 4, 9, and 10 (Fr. V). 2.9.1. Pinobanksin-3-O-butyrate (16). [α]25D +43.1 (c 0.25, MeOH); LC-UV

[MeOH in H2O−0.1% FA)] λMax 295 nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR

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(CD3OD, 150.9 MHz) data are reported in Table 3; Negative ESI-HRMS m/z

341.31033 [M-H]- (calcd for C19H17O6, 341.1020). ESI-MS/MS data are reported in

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Table 2.

2.9.2. Pinobanksin-3-O-pentanoate (17). [α]25D +44.0 (c 0.23, MeOH); LC-UV [MeOH in H2O−0.1% FA)] λMax 295 nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150.9 MHz) data are reported in Table 3; Negative ESI-HRMS m/z 355.1186

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[M-H]- (calcd for C20H19O6, 355.1176). ESI-MS/MS data are reported in Table 2. 2.9.3. Pinobanksin-3-O-hexanoate (18). [α]25D +42.1 (c 0.26, MeOH); LC-UV [MeOH in H2O−0.1% FA)] λMax 295 nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR

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(CD3OD, 150.9 MHz) data are reported in Table 3; Negative ESI-HRMS m/z 369.1344 [M-H]- (calcd for C21H21O6, 369.1333). ESI-MS/MS data are reported in Table 2.

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2.9.4. Compounds 1, 3-4, 9-10, 12, and 14. 1H and 13C NMR data were

consistent with those previously reported; Pinobanksin-5-methyl ether (1) [26]; Pinocembrin-5-methyl ether or alpinetin (3) [12]; Pinobanksin (4), Chrysin (9) [28]; Pinocembrin (10) [3]; Pinobanksin-3-acetate (12) [28]; Pinobanksin-3-O-propanoate (14) [15]. For ESI-MS data see Table 2. 2.10. Statistical Analysis. All results are shown as mean ± SEM of at least three independent experiments performed in triplicate. Data were graphed using Prism 5

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ACCEPTED MANUSCRIPT software version 5.01 (2007). Data were analyzed using analysis of variance (ANOVA) with Tukey-Kramer and Duncan’s multiple comparison tests (Number Cruncher

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Statistical Software, NCSS 2000).

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ACCEPTED MANUSCRIPT 3. Results 3.1. The antiproliferative activity of SP in M12.C3.F6 cells is by induction of apoptosis.

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In a previous study, we reported that SP had strong antiproliferative activity on different cancer cell lines in comparison with normal cells, suggesting a possible selectivity of SP against cancer cells [14,29,30]. In order to know whether the

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growth-inhibitory activity of SP in B-cell lymphoma M12.C3.F6 cells (SP IC50: 20.6

± 0.5 µg/mL) was part due to induction of apoptosis, we performed a double annexin

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V/propiduim iodide staining. M12.C3.F6 cells were treated with SP (50 µg/mL) during 12 h, and the presence of apoptotic cells was evaluated by flow cytometry. The extract of SP induced apoptosis in M12.C3.F6 cells (46.2 %), which represents an increase in apoptotic cells by four times more than negative control of normal cell

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growth (11.8 %). CAPE, a Sonoran propolis chemical constituent, was included as a positive control for apoptosis induction (81.9 % at 3.75 µM). Ethanol (used as a solvent) did not induce apoptosis in M12.C3.F6 cells as compared with control

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M12.C3.F6 cell culture (12.8 % and 11.8 %, respectively).

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ACCEPTED MANUSCRIPT Table 1. Evaluation of induced apoptosis in cancer cells M12.C3.F6 by SP

Total apoptotic cells

Death cells

Cells in early apoptosis

Cells in late apoptosis (secondary necrosis)

Normal growth control

85.4 ± 2.2

2.8 ± 1.0

4.0 ± 1.9

7.8 ± 1.0

11.8 ± 2.9

EtOH

83.3 ± 4.5

5.9 ± 0.8

6.2 ± 3.9

6.6 ± 1.7

12.8 ± 5.4

CAPE

16.0 ± 0.4

3.0 ± 1.3

7.5 ± 5.3

74.5 ± 4.6

81.9 ± 0.8

SP

49.7 ± 4.7

6.4 ± 4.6

23.0 ± 8.1

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Normal cells

46.2 ± 1.5

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23.2 ± 9.1

EtOH was uses as a dissolvent control. CAPE was used as a positive apoptosis inducer (7.5 µM). Propolis samples were evaluated at 50 µg/mL concentration. All

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the values represent the average of three independent experiments ± SD.

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ACCEPTED MANUSCRIPT 3.2. SP induces loss of mitochondrial membrane potential and caspase activation in M12.C3.F6 cells. The cell death process mediated by apoptosis can be initiated by two different

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signaling pathways, the extrinsic and intrinsic pathways. The loss of mitochondrial membrane potential (∆Ψm) is associated with early events in the activation of intrinsic pathway of apoptosis process. In order to measure the electrochemical collapse of

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mitochondrial membrane gradient, we performed a JC-1 staining of apoptotic cells

using the cancer cell line M12.C3.F6 treated with SP (50 µg/mL) or CAPE (7.5 µM,

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used as a positive control of induction of apoptosis), at different periods of times (0.25 – 24 h). The dual JC-1 fluorescence intensity ratio emitted by cells treated with SP and CAPE is shown in Figure 1 by biparametric dot plot cytograms (A and B) and graphics (C and D). A displacement from red fluorescence towards green fluorescence can be

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observed for both, indicating a gradual and imminent decrease of ∆Ψm that was dependent of apoptosis time induction.

With the aim to determine whether the apoptosis induced by SP is activated by

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the intrinsic (mitochondrial pathway) or the extrinsic pathway, caspases activity assays were carried out. Intrinsic pathway is generally activated with an association of caspase

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9 activation (initiator) and the subsequent activation of caspase 3 (effector). M12.C3.F6 cells were incubated with SP (50 µg/mL) and CAPE (7.5 µM) at different periods of time (0.25 – 12 h). SP treatment in M12.C3.F6 cells induced the activation of caspases 3 and 9. The activation of both enzymes resulted evident at 6 and 12 h after SP treatment as compared with control cell culture (P<0.05) (Figure 2). Cancer cells treated with CAPE shown a perceptible activation of caspases at 1h of incubation.

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ACCEPTED MANUSCRIPT Since caspase 8 is the responsible protein to initiate the signaling pathway that triggers the apoptosis process via extrinsic pathway, we decided to evaluate the activation of caspase 8 with the aim to determine whether the extrinsic pathway is

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activated by SP treatment in M12.C3.F6 cells. The most evident activation of caspase 8 was observed at 6 h and 12 h of incubation with propolis (Figure 2). Statistically

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significant differences not were observed among the activation of the tested caspases.

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Figure 1. Changes in mitochondrial membrane potential (∆Ψm) of M12.C3.F6 cells treated with SP and CAPE. The displacement of red fluorescence towards green fluorescence (RF/GF rate) is shown for M12.C3F6 cells treated with SP (50 µg/mL; A and C) or CAPE (7.5 µM or 2.13 µg/mL; B and D) at different periods of time (0, 0.25, 1, 3, 6, 12 and 24 h). The JC-1 fluorescence intensity ratio for SP and CAPE treatment is represented in biparametric dot plot cytograms (A and B) and in graphics (C and D). Data is representative of at least three independent experiments.

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A

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60

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0 Control

0.25

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3

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Time (h)

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Cell (%) with activity of caspases

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Caspase 8

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Cell (%) with activity of caspases

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Time (h)

Caspase 9

Caspase 3

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Figure 2. Apoptosis induction in M12.C3.F6 cell line is activated by caspase-signaling pathways after SP treatment. Cells were harvested and analyzed to determine caspase 3,

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8 and 9 activation at different treatment times (0.25 to 24 h). A: treatment with SP (50 µg/mL). B: Treatment with CAPE was used as positive control of induction of apoptosis (7.5 µM). All the values shown represent at least the mean of three independent experiments ± standard deviation. Statistical differences (P < 0.05) from the DMSO control (0.06-0.05 %) are marked with asterisk.

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ACCEPTED MANUSCRIPT 3.3. HPLC-PDA-ESI-MS Analysis of SP. In order to better characterize the SP chemical constituents that induce apoptosis in cancer cells, an HPLC-PDA-ESI-MS/MS analytical procedure was applied to supply

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the general chemical fingerprint of SP and to identify a wide range of compounds with varying polarity. Figure 3 (A and B) showed the HPLC-UV and HPLC-ESI-MS

profiles, in negative ionization modes, of SP extract. Eighteen major peaks (1-18) were

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characterized, their retention times, λmax values, deprotonated molecular ions, and

MS/MS fragments are listed in Table 2. UV spectra of peaks 2, 5, 6, 9 and 11 provided

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two maxima at 310-385 nm (band I), and 250-280 (band II) coincident with flavonols and flavones, whereas peaks 1, 3, 4, 7-8, 10, and 12-18 showed UV spectra (maxima at 277-295 nm and shoulder at 330−335 nm) characteristic of dihydroflavonols and flavanones [31].

ESI-MS spectra of compounds 1−18, acquired in negative ionization (NI)

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modes for sensitivity and fragmentation specificity of polyphenols, showed [M − H]− ion as the base peak, selected for successive MS/MS experiments through collision

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energy ranging from 40% to 45%. (−)-MS/MS spectra of peaks 4, 9-11 ([M − H]− at m/z 271, 253, 255, and 269, respectively) presented the typical fragmentation pattern of

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flavonoids and were identified as pinobanksin (4), chrysin (9), pinocembrin (10), and galangin (11) by the comparison with reference standards.

20

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Figure 3. Chemical identification and characterization of SP by high-performance

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liquid chromatography paired with photodiode array and electrospray ionization tandem mass spectrometry detectors (HPLC-PDA-ESI-MS/MS). The major constituents of SP (peaks 1-18) were analyzed by HPLC-PDA-ESI-MS/MS in negative ionization mode. A: Chromatographic profile recorded at 280 nm. B: Chromatographic profile recorded at 330 nm. C: Chemical structures of compounds characterized from SP by HPLCPDA-ESI-MS/MS analysis.

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ACCEPTED MANUSCRIPT Table 2. Retention times and UV and ESI-MS data of compounds 1−18 of SP.

5 6 7 8 9 10 11 12 13 14 15 16 17 18

14.9 15.2 16.6 21.3 22.5 24.2 24.3 25.6 27.0 31.1 31.5 35.4 38.8 42.1

275, 355 265, 360 295 295 280, 325 295 270, 365 295 295 295 295 295 295 295

299 329 327 341 253 255 269 313 355 327 369 341 355 369

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Relative abundance > 2 (%).

(-)-MS/MS (m/z, relative abundance)a 267 (100), 257 (4), 252 (9), 241 (4), 239 (24), 229 (4), 179 (2) 300 (100) 254 (45), 227 (100), 212 (2), 165 (21), 139 (3) 271 (100), 253 (56), 243 (6), 241 (4), 225 (13), 215 (7), 199 (2), 197 (5), 185 (3), 165 (3), 151 (5) 284 (100) 314 (100) 285 (100), 267 (14), 239 (17) 285 (100), 267 (38), 239 (44) 213 (26), 211 (8), 187 (5), 151 (6) 271 (22), 253 (100) 285 (100), 267 (33), 239 (44) 271 (10), 253 (100) 285 (100), 267 (40), 239 (53) 271 (10), 253 (100) 271 (7), 253 (100) 271 (11), 253 (100)

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a

[M-H](m/z) 285 315 269 271

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4

λMax (nm) 295 265, 360 295 300

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1 2 3

tR (min) 10.2 10.7 13.4 13.9

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N°

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ACCEPTED MANUSCRIPT Peaks 1-3, and 5-6 showed, in the MS/MS spectra, as base peak the [M–H– CH3]− product ion (Table 2) characteristic of the methylated flavonoids. On the basis of these data, UV spectra, and MS data reported in literature there were tentatively

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identified as pinobanksin-5-methyl-ether (1), quercetin-3-methy-ether (2), pinocembrin-5-methyl-ether (3), luteolin-5-methyl-ether (5), and quercetin-5,7dimethyl-ether (6) [28,32,33].

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In the (−)-MS/MS spectra of peaks 12, 14, and 16-18 ([M − H]− at m/z 313, 327, 341, 355, and 369, respectively) were observed the characteristic fragment ions at m/z

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271 [pinobanksin - H]− and 253 [chrysin - H]− (Table 2) that are consisted with the fragmentation pathway of the esterified pinobanksin derivatives generated by the loss of the alkanoate group [32,33]. In according to the literature data, 12, 14, and 16-18 were identified as pinobanksin-3-O-acetate (12), pinobanksin-3-O-propanoate (14),

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pinobanksin-3-O-butyrate (16), pinobanksin-3-O-pentanoate (17), and pinobanksin-3O-hexanoate (18), respectively [32,33].

The peaks 7-8, 13, and 15 showed isobaric deprotonated molecular ions ([M −

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H]− at m/z 327, 341, 355, and 369, respectively) of peaks 14, and 16-17. A base peak at m/z 285 and product ions at m/z 267 and 239 in their MS/MS spectra (Table 2) were in

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accordance with the structure of esterified derivatives of pinobanksin-5-methylether [32]. Thus, these data suggested that these compounds were pinobanksin-5methylether-3-O-acetate (7), pinobanksin-5-methylether-3-O-propanoate (8), pinobanksin-5-methylether-3-O-butyrate (13) and pinobanksin-5-methylether-3-Opentanoate (15). The supposed structures were reinforced by the MS3 spectra of the product ions at m/z 285 superimposable to that of pinobanksin-5-methylether (1).

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ACCEPTED MANUSCRIPT 3.4. Isolation and Structure Determination of Compounds. To confirm the structures of major compounds proposed by HPLC-DAD-MS, the isolation procedure of SP was undertaken. A part of the SP extract was subjected to

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purification by Sephadex LH-20 followed by HPLC reversed-phase to yield seven dihydroflavonols (1, 4, 12, 14, and 16-18), two flavanones (3, and 10), and one flavone (9) confirmed by NMR spectroscopy.

The structure elucidation of compounds 16-18 proceeded as follows. The

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molecular formula of 16 was determined to be C19H18O6 by HRESIMS analysis. The 1H

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NMR spectrum of 16 displayed characteristic resonances corresponding to a pinobanksin moiety [28,34], showing two oxymethines at δH 5.42 (1H, d, J=12 Hz) and 5.86 (1H, d, J=12 Hz), aromatic proton at δH 5.95 (x2), and a phenyl group at δH 7.47.6. In addition, one terminal methyl (δH 0.75), a methylene (δH 1.46), and an acetyl

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methylene (δH 2.18 and 1.40) group, in aliphatic region, were also observed. The 13C spectrum (Table 3) showed 19 signals, 15 of them corresponding to pinobanksin moiety and remaining ascribable to an alkanoyl residue (δC 174.3, 30.4, 13.6, 19.2). Full

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assignment of the proton and carbon resonances were obtained by 1H-1H DQF-COSY, HSQC and HMBC experiments indicating that compound 16 was pinobanksin [34],

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esterified with a butyric acid. Long-range C-H correlations in HMBC spectrum of 16 between the acetyl methylene signal at δH 2.18 and 1.40 (H-2’’) and resonances of C3’’ (CH2 δ 19.2), C-4’’ (CH3 δ 13.6), and carbonyl (C-1’’, δ 174.3), confirmed the presence of a butanoyl chain as well as the HMBC correlation observed between the oxymethine signal (δ 5.86) and carbonyl group C-1’’ (δ 174.3) established unambiguously it location at C-3 of aglycone. On the basis of these data the structure of 16 was determined as Pinobanksin-3-O-butyrate (Table 3).

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ACCEPTED MANUSCRIPT

a

2.18, m 1.40, q (6.4) 1.46, m 0.75, t (6.9)

δH (J in Hz)b 5.42, d (12.0) 5.89, d (12.0)

5.93, d (1.8)

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7.53, m 7.43, m 7.41, m

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19.2, CH2 13.6, CH3

5.95, d (2.0)

δC 83.4, CH 73.0, CH 192.6, C 165.5, C 96.8, CH 172.9, C 164.0, C 102.0, C 137.6, C 128.7, CH 129.6, CH 129.0, CH 172.9, C 43.2, CH2 27.5, CH2 26.5, CH2 11.2, CH3

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3’’ 4’’ 5’’ 6’’

17 δH (J in Hz)b 5.42, d (12.1) 5.86, d (12.1)

7.54, m 7.45, m 7.43, m 2.14, q (6.0) 2.35, m 1.50, m 1.90, m 0.62, t (6.5)

18 δC 83.4, CH 73.2, CH 191.8, C 165.6, C 97.0, CH 167.9, C 163.3, C 102.4, C 137.5, C 128.8, CH 129.4, CH 130.3, CH 173.5, C 34.5, CH2 24.7, CH2 31.7, CH2 22.5, CH2 13.5, CH3

δH (J in Hz)b 5.34, d (12.3) 5.89, d (12.3)

5.94, d (1.9)

7.50, m 7.40, m 7.37, m 2.20, m 2.27, m 1.41, m 1.06, m 1.19, m 0.82, t (6.3)

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2 3 4 5 6, 8 7 9 10 1’ 2’, 6’ 3’, 5’ 4’ 1’’ 2’’

δC 82.6, CH 73.5, CH 193.2, C 165.6, C 96.6, CH 172.0, C 163.5, C 102.3, C 137.5, C 128.5, CH 129.7, CH 130.3, CH 174.3, C 30.4, CH2

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16 Position

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Table 3. 13C and 1H NMR data for compounds 16 -18 in CD3ODa

Assignments confirmed by 2D COSY, HSQC, HMBC experiments. H-1H coupling constants were measured from COSY spectra in Hz.

b1

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ACCEPTED MANUSCRIPT The molecular formula of 17 and 18 were determined as C20H20O6, and C21H22O6, respectively by HPLC-PDA-ESI-MS and 13C analysis. The NMR data (1H, 13

C, DQF-COSY, HSQC, and HMBC) revealed that compounds 17-18 differed from 16

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only in the carbon chain length of the ester group at C-3 of pinobanksin moiety. The proton coupling network within each of the n-alkanoyl residues was established using a combination of 1D TOCSY and DQF-COSY methods; direct evidence for the n-

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alkanoyl sequence and its linkage sites was derived from HSQC and HMBC data

(Table 3). Thus, compounds 17-18 were elucidated as Pinobanksin-3-O-pentanoate, and

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Pinobanksin-3-O-hexanoate, respectively.

The structure of compounds 1, 3-4, 9-10, 12, and 14 were identified by comparison of their NMR data with those from the literature as pinobanksin-5-methylether (1) [12], pinocembrin-5-methyl-ether (3) [35], pinobanksin (4) [28], chrysin (9)

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[28], pinocembrin (10) [3], pinobanksin-3-acetate (12) [28], pinobanksin-3-Opropanoate (14) [15]. For compound 1 and 3 the presence of the methoxyl group and its location at the C-5 position of ring A were established on the basis of the 13C NMR

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spectra and HMBC data with respect to an unmethoxylated model. The chemicals structures of isolated compounds are shown in Figure 3 (C).

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3.5. Antiproliferative activity and apoptosis induction by SP chemical constituents in M12.C3.F6 cells.

In order to evaluate the biological effect of isolated compounds of SP, the

antiproliferative activity of them was assessed against M12.C3.F6 using the MTT cell viability assay. Results showed that chrysin (9; IC50 = 49.1 µM or 12.5 µg/mL), pinobanksin (4; 52.1 µM or 14.2 µg/mL), and some of its ester derivatives: 14 (67.0 µM or 22.0 µg/mL), 16 (49.9 µM or 17.0 µg/mL), 17 (51.3 µM or 18.3 µg/mL) and 18

26

ACCEPTED MANUSCRIPT (76.6 µM or 28.3 µg/mL), exhibited significant dose-dependent inhibitory effect on cancer cells growth. No antiproliferative activity was shown by 1, 3, 10, and 12. Galangin (11; 17.3 µM or 4.7 µg/mL) and CAPE (1.6 µM or 0.4 µg/mL), both SP

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chemical constituents, were included as positive controls since its inhibitory effect on cell proliferation [14]. Cell cultures treated only with vehicle (DMSO at 0.05 % v/v) did not show any evidence of cell damage and were considered as negative control.

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In order to assess the induction of apoptosis by the antiproliferative compounds of SP, a double annexin V-FITC/propidium iodide staining by flow cytometry was

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used. M12.C3F6 cells were treated with compounds 4, 9, 14, and 16-18 (each at 50 µM) for 12 h. All the evaluated antiproliferative compounds exhibited interesting proapoptotic effect, and the compounds 4, 9, 14, 16 and 17 showed statistical differences (P < 0.05) from the DMSO control (Figure 4). Notably, pinobanksin-3-O-propanoate

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showed the higher apoptotic induction in comparison to the other esterified pinobanksin

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derivatives. CAPE and galangin were used as positive apoptosis inducers.

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* *

80

40

*

20

*

*

0 C

DMSO

18

16

4

17

*

9

14

11

CAPE

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Treatment

*

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60

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% Total Apoptotic Cells

100

Figure 4. Chemical constituents of SP induce apoptosis in M12.C3.F6 cells. Apoptosis induction by SP constituents was evaluated at the solely concentration of 50 µM. CAPE

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was used as a positive apoptosis inducer (3.75 µM or 1.06 µg/mL). C represents the cells treated only with D5F medium. DMSO (at 0.06-0.05 %) represents the cells incubated with the vehicle. All the values represent at least the mean of three

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independent experiments ± standard deviation. Statistical differences (P < 0.05) from

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the DMSO control are marked with asterisk.

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ACCEPTED MANUSCRIPT 4. Discussion In this study we determined that the antiproliferative activity of SP in a Bcell lymphoma cancer cell line (M12.C3.F6) is induced through apoptosis, which

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resulted to be mediated by modulations in the loss of mitochondrial membrane potential orchestrated with the activation of both the intrinsic and extrinsic caspase

signaling pathways. In addition, we chemically characterized the major compounds

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of SP, and we achieved the isolation of some of its apoptotic inducer constituents, including pinobanksin and its ester derivatives which never have been previously

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reported as bioactive and as apoptotic inducers.

Apoptosis induction is one of the proposed mechanisms for the biological effects of propolis as previously described elsewhere [36,37]. In general, apoptosis may occur through two main specific signaling pathways, the extrinsic and intrinsic

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pathway. The extrinsic pathway is induced by an external signal stimulated by receptors such as death receptors: Fas (TNF receptor superfamily, member 6), TRAIL-R1 and R-2 (TNF-related apoptosis-inducing ligand-R1 and R2). This

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pathway is generally triggered in association of activation of caspase 8 (initiator) and caspase 3 (effector), subsequently. The intrinsic pathway is mediated by

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mitochondria and releasing of pro-apoptotic proteins including cytochrome c into the cytoplasm [16, 37] that activate caspase 9 signaling that triggers caspase 3 activation. Caspase-3 has been suggested to be a key role of the apoptotic machinery [16,38].

In previous works, we have reported that cancer cells treated with SP and CAPE, exhibit morphological changes and a characteristic DNA fragmentation pattern related to apoptosis [14]. In order to analyze how the apoptosis process is

29

ACCEPTED MANUSCRIPT initiated and developed in the cancer cells after treatment with SP, we attempted to investigate some of the molecular mechanisms of apoptosis. According to our results, the percentage of M12.C3.F6 cells with activated caspases (3, 8 and 9)

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became significant at 6 h after SP treatment, which suggested the coordinated enzymatic activation of both pathways of apoptosis response process after SP

treatment. Interestingly, the loss of ∆Ψm in treated cells with SP became significant

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at 12 h, aspect that could be explained by the maintained integrity of ∆Ψm, whereas the apoptotic cytochrome c was released into the cytosol and consequently the

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intrinsic pathway of apoptosis process was activated [39]. In addition, it has been reported that caspase 8 cleaves Bid protein that triggers Bax and Bak stimulation, and the subsequently activation of the intrinsic pathway of apoptosis [40]. These results are in agreement to those obtained and reported for CAPE,

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which induces apoptosis in cancer cells by the activation of a complex molecular machinery including cell cycle regulator proteins, signaling kinases, the activation of caspases through TRAIL and Fas receptor (extrinsic pathway), and through the

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mitochondrial pathway [16,19]. Interestingly, the activation of caspases after the treatment with CAPE became significant at 6 hours, same time as SP activates

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caspases pathway as well.

Chrysin and galangin are already reported as apoptosis inducers. Although

the molecular mechanisms by which those compounds induce apoptosis are still not clear, it is reported that they activate caspase 3 [16,20]. The activation of both apoptosis pathways by SP treatment is possibly related to the presence of CAPE, chrysin and galangin.

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ACCEPTED MANUSCRIPT The analytical approach used in the present study for SP allowed the identification of 18 flavonoids. Some of them were previously reported in SP (1, 4, 912, and 14) [14,15,29]. Compounds 2-7 and 15-18 already described in European

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propolis, were identified for the first time in SP. In addition, two esters of pinobanksin (8 and 13) are new for propolis in general. Practically, two different families of

esterified pinobanksin derivatives were found in SP, both are determined by the

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esterification at hydroxyl group positioned at C3 from pinobanksin and pinobanksin-5methylether, respectively.

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Pinobanksin ester derivatives (12, 14, and 16-18) are usually identified by GC and ESI/MS analysis, and many authors do not isolate the compounds and do not determine the full structure on the basis of NMR data. Herein, we isolated and characterized these compounds, and the full assignment of all 1H and 13C resonances

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were carried out by extensive analysis of their NMR spectra.

Interestingly, pinobanksin (52.1 µM or 14.2 µg/mL) but not pinobanksin-5methylether exhibited antiproliferative activity on M12.C3.F6 cells. In addition, the

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isolated pinobanksin ester derivatives (14, 16-18) exhibited a moderate antiproliferative activity on M12.C3.F6 cells, in exception to pinobanksin-3-O-acetate (12).

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In regard to the chemical constituents of SP that induce apoptosis in M12.C3.F6 cells, pinobanksin-3-O-propanoate (14) showed the highest percentage of apoptotic cells after the treatment, even more than pinobanksin and its other ester derivatives (1618). In addition, the treatment with 14 was slightly more effective than chrysin, meanwhile pinobanksin-3-O-pentanoate (17) showed a remarkable apoptotic induction activity as effective as chrysin. More studies focused on the structure-activity relationship (SAR) are required in order to understand how the elongation of the

31

ACCEPTED MANUSCRIPT aliphatic chain in the ester part of these derivatives of pinobanksin impacts on the induction of antiproliferative activity by apoptosis. On the basis of these observations, the antiproliferative activity of SP on M12.C3.F6 cancer cells could be due to some of

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its constituents, such as 4, 9, 11, 14, 17 and CAPE through induction of apoptosis. In conclusion, our data revealed that the antiproliferative activity of SP is induced through apoptosis mediated by modulations in the loss of mitochondrial

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membrane potential and caspases activation pathway. Some of SP chemical

constituents showed to induce apoptosis in M12.C3.F6 cells, including pinobanksin and

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some of its ester derivatives (4, 14, and 16 and 17), in addition to the well described apoptisis inducers CAPE, chrysin (9) and galangin (11), constituents that could be implied in the pro-apoptotic activity of SP. In this study, the compounds 2-7 and 15-18 were identified for the first time in SP, which are flavonoids already described in

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European propolis. In addition, the presence of two esters of pinobanksin (8 and 13) is described by first time in propolis samples in general. Further studies are needed to advance in the understanding of the molecular basis of apoptosis induction by SP

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constituents and the structure-activity relationship of them. SP should be considered as an important source of natural bioactive compounds in cancer research, due to the

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potent apoptotic effect of SP and some of its chemical constituents in cancer cells.

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ACCEPTED MANUSCRIPT 5. Acknowledgements We would thank the professional beekeeper Gilberto Valenzuela for his help, as well as Lucila Rascon and Judith Valdez for their collaboration in providing all the facilities in

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the development of this work. This project was partially supported by a Grant from National Council for Science and Technology of Mexico (CONACYT, 83462), and FOMIX VER-2009-C03-127523 of COVECyT México.

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6. Conflict of interest

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The authors have declared that there is not conflict of interest.

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29. D. Valencia, E. Alday, R. Robles-Zepeda, A. Garibay-Escobar, J.C. Galvez-Ruiz,

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30. BJ. Conti, KB. Santiago, MC. Búfalo, YF. Herrera, E. Alday, C. Velazquez, J. Hernandez J, JM. Sforcin, Modulatory effects of propolis samples from Latin

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America (Brazil, Cuba and Mexico) on cytokine production by human monocytes. J Pharm Pharmacol (2015) doi: 10.1111/jphp.12431.

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ACCEPTED MANUSCRIPT 33. C. Gardana, M. Scaglianti, P. Pietta, P. Simonetti, Analysis of the polyphenolic fraction of propolis from different sources by liquid chromatography–tandem mass spectrometry. J. Pharm. Biomed. Anal. 45 (2007) 390-399.

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34. S. Kumazawa, K. Hayashi, K. Kajiya, T. Ishii, T. Hamasaka, T. Nakayama, Studies of the constituents of Uruguayan Propolis. J. Agric. Food Chem. 50 (2002) 47774782.

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35. D.R. Katerere, A.I. Gray, A.R. Kennedy, R.J. Nash, R.D. Waigh, Cyclobutanes from Combretum albopunctatum, Phytochemistry. 65 (2004) 433-438.

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36. S. Mishima, Y. Narita S. Chikamatsu, Y. Inoh, S. Ohta, V. Yoshida, Y. Araki, K.M. Suzuki, Y. Nozawa, Effects of propolis on cell growth and gene expression in HL60 cells, J. Ethnopharmacol. 99 (2005) 5-11.

37. M. Motomura, K.M. Kwon, S.J. Suh, Y.C. Lee, Y.K. Kim, I.S. Lee, M.S. Kim, D.Y.

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Kwon, I. Suzuki, C.H. Kim, Propolis induces cell cycle arrest and apoptosis in human leukemic U937 cells through Bcl-2/Bax regulation, Environ. Toxicol. Pharmacol. 26 (2008) 61-67.

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38. T. Kamiya, H. Nishihara, H. Hara, T. Adachi, T, Ethanol extract of Brazilian red propolis induces apoptosis in human breast cancer MCF-7 cells through

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endoplasmic reticulum stress. J. Agric. Food Chem. 44 (2012) 11065-11070. 39. J. Cai, J. Yang, D.P. Jones, Mitochondrial control of apoptosis: the role of cytochrome c. Biochimica et Biophysica Acta. 1366 (1998) 139-149.

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Antiproliferative effect of SP on M12.C3.F6 cells is through apoptosis induction. Apoptosis induced by SP is mediated by modulations on ∆Ψm and caspases activation. Chemical composition of SP was analyzed by HPLC-PDA-ESI/MS/MS and NMR. Eighteen flavonoids described in propolis from temperate regions were characterized. Pinobanksin and some its derivatives from SP were apoptotic inducer compounds.

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