semi-volatile compounds and in vitro bioactive properties of Chilean Ulmo (Eucryphia cordifolia Cav.) honey

Food Research International 94 (2017) 20–28

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Volatile and non-volatile/semi-volatile compounds and in vitro bioactive properties of Chilean Ulmo (Eucryphia cordifolia Cav.) honey Francisca Acevedo a,b,⁎, Paulina Torres a, B. Dave Oomah c, Severino Matias de Alencar d, Adna Prado Massarioli d, Raquel Martín-Venegas g, Vicenta Albarral-Ávila e, César Burgos-Díaz f, Ruth Ferrer g, Mónica Rubilar a,h a

Scientific and Technological Bioresource Nucleus, BIOREN, Universidad de La Frontera, Casilla 54-D, Temuco, Chile Department of Basic Sciences, Faculty of Medicine, Universidad de La Frontera, Casilla 54-D, Temuco, Chile Retired, formerly with the National Bioproducts and Bioprocesses Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC, V0H 1Z0, Canada d Escola Superior de Agricultura Luiz de Queiroz (ESALQ), Universidade de São Paulo (USP), Piracicaba, SP, Brazil e Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain f Agriaquaculture Nutritional Genomic Center CGNA, Technology and Processes Unit, Temuco 4791057, Chile g Department de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XIII s/n, 08028 Barcelona, Spain h Department of Chemical Engineering, Universidad de La Frontera, Casilla 54-D, Temuco, Chile b c

a r t i c l e

i n f o

Article history: Received 10 April 2016 Received in revised form 28 October 2016 Accepted 24 January 2017 Available online 27 January 2017 Keywords: Antibacterial Antiproliferative Antioxidant Biological properties Functional food

a b s t r a c t Ulmo honey originating from Eucryphia cordifolia tree, known locally in the Araucania region as the Ulmo tree is a natural product with valuable nutritional and medicinal qualities. It has been used in the Mapuche culture to treat infections. This study aimed to identify the volatile and non-volatile/semi-volatile compounds of Ulmo honey and elucidate its in vitro biological properties by evaluating its antioxidant, antibacterial, antiproliferative and hemolytic properties and cytotoxicity in Caco-2 cells. Headspace volatiles of Ulmo honey were isolated by solid-phase microextraction (SPME); non-volatiles/semi-volatiles were obtained by removing all saccharides with acidified water and the compounds were identified by GC/MS analysis. Ulmo honey volatiles consisted of 50 compounds predominated by 20 flavor components. Two of the volatile compounds, lyrame and anethol have never been reported before as honey compounds. The non-volatile/semi-volatile components of Ulmo honey comprised 27 compounds including 13 benzene derivatives accounting 75% of the total peak area. Ulmo honey exhibited weak antioxidant activity but strong antibacterial activity particularly against gram-negative bacteria and methicillin-resistant Staphylococcus aureus (MRSA), the main strain involved in wounds and skin infections. At concentrations N 0.5%, Ulmo honey reduced Caco-2 cell viability, released lactate dehydrogenase (LDH) and increased reactive oxygen species (ROS) production in a dose dependent manner in the presence of foetal bovine serum (FBS). The wide array of volatile and non-volatile/semi-volatile constituents of Ulmo honey rich in benzene derivatives may partly account for its strong antibacterial and antiproliferative properties important for its therapeutic use. Our results indicate that Ulmo honey can potentially inhibit cancer growth at least partly by modulating oxidative stress. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Honey has been used since antiquity to treat wounds and ailments, and only recently scientists have begun to explain the precise antiseptic and antibacterial effects on human health. Ulmo honeys are considered to be of high quality and are highly esteemed in international markets, although their production area is limited to three countries (Chile, Australia and Tasmania) (Horn & Aira, 2009). Chilean Ulmo honey is pale ⁎ Corresponding author at: Scientific and Technological Bioresource Nucleus, BIOREN, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. E-mail address: [email protected] (F. Acevedo).

http://dx.doi.org/10.1016/j.foodres.2017.01.021 0963-9969/© 2017 Elsevier Ltd. All rights reserved.

amber in color, very aromatic, almond-like taste, compact crystallization with 115 identified pollen types, 84% of which are from nectar producing plants, with a predominance of Lotus and Weinmmannia. It is best identified by the following pollen combinations: Eucryphia (predominant pollen)-Lotus (important pollen)-Eucalyptus (minor pollen) based on the pollen analysis of 37 Ulmo honeys from the Los Lagos region of southern Chile (Horn & Aira, 2009). Ulmo honey originating from Eucryphia cordifolia tree, known locally in the Arucania region as the Ulmo tree has been and continues to be used in traditional medicine in the Mapuche culture (unrecorded, but verbally transmitted) to treat infections and other therapeutic treatments. Its commercialization around the world has increased in the last decade.

F. Acevedo et al. / Food Research International 94 (2017) 20–28

Recently, Ulmo honey supplemented with ascorbic acid was effective in healing wounds caused by burns in guinea pigs (Schencke, Vasconcellos, Salvo, Veuthey, & Del Sol, 2015). The rapid healing was due to enhanced tissue formation, fibroblast activation, and fast keratinocyte/connective tissue re-epithelialization observed at 14 days post injury. However, unsupplemented Ulmo honey alone did not regenerate epidermal layer and showed an initial proliferative phase (Schencke et al., 2015) suggesting that ascorbic acid scavenged reactive oxygen species (ROS) enhancing the anti-inflammatory effect of honey. The same investigators demonstrated the therapeutic benefits of Ulmo honey and oral vitamin C in completely healing venous ulcers in adult patients (Del Sol, Schencke, Salvo, Hidalgo, & Ocharan, 2015). Ulmo honey reduced the possibility of infection, inflammation and edema, leading to rapid healing; it also displayed debriding and non-adherent properties, was easy to apply and remove and was well accepted by the users. The healing properties of Ulmo honey have been ascribed to its antibacterial action, osmolarity, acidity, presence of phytochemicals and enzymes catalyzing hydrogen peroxide production (Del Sol et al., 2015). Besides, due to its antimicrobial properties, honey may serve as a natural food preservative in different products. Many studies have shown the antibacterial activity of different types of honey obtained from various sources. The antibacterial activity of Ulmo honey was superior particularly against methicillin resistant Staphylococcus aureus (MRSA) compared to Manuka honey, the most studied medical grade honey. Ulmo honey also exhibited stronger peroxide attributable antimicrobial effect against five out of seven bacterial isolates tested than Manuka honey (Sherlock et al., 2010). Further investigation on phytochemical of Ulmo honey with antibacterial properties led to the development of disinfectant, sanitizer, bactericide and fungicide using extracted honey phenolics for topical applications in animals and humans (Montenegro & Ortega, 2011). All medical grade honeys are effective in wound healing with significant differences among honeys in their cellular mechanisms depending partly on their botanical origin (Ranzato, Martinotti, & Burlando, 2012). In this regard, Ulmo honey was compared to two unifloral Chilean honeys for their volatile compounds and sensory properties by gas chromatography/mass spectrometry (GC/MS) analysis (Montenegro, Gómez, et al., 2009). Ulmo honey was characterized by its anise scent and floral jasmine note, the distinctive volatiles isophorone and ketoisophorone and predominantly abundant lilac aldehyde and alcohol (Montenegro, Gómez, et al., 2009). However, the antibacterial potential of honey may not be the sole criterion for selecting medical grade honeys (Gómez-Caravaca, Gómez-Romero, Arráez-Román, Segura-Carretero, & Fernández-Gutiérrez, 2006; Majtan, 2014). Hence, the identification of compounds and their contribution to the bioactivity is essential in understanding the mechanisms behind honey-mediated health benefits. Our investigation was therefore aimed to identify the volatile and non-volatile/semi-volatile compounds of Ulmo honey and elucidate its in vitro biological properties by evaluating its antioxidant, antibacterial, cytotoxic, antiproliferative and hemolytic properties. Volatile analysis with solid phase microextraction (SPME)-GC/MS is useful in estimating Ulmo honey authenticity and detecting its adulteration. The results of this work will provide better understanding for increased utilization of Ulmo honey as a functional food. Nowadays, functional foods are an emerging field in food science due to their increasing popularity with health-conscious consumers. Additionally, knowledge of Ulmo honey's biological properties may contribute to our understanding of its antiproliferative mechanism and potential therapeutic benefits in human medicine. 2. Materials and methods Ulmo honey was harvested from various apiaries in Puerto Varas, southern Chile in March 2014. Honeys were stored at 4 °C and homogenized with a stirrer prior to measurements.

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2.1. Extraction of volatiles Ulmo volatiles were determined based on a previously established procedure (Bianchi et al., 2011) with some modifications. Briefly, honey (2 g) and ultrapure water (2 mL) was transferred to a 4 mL vial hermetically capped with PTFE/silicon septum (Chromatographic Specialties Inc.). Samples were allowed to equilibrate at 60 °C for 20 min using a heating module (Heidolph MR 3002; Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). Volatiles were extracted by exposing a 2 cm – 50/30 μm divenylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) Stable Flex SPME-fiber (Supelco, Bellefonte, PA, USA) at 60 °C for 40 min. Prior to each extraction, the fiber was conditioned (270 °C, 1 h) in the injection port of the gas chromatography (GC) as recommended by the supplier. The extraction process was performed in triplicate. 2.2. Extraction of non-volatile/semi-volatile compounds Honey (60 g) was dissolved in 300 mL acidified distilled water (pH 2 adjusted with 1 M HCl) and the solution filtered through cotton wool in a funnel to remove solid particles. The filtrate was mixed with 90 g Amberlite XAD 2 (pore size 9 nm, particle size 0.3–1.2 mm), the mixture transferred to a glass column (35 × 3.4 cm) and eluted with acidified water (150 mL, pH 2) followed by distilled water (180 mL) to remove all saccharides. Compounds absorbed on the solid phase were eluted with 240 mL methanol and the methanol extract was evaporated to dryness in a rotary vacuum evaporator (50 °C). The solids were dissolved in distilled water (3 mL) and extracted three times with diethyl ether (3 mL). The diethyl ether extracts were combined, dried with anhydrous sodium sulphate, the ether removed under nitrogen, weighed and stored at 4 °C. The non-volatile extract was trimethylsilylated with a mixture of pyridine and N-methyl-N-(trimethylsily) trifluoroacetamide (MSTFA, Sigma-Aldrich, USA) (1:1) and kept for 30 min – 8 h at room temperature prior to gas chromatography–mass spectrometry (GC– MS) analysis. The extraction process was duplicate. 2.3. Gas chromatography-mass spectrometry (GC–MS) analyses Extracted volatiles were thermally desorbed at 240 °C for 5 min in the injection port of a GC-2010 coupled with a GCMS-QP2010 Plus (Shimadzu Corp., Tokyo, Japan) and separated on a Rtx®-5MS (5% diphenyl/95% dimethyl polysiloxane, 30 m × 0.25 mm with a 0.25 μm film thickness, Teknokroma Analitica, SA, Barcelona, Spain) fused silica capillary column (da Cunha et al., 2013). The injection port was operated in a splitless mode subjected to a constant flow of ultrahigh-purity helium (36.8 cm/s linear velocity). The initial oven temperature of 40 °C (held for 5 min) was ramped at 5 °C/min to 250 °C, then 10 °C/min increase rate up to 290 °C and held at that temperature for 15 min for a total run time of 47 min. The quadrupole mass spectrometer was operated in electron ionization mode at 70 eV, a source temperature of 230 °C, quadrupole at 200 °C, in the scan range m/z 20–550. The extracted volatiles were injected in triplicate. For the non-volatiles/semi-volatiles, silyated derivatives (100 nL) were injected in triplicate in a heated (280 °C) injection port under split mode (split ratio1:30) and the resulting peak area was used as analytical signal for quantitation. The initial oven temperature of 80 °C (held for 1 min) was ramped at 5 °C/min to 200 °C, held at that temperature for 5 min, then 5 °C/min increase rate up to 250 °C, followed by 3 °C/min increase rate to 300 °C, held at that temperature for 5 min, then 10 °C/min increase rate to 320 °C and held at that temperature for 10 min for a total run time of 66 min. Helium was used as a carrier gas at a constant linear velocity of 36.8 cm/s. The quadrupole mass spectrometer was operated in electron ionization mode at 70 eV, a source temperature of 280 °C, quadrupole at 200 °C, in the scan range m/z 30–600.

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F. Acevedo et al. / Food Research International 94 (2017) 20–28

Spectra were acquired with Shimadzu GCMS solution software and searched against the FFNSC1.3 (Flavor and Fragrance Natural and Synthetic Compounds) and Wiley 8 libraries (Pallisade Corp., Newfield, NY, USA). Compounds were identified through mass spectra (MS) library search and verified by comparison of the relative retention (RIs) using linear retention indices. RIs were determined using a homologous series of C7-C30 n-alkanes based on the calculation of Van den Dool and Kratz (1963). Levels of volatile and non-volatile/semi-volatile components were determined from the average of the replicate chromatograms, calculated and expressed as the relative area values (percentage of total volatile and non-volatile/semi-volatile composition) obtained directly from their total ion current (TIC). 2.4. In vitro bioactive properties 2.4.1. Antioxidant properties The antioxidant activity of Ulmo honey non-volatile extracts was measured against free radical DPPH• and reactive oxygen species (ROO•, HOCl and O2•−) according to Melo et al. (2015). A microplate reader (SpectraMax M3, LLC, Molecular Devices Corp., Sunnyvale, CA, USA) was used to measure absorbance (560 nm for superoxide scavenging radical O2•−) and fluorescence (485 and 528 nm excitation and emission, respectively for ROO• scavenging capacity and HOCl). Results for DPPH• and Peroxyl radical (ROO•) scavenging assays were expressed as Trolox equivalents (μmol Trolox/mg or g honey extract) and those for hypochlorous acid (HOCl) and superoxide anion radical (O2•−) scavenging as IC50 (μg/mL). The analyses were conducted in triplicate. 2.4.2. Antibacterial properties The antibacterial properties of Ulmo honey were tested against nine bacterial strains: Bacillus subtilis (ATCC 6633), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Staphylococcus aureus methicillin-resistant, (MRSA) (ATCC 4330); Aeromonas hydrophila (CECT 839T), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883T), Pseudomonas aeruginosa (ATCC 27853), Salmonella enterica (ATCC 14028) and one yeast Candida albicans (ATCC 10231). Bacterial strains were grown on Mueller Hinton broth (MHB) and Sabouraud dextrose broth (SDB) for Candida albicans, both medium acquired from Oxoid U.K. The minimum inhibitory concentration (MIC) of Ulmo honey samples were performed according to Sherlock et al. (2010). MIC defined as the lowest concentration of honey required to inhibit microbial growth were determined in sterile 96 well round bottomed polystyrene microtiter plates (Thermo Fisher Scientific, Ñuñoa, Santiago, Chile). Thus, 9 serial dilutions of honey were prepared aseptically for MIC assay, resulting in final concentrations of: 50 to 0.2% (v/v) in MHB or SDB. Bacterial and yeast cultures were prepared to equal 0.5 McFarland standard (1 × 108 cfu/mL). 10 μL of 0.5 McFarland standardized culture was added to 190 μL of test honey, at each concentration, in each well (4 replicates per dilution, 9 dilutions tested). Control wells contained only broth (negative control) or bacteria and broth (positive control). Plates were incubated in the dark at 37 °C. The optical density was determined at 600 nm just prior to (T0) and after 24 h incubation (T24). 2.4.3. Cytotoxic and antiproliferative properties Dulbecco's Modified Eagle's Medium (DMEM), trypsin, penicillin and streptomycin were supplied by GIBCO (Paisley, Scotland). Non-essential amino acids, foetal bovine serum (FBS), Dulbecco's phosphatebuffered saline (D-PBS) along with other chemicals, were supplied by Sigma (St. Louis, MO). Tissue culture supplies were obtained from Costar (Cambridge, MA). 2.4.3.1. Cell culture conditions of Caco-2 cells. Caco-2 cells were provided by the American Type Culture Collection (Rockville, MD) and cultured as previously described (Martín-Venegas et al., 2006). The cells (passages 67–75) were routinely grown in plastic flasks at 104 cells/cm2

density and cultured in standard DMEM (containing 4.5 g/L D-glucose, 2 mMol/L L-glutamine, 1% (v/v) non-essential amino acids, 10% (v/v) heat-inactivated FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in modified atmosphere (5% CO2 in air). Cells grown to approximately 80% confluence were released by trypsinization and subcultured at 104 cells/cm2 density on 24-well plate clusters. Experiments were performed on preconfluent cultures, 4 days after seeding. The cells were then incubated for 48 h in the presence of FBS with increasing Ulmo concentration (0.25 to 8% in DMEM). Cultures were also incubated in the absence of growth factors (without FBS) as a negative control. Eight replicates were conducted. 2.4.3.2. Cell proliferation and lactate dehydrogenase (LDH) activity. After the incubation period, the cultures were washed with D-PBS, trypsinized and viable cells counted under a microscope using ethidium bromide/acridine orange stain (Parks et al., 1979). Results were expressed as number of viable cells/cm2 of monolayer. Product cytotoxicity was assessed from lactate dehydrogenase (LDH) activity as an indicator of membrane integrity (Cook & Mitchell, 1989). LDH was quantified in the incubation media at the end of the experiment following the manufacturer's instructions (Life Technologies, Paisley, Scotland). Results were expressed as absorbance units. Eight replicates were run. 2.4.3.3. Intracellular reactive oxygen species (ROS) production. Intracellular reactive oxygen species (ROS) were quantified using a commercial Intracellular ROS assay kit (OxiSelect™ kit, Cell Biolabs Inc., San Diego, CA). The fluorescence was measured (480 and 530 nm, excitation and emission, respectively, Wallac 1420 Victor3, Perkin-Elmer) at 0 and 48 h of the incubation period. The increased fluorescence (FI) resulting from oxidation of the dye by intracellular ROS, was calculated as (F48 h − F0 min) / F0 min × 100. The analyses were performed in triplicate. 2.4.4. Hemolytic activity Hemolytic activity was determined as described previously (BurgosDíaz et al., 2013). Briefly, human erythrocytes were prepared just before the experiments using red blood cell concentrates supplied by Universidad de La Frontera (Chile). Cells were washed twice with a buffer (150 mM NaCl, 5 mM HEPES, pH 7.4). After centrifugation, the pellet was finally suspended in the same volume of buffer prior to use. The erythrocyte concentrate was diluted with a buffer (150 mM NaCl, 5 mM HEPES, pH 7.4) to obtain a suspension with A540 = 1. Hemoglobin release was determined after red blood cells incubation with Ulmo honey samples at different concentrations (0–75 g/L), by measuring the absorbance at 540 nm after pelleting the membranes by centrifugation (Neofuge 15R centrifuge, Heal Force, Victoria, Australia, 5000 ×g, 2 min). The total amount of hemoglobin was established by lysing the erythrocytes with distilled water. Ulmo honey solutions were added from a stock of honey solution in the same buffer. All operations were carried out at 4 °C and in triplicate. 2.4.5. Statistical analyses Data are expressed as mean ± standard deviation of the mean (M ± SD) and statistical comparison between groups was carried out utilizing analysis of variance (ANOVA) followed by Tukey's test. Significance was accepted at p ≤ 0.05. 3. Results and discussion 3.1. Gas chromatography-mass spectrometry (GC–MS) analyses A total of 50 headspace volatile compounds isolated by SPME were tentatively identified in Ulmo honey by GC–MS (Table 1). The primary group (peak area N 1%) consisting of 21 compounds accounted for 88.3% of the total peak area. The most important flavor components of Ulmo honey were (in % peak area): benzaldehyde, 14.12; octane, 8.16;

F. Acevedo et al. / Food Research International 94 (2017) 20–28

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Table 1 Linear retention indices, percent area and important ions present in the mass spectra of volatile compounds in Ulmo honey identified by GC–MS. No

Volatile compounds

RT (min)

RIc

1 2 3 4 5 6 7 8 9 10 11 12 13 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 42 43 44 45 46 47 48 49

Octane 2-Heptanol Benzaldehyde Hexadecanoic acid, ethyl ester Octanal α-Terpinene p-Methylanisole m-Cymene Limonene 2-Ethyl hexanol Benzyl alcohol Benzeneacetaldehyde Cyclohex-3-ene-1-methanol 1-Butanone, 3,3-dimethyl-1-phenyl Linalool oxide p-Cymenene 2-Nonanone Linalool Nonanal 2-Phenylethanol Isophorone Hexylbenzene Pinocarveol,transLinalool oxide, trans4-Ketoisophorone Isphorone, 2-hydroxy4-Vinylanisole Pinocarvone Ethyl linalool Ethyl benzoate Dill ether (−)-Beta-Fenchol Ethyl octanoate Myrtenal Decanal Bornylene 2-Hydroxyisophorone Benzeneacetic acid, ethyl ester 4-Methoxybenzaldehyde Anethole Ethyl nonanoate 2′-Aminoacetophenone Edulan, transHydrocinnamate, ethyl1,1,6-Trimethyl-1,2-dihydronaphtalene β-Damascenone Dodecanal Ethyl 4-methoxybenzoate Benzoic acid b2-[[[4-[4-hydroxy-4-methylpentyl]-, 3-cyclohexen-1-yl]methylene]amino]-, methyl-N ester (Lyrame) Cuminone

7.197 11.142 13.259 14.653 14.773 15.2 15.375 15.474 15.65 15.686 16.029 16.193 16.834 17.003 17.114 17.665 17.753 17.983 18.122 18.567 18.699 18.981 19.275 19.375 19.409 19.533 19.676 20.042 20.076 20.227 20.71 20.863 20.947 21.052 21.213 21.99 22.143 22.408 22.706 23.591 23.758 24.104 24.385 25.291 25.463 26.263 26.78 27.948 31.4

802 906 963 1002 1005 1018 1023 1026 1031 1032 1043 1048 1067 1072 1075 1092 1094 1101 1106 1120 1125 1134 1144 1147 1148 1152 1157 1169 1170 1175 1191 1196 1198 1202 1207 1235 1240 1250 1260 1291 1297 1310 1320 1355 1361 1391 1411 1458 1602

32.755

1663

50

RIr 899.4 962.7 1002.8 1017.1 1023.8 1022 1029.5 1030.6 1036.9 1045.9

1075.1 1087.9 1092.5 1099.0 1103.3 1114.9 1127.2 1140.0 1147.4

1160.6 1171.3

1196.2 1192.0 1205.4

1285.2

1385.5 1408.1

% Area

SD

SE

Ion (m/z, abundance in parentheses)

8.16 0.56 14.12 1.56 0.72 0.12 b0.1 0.19 0.17 1.57 1.59 3.58 0.87 b0.1 3.54 1.70 b0.1 3.62 7.97 0.42 6.38 0.34 0.18 0.30 3.67 0.23 4.58 0.21 0.78 2.44 0.59 0.32 0.55 1.94 1.92 0.36 0.76 0.41 7.02 0.97 1.09 0.76 0.31 0.28 0.34 5.50 0.23 1.16 5.22

1.19 0.09 1.51 0.67 0.15 0.01 – 0.03 0.12 0.62 0.15 0.36 0.02 – 0.39 0.05 – 0.08 1.40 0.04 0.09 0.03 0.02 0.04 0.58 0.03 0.37 0.03 0.05 0.99 0.06 0.03 0.09 0.02 0.47 0.01 0.11 0.03 0.70 0.02 0.19 0.14 0.01 0.04 0.02 0.04 0.04 0.07 2.63

0.69 0.05 0.87 0.39 0.09 b0.01 – 0.02 0.07 0.36 0.09 0.21 0.01 – 0.23 0.03 – 0.04 0.81 0.02 0.05 0.02 0.01 0.02 0.33 0.02 0.21 0.02 0.03 0.57 0.04 0.02 0.05 0.01 0.27 b0.01 0.06 0.02 0.40 0.01 0.11 0.08 b0.01 0.02 b0.01 0.02 0.02 0.04 1.51

43 (100), 85 (52), 57 (44), 41 (42), 71 (30), 56 (24) 28 (100), 45 (74), 55 (32), 70 (19), 83 (9), 98 (5) 106 (100), 77 (98), 105 (97), 51 (39), 50 (20), 78 (15) 88 (100), 43 (62), 99 (52), 70 (34), 61 (22), 115 (10) 41 (100), 43 (99), 56 (96), 57 (91), 44 (79), 55 (68) 93 (100), 121 (86), 136 (52), 105 (43), 119 (38), 77 (32) 122 (100), 121 (51), 77 (47), 107 (40), 79 (29), 91 (28) 119 (100), 91 (37), 43 (35), 134 (30), 123 (17), 120 (17) 93 (100), 68 (88), 67 (77), 79 (44), 91 (41), 94 (39),136 (23) 57 (100), 41 (37), 70 (35), 43 (29) 55 (28), 56 (22) 79 (100), 108 (74), 77 (61), 107 (47), 51 (22), 91 (14) 91 (100), 92 (29), 65 (22), 120 (20), 39 (9), 63 (6) 107 (100), 81 (26), 79 (24), 125 (19), 122 (18), 55 (18) 105 (100), 77 (80), 120 (32), 51 (25), 43 (14), 57 (14) 79 (100), 93 (80), 43 (76), 59 (66), 94 (64), 55 (37) 117 (100), 132 (97), 115 (59), 91 (49), 59 (34), 92 (21) 142 (4), 58 (100), 43 (82), 71 (24), 57 (23), 59 (21), 41 (15) 93 (100), 71 (76), 41 (67), 55 (53), 43 (50), 69 (49) 57 (100), 43 (87), 41 (82), 71 (79), 82 (77), 56 (61) 91 (100), 92 (62), 122 (26), 65 (20), 39 (6), 51 (6) 82 (100), 138 (21), 54 (17), 39 (11), 95 (6), 83 (5) 91 (100), 92 (81), 43 (21), 79 (13), 77 (12), 39 (12) 91 (100), 92 (79), 55 (67), 119 (53), 41 (52), 134 (34) 55 (100), 43 (66), 93 (56), 67 (50), 41 (39), 11 (38) 68 (100), 96 (76), 152 (28), 40 (25), 39 (22), 41 (12) 70 (100), 154 (55), 98 (44), 55 (40), 139 (28) 134 (100), 119 (60), 91 (51), 65 (25), 135 (10), 39 (8) 53 (100), 81 (95), 108 (71), 79 (55), 107 (52), 41 (49), 150 (8) 55 (100), 43 (53), 93 (48), 71 (41), 41 (39), 67 (38) 105 (100), 77 (46), 122 (32), 150 (17), 51 (15), 56 (10) 137 (100), 69 (39), 109 (34), 41 (21), 67 (20), 91 (19), 152 (3) 59 (100), 93 (84), 121 (62), 136 (51), 81 (41), 67 (36) 88 (100), 57 (38), 101 (37), 70 (34), 73 (28), 127 (27) 79 (100), 107 (60), 91 (41), 77 (38), 108 (31), 105 (28) 57 (100), 43 (87), 41 (87), 55 (78), 82 (57), 70 (56) 93 (100), 121 (76), 108 (48), 43 (26), 79 (26), 91 (25) 84 (100), 56 (82), 126 (65), 140 (47), 69 (46), 83 (46), 168 (38) 91 (100), 164 (15), 129 (15), 65 (13), 92 (11), 29 (8) 135 (100), 136 (73), 77 (34), 107 (22), 92 (14), 64 (9) 148 (100), 147 (54), 117 (38), 105 (31), 91 (27), 77 (27) 88 (100), 101 (39), 70 (32), 73 (27), 43 (23), 41 (22) 120 (100), 135 (68), 92 (56), 65 (36), 43 (14), 39 (14) 177 (100), 91 (33), 133 (29), 105 (28), 57 (23), 43 (23) 104 (100), 91 (50), 105 (45), 107 (43), 178 (24), 79 (23) 157 (100), 142 (72), 141 (31), 172 (26), 115 (18), 158 (12) 69 (100), 121 (70), 105 (28), 41 (24), 120 (16), 91 (14), 190 (13) 57 (100), 41 (83), 55 (82), 43 (73), 82 (70), 69 (54) 135 (100), 163 (60), 77 (31), 152 (20), 180 (15) 92 (15) 149 (100), 177 (23), 176 (14), 150 (10), 65 (9), 93 (9)

0.66

0.22

0.12

147 (100), 43 (82), 162 (72), 119 (26), 91 (20), 77 (18)

RT: retention time. RI: retention indices-linear indices (RIc: calculated; RIr: Babushok, Linstrom, and Zenkevich (2011)). SD: standard deviation; SE: standard error.

nonanal, 7.97; 4-methoxybenzaldehyde, 7.02; isophorone, 6.38; βdamascenone, 5.5; lyrame (benzoic acid-2-[[[4-[4-hydroxy-4methylpentyl]-,3-cyclohexen-1-yl]methylene]amino]-, methyl- ester), 5.22 and 4-vinylanisole, 4.58. These eight compounds (peak area N 4%) together comprised over 1/ 2 (58%) of the total peak area. Twenty-six compounds were present at low levels (0.1 to b1% peak area) accounting for 11.6% of total peak area, whereas another 3 compounds occurred at extremely low/trace amounts (b0.1% peak area). Volatile benzene derivatives/aromatics (16 compounds) were dominant in Ulmo honey. Examples of such compounds included well-known flavor chemicals such as 4-vinylanisole (1-methoxy-4-vinyl-benzene), benzylaldehyde, ethyl benzoate, ethyl anisate (ethyl 4-methoxybenzoate), lyrame, linalool (3,7-dimethylocta-

1,6-dien-3-ol) and damascenone. A previous study comparing the volatiles of Ulmo honey with two Chilean honeys showed that it consisted of 28 compounds including 15 terpenes, 5 norisoprenoids and 8 phenolics with the predominant lilac aldehyde and alcohol accounting for about 75% of the total peak area (Montenegro, Gómez, et al., 2009). Five volatile compounds, isophorone, ketoisophorone, damascenone, limonene (1-methyl-4-(1-methylethenyl)-cyclohexene) and anethol (1-methoxy-4-(1-propenyl)benzene) were common to Ulmo honey in our study and those of Montenegro, Gómez, et al. (2009). Benzylaldehyde, benzeneacetaldehyde, benzyl alcohol, nonanal and linalool have been identified in several honeys and particularly from the central valley of Ñuble province in Chile (Barra, Ponce-Díaz, & Venegas-Gallegos, 2010). Benzeneacetaldehyde is considered one of

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the compounds that give honey its characteristic honey aroma (CastroVázquez, Díaz-Maroto, & Pérez-Coello, 2007). The high abundance of lyrame, 4-vinylanisole, benzaldehyde together with nonanal, damascenone, decanal, anethol and other odor-active compounds characterizes the specific aroma of Ulmo honey. The fragrance of lyrame, 4vinylanisole and anethol correspond to the primary sensory descriptive aroma attributed to those of anise and jasmin in Ulmo honey (Montenegro, Gómez, et al., 2009). 4-Vinylanisole occurs naturally in Thymus, Ramulus, Sambucus, Hedyotis and lignin degradation by mushroom (Tellez et al., 2004). Lyrame is a fragrant (bright yellow viscous liquid) with a floral (fresh lily floral sweet orange blossom odor) perfuming agent used widely in deodorants and fine fragrances up to 5%. However, a natural source of lyrame has never been identified, although other polyhydroxy benzoic acid derivatives occur in diverse plants/fungal/bacterial species primarily protecting the organisms from their biological environment (Khadem & Marles, 2010). Compounds such as aminoacetophenone, benzaldehyde, benzylalcohol, decanal, linalool (3,7-Dimethyl-1,6-octadien-3-ol), linalool oxide (2,6-dimethyl-2,7octadien-6-ol), nonanal, octanal and 2-phenylethanol have been previously reported in honey (Alissandrakis, Kibaris, Taranatilis, Harizanis, & Polissiou, 2003; Piasenzotto, Gracco, & Conte, 2003). Three compounds, cis-linalool oxide, 2-phenylethanol and p-anisic acid were also present in and characteristic of kanuka honey (Beitlich, Koelling-Speer, Oelschlaegel, & Speer, 2014). Two of the volatile compounds, lyrame and (E)-anethol have never been reported before as honey compounds. Honey produced in different areas of the central valley of Ñuble province in Chile also had four volatile compounds (1,3-propanodiol; 2methyl butanoic acid; 3,4-dimethyl-3-hexen-2-one and 6-methyl-5octen-2-one) never reported before as honey compounds (Barra et al., 2010). The non-volatile/semi-volatile components of Ulmo honey comprised 27 compounds including 13 benzene derivatives accounting 75% of the total peak area (Table 2). The most abundant benzene derivatives; p-anisylacetic acid (benzeneacetic acid, 4-methoxy-), ethyl benzoate, p-anisic acid (4-methoxybenzoic acid) and benzenepropanoic

acid, α-oxo-contributed 76% to the total non-volatile/semi-volatile peak area. The primary non-volatile/semi-volatile constituents (peak area N 1%) consisted of 14 compounds accounted for 98% of the total peak area; 10 compounds were present at low levels (0.1 to b 1% peak area) and 3 compounds at extremely trace amounts (b0.1% peak area). Ethyl benzoate, p-anisylacetic acid, benzeneacetic acid and other benzene derivatives have previously been identified in other honey (Alissandrakis et al., 2003; Jerković, Tuberso, Gugić, & Bubalo, 2010; Moniruzzaman et al., 2014; Piasenzotto et al., 2003).

3.2. Bioactive properties 3.2.1. Antioxidant properties Ulmo honey generally exhibited weak antioxidant or antiradical activity (Table 3). However, the DPPH value of Ulmo honey was over twice (2.7 ×) those of Heather honey originating from Małoposka, Poland (Wilczyńska, 2010). The IC50 value for the hypochlorous acid scavenging assay obtained in this study was 24.56 mg/mL, similar to Eucalyptus honey from Republic of Mauritus (24.03 mg/mL) reported by Dor & Mahomoodally (2014). The antioxidant activity of Ulmo honey resided within the non-volatile/semi-volatile extract indicated by its higher potency compared to the honey (Table 3). This probably reflects the phenolic contribution to the antioxidant activity since the non-volatile/semi-volatile extract was obtained similar to the procedure described for phenolic-enriched extract of Ulmo honey (Montenegro & Ortega, 2011). Thus, the HOCl scavenging activity of the non-volatile/ semi-volatile extract was tenfold more potent than those of the phenolic acid p-coumaric acid. The ORAC value of the non-volatile/semivolatile was comparable to those of low-antioxidant honeys (acacia, dandelion, and clover) and to some from the metropolitan region of Chile (Beretta, Granata, Ferrero, Orioli, & Facino, 2005; Muñoz, Copaja, Speisky, Peña, & Montenegro, 2007). Superoxide radical scavenging activity was not detected in Ulmo honey or its non-volatile/semi-volatile fraction.

Table 2 Linear retention indices, percent area of each non-volatile/semi-volatile compound and important ions present in the mass spectra of silylated compounds in Ulmo honey identified by GC– MS. No

Non-volatile/semi-volatile compounds

RT (min)

RI

% Area

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Methyl n-acetylcarbamothioate Acetic acid, ester 1,1,1-Triethoxyethane 2-Hexene, 1-(1-ethoxyethoxy) Benzoic acid, ester Benzeneacetic acid Phenoxyacetate 2-Hydroxy-2-phenylacetic acid 1,2-Benzenedicarboxylic acid, dimethyl ester p-Anisic acid Benzenepropanoic acid, alpha 4-Hydroxybenzoic acid Cinnamic acid, p-methoxy-, ester Benzeneacetic acid, 4-methoxyVanillic acid 4-(2-Acetyl-5,5-dimethyl-2-cyclopenten-1-ylidene)-2-butanone 5-Isopropenyl-2,3-dimethyl-1-cyclohexen-1-yl ether [2.2]Paracyclophane-1,9-diene p-Coumaric acid Non-8-en-1-yn-3-one Hexadecanoic acid, ester Methyl-5-[1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl]-3methyl-2,4-pentadienoate 2-(3,5-Di-tert-butyl-4-phenyl)-n-methylpyrrole Octadecanoic acid, ester 3,6,9,12-Tetraoxatetracosan-1-ol 1-(1,3-Benzodioxol-5-ylmethyl)-4-(3,4-dimethoxyphenyl)sulfonylpiperazine Benzeneethanamine, 3-methoxy-n-[(pentafluorophenyl)methylene]-beta

5.08 6.75 8.88 10.23 10.84 12.14 12.81 15.48 16.12 17.62 19.23 20.08 21.67 21.92 23.25 24.08 25.24 25.40 26.75 27.85 28.84 32.04

944 1034 1128 1183 1207 1258 1284 1389 1415 1476 1544 1580 1650 1661 1722 1761 1814 1821 1883 1933 1979 2100

4.72 0.39 3.32 2.21 12.12 0.24 0.33 1.27 0.31 11.06 3.14 1.88 2.20 41.29 0.77 5.43 0.95 4.80 0.87 0.46 1.13 4.51

32.38 32.62 34.88 34.99 38.83

2129 2150 2294 2299 2506

0.08 0.28 0.02 0.04 0.55

23 24 25 26 27

RT: retention time; SD: standard deviation; RI: linear retention indices.

SD

0.11 0.30 6.19

0.55 0.17 1.40 0.19 0.92 0.74 13.12 0.47 0.06 1.22 6.63 0.06 0.45 0.32 3.49 0.03 0.04 0.04 0.57

Ion (m/z, abundance in parentheses) 43 (100), 91 (50), 133 (43), 59 (39), 61 (37), 75 (28) 147 (100), 73 (92), 177 (20), 66 (19), 45 (16), 148 (15) 61 (100), 75 (58), 89 (55), 45 (51), 59 (49), 43 (41) 73 (100), 45 (62), 75 (15), 103 (10), 133 (9), 89 (7) 179 (100), 135 (92), 105 (85), 77 (63), 180 (14), 194 (9) 73 (100), 75 (30), 164 (19), 91 (18), 193 (12), 74 (11) 73 (100), 224 (44), 209 (25), 75 (22), 181 (19), 45 (17) 179 (100), 73 (87), 207 (57), 133 (47), 163 (42), 147 (35) 163 (100) 133 (37), 207 (31), 77 (25), 76 (12), 164 (11) 135 (100), 165 (90), 209 (83), 77 (26), 92 (20), 224 (20) 73 (100), 193 (71), 147 (57), 220 (17), 45 (13), 194 (12) 167 (100), 223 (93), 73 (81), 193 (43), 268 (24), 282 (22) 161 (100), 235 (87), 191 (77), 250 (41), 133 (34), 121 (30) 209 (100), 73 (50), 210 (16), 147 (12), 283 (10), 135 (8) 297 (100), 267 (94), 73 (58), 312 (53), 253 (47), 223 (44) 121 (100), 43 (72), 73 (69), 163 (51), 75 (31), 206 (2) 73 (100), 353 (68), 223 (64), 195 (44), 43 (41), 238 (9) 73 (100), 333 (58), 348 (46), 334 (17), 349 (14), 75 (13) 293 (100), 308 (85), 73 (78), 219 (75), 249 (62), 309 (22) 125 (100), 140 (73), 73 (69), 75 (36), 43 (31), 156 (28) 328 (5), 117 (100), 313 (69), 73 (63), 75 (53), 132 (40) 278 (4), 190 (100), 73 (90), 134 (85), 183 (63), 162, 75 (46) 357 (100), 73 (38), 358 (32), 372 (12), 359 (11), 207 (7) 117 (100), 341 (77), 73 (67), 75 (52), 132 (47), 129 (42) 116 (100), 117 (79), 73 (78), 57 (69), 71 (50), 43 (50) 135 (100), 105 (31), 219 (24), 43 (20), 85 (13), 79 (12) 192 (100), 297 (65), 73 (63), 401 (44), 416 (39), 193 (25)

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Table 3 Scavenging activity of Ulmo honey and Ulmo honey non-volatile/semi-volatile extract, measured through synthetic DPPH free-radical scavenging and reactive oxygen species (ROO•, HOCl and O2•−).

Ulmo honey 24.56 × 103 ± 1.04 × 103 Ulmo honey non-volatile/semi-volatile extract a

ROO• (μmol Trolox/mg)

HOCl IC50 (μg/mL)

O2•− IC50 (μg/mL)

DPPH• (μmol Trolox/g)

0.91 × 10−3 ± 0.00 nd 2.02 ± 0.21

0.039 ± 0.00 11.06 ± 0.47

nda

87.14 ± 1.13

nd: no activity was determined at maximum concentration of 1666 μg/mL.

3.2.2. Antibacterial properties Ulmo honey exhibited strong antibacterial activity against Klebsiella pneumoniae, Salmonella enterica and Staphylococcus aureus MRSA (MIC 3.1%, v/v) (Table 4). Other bacterial strains were sensitive at moderate (6.3%, v/v) (Staphylococcus aureus) or higher (12.5%, v/v) concentrations indicating that Ulmo honey displayed strong inhibitory activity against most pathogenic microorganism generally found in wounds and ulcers (Mihai et al., 2014; Janda & Abbott, 2010). The antibacterial activity of Ulmo honey against Staphylococcus aureus MRSA was twice as potent as those of Ulmo 90 and four times those of Manuka honey, although it had the same potency against Escherichia coli and Pseudomonas aeruginosa (Sherlock et al., 2010). This may probably be due primarily to hydrogen peroxide production advocated by Sherlock et al. (2010) and demonstrated in our study by the absence of superoxide radical activity (Table 3). The results indicate that Ulmo honey can potentially be used as a therapeutic agent to treat drug-resistant bacteria such as Staphylococcus aureus MRSA, usually the main bacterial strain involved in difficult to treat skin infections (David & Daum, 2010). Ulmo honey also inhibited the growth of the most common food transmitted bacteria such as Salmonella enterica and Klebsiella pneumoniae that cause gastrointestinal infections. These pathogens were more sensitive to Ulmo honey with lower MIC compared to other type of honeys (6.25–12.5%, v/v) (Ewnetu et al., 2013; Lin et al., 2011). Bacillus subtilis, Enterococcus faecalis and Candida albicans growth inhibition were unaffected by Ulmo honey similar to those observed with other honeys (Taormina et al., 2001; Omafuvbe, & Akanbi, 2009). Ulmo honey was most effective on gram-negative bacteria inhibiting five strains at different concentrations, but only two gram-positive bacteria. These results are similar to those observed in Malaysian honey from different floral sources (Vallianou et al., 2014). Ulmo honey phenolic extract had lower bacteriostatic activity (MIC 18.8%, v/v) against human pathogens Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica (Rizzardini, 2007) than Ulmo honey suggesting that phenolic compounds were not the main contributor to its antimicrobial properties as proposed by Estevinho et al. (2008). Furthermore, other unifloral Chilean honeys had higher MIC for Pseudomonas aeruginosa and Staphylococcus aureus than our Ulmo honey and their phenolic extracts were inactive against Escherichia coli (Montenegro, Salas, Pena, & Pizarro, 2009).

Volatile and non-volatile/semi-volatile compounds probably contributed partly to the antibacterial activity of Ulmo honey. Many of these compounds used as flavor and aroma ingredients exhibit strong antibacterial properties. For example, major compounds identified in our study, benzaldehyde, ethyl benzoate, linalool, benzyl alcohol and ethyl linalool were effective (MIC 0.01%, v/v) against E. coli and S. aureus (Morris, Khettry, & Seitz, 1979). Furthermore, the abundance of benzaldehyde derivatives and p-anisic acid in Ulmo honey may confer additive and/or synergistic antibacterial activity observed in this study (Friedman, Henika, & Mandrell, 2003; Van Chuyen, Kurata, Kato, & Fujimaki, 1982). Some volatile benzene derivatives dominant in Ulmo honey may also exert antibacterial activity similar to those demonstrated in New Zealand native honeys (Manyi-Loh, Ndip, & Clarke, 2011). 3.2.3. Antiproliferative effect and ROS induced by Ulmo honey in Caco-2 cancer cells Caco-2 cell viability in the presence of FBS (positive control containing growth factors) was dependent on Ulmo honey concentration (Fig. 1). Cell viability was unaffected at low honey concentrations (0.25 and 0.50%), but considerably reduced (48 and 92%) at higher concentrations (1 and 2% respectively). Similar cytotoxic effects have been reported for Gelam honey extract at high concentrations and no effect on HIT-T15 cell viability at low concentrations (Batumalaie, Qvist, Yusof, Ismail, & Sekaran, 2014). LDH release (Fig. 2) an indicator of cell membrane integrity was significantly lower in cell cultures preincubated with 0.25 or 0.50% Ulmo honey than in those treated at higher concentrations. Furthermore, LDH release was concentration dependent and increased linearly (Y = 0.2851x + 0.4415, r2 = 0.9326) with Ulmo honey concentration (0.25–4%). The absence of cytotoxic damage and viability in Caco-2 cell suggest that at low concentration Ulmo honey can act as a free-radical scavenger with potential for direct topical application. In contrast, Caco-2 cells were detached from the substrate at the highest concentrations (4% and 8%) indicating total cell viability reduction (presumably subsequent apoptotic cell death) corroborated by significant increase in LDH release. The LDH release at low Ulmo

Table 4 Minimum inhibitory concentration (% v/v) of Ulmo honey against different microorganism. NA: non-activity. Test organisms

MIC (%)

Aeromonas hydrophilia CECT 839T Escherichia coli ATCC 25922 Klebsiella pneumoniae ATCC 13883T Pseudomonas aeruginosa ATCC 27853 Salmonella enterica ATCC 14028 Staphylococcus aureus ATCC 25923 Staphylococcus aureus ATCC 4330 MRSA Bacillus subtilis ATCC 6633 Candida albicans ATCC 10231 Enterococcus faecalis ATCC 29212

12.50 12.50 3.10 12.50 3.10 6.30 3.10 NA NA NA

NA: non-activity.

Fig. 1. Number of viable cells in the presence of increasing concentrations of Ulmo honey (0.25–8%) in non-differentiated Caco-2 cells. Results in the absence and presence of FBS were included as negative and positive control, respectively. Results are expressed as mean ± SEM of n = 8 monolayers. The asterisk denotes significant difference compared to the positive control (FBS) condition (p b 0.05).

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Fig. 2. A. Lactate dehydrogenase (LDH) activity in the presence of increasing Ulmo honey concentrations (0.25–8%) in non-differentiated Caco-2 cells. Results in the absence and presence of FBS were included as negative and positive control, respectively. Results are expressed as mean ± SEM of n = 8 monolayers. The asterisk denotes significant difference compared to the positive control (FBS) condition (p b 0.05). B. Intracellular ROS production (FI, fluorescence increase) in the presence of increasing concentrations of Ulmo honey (0.25–2%) in nondifferentiated Caco-2 cells. C. Results in the absence and presence of FBS were included as negative and positive control, respectively. The results are calculated as (F48 h − F0 min) / F0 min × 100 and expressed as mean ± SEM of n = 8 cultures. Mean values labeled with different letters are significantly different (p b 0.05).

honey concentrations corresponds to the LDH reduction observed in male albino rats treated with 5% Palestinian honey for 20 days resulting in spermatogenesis induction (Abdul-Ghani, Dabdoub, Muhammas, Abdul-Ghani, & Qazzaz, 2008). Ulmo honey reduced ROS production considerably compared to the negative control (Fig. 2A). Our result corresponds to the ability of 10% Brazilian Pampa biome honey in blocking ROS production and other deleterious outcomes of hypoxia (alterations in lifespan, locomotor ability and enzyme activities) in the fly model (Cruz et al., 2015). ROS production was negligible at 0.25% Ulmo honey concentration and increased linearly (Y = 14.896× + 7.86, r2 = 0.9628) with concentration in the presence of FBS (Fig. 2B). This ROS production corresponding to the linear LDH release may be indicative of the antimicrobial activity (Cooke, Dryden, Patton, Brennan, & Barrett, 2015) and/or the Caco-2 cell membrane integrity or sensitivity to Ulmo honey. ROS production in

hamster pancreatic (HIT-T15) cells cultured under hyperglycemic condition increased with glucose alone (20 and 50 mM) compared to control and was significantly reduced by Gelam honey extract and flavonoids (Batumalaie et al., 2014). ROS production in Ulmo honey treated Caco-2 cells in the presence of FBS was similar to those observed in colon cancer (HCT-15 and HT-29) cells presumed to be associated with p53 induction leading to apoptosis in treated cells (Jaganathan & Mandal, 2009). The antiproliferative effects of honey has mostly been ascribed to its antioxidant properties, although the antiproliferative mechanisms on tumor cells may probably be due to combined effects of different components present in the whole honey (Staver et al., 2014). Several compounds identified in Ulmo honey such as ketoisophorone, 2acetylfuran, fatty acids and other compounds have been reported to induce antiproliferative effects on keloid fibroblasts (Nurul Syazana,

F. Acevedo et al. / Food Research International 94 (2017) 20–28

Halim, Gan, & Shamsuddin, 2011). Our results indicate that Ulmo honey can inhibit cancer growth at least partly by modulating oxidative stress. 3.2.4. Hemolytic activity The hemolytic activity of Ulmo honey was investigated in human erythrocytes considered as a simple cellular model and a convenient membrane system to evaluate honey-membrane interactions. Ulmo honey had no hemolytic activity on human red blood cells at concentration up to 7.5% (w/v) after 1 h contact at 37 °C (data not shown). The absence of hemolytic activity may partly be due to the low antioxidant and free radical scavenging activity of Ulmo honey inability to reduce lipid peroxidation thereby destabilizing cell membranes (Chaudhuri et al., 2007). Similar erythrocyte protection has been reported against hemolysis that was not associated with antioxidant and/or other parameters in South African honey (Serem & Bester, 2012). Our results suggest that cell membrane disruption may not be the targeted mechanism by which Ulmo honey exert its antibacterial/antiproliferative properties. 4. Conclusions This is the first report defining Ulmo honey by its wide array of volatile and non-volatile/semi-volatile constituents rich in benzene derivatives that may partly account for its strong antibacterial and antiproliferative properties. The abundant benzaldehyde and benzene derivatives in Ulmo honey may be partly responsible for its strong bioactivity (antimicrobial, antibacterial, antiproliferative and cytotoxic properties). Ulmo honey can potentially be used as a therapeutic agent to treat drug-resistant bacteria such as Staphylococcus aureus MRSA and gastrointestinal infections caused by Salmonella enterica and Klebsiella pneumoniae. It can also act as a free-radical scavenger with potential for direct topical application at low concentration and induce apoptotic cell death at high concentrations due to increase in LDH release. Our study also explored in vitro biological activities of Ulmo honey that can differentiate it from other medical grade honey thereby enhancing its therapeutic potential and use. Acknowledgements This work was supported by the Project CORFO 13 IDL2-23290. References Abdul-Ghani, A. -S., Dabdoub, N., Muhammas, R., Abdul-Ghani, R., & Qazzaz, M. (2008). Effect of Palestinian honey on spermatogenesis in rats. Journal of Medicinal Food, 11(4), 799–802. Alissandrakis, E., Kibaris, A. C., Taranatilis, P. A., Harizanis, P. C., & Polissiou, M. (2003). Flavour compounds of Greek cotton honey. Journal of the Science of Food and Agriculture, 85, 144–1452. Babushok, V. I., Linstrom, P. J., & Zenkevich, I. G. (2011). Retention indices for frequently reported compounds of plant essential oils. Journal of Physical and Chemical Reference Data, 40(4), 1–47. Barra, M. P. G., Ponce-Díaz, M. C., & Venegas-Gallegos, C. (2010). Volatile compounds in honey produced in the central valley of Ñuble Province, Chile. Chilean Journal of Agricultural Research, 70(1), 75–84. Batumalaie, K., Qvist, R., Yusof, K. M., Ismail, I. S., & Sekaran, S. D. (2014). The antioxidant effect of the Malaysian Gelam honey on pancreatic hamster cells cultured under hyperglycemic conditions. Clinical and Experimental Medicine, 14, 185–195. Beitlich, N., Koelling-Speer, I., Oelschlaegel, S., & Speer, K. (2014). Differentiation of Manuka honey from kanuka honey and from jelly bush honey using HS-SPME-GC/ MS and UHPLC-PDA-MS/MS. Journal of Agricultural and Food Chemistry, 62, 6435–6444. Beretta, G., Granata, P., Ferrero, M., Orioli, M., & Facino, R. M. (2005). Standardization of antioxidant properties of honey by a combination of spectrophotometric/fluorimetric assays and chemometrics. Analytica Chimica Acta, 533, 185–191. Bianchi, F., Mangia, A., Mattarozzi, M., & Musci, M. (1 December 2011). Characterization of the volatile profile of thistle honey using headspace solid-phase microextraction and gas chromatography–mass spectrometry. Food Chemistry, 129(3), 1030–1036. Burgos-Díaz, C., Pons, R., Teruel, J. A., Aranda, F. J., Ortiz, A., Manresa, A., & Marqués, A. M. (2013). The production and physicochemical properties of a biosurfactant mixture obtained from Sphingobacterium detergens. J Colloid Interface Sci., 394, 368–379. Castro-Vázquez, L., Díaz-Maroto, M. C., & Pérez-Coello, M. S. (2007). Aroma composition and new chemical markers of Spanish citrus honeys. Food Chemistry, 103, 601–606.

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