Phenolic profile, antibacterial, antimutagenic and antitumour evaluation of Veronica urticifolia Jacq. Jelena Živkovića*, João C.M. Barreirab,c,d, Dejan Stojkoviće, Tatjana Ćebovićf, Celestino Santos-Buelgac,*, Zoran Maksimovićg, Slobodan Davidovićh, Isabel C.F.R. Ferreirab aInstitute for Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia. bMountain Research Centre (CIMO), ESA, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Ap. 1172, 5301-855 Bragança, Portugal. cGrupo de Investigación en Polifenoles (GIP-USAL), Faculty of Pharmacy, University of Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain. dChemical Sciences Department, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal. eUniversity of Belgrade, Institute for Biological Research “Siniša Stanković”, Department of Plant Physiology, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia. fUniversity of Novi Sad, Department of Biochemistry, School of Medicine, Hajduk Veljkova 3, 21102 Novi Sad, Serbia. gUniversity of Belgrade, Department of Pharmacognosy, School of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia. hUniversity of Belgrade, Institute of Molecular Genetics and Genetic Engineering, 11010 Belgrade, Serbia. *Authors to whom correspondence should be addressed: (Jelena Živković; e-mail: [email protected]. Phone: +381 648674921; Fax: +381 113031655; Celestino Santos-Buelga; e-mail: [email protected]. Phone: +34 923294537; Fax: +34 923294515). 1 Abstract This study was designed to characterize the phenolic profile and evaluate the antibacterial, antimutagenic and antitumor activities of Veronica urticifolia Jacq. methanolic extract. HPLC-DAD/ESI-MS analysis revealed the presence of phenolic acids, flavonoids and phenylethanoids, with acteoside as the main component (14.9 mg/g of extract). Antibacterial effect was determined using the microbroth dilution assay and Staphylococcus aureus was the most sensitive strain (MIC and MBC = 7.5 mg/mL). Antimutagenic activity was evaluated by Ames mutagenicity assay. At 1 mg/plate, the tested extract afforded high protection against the mutagenicity of nitroquinoline-N-oxide (4NQO) to Salmonella typhimurium strain TA100 (inhibition rate 48.3%). Antitumor activity was screened in Ehrlich ascites carcinoma (EAC) model. Pretreatment with 2 mg/kg body weight showed statistically significant decrease in tumor cell viability, while ascites volume and tumor cell count became slightly decreased, but not to a statistically significant extent. Above results indicate that V. urticifolia deserves further research into chemoprevention. Keywords: Veronica urticifolia; Phenolic profile; HPLC-DAD/ESI-MS; Antibacterial; Antimutagenicity; Ehrlich carcinoma. 2 1. Introduction Cancer, the second leading cause of death worldwide next to cardiovascular diseases, is a group of more than 100 different diseases, characterized by uncontrolled cellular growth, local tissue invasion and distant metastases (Madhusudan and Middleton, 2005). According to World Health Organization, more than 10 million new cases of cancer are diagnosed every year, and the statistical trends indicate that this number would double by 2020 (Mignogna et al., 2004). The demand for new anticancer agents is escalating due to the accumulation of carcinogenic and mutagenic agents in the environment. Although many synthetic anticancer agents are used in the treatment of cancer, side effects and emergence of synthetic drug resistant cancer cells among patients limits their purpose (Madhusudan and Middleton, 2005). Recent studies have shown that cancer management by phytochemicals from vegetables, fruits and medicinal plants is one of the most feasible approaches to treat this cascading disease (Dhamija et al., 2013). In the fight against cancer, the search for antimutagenic agents is essential, since mutagenic and carcinogenic factors are present in the human environment and their total elimination seems to be impossible (Abdillahi et al., 2012). Humans are provided with many defences and their failure could lead to DNA damage and consequently to cancer (El-Sayed and Hussin, 2013). Carcinogenesis is generally a slow process and often takes decades from tumor initiation to diagnosis, offering a considerable time frame for chemopreventive approaches. Chemoprevention is defined as the use of specific natural or synthetic agents to prevent, delay, or slow the carcinogenic process (Martin et al., 2013). The prevention of cancer can be achieved by avoiding exposures to mutagens, by fortifying physiological defence mechanisms, or by supplying the intake of protective factors (De Flora, 1998). Human epidemiology and animal studies have indicated that cancer risk may be modified by changes in dietary habits or by dietary supplements (Miyazawa and Hisama, 2004). 3 More than 15% of malignancies worldwide are established to have an infectious cause (Mager, 2006). Therefore, application of methods to prevent and/or treat infection, such as the use of antimicrobial treatments, could have an effect on future burden of cancer worldwide. Although antibiotics decreased the spread and severity of a wide variety of infectious diseases, bacteria and fungi have developed numerous mechanisms to escape old and new antimicrobial agents due to their uncontrolled use. In this concern, the proper treatment of cancer and microbial infections would have a great impact on the population’s health (Assaf et al., 2013). The identification of dietary components as potential cancer chemopreventive agents in the form of functional foods or as nutraceuticals has become an essential subject of many studies. One of the best approaches in search for anticancer agents from plant resources is the selection of plant species based on ethnomedical leads and testing the selected species efficacy in light of modern science. In traditional medicine, several Veronica species are used to treat cancer (Harput et al., 2002a). As currently circumscribed, Veronica (Plantaginaceae) is a genus of 450 species found in temperate regions of both hemispheres. Regardless of widespread use, mostly as diuretics, for their wound-healing properties and in medical treatment of respiratory diseases, there is a scarcity of physiological evidence to support any claim of therapeutic values for Veronica species. Only few studies confirm that certain Veronica genus reveal considerable bioactivity such as antibacterial (Stojković et al., 2013; Gusev et al., 2012), antioxidant (Živković et al., 2012), anti-inflammatory (Harput et al., 2002a) and cytotoxic (Harput et al., 2002a; Saracoglu and Harput, 2012) activities. On the other hand, the phytochemistry of the genus has been extensively studied with many species surveyed for their iridoid (Jensen et al., 2005) and phenylethanoid derivatives 4 (Taskova et al., 2006) and flavonoid glycosides (Albach et al., 2003, 2005; Saracoglu et al., 2004; Taskova et al., 2008) with most of these being considered beneficial for human health. Previously we reported in vitro and in vivo antioxidant activity of Veronica urticifolia Jacq. (Živković et al., 2012). Nevertheless, to the best of our knowledge, there are no reports on other biological activities of the selected species. Phenolic profile of the species V. urticifoila is presented for the first time. The aim of this study was profiling the phenolic compounds of V. urticifolia and evaluating the antibacterial, antimutagenic and antitumor effects of its methanolic extract. 2. Materials and methods 2.1. Plant samples The aerial parts of Veronica urticifolia were collected during their flowering period in June 2008 from Mountain Goč in central Serbia. Plant material was taxonomically determined and deposited in the Herbarium collection of the Institute of Botany, School of Pharmacy, Belgrade (voucher specimen numbers were VR 165). 2.2. Standards and reagents HPLC-grade acetonitrile was obtained from Merck KgaA (Darmstadt, Germany). Dimethylsulfoxide (DMSO) (Merck KGaA, Germany) was used as a solvent in antimicrobial assays. Formic acid was purchased from Prolabo (VWR International, France). N-acetyl-L- cysteine and Trypan blue solution were obtained from Sigma-Aldrich (Steinheim, Germany). Nitroquinoline-N-oxide (4NQO) was purchased from Sigma, USA. The phenolic compound standards were from Extrasynthese (Genay, France). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, USA). 5 2.3. Extraction procedures The powdered plant sample (~1 g) was extracted by stirring with 30 mL of methanol:water 80:20 (v/v), at room temperature, 150 rpm, for 1 h. The extract was filtered through Whatman no. 4 paper. The residue was then re-extracted twice with additional portions (30 mL) of methanol:water 80:20 (v/v). The combined extracts were evaporated at 35 °C (rotary evaporator Büchi R-210, Flawil, Switzerland) to remove methanol. The aqueous phase was lyophilized and re-dissolved in 20% aqueous methanol at 5 mg/mL and filtered through a 0.22-µm disposable LC filter disk for high performance liquid chromatography (HPLC-DAD- MS) analysis. For biological activity evaluations, the air dried plant material was reduced to a fine powder and extracted by maceration with methanol in a solid/solvent ratio of 1:20 at room temperature for 48 h. The obtained extract was evaporated under reduced pressure and further kept in a vacuum desiccator to remove traces of solvents. 2.3. Elucidation of phenolic compounds structures The extracts were analysed using a Hewlett-Packard 1100 chromatograph (Agilent Technologies) with a quaternary pump and a diode array detector (DAD) coupled to an HP Chem Station (rev. A.05.04) data-processing station. A Waters Spherisorb S3 ODS-2 C , 3 18 µm (4.6 mm × 150 mm) column thermostatted at 35 °C was used. The solvents used were: (A) 0.1% formic acid in water, (B) acetonitrile. The elution gradient established was isocratic 15% for 5 min, 15% B to 20% B over 5 min, 20-25% B over 10 min, 25-35% B over 10 min, 35-50% for 10 min, and re-equilibration of the column, using a flow rate of 0.5 mL/min; the injection volume was 100 µL. Double online detection was carried out in the DAD using 280 6 nm and 370 nm as preferred wavelengths and in a mass spectrometer (MS) connected to HPLC system via the DAD cell outlet. MS detection was performed in an API 3200 Qtrap (Applied Biosystems, Darmstadt, Germany) equipped with an ESI source and a triple quadrupole-ion trap mass analyzer that was controlled by the Analyst 5.1 software. Zero grade air served as the nebulizer gas (30 psi) and turbo gas for solvent drying (400 ºC, 40 psi). Nitrogen served as the curtain (20 psi) and collision gas (medium). The quadrupols were set at unit resolution. The ion spray voltage was set at -4500 V in the negative mode. The MS detector was programmed to perform a series of two consecutive modes: enhanced MS (EMS) and enhanced product ion (EPI) analysis. EMS was employed to record full scan spectra to obtain an overview of all of the ions in sample. Settings used were: declustering potential (DP) -450 V, entrance potential (EP) -6 V, collision energy (CE) -10 V. Spectra were recorded in negative ion mode between m/z 100 and 1000. Analysis in EPI mode was further performed in order to obtain the fragmentation pattern of the parent ion(s) detected in the previous experiment using the following parameters: DP -50 V, EP -6 V, CE -25 V, and collision energy spread (CES) 0 V. The phenolic compounds present in the samples were characterized according to their UV and mass spectra and retention times compared with commercial standards when available. For the quantitative analysis of phenolic compounds, a calibration curve was obtained (using the UV detector with λ = 280 nm for phenolic acids and λ = 370 nm for flavonoids) by injection of known concentrations (1-100 µg/mL) of different standards compounds: luteolin-7-O- glucoside (y = 80,829ϰ - 21,291; R2 = 0.999); apigenin-7-O-glucoside (y = 159.62ϰ + 7.5025; R2 = 0.999); 5-O-Caffeoylquinic acid (y = 313.03ϰ - 58.2; R2 = 0.999); caffeic acid (y = 611.9ϰ -4.5733; R2 = 0.999); p-coumaric acid (y = 884,6ϰ + 184,49; R2 = 0.999). 2.4. Antibacterial activity 7 2.4.1. Microorganisms and culture conditions.     For the bioassays seven bacterial strains were used, four Gram-positive: Staphylococcus aureus (ATCC 6538), Bacillus cereus (human isolate), Micrococcus flavus (ATCC 10240) and Listeria monocytogenes (NCTC 7973), and three Gram-negative: Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (human isolate) and Escherichia coli (ATCC 35210).   All of the tested microorganisms were obtained from the Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”, Belgrade, University of Belgrade, Serbia. The bacteria were cultured on Mueller-Hinton agar (MH) and cultures were stored at +4 °C and subcultured once a month. 2.4.2. Microdilution method. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined by the modified microdilution method (Espinel-Ingroff, 2001). Briefly, fresh overnight culture of bacteria was adjusted to a concentration of 1×105 CFU/mL by counting the spores in suspension. The inocula were prepared daily and stored at +4 ºC until use. Dilutions of inocula were cultured on solid medium to verify the absence of contamination and check the validity of the inoculum. Different solvent dilutions of methanolic extract were carried out over the wells containing 100 µL of Tryptic Soy Broth (TSB) and afterwards, 10 µL of inoculum was added to all the wells. The microplates were incubated for 24 h1 at 37 ºC. The MIC of the samples was detected following the addition of 40 µL of iodonitrotetrazolium chloride (INT) (0.2 mg/mL) and incubation at 37 °C for 30 min. MBC was determined by serial sub-cultivation of 10 µL into microplates containing 100 µL of TSB. The lowest concentration that shows no growth after this sub-culturing was read as the MBC. 8 Streptomycin (Sigma P 7794) (0.05-3 mg/mL) was used as positive control for bacterial inhibition. A 5% solution of DMSO in water was used as negative control. 2.5. Antimutagenicity A variation of the Ames test was used to screen for antimutagenic activity of Veronica urticifolia methanolic extract. For this activity, Salmonella typhimurium TA100 (Molecular Toxicology, Inc) without S9 metabolic activation were used due to cost implications. It is also known that these two strains are capable of identifying up to 90% of the mutagens (Mortelmans and Zeiger, 2000). Here, 500 µL of 0.1 M phosphate buffer was added to 50 µL of test sample in a test-tube. Fifty microlitres of the nitroquinoline-N-oxide (4NQO) in DMSO (20 µg/mL) was added to the mixture and then pre-incubated for 3 min before the addition of 100 µL of overnight TA100 strains of bacterial culture. After incubation for 48 h at 37 °C, the number of viable cells (cultured on MH) and revertant colonies were determined (on minimal glucose agar) and percentage of inhibition (Index of antimutagenicity) calculated using the formula shown below. The extract was tested in triplicate and it was repeated twice. The percentage inhibition was calculated by the following formula: Percent of inhibition (%) = (1 - T/M) × 100, where T is the number of revertants per plate in the presence of the mutagen 4NQO, and M is the number of revertants per plate in the positive control (mutagen). The mutagenic index (MI) was also calculated for each concentration tested, this being the average number of revertants per plate with the tested extract divided by the average number of revertants with the negative (solvent) control. A tested extract was considered mutagenic when a dose-response relationship was detected and a two-fold increase in the number of mutants (MI≥2) was observed for at least one concentration (Resende et al., 2012). 9 Cell viability was also determined to assess the potential bactericidal effect of the mutagens. A substance was considered bactericidal when the bacterial survival was less than 60% of that observed in the negative control (Resende et al., 2012). 2.6. Cytotoxic activity against Ehrlich’s tumor cells in mice 2.6.1. Animals and experimental procedure. Dry extract was dissolved under sonication in water to make 5% (w/v) solution, and filtered through a 0.45 µm membrane filter. The resulting solutions were kept in the refrigerator (4-8 °C) until applied. Animal care and all experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animal Resources edited by the Commission of Life Sciences, National Research Council and according to approval from ethical committee for animals care at University of Novi Sad. Male and female Hannover National Medical Institute (Hann:NMRI) mice were obtained from the Biochemical Laboratory, Clinical Centre Novi Sad (Novi Sad, Serbia). Animals were fed standard mouse chow (LM2, Veterinarski zavod, Subotica, Serbia) with free access to tap water, in a temperature (25 °C) and humidity- controlled (30-50%) animal house under 12 h night/day cycles. NMRI mice of both sexes (6-8 weeks old) weighing 25±2.5 g were used in the experiments. Animals were divided into three groups of six mice under the following conditions and treatments: I, EAC group (mice with implanted EAC cells), (n =6); II, mice pre-treated with the Veronica urticifolia extract, 2 mg/kg b.w. per day, i.p., starting 7 days before the EAC implantation (n = 6), III, NALC group, mice pre-treated with the N-acetyl-L-cysteine (5 mM), 2 mL/kg b.w. per day, i.p., starting 7 days before the EAC implantation (n = 6). Fourteen days after EAC implantation, all mice were killed, and the ascites of the carcinoma were collected for further experiments. 10