Antidepressive Effects of a Chemically Characterized Maqui Berry Extract

Food and Chemical Toxicology 129 (2019) 434–443

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Food and Chemical Toxicology

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Antidepressive eﬀects of a chemically characterized maqui berry extract (Aristotelia chilensis (molina) stuntz) in a mouse model of Post-stroke T depression

Arianna Di Lorenzoa,1, Anatoly P. Sobolevb,1, Seyed Fazel Nabavic,1, Antoni Suredad, Akbar Hajizadeh Moghaddame, Sedigheh Khanjanif, Carmen Di Giovannij, Jianbo Xiaog, Samira Shirooieh, Arold Jorel Tsetegho Sokenga, Alessandra Baldia, Luisa Manninab,i,2, ∗ Seyed Mohammad Nabavic,2, Maria Dagliaa, ,2 a Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section, Pavia University, Viale Taramelli 12, Pavia 27100, Italy b Istituto di Metodologie Chimiche, Laboratorio di Risonanza Magnetica “Annalaura Segre”, CNR, 00015, Monterotondo (Rome), Italy c Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, P.O. Box, 19395-5487, Tehran, 19395-5487, Iran d Grup de Nutrició Comunitària I Estrès Oxidatiu and CIBEROBN (Physiopathology of Obesity and Nutrition), Universitat de les Illes Balears, Palma de Mallorca, E-07122, Spain e Department of Biology, Faculty of Sciences, University of Mazandaran, Babolsar, Iran f Department of Physiology, Faculty of Biological Sciences, Shahid Behshti University, P.O. Box, 19615-1178, Tehran, 19615-1178, Iran g Institute of Chinese Medical Sciences, State Key Laboratory of Quality Control in Chinese Medicine, University of Macau, Macau SAR, China h School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, 6734667149, Iran i Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le Aldo Moro 5, 00185, Rome, Italy j Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano, 49, 80131, Napoli, Italy

ABSTRACT Keywords: Maqui berry extract Mood disorders occur in 30% of stroke patients, and of these post-stroke depression (PSD) is the most significant. Post-stroke depression This study aimed to evaluate the antidepressive-like effects and in vivo antioxidant activity of a chemically In vivo antioxidant activity characterized maqui berry (Aristotelia chilensis (Molina) Stuntz) extract obtained from an optimized extraction Murine model system Chemical characterization method, on a murine PSD model. The extraction process was optimized to maximize anthocyanin content, and the phytochemical profile of the extract was evaluated using a multi-methodological approach including a liquid chromatographic method coupled with mass spectrometry and nuclear magnetic resonance spectroscopy. The antidepressive-like activity was investigated through despair swimming and tail suspension tests. The in vivo antioxidant activity was evaluated in mouse brain tissue by measuring the activity of antioxidant enzymes and lipid peroxidation products. A number of compounds have been first identified in maqui berry here, including malvidin-glucoside, GABA, choline and trigonelline. Moreover, the results showed that the antidepressive-like activity exerted by the extract, which was found to restore normal mouse behavior in both despair swimming and tail suspension tests, could be linked to its antioxidant activity, leading to the conclusion that maqui berries might be useful for supporting pharmacological therapy of PSD by modulating oxidative stress.

Abbreviations: PSD, Post-stroke depression; DoE, Design of experiments; MBE, Maqui berry extract; TMA, Total monomeric anthocyanin; SD, Standard deviation; TSP, 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt; GA, Gallic acid; PA, Protocatechuic acid; DST, Despair swimming test; TST, Tail suspension test; TBARS, Thiobarbituric acid-reactive substance; SOD, Superoxide dismutase; GSH, Reduced glutathione; CAT, Catalase; GABA, γ-Aminobutyrate; C3G, Cyanidin-3-glucoside; D3G, Delphinidin-3-glucoside; BCCAO, Bilateral common carotid artery occlusion ∗ Corresponding author. E-mail addresses: [email protected] (A. Di Lorenzo), [email protected] (A.P. Sobolev), [email protected] (S.F. Nabavi), [email protected] (A. Sureda), [email protected] (A.H. Moghaddam), [email protected] (S. Khanjani), [email protected] (C. Di Giovanni), [email protected] (J. Xiao), [email protected] (S. Shirooie), [email protected] (A.J. Tsetegho Sokeng), [email protected] (A. Baldi), [email protected] (L. Mannina), [email protected] (S.M. Nabavi), [email protected] (M. Daglia). 1 These three authors share the ﬁrst authorship. 2 These three authors share the senior authorship. https://doi.org/10.1016/j.fct.2019.04.023 Received 14 January 2019; Received in revised form 10 April 2019; Accepted 15 April 2019 Available online 22 April 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved. A. Di Lorenzo, et al. Food and Chemical Toxicology 129 (2019) 434–443

1. Introduction levels of nitric oxide and prostaglandin E2, and cycloxygenase-2 ex- pression in lipopolysaccharide-stimulated RAW 264.7 macrophages Stroke is the second leading cause of disability in Europe, preceded (Tanaka et al., 2013). Based on the information on maqui found in the by ischaemic heart disease, and the sixth worldwide. Recent estimates literature, it can be considered to be a new functional food due to its predict a continuing increase in stroke events targeting the elderly, range of additional biological properties, further to its nutritional leading up to 1.5 million strokes per year by 2025, with an increase of properties. 0.4 million/year since 2000 in Europe alone (http://www.who.int/ Our research group previously postulated the hypothesis that medicines/areas/priority_medicines/en/). polyphenols may exert positive effects for the management of post- Epidemiological studies show that mood disorders, with depression stroke depression (Nabavi et al., 2015), due to their antioxidant ac- occurs in 30% of stroke patients. The first clinical recognition of post- tivity. We considered maqui berry to be a promising candidate for this stroke depression (PSD) dates back by over 100 years. PSD is associated investigation due to its high content of polyphenols responsible for in with an increase in disability, worsening the patient's quality of life and vitro and in vivo antioxidant properties, in vivo cardioprotective activity, increasing mortality. An improvement in depressive symptoms is often and richness in anthocyanins which inhibit both MAO-A and MAO-B linked to improved cognitive and functional recovery. Despite this isoforms in vitro, thus exerting a crucial role against the pathogenesis of finding, depression is only diagnosed in a small number of stroke pa- depression (Dreiseitel et al., 2009) Therefore, the aim of this in- tients, and few of these received common antidepressant therapies, vestigation is to evaluate the antidepressive-like effects and in vivo with unverified results (Paolucci, 2008). antioxidant activity of a chemically characterized maqui berry (Aris- Prospective studies have identified a range of risk factors in the totelia chilensis (Molina) Stuntz) extract, obtained by an optimized ex- development of PSD, such as genetic factors, gender, age, medical and traction method, on a murine PSD model. The phytochemical profile of psychiatric history, severity and type of stroke. Moreover, investiga- maqui berry extract was investigated with using a multi-methodological tions conducted in in vitro and in vivo studies have revealed that oxi- approach previously used for the study other food matrices (Mannina dative stress and inflammation, which occur in stroke patients, also play et al., 2015, 2016; Marchese et al., 2014), which consists of the com- key roles in the development of depression (Robinson and Jorge, 2016). bination of two different analytical techniques, targeted RP-HPLC-PDA- Growing evidence suggests that plant foods may exert antioxidant ESI-MSn and untargeted NMR spectroscopy. These results will enable us and anti-inflammatory effects because of their content of antioxidant to link a specific chemical composition to the biological properties vitamins and secondary metabolites with beneficial effects on oxidative measured. stress. Maqui (Aristotelia chilensis (Molina) Stuntz), of the Elaeocarpaceae 2. Materials and Methods family, is an evergreen dioecious plant native to temperate forests of Central and Southern Chile and Western Argentina (Fredes et al., 2012). 2.1. Chemicals and reagents It produces small edible berries characterized by an intense purple/ black color due to an abundant presence of anthocyanins. Maqui berries Three different batches of commercial maqui berry powder were are used for the treatment of various diseases as part of Chilean folk sourced from CiboCrudo srl (Ciciliano, Rm, Italy). LC/MS-grade acet- medicine, including digestive disorders, haemorrhages and inflamma- onitrile, HPLC-grade methanol, formic acid solution 1 M, proto- tion (Escribano-Bailón et al., 2006). Over the past decade, a growing catechuic acid, gallic acid, 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid interest in berries and related products has led to the publication of sodium salt, D2O and phosphate buffer were acquired from Sigma scientific reports on maqui, aimed at elucidating its phytochemical Aldrich Chemical Company (St. Louis, MO, USA). Delphinidin-3-glu- composition and biological and functional properties. coside and cyanidin-3-glucoside were sourced from PhytoLab GmbH & Early investigations into the phytochemical composition of maqui Co. KG (Vestenbergsgreuth, Germany). A LC-PakTM Millex system focused on the identification of anthocyanins. In 2006, Escribano- (Millipore Coorporation, Billerica, MA) was used for the production of Bailón et al. first elucidated the anthocyanin profile of the berries, re- HPLC grade water. Sodium acetate, acetic acid, hydrogen chloride and vealing the presence of 8 anthocyanins: delphinidin 3,5-diglucoside, potassium chloride were obtained from Carlo Erba (Milan, Italy). delphinidin 3-sambubioside-5-glucoside, cyanidin 3,5-diglucoside, cy- Further solvents and reagents were acquired from Merck Chemical anidin 3-sambubioside-5-glucoside, delphinidin 3-sambubioside, cya- Company (Darmstadt, Germany). nidin 3-glucoside, delphinidin 3-glucoside, and cyanidin 3-sambubioside (Escribano-Bailón et al., 2006). According to available literature 2.2. Optimization and characterization of MBE data, the anthocyanin content of maqui berries is extremely variable ranging from 2.12 mg/kg to 45.7 mg/g of dry weight (Schreckinger 2.2.1. Optimization of maqui berry extraction by design of experiments et al., 2010; Céspedes, 2010; Fredes et al., 2014; Gonzàlez, 2015; (DoE) Genskowsky et al., 2016; Rodriguez, 2016; Brauch et al., 2016). The Maqui berry extract (MBE) was obtained from a mixture of three reason of a such high variability is linked to variations in geographical different batches of commercial samples of dehydrated maqui berry origins, growing conditions, times of harvest, plant genotype (Fredes powder, by means of solid/liquid (S/L) extraction. The extraction was et al., 2014), and the different extraction procedures applied to obtain performed in triplicate in an ice bath under a nitrogen atmosphere and the tested extract. constant stirring, and was protected from light to prevent the de- The main non-anthocyanin components identified have been phe- gradation of anthocyanins. Some factors affecting the extraction yield nolic acids, mainly hydroxycinnamic acids, and flavonoids, with a great were investigated by means of a DoE methodology, using MODDE Pro prevalence of quercetin and myricetin derivatives, and ellagic acids, 11 Software (Umetrics, Umeâ, Sweden). A three factor, two level, full (i.e. ellagic acid, granatin B, and HHDP-hexose) (Céspedes, 2010; factorial design study was applied to study the impact of certain vari- Brauch et al., 2016; Genskowsky et al., 2016; Ruiz, 2016). ables, such as S/L ratio (1:5, 1:20 g/mL), percentage of acidification of The antioxidant activity of maqui is its most studied property, which the extraction mixture (0.1–1% formic acid) and percentage of me- has been demonstrated through some of the most common in vitro as- thanol in the extraction mixture (10–90%), on the yield of anthocyanins says and in cell culture and experimental animal model systems from maqui berry powder, expressed as total monomeric anthocyanin (Rodriguez, 2016; Céspedes, 2010; Schreckinger et al., 2010; Miranda- (TMA) content. A total number of 11 experiments were carried out, Rottmann et al., 2002). Other properties have also been ascribed to including a triplicate measurement at the center point used to estimate maqui, such as anti-diabetic properties (Rojo et al., 2012; Rubilar et al., the experimental error, all reported in Supplementary Materials (Table 2011), and inhibition of adipogenesis and inflammation by decreasing S3).

435 A. Di Lorenzo, et al. Food and Chemical Toxicology 129 (2019) 434–443

2.2.2. Total monomeric anthocyanin content 2.2.5. RP-HPLC-PDA quantitative analysis The total monomeric anthocyanins (TMA) were evaluated using a The quantification of gallic acid (GA), protocatechuic acid (PA) and pH-differential method, as presented by Di Lorenzo et al. (Di Lorenzo anthocyanins in MBE was performed by means of the RP-HPLC-PDA et al., 2016). MBE was diluted using 0.025 M potassium chloride buffer method previously reported, using the external standard method. at pH 1.0 and a 0.4 M sodium acetate buffer at pH 4.5, after the proper Before quantification, the analytical method was validated according to dilution factors had been identified. Sample absorbance was read at ICH guidelines. 520 nm and 700 nm in each buffer with a FLUOstar Omega microplate reader (BMG Labtech GmbH, Ortenberg, Germany). The concentration 2.2.6. MBE statistical analysis of TMAs was evaluated in the form of cyanidin-3-glucoside equivalent The experimental design was developed using MODDE Pro 11.1 (CGE) using the equation reported by Siddiq et al. (2018). Data are software (Umetrics, Umeȃ, Sweden). Results have been presented as given as the mean of three separate measurements, ± the standard means ± SD. deviation (SD). 2.3. In vivo antidepressive-like and antioxidant activities of MBE 2.2.3. RP-HPLC-PDA-ESI-MSn analysis RP-HPLC-PDA-ESI-MSn analysis was executed using the same 2.3.1. Animals equipment as reported by Daglia et al. (2017). To perform the study, five-week old male balb/c mice between 20 g Separation used a Synergi Fusion RP-18 column (150 × 4.6 mm, and 25 g were acquired from the Pasteur Institute of Iran and kept at 5 μm), with a Hypersil Gold C18 precolumn (10 × 2.1 mm, 5 μm), both 24 ± 2 °C with a 12/12 h light/dark cycle and a humidity of sourced from Phenomenex (Torrance, USA). 60% ± 5%. Water and food were supplied ad libitum. Mice were al- The mobile phase was water, with 0.5% formic acid (eluent A) and lowed to acclimatize to the testing area for 24 h prior to behavioral acetonitrile (eluent B), eluted in a gradient as follows: 2% B for 10 min, testing, which occurred between the hours of 10:00 a.m. and 2:00 p.m. from 2% to 15% B in 55 min, from 15% to 40% B in 35 min, from 40% In vivo experimentation followed international ethical guidelines for the to 100% B in 15 min, ending with a 15 min isocratic run at 100% B. The care of laboratory animals, as according to Principles of Laboratory run time was 120 min in total, which includes reconditioning of the Animals Care (NIH Publication No. 85-23, revised 1996). Ethical ap- column. Flow rate was kept to 0.4 mL/min, the autosampler was set to proval number “81/021, 10 July 2002”. 4 °C in temperature, and the column was set to 45 °C. A 5 μL injection volume was used. Chromatogram measurements were taken at 280, 2.3.2. Induction of stroke 330, and 520 nm; spectral data were taken between 200 and 800 nm for The induction of stroke was carried out using the procedures re- every peak. ported by Nabavi et al., 2013, 2018. HPLC-ESI-MSn data were obtained in both positive and negative ionization modes using the Xcalibur software. The ion trap was set to 2.3.3. MBE administration full scan (100–1500 m/z), data-dependent scan and MSn modes. For the The chemically characterized MBE was used in the in vivo experi- purposes of MSn data, collision energy was chosen to be 35% with an ments. Mice were assigned into 5 groups for this study, each made up of isolation width of 2 m/z. A preliminary experiment was executed for the 10 animals: i) a healthy mouse control group; ii) a BCCAO group, made purpose of optimizing MS operating parameters: 5 μg/mL delphinidin- up of mice which had been subjected to bilateral common carotid artery 3-glucoside (50:50 0.1% formic acid:methanol, % v/v) and 10 μg/mL occlusion (BCCAO) induced stroke with no further treatment; and iii) gallic acid (50:50 0.1% formic acid:methanol, % v/v) solutions, infused three groups of BCCAO mice treated with MBE. Mice were placed into through the ESI interface directly into the mass spectrometer, with a one of these groups at random. The animal equivalent dosage was flow rate of 25 μL/min. Optimized parameters were as follows: tem- calculated from the human equivalent dose. We considered an average perature 220 °C, spray voltage 4.5 and 5.0 kV, sheath gas 30 and 60, daily human consumption of berries to be 20–50 g. Based on the TMA capillary auxiliary gas 10 and 20, capillary voltage −23.15 V and 45 V, content occurring in our MBE, daily ingestion of anthocyanins ranges for negative and positive ionization modes, respectively. from 114 to 285 mg, and the human equivalent dose (HED) thus varies between 1.9 and 4.74 mg/kg of anthocyanins for an adult weighing 2.2.4. NMR analysis 60 kg. To extrapolate the animal dose from the HED, doses were nor- NMR analysis was carried out using a Bruker AVANCE 600 spec- malized according to body surface area (BSA) through the equation: trometer with a proton frequency of 600.13 MHz, using a Bruker mul- animal dose = HED x Human Km/ Animal Km (with humans having a tinuclear z-gradient inverse probehead. Km factor of 37 and the animal Km being given a value of 3 for mice). Maqui berry extract (5–10 mg) was dissolved in 0.7 mL of D2O with The translated animal dose ranges from 22 to 60 mg/kg. MBE was ad- phosphate buffer (400 mM, pH = 7.0) with 1 mM of 3-(trimethylsilyl)- ministered intraperitoneally for 7 days at three doses (25, 50 and propionic-2,2,3,3-d4 acid sodium salt (TSP) used as an internal stan- 100 mg/kg body weight), starting the day following induction of stroke. dard. Depressive-like behaviors were evaluated using the tests reported The 1H spectrum was acquired at 27 °C adding 512 transients using below, 30 min after final MBE administration. a recycle delay of 9.0 s, with a 90°, 14.25 μs pulse and 16 K data points. The suppression of the water signal was achieved by solvent pre- 2.3.4. Examination of stroke-induced anhedonia saturation using a soft pulse for 2 s immediately before acquisition. Anhedonia, defined as a loss of interest and inability to feel plea- Bruker TOPSPIN 1.3 software was used to process data. An exponential sure, is one of the most common features of depression. Stroke-induced function was used with LB set to 0.3 Hz. After Fourier transformation of anhedonia was evaluated in experimental animals by determining the the free induction decay, the spectra were phased manually using volumes of water and sucrose solution consumed by mice, as reported polynomial baseline correction. by Daglia et al., and Di Lorenzo et al. (Di Lorenzo et al., 2016; Daglia Maqui 1H NMR spectra was assigned using a set of 2D NMR tests, et al., 2017). namely 1H-1H TOCSY, 1H-13C HSQC and 1H-13C HMBC, which were executed as previously stated (Mannina et al., 2008). Mixing time for 2.3.5. Despair swimming test (DST) 1H–1H TOCSY was 80 ms. 150 Hz was used as the coupling constant DST is among the most important animal models used for evaluation 1JC–H for the 1H–13C HSQC experiment, whereas an 80 ms delay was of depressive-like behavior. During the 6 min test, animals are placed in used for the evolution of long-range couplings in the 1H–13C HMBC a cylindrical container and three parameters are recorded: climbing, experiment. swimming and immobility times. The DST was carried out using the

436 A. Di Lorenzo, et al. Food and Chemical Toxicology 129 (2019) 434–443 methods reported by Moghaddam et al. (Moghaddam, 2014). while three repeats were made at the center point to evaluate experimental error. 2.3.6. Tail suspension test (TST) Regression analysis of the model showed that it is characterized by a The second common and validated test used for behavioral assess- good fit, strength and predictive power. As expected, two variables (% ment in this animal model is the TST. Immobility time is registered over of methanol in the extraction mixture and S/L ratio) had a positive the 6 min testing period. The TST was carried out using the methods in fluence on the response, while the third variable (% of acidification of reported by Nabavi et al., 2013, 2018. the extraction mixture) did not influence the response itself, but ac- quires a greater importance when considered in association with the 2.3.7. Anesthesia and tissue Collection other two variables. In this case, both associations had a negative in- At the conclusion of experimentation, mice were feeded for 12 h and fluence on the TMA content. The 3D response surface plot (Fig. S1) then sedated as reported by Nabavi et al. (2018). showed a linear model which reveals that, within the experimental domain studied, the best conditions to obtain a MBE with the highest 2.3.8. Preparation of tissue homogenate and determination of protein content of TMA (8.87 ± 0.1 mg/g of cyanidin-3-glucoside equivalents) content are: 1) the lowest acidification of the extraction mixture (0.1% of A brain tissue homogenate was produced from each mouse, using formic acid), 2) the highest S/L ratio (1 g in 20 mL) and 3) the highest the procedures reported by Nabavi et al. (2018). amount of methanol in the extraction mixture (90%) (Table S3). The Bradford method was used to assess the levels of protein in each homogenate, using bovine serum albumin as the standard (Bradford, 3.1.2. Maqui berry metabolite profiling through RP-HPLC-PDA-ESI-MSn 1976). analysis The metabolite profiling of the optimized MBE was determined 2.3.9. Estimation of lipid peroxidation using RP-HPLC-PDA-ESI-MSn analysis, yielding 32 identified com- Thiobarbituric acid-reactive substance (TBARS) formation, a mea- pounds, as given in Table 1. The chromatogram of MBE is shown in sure of lipid peroxidation, was quantified using a technique previously Supplementary Materials (Fig. S2). This identification was accom- developed by our research group (Di Lorenzo et al., 2016). plished by comparing experimental data (retention time, MS and MSn spectra) with those available in literature and with commercial stan- 2.3.10. Determination of superoxide dismutase activity dard compounds where possible. Superoxide dismutase (SOD) activity was evaluated using a tech- The identified compounds consist of nine anthocyanins, six organic nique described by Misra and Fridovich (1972). and phenolic acids, three derivatives of ellagic acid, and 14 flavanols with two unidentified compounds. 2.3.11. Determination of catalase activity Peaks 6, 7, 8, 9, 11, 12, 14, 16 and 26 were identified as antho- Catalase activity was assessed using a technique previously estab- cyanins due to their high absorbance at 520 nm. Peaks 6, 7, 11, and 12 lished by our research group (Nabavi, 2012). were assigned to delphinidin derivatives, because they presented a common fragment ion at m/z 303, acquired under positive ionization, 2.3.12. Determination of reduced glutathione level which corresponds to delphinidin aglycone. Peaks 6 and 7, co-eluting at Ellman's method was used to determine reduced glutathione (GSH) 40.5 min, were identifiable by a pseudomolecular ion at m/z 627 and levels (Ellman, 1959). 759, respectively. Peak 6, leading to fragment ions at m/z 465 and 303 thanks to the subsequent loss of two hexosyl moieties ([M-162]+ and 2.3.13. Statistical analysis of animal test results [M-162-162]+), was assigned to delphinidin-3,5-dihexoside. Peak 7 Statistical analysis used the SPSS statistical software package ver- produced an abundant fragment ion at m/z 597 in losing a pentosyl sion 21.0 (SPSS Inc., Chicago, IL, USA). Results have been given in the sugar moiety, together with fragment ions at m/z 465 and 303: thus, it form of means ± SD, with p < 0.05 being taken to be statistically was assigned to delphinidin dihexoside pentoside. Peak 11, with a significant. This was calculated using one-way variance analysis. pseudomolecular ion at m/z 465 and the most abundant fragment ion at Bonferroni post-hoc testing was conducted on significant differences, to m/z 303, corresponded to delphinidin glucoside; while peak 12, evaluate relationships between involved groups. showing a loss of 294 amu due to a sambubiose moiety, was assigned to delphinidin sambubioside. Peaks 8, 9, 14 and 16 were assigned to cy- 3. Results anidin derivatives, because of the common presence in their MS/MS spectra of cyanidin aglycone ions at m/z 287. Peak 8, leading to frag- The present study aims to assess the in vivo antidepressive-like and ment ions at m/z 449 and 287 thanks to the subsequent loss of two antioxidant effects of a characterized anthocyanin rich MBE in a model hexosyl moiety ([M-162]+ and [M-162-162]+), was assigned to cya- of post-stroke depression in mice. To enhance the anthocyanin content nidin-3,5-dihexoside. Peak 9 produced an abundant fragment ion at m/ of MBE, three commercially available batches of dehydrated, powdered z 581, from cyanidin sambubioside, because of the loss of a hexosyl maqui berries were submitted to S/L extraction, and certain variables sugar moiety from the parent ion: thus, it was assigned to cyanidin 3- influencing the extraction yield were optimized by means of DoE. sambubioside-5-glucoside. Peaks 14 and 16 corresponded to cyanidin Afterwards, a multi-methodological approach was used in order to in- glucoside and cyanidin sambubioside respectively, as the fragment ion vestigate the phytochemical profile of the optimized MBE. at m/z 287 is derived from losing a hexosyl sugar moiety and a sambubiose sugar moiety, respectively. The eight identified anthocyanins, 3.1. Optimization and characterization of MBE already detected in maqui berries by different research groups (Schreckinger et al., 2010; Céspedes, 2010; Fredes et al., 2014; 3.1.1. Optimization of maqui berry extraction by design of experiments González, 2015; Genskowsky et al., 2016; Rodriguez, 2016; Brauch (DoE) et al., 2016), are considered distinguishing compounds of maqui ber- A full factorial design technique was applied to investigate the in- ries. Our analysis revealed the presence of another anthocyanin, which dependent variables 1) S/L ratio, 2) percentage of acidification of the elutes later than the others (RT: 80.4 min). Peak 26 MS/MS spectra was extraction mixture and 3) percentage of methanol of the extraction characterized by the presence of m/z 331 as the most abundant frag- mixture, acting on the dependent variable, the total quantity of ment ion, which corresponds to malvidin aglycone. The loss of a glu- monomeric anthocyanins occurring in maqui berry extract. The effects cosyl moiety ([M-162]+) from the parent ion allowed us to identify of each independent variable were evaluated at two different levels, malvidin glucoside (Di Lorenzo et al., 2016). According to our

437 A. Di Lorenzo, et al. Food and Chemical Toxicology 129 (2019) 434–443

Table 1 Chromatographic behavior (retention time, RT), MS and MSn data of the compounds identiﬁed in MBE.

Peak N. RT (min) m/z MSn Proposed structure

Organic and phenolic acids 1 6.5 353 111(65), 173(100),191(2) 4-caffeoylquinic acid 2 6.8 191 111(100), 173(10), 87(2) citric acid 3 14.1 169 125(100) gallic acid 4 22.5 343 191(100) galloylquinic acid 5 26.4 153 109(100) protocatecuic acid 13 59.9 371 209(100), 191(10) caffeoylglucaric acid Ellagic acid derivatives 10 55.4 481 301(100), 319(30) HHDP-Hexose 15 63.1 633 301(100), 463(40) galloyl-HHDP-glucose 19 75.8 951 933(100), 301(10), 613(5) granatin B Flavonols 18 68.3 625 463(100), 300(10) quercetin dihexoside 20 77.6 595 433(100), 463(50), 301(10) quercetin hexosyl pentoside 21 77.8 631 479(100), 316(10) myricetin-galloyl-hexoside 22 78.3 463 301(100) quercetin hexoside 23 78.4 631 479(100), 316(10) myricetin-galloyl-hexoside 24 79.9 479 316(100),271(50), 179(20), 151(10) myricetin hexoside 25 80.2 479 316(100), 271(50), 179(20), 151(10) myricetin hexoside 27 82.1 615 463(100), 301(10) quercetin-galloyl-hexoside 28 83.1 433 301(100), 300(60) quercetin pentoside 29 83.2 609 301(80), 300(100) rutin 30 83.5 463 301(100) quercetin hexoside 31 84.2 301 179(60), 151(50), 108(30), 217(15) quercetin 32 85.9 433 301(100), 300(60) quercetin pentoside 33 87.8 447 285(100) kaempferol hexoside Anthocyanins 6 40.5 627+ 465(100), 303(50) delphinidin 3,5-dihexoside 7 40.5 759+ 597 (100), 465 (40), 303 (30) delphinidin dihexoside pentoside 8 45.9 611+ 449(100), 287(40) cyanidin 3,5-dihexoside 9 48.4 743+ 449(100), 287(100), 581(65) cyanidin-3-sambubioside-5-glucoside 11 56.8 465+ 303(100) delphinidin glucoside 12 56.8 597+ 303(100) delphinidin sambubioside 14 61.1 449+ 287(100) cyanidin glucoside 16 64.5 581+ 287(100) cyanidin sambubioside 26 80.4 493+ 331(100) malvidin glucoside Unknown compounds 17 67.5 775 463(100), 300(10) Unknown compound 34 88.1 329 MS2 314(100), 286, 271, 193, 165 Unknown compound MS3 314: 271(100), 164, 257, 286 MS4 271: 243(100), 227, 199, 183 knowledge, this compound has not been detected in maqui berries by (2016). Peak 15 was characterized by a fragmentation pattern resem- previous studies. bling those of ellagic acid derivatives (fragment ions at m/z 463, 301, Six organic and phenolic acids have been identified in MBE. Peaks 3 and 300). Based on previously published data, peak 15 was assigned to and 5 were assigned to gallic acid and protocatechuic acid respectively, galloyl-HHDP-hexoside: starting from the parent ion (m/z 633), the through the comparison of experimental data (retention time, MS and fragment ion at m/z 463 was produced after the loss of both a galloyl MS/MS spectra) with those obtained from commercial standards. Peak moiety and a water molecule, while the fragment ion at m/z 301 is 1 was assigned to 4-caffeoylquinic acid, because of its peculiar MS/MS derived from the subsequent loss of a glucosyl moiety. Galloyl-HHDP- spectra: the most abundant fragment ion is m/z 173, following by ions hexoside is already known in literature, but no one has reported its at m/z 111 and 191, as demonstrated by Clifford et al. (2003). Peak 4 identification in maqui berry extract. was assigned to galloylquinic acid: the pseudomolecular ion at m/z 343 Finally, 14 flavonols have been identified: nine are quercetin deri- [M-H]- yielded a fragment ion at m/z 191, corresponding to quinic acid, vatives, four are myricetin derivatives and one is a kaempferol deri- through the loss of a galloyl moiety (−152 amu). Peak 13 was identi- vative. Peak 33 was assigned to kaempferol hexoside, providing a fied as caffeoylglucaric acid, through comparison of its MS and MS/MS fragment ion at m/z 285 because of the loss a hexosyl moiety (−162 spectra with those found in literature (Ruiz et al., 2014). Finally, peak 2 amu). Peaks 21, 23, 24 and 25 have been identified as derivatives of showed a short elution time, suggesting its polar structure, a parent ion myricetin as there is a fragment ion at m/z 316, corresponding to at m/z 191 and fragment ions at m/z 111, 173, 87. From analysis of the myricetin aglycone, in all their MS/MS spectra. Peaks 21 and 23 were MS/MS spectra, we hypothesize that peak 2 might be citric acid. Direct characterized by the same pseudo-molecular ion, the same MS/MS analysis of a commercially available standard led to structural identi- fragmentation pattern, but different retention times: they have been fication. As far as we are aware, caffeoylglucaric acid and citric acid identified as two isoforms of myricetin-galloyl-hexoside, as already have not yet been reported in a maqui berry extract, and thus this study reported by Genskowsky et al. (2016). Peaks 24 and 25 were instead represents the first report of these two acids as present in a MBE. assigned to myricetin hexoside: the pseudomolecular ion [M-H]- at m/z Moreover, RP-HPLC-PDA-ESI-MSn analysis revealed 3 ellagic acid 479 yielded a main fragment ion at m/z 361 due to the loss of a hexosyl derivatives, which are known to occur in maqui berries (Brauch et al., moiety (−162 amu). 2016). Peaks 10 and 19 were assigned to hexahydroxydiphenoyl- Quercetin derivatives are the most abundantly identified flavonoids hexoside (HHDP-hexoside) and granatin B respectively, by comparison in MBE. Peaks 22 and 30 were characterized by the same pseudo-mo- of experimental spectra with those already published by Brauch et al. lecular ion, the same MS/MS fragmentation pattern, but different

438 A. Di Lorenzo, et al. Food and Chemical Toxicology 129 (2019) 434–443 retention times: they have been identified as two isoforms of quercetin Table 2 hexoside due to the loss of a hexosyl moiety, which leads to the pro- Summary of the metabolites identified in the 600.13 MHz 1H NMR spectra of duction of a fragment ion at m/z 301 from a parent ion at m/z 463. Two MBE. fi isoforms of quercetin pentoside were also identi ed: they were char- Compound Assignment 1H (ppm) Multeplicity: J 13C (ppm) acterized by a parent ion at m/z 433 and a main fragment ion at m/z (Hz) 301 due to losing a pentosyl sugar moiety (−142 amu). Quercetin Carbohydrates pentoside were detected as peaks 28 and 32. In MBE rutin (quercetin α fi -Glucose CH-1 5.24 d [3.8] 93.1 rutinoside, peak 29) and quercetin (peak 31) were also identi ed, as CH-2 3.55 dd [9.8;3.8] 72.5 demonstrated by Genskowsky et al. (2016). Peak 27 showed a parent CH-3 3.72 73.7 ion at m/z 615 and two main fragment ions, 1 at m/z 463 thanks to CH-4 3.42 70.6 losing a galloyl moiety (−152 amu) and 1 at m/z 301 thanks to sub- CH-5 3.84 72.5 ′ − CH2-6,6 3.84;3.78 61.6 sequent loss of a sugar moiety ( 162 amu). Peak 27 was therefore β-Glucose CH-1 4.66 d [7.9] 96.9 characterized as quercetin-galloyl-hexoside. All these flavonoid com- CH-2 3.26 dd [9.3;8.0] 75.2 ponents have been previously identified in maqui berry extract, with CH-3 3.51 t [9.1] 76.7 the exception of peaks 18 and 20, which were attributed to quercetin CH-4 3.41 70.6 dihexoside and quercetin hexosyl pentoside, respectively. Peak 18 was CH-5 3.47 77.0 CH2-6,6′ 3.90;3.74 61.7 assigned to quercetin dihexoside, as the parent ion produced two main β-D-Fructofuranose CH-1,1′ 3.61;3.57 63.7 fragment ions thanks to the subsequent loss of two hexosyl sugar C-2 102.5 moieties (m/z 463 and m/z 301), probably linked to two different hy- CH-3 4.12 76.5 droxyl substituents on quercetin (Ruiz, 2016). Peak 20 was identified as CH-4 4.12 75.4 CH-5 3.84 81.7 quercetin hexosyl pentoside, having a parent ion at m/z 595 and two CH2-6,6′ 3.81;3.68 63.4 main fragment ions at m/z 463 and 433, due to losing a pentosyl sugar α-D-Fructofuranose CH-3 4.13 82.9 moiety and a hexosyl sugar moiety respectively. CH-5 4.07 82.3 In summary, RP-HPLC-PDA-ESI-MSn analysis allowed us to first β-D-Fructopyranose CH-1,1′ 3.57;3.73 64.9 identify five compounds present in our maqui berry extract. C-2 99.0 CH-3 3.81 68.6 CH-4 3.90 70.6 3.1.3. Maqui berry metabolite profiling by NMR analysis CH-5 4.01 70.2 ′ The metabolites reported in Table 2 were assigned using 2D ex- CH2-6,6 3.72;4.03 64.4 periments (1H-1H TOCSY, 1H-13C HSQC, and 1H-13C HMBC) and data Organic acids Citric acid α,γ-CH 2.55 d [15.3] 46.7 from the literature (Capitani et a., 2010; Capitani et al., 2014; Mannina α′,γ′-CH 2.68 d [15.3] 46.7 et al., 2012). Proton NMR spectrum of MBE in D2O phosphate buffer is shown in Supplementary Materials (Fig. S3). Formic acid HCOOC 8.47 As can be seen in Fig. S3A, the 1H NMR spectra of MBE has domi- Gallic acid CH-2,5 7.05 S 110.5 α nant citrate and sugar signals (glucose and fructose isomeric forms). Malic acid -CH 4.30 dd [9.9;3.2] 71.4 β-CH 2.68 43.6 The spectrum with the expanded vertical scale (Fig. S3B) shows the β′-CH 2.38 dd [9.9;15.4] 43.6 presence of other signals from minor components, for example, amino Quinic acid CH2-1,1′ 1.88; 2.07 41.8 acids, organic acids and similar compounds. Besides citric acid, the CH-2 3.99 67.9 signals from formic, gallic, quinic, malic, succinic and shikimic acids CH-3 3.56 76.5 CH-4 4.15 71.5 were identified. These acids are present in many fruits and vegetables, CH -5,5′ 1.98 2.05 38.5 fi 2 although this is the rst time their occurrence has been reported in Succinic acid α,β-CH2 2.42 S 35.0 maqui berries. Four amino acids (Ala, Ile, Val, and Leu), GABA, choline Shikimic acid CH2-7,7′ 2.77; 2.20 33.7 and trigonelline were also identified in the 1H NMR spectrum as minor CH-6 3.72 components. CH-5 3.99 – CH-4 4.41 67.3 Additional unassigned signals in the 7.7 6.5 ppm range of 1H NMR CH-3 6.45 131.6 spectra (Fig. S3B) indicate the presence of other aromatic compounds Amino acids besides gallic acid and trigonelline. It is noteworthy that the experi- Alanine α-CH 3.79 51.5 β mental conditions (pH, solvent, buffer addition) were optimized for the -CH3 1.49 d [7.3] 17.2 Isoleucine γ′-CH 1.47 observation and identification of water-soluble metabolites (sugars, γ-CH 1.01 d [7.0] fl 3 organic acids, amino acids). In the case of avonols and anthocyanins, δ-CH3 0.94 t [7.4] lower solubility and pH-dependant transformations in these conditions Leucine β-CH2 1.72 δ precluded the observation of sufficiently resolved and intense NMR -CH3 0.97 d [6.1] 23.0 δ′ signals to associate with corresponding aromatic compounds. -CH3 0.96 d [6.0] 22.1 γ-Aminobutyrate α-CH2 2.32 t [7.4] 35.4 This study showed that NMR and RP-HPLC-PDA-ESI-MSn analyses (GABA) of MBE were complementary, providing a comprehensive metabolite β-CH2 1.91 M 24.7 γ profile of maqui berries not reported in the literature so far. -CH2 3.02 t [7.5] 40.3

Valine β-CH 2.27 3.2. RP-HPLC-PDA quantitative analysis γ-CH3 1.00 d [7.1] 17.9

γ′-CH3 1.05 d [7.1] 19.0 Four compounds, representative of different chemical classes of Other metabolites polyphenols (i.e benzoic acids, hydroxycinnamic acids and flavonoids) Choline N-CH3 3.20 S 55.0 were quantified in the maqui berry extract. For each analyte, the con- N-CH2 3.52 68.4 CH -OH 4.06 56.6 centration range, the calibration curve and the correlation coefficient 2 were determined as reported in Supplementary Materials (Table S1). Trigonelline N-CH3 4.43 49.3 The analytical method was linear within the following concentra- CH-2 9.12 S tion ranges: from 10 to 200 μg/mL for cyanidin-3-glucoside (C3G) and CH-4 8.83 delphinidin-3-glucoside (D3G), and from 5 to 200 μg/mL for GA and (continued on next page)

439 A. Di Lorenzo, et al. Food and Chemical Toxicology 129 (2019) 434–443

Table 2 (continued) validation are reported in Supplementary Materials (Table S2). The D3G calibration curve was used for the quantification of del- 1 13 Compound Assignment H (ppm) Multeplicity: J C (ppm) phinidin derivatives, while the C3G calibration curve was used for the (Hz) quantification of cyanidin derivatives. The results are reported in CH-5 8.08 t [7.0] Table 3. CH-6 8.83 The results demonstrated that MBE showed a higher amount of delphinidin derivatives (4.932 ± 0.4 mg/g) rather than cyanidin derivatives (1.127 ± 0.14 mg/g). In addition, maqui berry extract can be Table 3 considered a good source of gallic acid, which resulted to be exert Concentrations (mg/g of dried extract) of maqui berry extract com- beneficial effects against post stroke depression as reported by our pounds representative of different chemical classes. Data are expressed previous investigations (Nabavi et al., 2016). as mean ± standard deviation of three independent measurements.

Metabolite mg/g of dried extract 3.3. In vivo antidepressive-like and antioxidant activities of MBE Gallic acid 0.75 ± 0.08 Protocatechuic acid 0.251 ± 0.02 Cyanidin glucoside 0.679 ± 0.08 The chemically characterized MBE was used to perform the in vivo Cyanidin sambubioside 0.448 ± 0.06 experiments, as shown in Materials and Methods section. Delphinidin glucoside 3.899 ± 0.33 Mice affected by ischemic stroke (BCCAO group) showed a sig- Delphinidin diglucoside 1.033 ± 0.07 nificant reduction (p < 0.05) in their consumption of sucrose solution, and a significant increase (p < 0.05) in water consumption in comparison with the control group, showing depressed behavior PA. The correlation coefficients were all higher than 0.99. To evaluate (Supplementary Materials, Fig. S4). The administration of MBE miti- the accuracy and precision of the method, different concentrations of gated mouse anhedonia, since it produced dose-dependent increases in GA, PA, C3G and D3G solutions were analyzed in triplicate. The ob- consumption of sucrose solution and a dose-dependent decrease in tained results indicate that the developed method is accurate, providing consumption of water, when compared to the BCAAO group. Despite its recoveries ranging from 90.3% to 114.8%; and precise, since the in- efficacy, MBE was unable to rehabilitate mice to normal behavior, even traday and interday variation were lower than 10% for all the con- at the highest dose (100 mg/kg). centration levels. As far as sensibility is concerned, LOQ and LOD values Regarding DST, as reported in Fig. 1A, B, and 1C, a statistically determined for PA and GA were 1 and 0.3 μg/mL, respectively; while significant difference (p < 0.05) was found across the BCCAO and LOQ and LOD values determined for C3G and D3G were 10 and 5 μg/ control groups, with the BCCAO group being characterized by de- mL, respectively, suggesting that the analytical method developed is pressed behavior revealed by lower times for climbing and swimming, more sensitive for the detection of hydroxybenzoic acids rather than and a higher immobility time. anthocyanins. All the results obtained from the analytical method Intraperitoneal administration of MBE exerted a dose-dependent

Fig. 1. Effects of intraperitoneal administration of MBE on climbing time in the despair swimming model (A); effects of intraperitoneal administration of MBE on swimming time in the despair swimming model (B); effects of intraperitoneal administration of MBE on immobility time in the forced swimming model (C); behavioral effects of intraperitoneal administration of MBE in TST (D). Data are mean (s) ± SD (n = 7). Different letters indicate statistically significant differences (p < 0.05) between the two groups.

440 A. Di Lorenzo, et al. Food and Chemical Toxicology 129 (2019) 434–443

Fig. 2. Effects of intraperitoneal administration of MBE on oxidative stress levels in mouse brain tissue. In detail; TBARS levels, expressed as nmol MDA eq/g tissues (a); SOD activity, expressed as U/mg protein (b); GSH levels, expressed as μg/mg protein (c); and CAT activity, expressed as U/mg protein (d). Data are mean ± SD (n = 7); different letters indicate statistically significant differences (p < 0.05) between the two groups. antidepressive-like effect, significantly (p < 0.05) reducing immobility These two showed opposing trends, with TBARS inversely related to time and increasing both movement parameters. For all cases, the movement parameters and positively related to immobility times, and normal values, as represented by the control group, were not only re- Catalase activity positively related to climbing and swimming times and stored but also improved. inversely related to immobility times. In the case of the TST behavioral test, Fig. 1D highlights a statistically significant variation (p < 0.05) between BCCAO and control groups in terms of immobility time, confirming that animals affected by 4. Discussion and conclusions ischemic stroke present depressive-like behavior. Immobility time was significantly (p < 0.05) decreased by intraperitoneal administration of This study reports the in vivo antidepressive-like activity and anti- MBE, which had an effect on animal behavior with intensity depending oxidant capacity of a fully chemically characterized MBE rich in an- fi on dose. Once again, the normal value was restored and improved by thocyanins. The metabolite pro ling of MBE adds new information on fi fi administration of MBE. the phytochemical pro le of these berries, identifying ve new com- ff MBE was tested against the hypothesis that polyphenols might be pounds (malvidin glucoside, citric acid, ca eoylglucaric acid, galloyl- fi able to assist in managing PSD symptoms by modulating oxidative HHDP-glucose, and quercetin dihexoside) for the rst time, thanks to stress. To this end, some of the biomarkers of oxidative stress were HPLC-MS/MS analysis highlighting the presence in the extract of many measured in mouse brains following the behavioral assessment: these classes of antioxidant compounds besides anthocyanins, which could be fi ff biomarkers consisted of products of lipid peroxidation, expressed as responsible for the bene cial e ects shown through the restoration of TBARS, catalase and superoxide dismutase activity, and GSH levels. The normal mouse behavior, the decrease of TBARS levels and the increase fi results, reported in Fig. 2, highlighted the presence of intense oxidative of antioxidant defenses. Furthermore, NMR allowed the identi cation stress occurring in mouse brains following the induction of stroke. The of other small water-soluble compounds not easily detectable through BCCAO group showed higher TBARS levels and lower levels of anti- HPLC. NMR spectra showed the presence of six organic acids for the fi oxidant defenses (GSH levels, activity of SOD and CAT), than the con- rst time (i.e. gallic acid, formic acid, quinic acid, succinic acid, malic trol group (p < 0.05). The intraperitoneal administration of MBE was acid and shikimic acid), as well as GABA, choline and trigonelline. In found to mitigate oxidative stress, decreasing TBARS levels and in- conclusion, the combination of HPLC-MS and NMR was found to be fi creasing antioxidant defenses, both in a dose-dependent manner. The useful in obtaining a complete picture of the phytochemical pro le, restoration of oxidative stress biomarkers to normal levels was not re- focusing not only on polyphenol compounds but also on small water- gistered in any case. To find the potential correlation between oxidative soluble metabolites. stress biomarkers and behavioral parameters, Pearson's correlation Moreover, this investigation demonstrates that MBE is able to exert coefficients were calculated between possible variables. Looking at the in vivo antioxidant and antidepressive-like activities in a model system results reported in Table 2, it is evident that the parameters most re- of post-stroke depression in mice. Whilst the in vivo antioxidant activity fi lated to depressive symptoms are TBARS levels and Catalase activity. of maqui berries has already been investigated, this is the rst report, to the best of our knowledge, which demonstrates the in vivo antioxidant

441 A. Di Lorenzo, et al. Food and Chemical Toxicology 129 (2019) 434–443 activity of maqui berries in mouse brain and their antidepressive-like Berry anthocyanins and their aglycons inhibit monoamine oxidases A and B. properties. A correlation was found between oxidative stress bio- Pharmacol. Res. 59 (5), 306–311. https://doi.org/10.1016/j.phrs.2009.01.014. – fi Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82 (1), 70 77. markers and depressive symptoms, con rming our hypothesis, ac- https://doi.org/10.1016/0003-9861(59)90090-6. cording to which polyphenols are able to manage PSD symptoms Escribano-Bailón, M.T., Alcalde-Eon, C., Muñoz, O., Rivas-Gonzalo, J.C., Santos-Buelga, through their antioxidant activity. Moreover, this correlation means C., 2006. Anthocyanins in berries of maqui (aristotelia chilensis (mol.) Stuntz). Phytochem. Anal. 17 (1), 8–14. https://doi.org/10.1002/pca.872. that measured oxidative stress biomarkers might be predictive markers Fredes, C., Montenegro, G., Zoffoli, J., Gomez, M., Robert, P., 2012. Polyphenol content of depressive symptoms at preclinical levels, to be confirmed in clinical and antioxidant activity of Maqui (Aristotelia chilensis [Molina] Stuntz) during fruit trials. While we have obtained interesting results, our study presents development and maturation in central Chile. Chil. J. Agric. Res. 72 (4), 582–589 limitations due to the chosen route of administration. Nevertheless, we 0.4067/S0718-58392012000400019. Fredes, C., Yousef, G.G., Robert, P., Grace, M.H., Lila, M.A., Gómez, M., et al., 2014. expected that using intraperitoneal administration the pharmacoki- Anthocyanin profiling of wild maqui berries (Aristotelia chilensis [Mol.] Stuntz) from netics of maqui bioactive compounds are similar to those registered different geographical regions in Chile. J. Sci. Food Agric. 94 (13), 2639–2648. after oral administration. In fact, the primary route of absorption https://doi.org/10.1002/jsfa.6602. Genskowsky, E., Puente, L.A., Pérez-Álvarez, J.A., Fernández-López, J., Muñoz, L.A., happens at the mesenteric vessels level, which drain the molecules into Viuda-Martos, M., 2016. Determination of polyphenolic profile, antioxidant activity portal vein to achieve the liver where the liver metabolism takes in and antibacterial properties of maqui [Aristotelia chilensis (Molina) Stuntz] a Chilean – place (Lukas et al., 1971). Despite these considerations, whilst in- blackberry. J. Sci. Food Agric. 96, 4235 4242. https://doi.org/10.1002/jsfa.7628. González, B., 2015. Polyphenol, anthocyanin and antioxidant content in different parts of traperitoneal administration is widely used in animal studies, especially maqui fruits (Aristotelia chilensis) during ripening and conservation treatments after with small-size experimental animals, it does not accurately resemble harvest. Ind. Crops Prod. 76, 158–165. https://doi.org/10.1016/j.indcrop.2015.06. food consumption. Thus, there is the need for ongoing investigation, 038. Lukas, G., Brindle, S., Greengard, P., 1971. The route of absorption of intraperitoneally studying the results of oral administration of MBE on mood status and administered compounds. J. Pharmacol. Exp. Ther. 178, 562–566. oxidative stress. Mannina, L., Sobolev, A.P., Capitani, D., Iaffaldano, N., Rosato, M.P., Ragni, P., et al., 2008. NMR metabolic profiling of organic and aqueous sea bass extracts: implications in the discrimination of wild and cultured sea bass. Talanta 77, 433–444. https://doi. Author contributions org/10.1016/j.talanta.2008.07.006. Mannina, L., Sobolev, A.P., Viel, S., 2012. Liquid state 1H high field NMR in food analysis. M.D., S.M.N. and L.M. conceived the study; A.D.L., A.P.S., S.F.N., Prog. Nucl. Magn. Reson. Spectrosc. 66, 1–39. https://doi.org/10.1016/j.pnmrs. A.H.M., S.K., S.S., A.J.T.S. performed the chemical and biological ex- 2012.02.001. Mannina, L., Sobolev, A.P., Di Lorenzo, A., Vista, S., Tenore, G.C., Daglia, M., 2015. periments; A.S. analyzed the data and performed statistical analysis; Chemical composition of different botanical origin honeys produced by Sicilian black M.D., L.M., A.D.L., A.B. wrote the paper. J.X. revised critically the honeybees (Apis mellifera ssp. sicula). J. Agric. Food Chem. 63, 5864–5874. https:// paper. All authors approved the final version. doi.org/10.1021/jf506192s. Mannina, L., Sobolev, A.P., Coppo, E., Di Lorenzo, A., Nabavi, S.M., Marchese, A., et al., 2016. Antistaphylococcal activity and metabolite profiling of manuka honey Funding (Leptospermum scoparium L.) after in vitro simulated digestion. Food Funct 7, 1664–1670. https://doi.org/10.1039/c5fo01409c. Marchese, A., Coppo, E., Sobolev, A.P., Rossi, D., Mannina, L., Daglia, M., 2014. Influence This research received no external funding. of in vitro simulated gastroduodenal digestion on the antibacterial activity, metabolic profiling and polyphenols content of green tea (Camellia sinensis). Food Res. Int. 63, Conflicts of interest 182–191. https://doi.org/10.1016/j.foodres.2014.01.036. Miranda-Rottmann, S., Aspillaga, A.A., Pérez, D.D., Vasquez, L., Martinez, A.L., Leighton, F., 2002. Juice and phenolic fractions of the berry Aristotelia chilensis inhibit LDL The authors declare no conflict of interest. oxidation in vitro and protect human endothelial cells against oxidative stress. J. Agric. Food Chem. 50 (26), 7542–7547. https://doi.org/10.1021/jf025797n. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autoxidation of Appendix A. Supplementary data epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247 (10), 3170–3175. Supplementary data to this article can be found online at https:// Moghaddam, A.H., Sobarzo-Sánchez, E., Nabavi, S.F., Daglia, M., Nabavi, S.M., 2014. ff doi.org/10.1016/j.fct.2019.04.023. Evaluation of the antipsychotic e ects of 2-(dimethylamino)- and 2-(methylamino)- 7H-naphtho[1,2,3-de]quinolin-7-one derivatives in experimental model of psychosis in mice. Curr. Top. Med. Chem. 14 (2), 229–233. References Nabavi, S.M., 2012. In vivo protective effects of quercetin against sodium fluoride-induced oxidative stress in the hepatic tissue. Food Chem. 132 (2), 931–935. https:// doi.org/10.1016/j.foodchem.2011.11.070. Bradford, M.M., 1976. 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