MS Profiling of a Mastic Leaf Phenol Enriched Extract and Its

Article pubs.acs.org/JAFC

LC-MS/MS Profiling of a Mastic Leaf Phenol Enriched Extract and Its Effects on H2O2 and Aβ(25−35) Oxidative Injury in SK-B-NE(C)‑2 Cells Severina Pacifico,* Simona Piccolella, Sabina Marciano, Silvia Galasso, Paola Nocera, Vincenzo Piscopo, Antonio Fiorentino, and Pietro Monaco Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, I-81100 Caserta, Italy ABSTRACT: The development of polyphenol neuroprotective nutraceuticals useful for functional foods could be a valuable strategy for counteracting oxidative stress relative diseases as Alzheimer’s Disease (AD). Oxidative stress is one of the AD earliest event and seems to play a central role in Aβ generation, neuroinflammation, and neuronal apoptosis. In order to counteract AD neurodegeneration, the inhibition of the vicious cycle of Aβ generation and oxidation is an attractive therapeutic strategy, and antiamyloidogenic and antioxidant plant drugs could represent an alternative and valid approach. In this context, an alcoholic extract (Pl-M) from deterpenated Pistacia lentiscus L. leaves was investigated for its phenol composition through LC-ESI-MS/MS analysis. Besides the identified metabolites, ten compounds were reported for the first time as constituents of Pistacia lentiscus leaves. Through DPPH, ABTS, and ORAC methods, the antioxidant potential of the extract was initially investigated. In order to evaluate the preparation of a safe and no toxic extract, MTT, SRB, and LDH assays toward SH-5YSY, and SK-N-BE(2)-C human neuronal cell lines, as well as on C6 mouse glial cell line, were performed. Evaluating the protective effects from oxidant injury in SK-N-BE(2)-C cells cotreated with the plant complex and H2O2, or Aβ(25−35) fragment, it was observed that Pl-M extract exerted a significant cytoprotective response in both the oxidized cell systems. In particular, Pl-M extract was able to reduce by nearly 50% the Aβ(25−35) induced toxicity at 25.0 μg/mL dose level, whereas it counteracted almost completely the cytotoxic action at 100.0 μg/mL. Data obtained allow us to hypothesize the use of Pistacia lentiscus leaves, a broadly available and renewable source, as an alternative strategy for the enrichment of food matrices with polyphenol bioactives. The present study put the basis for bioavailability and preclinical studies, able to define Pl-M extract safety and efficacy. KEYWORDS: Pistacia lentiscus, LC-MS/MS metabolic fingerprinting, polyphenols, antioxidant activity, neuroprotection

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INTRODUCTION The remarkable improvements in life expectancy over the past century are shifting the leading causes of diseases and death.1 Chronic and degenerative diseases, also known as noncommunicable diseases, currently contribute substantially to the global burden of diseases. Among them, dementia, including Alzheimer’s Disease (AD), is one of the biggest global public health challenges facing our generation. Dementia affects people in all countries: there were an estimated 44.4 million people with dementia worldwide in 2013.2 This number is expected to increase dramatically to over 115 million by 2050. Treating and caring for people with dementia currently costs the world more than US$ 604 billion per year. This includes the cost of providing health and social care as well as the reduction or loss of income of people with dementia and their caregivers. Alzheimer’s disease (AD) is the most common form of dementia and possibly contributes to 60−70% of cases. Nowadays, there is no known cure associated with such degenerative condition. Symptoms, as loss of episodic memory, cognitive decline, loss of identity, progressive behavioral and physical disability, end with severe and global impairment of cognition and mobility and ultimately lead to death. It is devastating not only for those persons who have it but also for their caregivers and families.3 Thus, discovering new treatments to prevent the disease is the challenge in AD research today. AD has multiple etiological © 2014 American Chemical Society

factors including genetics, environmental factors, and general lifestyles,4 and its hallmark pathology includes extracellular amyloid β protein (Aβ) deposition in the form of senile plaques and intracellular deposits of the microtubule-associated protein tau as neurofibrillary tangles in AD brain.5 Oxidative stress is one of the earliest events in AD and seems to play a central role in Aβ generation, neuroinflammation, and neuronal apoptosis.6,7 In order to counteract AD neurodegeneration, the inhibition of the vicious cycle of Aβ generation and oxidation is an attractive therapeutic strategy. Antiamyloidogenic and antioxidant plant extracts could represent an alternative and valid approach. In fact, their use for the preparation of fortified and stable functional products based on antioxidant plant extracts could provide efficacy to maintain and support normal cognitive function and to prevent or delay AD onset. Naturally occurring phenol phytochemicals as curcumin, resveratrol, and epigallocatechin-3-gallate (EGCG), with massive antioxidant activity and antiamyloidogenic properties, are of considerable interest in the current AD research.8−11 Similarly, the phenylethanoid oleocanthal, one of the main constituents of the extra virgin olive oil, is shown to increase the Aβ clearance.12 The potential therapeutic effects from plant Received: Revised: Accepted: Published: 11957

September 19, 2014 November 17, 2014 November 18, 2014 November 18, 2014 dx.doi.org/10.1021/jf504544x | J. Agric. Food Chem. 2014, 62, 11957−11966

Journal of Agricultural and Food Chemistry

Article

material underwent four sonication cycles (each sonication cycle was 40 min) using MeOH as extracting solvent. The supernatant was dried under vacuum to yield a crude extract (24.4 g, Pl-M). RP-HPLC-ESI-MS/MS Analyses. The chromatographic analyses were carried out using an HPLC Agilent 1200 system (Agilent Technologies Inc., Palo Alto, CA, U.S.A.), provided of binary pump, microvacuum degasser, and autosampler. The chromatographic separation was carried using the reverse phase column (Luna C18, 5.0 μm, 250 × 4.6 mm id, Phenomenex) and as a mobile phase a mixture of A (H2O/CH3COOH, 975:25) and B (MeOH). The following gradient was applied: a linear gradient from 5% to 40% of B in 10 min, a linear gradient from 40% to 51% of B in 20 min, a linear gradient from 51% to 68% of B in 4 min, a linear gradient from 68% to 95% of B in 3 min, a linear gradient from 95% to 100% of B in 4 min, an isocratic elution for 5 min at 100% of B and a last linear gradient from 100% to 5% of B in 5 min. The flow was set at 0.4 mL/min. The Pl-M injection volume (1.0 mg/mL) was of 10 μL. The LC system was coupled to the mass spectrometer API QTRAP 2000 (Applied Biosystem). The ESI mass spectra were recorded in negative mode. The HPLC eluates f ull scan MS mass spectra were acquired in the mass range 50−1000 m/z in electrospray mode with negative ions [MH]−. In order to obtain more structural information, MS/MS experiments (mass range: 20-value m/z of the parental ion) were performed. Assessment of the Antiradical Capacity. Three methods were used to assess the ability of the extract of being chain breaker, which means free radicals’ inactivating. MeOH extract, previously dissolved in DMSO as stock solutions of 125.0 mg/mL, was evaluated at different concentration levels (DMSO final concentration was equal to 0.1% (v/v)). Tests were carried out performing three replicate measurements for three samples (n = 3) of the extract (in total, 3 × 3 measurements). Recorded activities were compared to a blank. Results are the mean ± SD values. Student t-test was applied in order to determine statistical significance (significance level was set at P < 0.05).21,22 Determination of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Capacity. In order to estimate the DPPH• (2,2-diphenyl-1-picrylhydrazyl) scavenging capability, P. lentiscus leaf extract (2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 μg/mL, final concentration levels) was dissolved in a DPPH• methanol solution (9.4 × 10−5 M; 1.0 mL) at room temperature. After 10 min, when the reaction has gone to completion, the absorption at 515 nm was measured by a Shimadzu UV-1700 spectrophotometer in reference to a blank. The results were expressed in terms of the percentage decrease of the initial DPPH• radical absorption by the test samples. Determination of ABTS•+ Scavenging Capacity. The determination of ABTS•+ ([2,2′-azinobis(3-ethylbenzothiazolin-6-sulfonic acid)] radical cation) solution scavenging capacity was estimated as previously reported. ABTS radical cation was generated by reacting ABTS (7.0 mM) and K2S2O8 (2.45 mM). The mixture was allowed to stand in the dark at room temperature for 12h. Thus, the ABTS•+ solution was diluted with PBS (pH 7.4) in order to reach an absorbance of 0.70 at 734 nm. Pl-M extract (2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 μg/mL, final concentration levels) was dissolved in 1.0 mL of diluted ABTS•+ solution. After 6 min of incubation, the absorption at 734 nm was measured by a Shimadzu UV-1700 spectrophotometer in reference to a blank. The results were expressed in terms of the percentage decrease of the initial ABTS•+ absorption by the test samples. Determination of Oxygen Radical Absorbance Capacity (ORAC). The ORAC assay was performed as follows: Pl-M extract (20 μL; 1.0, 2.5, and 5.0 μg/mL, final concentration levels) and fluorescein (FL, 120.0 μL; 70 nM, final concentration) were preincubated for 15 min at 37 °C in 75 mM phosphate buffer (pH 7.4). Then 2,2′azobis(2-amidinopropane)-dihydrochloride (AAPH) solution (60.0 μL; 12 mM, final concentration) was rapidly added. In parallel with the test samples, a blank (FL + AAPH) and solutions of the reference antioxidant Trolox (1−8 μM, final concentration levels) were properly prepared in PBS. The fluorescence decay (λex = 485 nm, λem = 525 nm) was recorded every minute for 120 min using a Tecan

extracts enriched in phenolic antioxidants have also been broadly examined. It was reported, for instance, that Curcuma longa extract, besides acting as antioxidant, significantly increased anti-inflammatory cytokine IL-4 production and reduced Aβ and tau levels in Aβ-overexpressing mice.13 Ginkgo biloba extracts showed significant neuroprotective effects in murine models and improved cognitive function in AD patients.14 Aqueous garlic extracts showed antiamyloidogenic properties inhibiting Aβ fibril formation and also defibrillating Aβ preformed fibrils.15 In recent times, we have demonstrated the ability of a phenol-rich extracts from Laurus nobilis leaves to promote a dose-dependent regression of the formation of Aβ oligomers in neuronal SH-SY5Y and SK-N-BE(2)C cell lines, pretreated in the previous 24 h with the Aβ(25−35) neurotoxic fragment, expounding a promising neuroprotective effect.16 In this context, the neuroprotective capability of an alcoholic extract from Pistacia lentiscus leaves (Pl-M) was of interest. Pistacia lentiscus L., commonly known as mastic tree or lentisk, is an aromatic bush indigenous to Italy and other Mediterranean and Middle East countries. The plant, belonging to the Anacardiaceae family, is a dioecious species with red flowers and fruits, whose resin (Chios mastic gum), rich in terpene molecules, found extensive use in folk medicine for their antibacterial, antiasthmatic, hepatoprotective, sedative, anti-inflammatory, and antitumor activities.17 Pistacia lentiscus essential oil was showed to exert beneficial effect in cerebral hyperfusion/reperfusion for preventing early neuroinflammatory events. The chemical composition of alcoholic Pistacia lentiscus leaf extracts was also investigated.18,19 In particular, a comprehensive characterization of a methanol extract from P. lentiscus leaves, collected in Algeria, was recently reported:20 As geographical and climatic conditions have remarkable influence in the plant phenol production, the LC-ESI-MS/MS analysis of a polyphenol leaf extract (Pl-M) from P. lentiscus plants collected in Southern Italy was carried out. To this purpose, leaves were previously deprived of their apolar constituents through maceration in CHCl3. In order to hypothesize the setting up of an antioxidant preparation with important effects in counteracting invalidating neurodegenerations as Alzheimer’s disease, Pl-M antioxidant and antiamyloidogenic effectiveness was evaluated.

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MATERIALS AND METHODS

Reagents and Chemicals. All of the solvents and reagents used for assessing antioxidant screening were purchased from Sigma-Aldrich Chemie (Buchs, Switzerland) except ABTS [2,2′-azinobis(3-ethylbenzothiazolin-6-sulfonic acid)], which was from Roche Diagnostics (Roche Diagnostics, Mannheim, Germany). Cell culture media and reagents for cytotoxicity testing were purchased from Invitrogen (Paisley, U.K.), MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide], SRB (sulforhodamine B), INT [(2-(4iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride)], Aβ 25−35 fragment, phenazine methosulfate, and lactic acid were from Sigma-Aldrich Chemie. Plant Collection and Fractionation. Leaves from Pistacia lentiscus L. collected in May 2011 in Caserta (Italy) were identified by Dr. Assunta Esposito of the Second University of Naples. A voucher specimen has been deposited at the Herbarium of the Department of Environmental, Biological, and Pharmaceutical Sciences and Technologies of the Second University of Naples. These were dried in a thermo-ventilated oven at 40 °C for 7 days, powdered in a mill and extracted by sonication (Dr. Hielscher UP 200S) for 1 h, using CHCl3 as extracting solvent. Then, the samples were centrifuged at 2044g for 10 min in a Beckman GS-15R centrifuge (Beckman Coulter, Milano, Italy) fitted with rotor S4180. The deterpenated and dried plant 11958

dx.doi.org/10.1021/jf504544x | J. Agric. Food Chem. 2014, 62, 11957−11966


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Table 1. Main Constituents of the Alcoholic Extract from Pistacia lentiscus L. Leaves Detected by LC-ESI-MS/MS in Negative Ionization Mode peak no.

compd. assignment

tR [min]

MW [Da]

[M-H]−

MS/MS fragment ions [m/z]

peak no.

1 2

shikimic acid quinic acid

11.40 12.17

174 192

173 191

3 4 5 6 7 8

galloylhexose I galloylhexose II galloylhexose III prodelphinidin B-4 galloylhexose IV galloylhexose V

13.30 13.78 14.40 14.65 15.15 15.58

332 332 332 610 332 332

331 331 331 609 331 331

9

16.29

316

315

17.50 18.50 19.36

306 442 344

305 441 343

261, 179, 137, 125 331, 169 325, 191, 169

34 35

19.50

344

343

191, 169

36

19.64

344

343

191, 169

37

20.20 20.85

290 344

289 343

245, 203, 179 191, 169

38

17

gentisic acid hexoside (epi)gallocatechin (epi)catechin Gallate 1-O-galloyl-quinic acid 3-O-galloyl-quinic acid 5-O-galloyl-quinic acid (epi)catechin 4-O-galloyl-quinic acid p-coumaroyl hexose

155, 137, 111, 173, 151, 127, 93, 85 271, 169, 125 271, 169 271, 211, 193, 441, 423, 305 271, 223, 211, 313, 271, 241, 169, 161 255, 225, 195,

21.46

326

325

18

gallocatechin gallate

21.53

458

457

44 45

10 11 12 13 14 15 16

19 20 21 22 23 24 25

93 111,

26 27 28 29

169

30

169 211,

31 32

153

33

39

ampelopsin Ohexoside digalloyl quinic acid I

21.90

482

481

23.31

496

495

digalloyl quinic acid II digalloyl quinic acid III digalloyl quinic acid IV trigalloyl quinic acid glabrol

23.50

496

495

265, 231, 187, 163, 145, 137, 119 375, 331, 305, 287, 217, 179, 169, 161, 125 463, 355, 319, 283, 193 435, 413, 343, 191, 169 343, 191, 169

24.81

496

495

343, 325, 191, 169

24.95

496

495

343, 191, 169, 113

27.00 27.95

648 392

647 391

495, 343 331, 309, 161

40 41 42 43

46

SpectraFluor fluorescence and absorbance reader. Antioxidant curves (fluorescence vs time) were normalized to the curve of the blank. From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated. Linear regression equations between net AUC (AUCantioxidant−AUCblank) and antioxidant concentration were calculated for all the samples. The antioxidant activity (ORAC value) was calculated by using the Trolox calibration curve. The ORAC values were expressed as Trolox equivalents (μM). Cytotoxicity Assessment. Cytotoxicity assays that use different parameters associated with cell death and proliferation were performed. Pl-M extract was prepared as a stock solutions of 125.0 mg/mL in DMSO and further diluted in cell culture medium to appropriate final dose levels (DMSO final concentration was equal to 0.1% (v/v)). Tests were carried out performing 12 replicate (n = 12) measurements for three samples of each extract (in total: 12 × 3 measurements). Recorded activities were compared to an untreated blank arranged in parallel to the samples. Results are the mean ± SD values. Student t-test was applied in order to verify statistical significance (significance level was set at P < 0.05).21,22 Cell Cultures. Rat C6 glioma cell line and human SH-SY5Y neuroblastoma cells were purchased from ATCC (American Type Culture Collection); SK-N-BE(2)-C human bone marrow neuroblastoma cells were purchased from ICLC (Interlab Cell Line Collection) at Istituto Nazionale per la Ricerca sul Cancro, Genoa (Italy). Rat C6 and SH-SY5Y cell lines were grown in DMEM high

compd. assignment taxifolin O-hexoside methyl digallate myricetin O-rutinose myricetin O-hexoside I myricetin O-hexoside II eriodictyol Ohexoside myricetin Odeoxyhexoside kaempferol (or Luteolin) Ohexoside rutin quercetin O-hexoside I gentisic acid pentoside quercetin O-hexoside II myricetin O-galloyldeoxyhexoside I myricetin O-galloyldeoxyhexoside II quercetin O-galloylpentoside I quercetin O-galloylpentoside II myricetin O-galloyldeoxyhexoside III quercetin Odeoxyhexoside quercetin O-galloyldeoxyhexoside kaempferol (or luteolin) Ohexoside kaempferol (or luteolin) Odeoxyhexoside

tR [min]

MW [Da]

[M-H]−

28.17 29.62 31.95 32.02

466 336 626 480

465 335 625 479

405, 275, 479, 419,

32.16

480

479

316, 299

34.71

450

449

35.49

464

463

35.56

448

447

389, 367, 287, 217, 151 381, 316, 299, 217, 179 387, 285,227, 193, 170

37.55 37.92

610 464

609 463

301 381, 301, 300, 217

38.63

286

285

225, 203, 153, 108

40.29

464

463

316, 301, 300, 217

40.77

616

615

316

40.84

616

615

463, 317

41.35

586

585

525, 301

41.50

586

585

525, 301, 179

41.82

616

615

463, 317

42.10

448

447

43.93

600

599

365, 301, 300, 283, 201, 179, 151 539, 447, 301

44.07

448

447

415, 365, 285, 201

44.82

432

431

371, 285, 284

MS/MS fragment ions [m/z] 303 253, 183 316 316, 299

glucose medium while SK-N-BE(2)-C cell line in RPMI 1640. All the culture media were supplemented with 10% fetal bovine serum, 50.0 U/mL penicillin, and 100.0 μg/mL streptomycin. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The cell count, for each assay, was carried out using Countess Automated Cell Counter (Invitrogen). MTT Cell Viability Test. The MTT is a colorimetric assay which values the activity of the mitochondrial dehydrogenase to reduce the tetrazolium ring of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), yellow colored, generating a chromogenic compound, which is formazan. C6, SH-SY5Y, and SK-N-BE(2)-C cell lines were seeded in 96-multiwell plates at a density of 1.0 × 104 cells/ well. After 24 h of incubation, cells were treated with Pl-M at five dose levels (2.5, 10.0, 25.0, 50.0, and 100.0 μg/mL). At 24, 48, and 72 h of incubation, cells were treated with 150 μL of MTT (0.50 mg/mL), dissolved in the culture medium, for 1 h at 37 °C in a 5% CO2 humidified atmosphere. The MTT solution was then removed and 100 μL of DMSO were added to dissolve the formazan originated. Finally, the absorbance at 570 nm of each well was determined using a Tecan Spectra Fluor fluorescence and absorbance reader. The results were expressed as percentage of mitochondrial redox activity of the cells treated with the extracts compared to the untreated control. SRB Cell Viability Test. C6, SH-SY5Y, and SK-N-BE(2)-C cell lines were seeded in 96-multiwell plates at a density of 1.0 × 104 cells/ well. After 24 h of incubation, cells were treated with Pl-M at five dose 11959



Article

levels (2.5, 10.0, 25.0, 50.0, and 100.0 μg/mL). At 24, 48, and 72 h of incubation, cells were fixed with ice-cold TCA (10% w/v, 40 μL) for 1 h at 4 °C. The plates were washed five times in distilled water and allowed to dry. Then, 50 μL of sulforhodamine B (SBR, 0.4% w/v in 1% aqueous acetic acid) solution was added to each well of the dried 96-well plates and incubated at room temperature for 30 min. In order to remove unbound dye, the plates were quickly washed with 1% aqueous acetic acid and dried subsequently. The bound SRB was solubilized by adding 100 μL of 10 mM unbuffered Tris Base (pH 10.5) to each well and shaking for 5 min on a shaker platform. Finally, the absorbance at 570 nm of each well was measured using a Tecan SpectraFluor fluorescence and absorbance reader. Lactate Dehydrogenase (LDH) Leakage Assay. The lactate dehydrogenase (LDH) leakage assay is a colorimetric assay for the quantification of cell death and cell lysis based on the measurement of LDH released into the supernatant from the cytosol of damaged cells. The oxidation reaction (LDH oxidizes lactate to pyruvate and converts NAD+ to NADH) catalyzed by this enzyme produces NADH that is reacted in vitro with a tatrazolium salt 2-(4-iodophenyl)-3-(4nitrophenyl)-5-phenyltetrazolium chloride (INT), yellow colored to generate a formazan salt purple/red colored; this coloration can be measured using a spectrophotometer. C6, SH-SY5Y, and SK-N-BE(2)C cell lines were seeded in 24-multiwell plates at a density of 4.0 × 104 cells/well. Twenty-four hours after seeding, the cells were treated Pl-M extract (100.0 μg/mL). After the exposure times of 24 h, 48 h, and 72 h, the supernatant (100 μL) was treated with 100 μL of the reaction mixture (0.7 mM INT; 54.0 mM lactic acid; 0.3 mM phenazine methosulfate; 0.8 mM NAD+ in 0.2 M Tris-HCl pH 8.0) in a 96-wells plate. The reaction was carried out for 45 min in the dark under gentle stirring at 37 °C and stopped by addition of 1 M HCl (50 μL). The absorbance was red at 492 nm by a Tecan SpectraFluor fluorescence and absorbance reader. Data obtained were expressed in reference to a blank prepared using a Triton X-100 (1%) solution. The increase in the amount of formazan detected in culture supernatant reflects the membrane integrity and directly correlates to the increase in the number of lysed cells. Hence, cell vitality is inversely proportional to the LDH released. Determination of Cytoprotective Effect in Oxidized Cell Systems. SK-N-BE(2)-C cell line was seeded in 96-multiwell plates at a density of 1.0 × 104 cells/well. After 24 h of incubation, cells were cotreated with Pl-M extract (25.0, 50.0, and 100.0 μg/mL, final concentration levels) and H2O2 (400 μM) or Aβ(25−35) peptide domain (100 μM) for 24 h. After the treatment period, cell viability inhibition (CV) was assessed by MTT assay.16

Compound 1 was identified on the basis of its mass spectrum, as shikimic acid.24 A [M-H]− pseudomolecular ion at m/z 173 and the corresponding dimeric deprotonated ion [2MH]− a m/z 347 were detectable. The selection of the [M-H]− precursor ion and the subsequent dissociation (MS2 experiment) generated a fragment ion at m/z 155, due to the loss of a water molecule, probably including the hydroxyl function at C5. A second fragment ion at m/z 111 was putatively attributed to the ion [M-H-CO2-H2O]−. Compound 2 was characterized as quinic acid on the basis of its fragmentation pattern. In fact, the collision activated decomposition of the deprotonated molecule at m/z 191, beside the ion at m/z 173 (- H2O), led to the formation of the fragment ion at m/z 127, corresponding to the loss of CO and of two water molecules.25 Metabolites 12−14 and 16 are quinic acid derivatives, and in particular they were identified as galloylquinic acid isomers, showing a deprotonated pseudomolecular ion at m/z 343, which in turn generated product ions at m/z 191 and 169, following the neutral loss of a quinic acid residue and a gallic acid moiety, respectively. The discrimination between the four isomers was performed on the basis of their relative retention times and according to the data previously reported.26 The Pl-M extract also contained four peaks characterized by a pseudomolecular deprotonated ion at m/z 495 (compounds 20-23). The fragmentation of the [M−H]− ion at m/z 495 provided the fragments at m/z 343 ([M-H152]−) and m/z 191 ([M-H-304]−) corresponding to the loss of one and two galloyl moieties, respectively. The fragment at m/z 169 was attributed to gallic acid anion. On the basis of these experimental evidence they were identified as isomers of digalloyl quinic acid, recently identified in P. lentiscus leaves.20 Metabolites 3−5, 7, 8 exibited the same pseudomolecolar deprotonated ion at m/z 331. On the basis of the data arising from MS2 experiments, compared with those present in literature,27 the compounds were tentatively identified as hexoside isomers of gallic acid. In fact, the collision activated decomposition of the precursor ions at m/z 331 led to the formation of product ions consistent with this hypothesis, namely the fragment ions at m/z 271 ([M-H-60]−), 241 ([MH-90]−), and 211 ([M-H-120]−), due to the cross-ring fragmentation of the hexose moiety. Furthermore, the product ion at m/z 169 ([M-H-C6H10O5]−) could be due to the neutral loss of the hexose residue, and could consequently be ascribed to the deprotonated gallic acid, which in turn lost a 44 Da moiety (likely CO2) to give the ion at m/z 125. Compound 6 exhibited a pseudomolecular deprotonated ion at m/z 609. The MS2 spectrum showed characteristic fragment ions that allowed us to identify the molecule as the proanthocyanidin prodelphinidin B-4, a dimer of gallocatechin and epigallocathechin.28 In fact, beside the ion at m/z 305, due to the loss of one monomer ([M-H-304]−), the ions at m/z 441 and 423 could be formed following a retro Diels−Alder fragmentation mechanism involving the C ring (cleavage of O1−C2 and C3−C4 bonds) and subsequent water loss. Metabolite 9, characterized by a [M-H]− pseudomolecular ion at m/z 315, was tentatively identified as gentisic acid hexoside.20 The product ions deriving from MS/MS experiments were detected at m/z 255, 225, 195, corresponding to cross-ring cleavage of the sugar moiety (−60, −90, and −120 Da, respectively), and at m/z 153, attributable to the deprotonated gentisic acid. The deprotonated compound 36 differed from 9 for 30 mass units, having a [M-H] − pseudomolecular ion at m/z 285. Its fragmentation pattern

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RESULTS AND DISCUSSION The diverse array of bioactive nutrients present in plant natural products plays a pivotal role in prevention and cure of various neurodegenerative diseases, such as Alzheimer’s disease (AD). Accumulated evidence suggests that naturally occurring phytocompounds, such as polyphenolic antioxidants may potentially hinder neurodegeneration, and improve memory and cognitive function.23 In order to investigate the neuroprotective potential of Mediterranean medicinal plants, Pistacia lentiscus was of interest. Leaf plant drug was analyzed for its antioxidant and cytoprotective effectiveness, whereas its chemical composition was characterized by means of a LC-MS/MS approach. LC-MS/MS Metabolic Profiling of the Alcoholic Extract from Pistacia lentiscus L. Leaves. LC-ESI-MS/MS techniques allowed us to obtain qualitative information about the biochemical constitution of Pl-M extract. Forty-six main secondary metabolites were tentatively identified by means of LC-MS/MS data (Table 1). Among them ten compounds (6, 11, 17−19, 27, 31, 36, 40, 41) were tentatively characterized for the first time in Pistacia lentiscus leaves. 11960



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rutinoside ([M−H]− at m/z 625). The presence of fragments at m/z 479 and 316 in MS2 spectrum was attributable to the loss of a deoxyhexose moiety [M−H−146]− and to the aglycone radical myricetin, respectively. The formation of this latter was probably due to the homolytic cleavage of the glycosidic bond. Compounds 29 and 30 were tentatively identified as myricetin O-hexoside isomers ([M−H]− at m/z 479). Also in this case, the formation of the aglycone radical [Aglicone−H]−• was observed. The metabolite 32 was putatively identified as myricetin O-deoxyhexoside. In fact, the homolytic cleavage of the [M−H]− ion at m/z 463 generated the aglycone radical and a glycanic radical (−147 Da) of deoxyhexose nature. Other three myricetin derivatives (38, 39, 42) were found to be present in Pl-M extract, characterized by the presence of a galloyl moiety as evidenced by neutral losses of 152 Da, beside a deoxyhexoside residue. They were tentatively identified as myricetin O-galloyl-deoxyhexoside isomers. On the basis of their fragmentation pattern metabolites 34, 35, 37, 43 were identified as quercetin glycosides, which differed for the sugar residue (rutinose, hexose, deoxyhexose). The product ions at 301 and 300 likely corresponded to deprotonated quercetin and quercetin radical, respectively. The two hexoside isomers (35 and 37) differed mainly for the relative abundance of these ions, whose ratio suggested the glycosylation position at the C-3 for compound 37, and C-7 or C-4′ for compound 35.35,36 Compounds 40 and 41 were putatively identified as quercetin O-galloylpentoside isomers. In fact, following the collision induced dissociation of the deprotonated molecules at m/z 585, the ions at m/z 301 were generated, likely due to the loss of a pentose sugar and a galloyl moiety (132 + 152 Da). Metabolite 44 was characterized as quercitrin gallate, according to MS/MS evidence. In fact, it showed a molecular deprotonated ion at m/z 599, which yielded fragment ions at m/z 447, due to the loss of the galloyl moiety, and m/z 301, likely the deprotonated quercetin arising from the further loss of a deoxyhexoside residue. This compound was recently reported as a component of P. lentiscus leaves.20 Compounds 19 and 26 were putatively ascribed to glycosylated dihydroflavonols. In particular the MS2 spectrum of metabolite 19, sharing a [M-H]− pseudomolecular ion at m/ z 481, showed the product ion at m/z 319 after the loss of a hexose residue (162 Da), likely corresponding to dihydromyricen (ampelopsin), whereas deprotonated compound 26 at m/z 465, once fragmented, gave rise to the ion at m/z 303, likely dihydroquercetin (taxifolin). According to these experimental data, compounds 19 and 26 were tentatively characterized as ampelopsin O-hexoside and taxifolin O-hexoside, respectively. Metabolite 31 was putatively identified as a hexoside of the flavanone eriodictyol ([M−H]− a m/z 449). The fragment ion at m/z 287 ([M-H-C6H10O5]−) suggested the presence of the flavonone for loss of a six carbon atoms glycidic moiety.37 Compounds 33 and 45 were tentatively identified as hexosides of the flavonoid kaempferol and/or luteolin. In fact, they showed the same [M-H]− molecular ion at m/z 447, and the same product ion at m/z 285, formed after the loss of a hexose residue (−162 Da). Unfortunately mass spectrometric data did not allow us to discriminate between the two aglycone isomers. For the same reason metabolite 46 was putatively identified as kaempferol-3-O-deoxyhexoside or luteolin-3-Odeoxyhexoside. The MS2 spectrum of the deprotonated molecular ion at m/z 431 provided a base peak at m/z 285,

showed product ions at m/z 225, 203, 153 and 108, consistent with the presence of a pentose instead of hexose. For this reason, it was putatively identified as gentisic acid pentoside. Compound 15 was characterized by a deprotonated pseudomolecular ion at m/z 289, attributable at least in principle to catechin or epicatechin. The relative intensity of the characteristic fragment ions detected in the fragmentation pattern of this molecule (ions at m/z 245, 203, 179), even if compared with literature,29 was insufficient to discriminate between the two epimers. Similarly the metabolite 10 was putatively identified as the flavonol (epi)gallocatechin. Indeed, the mass spectrum showed the peak of the [M-H]− ion at m/z 305 and characteristic fragments at m/z 261, 219, 179, and 125. The presence of the fragment at m/z 125 was identifiable in the intact flavonoid A ring.30 The metabolite 11, on the basis of data provided by mass spectrometry experiments, was putatively identified as (epi)catechin gallate. The fragmentation of the [M-H]− ion at m/z 441 generated two major fragments at m/z 331 and 169. The formation of the former fragment could be due to the loss of the only catechol B ring and introduction of a new unsaturation between carbons C2−C3 of the C ring. The fragment at m/z 169 consisted of gallic acid anion. The [M−H]− ion for the metabolite 18 at m/z 457 yielded main fragment ions at m/z 331, 305, and 169. The product ion at m/z 331 was due to the neutral loss of intact B-ring, whereas the ion at m/z 305 consisted of deprotonated epigallocatechin or gallocatechin, formed by the loss of the galloyl moiety (−152 Da). Finally, the ion at m/z 169 corresponded to the gallic acid anion.31 The abundance of this latter, respect to that at m/z 305, allowed us to unequivocally identify this compound as gallocatechin gallate.29 Compound 17 was identified as p-coumaroylhexose on the basis of its MS2 spectrum compared with literature.32 In fact, the deprotonated molecular ion at m/z 325, once fragmented, yielded product ions at m/z 163, likely corresponding to deprotonated p-coumaric acid formed after the loss of the hexose residue as 162 Da, and m/z 145 and 119 due to the subsequent loss of a water molecule and CO2, respectively. The compound 24, with the [M-H]− pseudomolecular ion at m/z 647, was identified as trigalloylquinic acid (e.g., pistafolin A).33 The fragments at m/z 343 and 495 were reported as successive losses of two molecules of gallic acid. The fragmentation pattern of the metabolite 25 was in agreement with the presence of glabrol, a molecule that reduces the absorption of cholesterol and modulates the expression of genes involved in lipid metabolism and recently was found in P. lentiscus leaves.20 The pseudomolecular ion at m/z 391 gave rise to the fragments at m/z 331 and 161. The latter, corresponding to a residue of 2-(3-methylbut-2-enyl)phenol, could be formed after the loss of a benzo-γ-pyrane core that brought an isoprenic chain in C-8. Metabolite 27 showed a deprotonated pseudomolecular ion at m/z 335 and a characteristic fragment ion at m/z 183, which allowed us to identify it as methyl digallate on the basis of literature data.27 It has previously reported that the Anacardiaceae family is characterized by the occurrence of both gallic acid and myricetin derivatives.34 Our data confirmed these evidence. In fact, beside the gallic acid derivatives discussed above, the alcoholic extract of P. lentiscus under study also contained myricetin glycosides (compounds 28−30, 32). In particular, metabolite 28 was identified as the flavonol myricetin O11961



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which allowed us to hypothesize the presence of kaempferol/ luteolin as aglycone, and a deoxyhexosyl glyconic moiety (−146 Da). Antioxidant and Cytoprotective Capabilities of Pl-M Extract. The antiradical properties of Pl-M extract were evaluated through three methods, useful to define the capability of the investigated plant complex to act as a source of secondary antioxidants (chain breaker), which are chemical species able to inactivate dangerous free radicals transferring them an hydrogen atom and/or a single electron (Figure 1).

Figure 1. Radical scavenging capacity (RSC, %) of P. lentiscus investigated extract toward DPPH radical, ABTS+ radical, and ROO• radicals. These latter are reported as the mean of ΔAUC values from ORAC assay. Values, reported as percentage vs a blank, are the mean ± SD of measurements carried out on 3 samples (n = 3) analyzed three times.

Figure 2. Pl-M cell viability inhibition (CVI, %) toward SK-N-BE(2)C, SH-SY5Y, and C6 cell lines at 24, 48, and 72 h exposure times by means of (A) MTT test results, (B) SRB test results. Values, reported as percentage vs an untreated control, are the mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times.

The extract showed an efficacious reducing power the target radical species ABTS•+ and DPPH•. In fact, 0.6 μg/mL Pl-M dose level was able to reduce ABTS•+ by 50%, whereas a dose equal to 2.9 μg/mL was required to scavenge the same percentage of DPPH radical. The scavenging effectiveness appeared to be strongly dependent on the dose tested. In particular, it was observed that the complete conversion of the radical cation ABTS in its reduced form was realized when the probe was exposed to an extract dose equal to 25.0 μg/mL. The massive antiradical activity was confirmed by ORAC data. The high sensitivity of the ORAC method required, in order to obtain useful information, the use of extract doses lower than those adopted in the colorimetric tests (1.0, 2.5, and 5.0 μg/ mL). The values of ΔAUC (area under the curve) shown in Figure 1 and ORAC values, reported as equivalents of Trolox, a water-soluble analogue of vitamin E commonly used as a standard positive, highlighted the antiradical effectiveness of the extract capable to exercise at a dose of 1.0 μg/mL an activity comparable to that exerted by 3.1 μg/mL of Trolox. The evaluation of the cytotoxic effects of the extract through MTT and SRB assays that it exerted only an mild cell viability inhibition (Figure 2A, B). In particular, it was observed that the extract influenced in a time dependent manner the proliferation of murine cell line, whereas both the human neuroblastoma cell lines showed a similar cytotoxic behavior at all the three exposure times. SK-N-BE(2)-C cell line appeared to be less sensitive to the addition of toxic. In fact, an inhibition of cell viability of approximately 10% was detected when SK-N-BE(2)C cells were treated with the extract highest tested dose (100 μg/mL). Data obtained emphasize that Pl-M complex did not adversely influence the growth and proliferation of tested cell lines opening a favorable scenario for an its likely therapeutic application.

In order to further explore the Pl-M toxic effects, lactate dehydrogenase (LDH) release was also investigated. The test (Figure 3) was performed toward the three cell lines using the single dose of 100.0 μg/mL. Detected data allowed us to state that the extract did not affect LDH release. The evaluation of the plant extract effect on H2O2-induced oxidative stress in SK-N-BE(2)-C cells was tested. Cotreatment with 25.0, and 50.0 μg/mL Pl-M extract showed viability values slightly lower than those elicited by H2O2treated group (Figure 4A). Only the cells treated with Pl-M

Figure 3. Pl-M induced LDH release (±SD) vs SK-N-BE(2)-C, SHSY5Y, and C6 cell lines. The evaluation of LDH release was carried out treating cells with 100.0 μg/mL extract dose at 24, 48, and 72 h exposure times. Values, reported as percentage vs an untreated control, are the mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times. 11962



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by nearly 50%, the Aβ 25−35-induced toxicity at the lowest dose level tested. A marked neuroprotective effect was observed when Pl-M 100.0 μg/mL dose level was screened. In fact, the results underlined the high antioxidant capacity of this Pl-M dose level able to counteract almost completely the cytotoxic action of the oxidant. There is ample scientific and empirical evidence supporting the use of plant-derived antioxidants for the control of neurodegenerative disorders. Antioxidants may have neuroprotective (preventing apoptosis) and neuroregenerative roles, by reducing or reversing cellular damage and by slowing progression of neuronal cell loss. Dementia pathologies such as Alzheimer’s disease (AD) are reaching epidemic proportions, yet they are not successfully managed by effective symptomatic treatments.40 Since two of the currently licensed drugs for AD are based on natural products (galantamine and rivastigmine), different plants are now being investigated as a potential source of new therapy for AD.41,42 Furthermore, different studies have revealed that natural polyphenols can reduce Aβ neurotoxicity43,44 possibly via an antioxidative mechanism and inhibition of Aβ oligomerization.45 The Aβ lowering properties of these substance could be mainly mediated via their effect on NF-κB signaling which in turn could affect the regulation of BACE-1 expression. A significant inverse association between phenolics intake and dementia risk, was found.46 In neuronal cell cultures, it was observed that EGCG was able to regulate the proteolytic processing of APP (amyloid precursor protein) and to reduce Aβ generation.47 The molecular mechanism involved in the neuroprotection effect of EGCG could also due to its ability to decrease the expression of pro-apoptotic genes.48 Upon their antioxidant properties, phenol compounds as chlorogenic acid, gallic acid, and quinic acid, together with various flavonoids, were reported to be potential inhibitors of acetylcholinesterase. Thus, the use of these substances could be used to counteract the AD deficiency in acetylcholine production.49 Furthermore, previous bioavailability studies allow us to encourage the use of Pistacia lentiscus leaf alcoholic extract as an alternative source for the preparation of functional food enriched in flavan-3-ols and galloyl esters. Indeed, although absorption, distribution, metabolism, and excretion (ADME) studies highlighted that polyphenols bioavailability varies widely from one compound to another, as it is influenced by their chemical structure and molecular weight,50 positive data are available on molecules identified in Pl-M extract. An acute feeding study, through HPLC-MS3 analysis of plasma and urine samples of healthy human subjects fed with green tea products, showed that (−)-epigallocatechin-3-O-gallate and (−)-epicatechin-3-O-gallate appeared unmetabolized in plasma.51 The passage of ECG and EGCG through the wall of the small intestine into the circulatory system without metabolism could be a consequence of the presence of the 3-O-galloyl moiety interfering with phase II metabolism. Gallic acid per se is readily absorbed with a reported urinary excretion of 37% of intake, and so, the gallate ester might exhibit improved absorption.52 The absence of detectable amounts of EGCG in urine was hypothesized to be due to the kidney inability to remove it from the bloodstream.53 Preclinical studies speculated that EGCG may be removed from the bloodstream in the liver and returned to the small intestine in the bile.54 The level of EGCG found in the major organs was found to be ∼1/10 that found in the serum. Most interestingly, this includes the brain, suggesting that EGCG passes through

Figure 4. Cell viability inhibition (CVI, %) toward SK-N-BE(2)-C cell line cotreated with Pl-M extract and (A) H2O2 (400 μM) or (B) Aβ(25−35) fragment (100 μM) at 24 h exposure time by means of MTT test results. Values, reported as percentage vs. an untreated control, are the mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times.

100.0 μg/mL dose level presented a high and significant value of cell viability compared to H2O2-treated group. In order to determine the possible beneficial effect of the extract against the phenomena of oxidative stress induced by amyloid plaques in Alzheimer’s dementia, its antioxidant capacity in a system utilizing the functional domain of the β-amyloid peptide, Aβ(25−35), as oxidizing agent, was tested. As the cellular reduction of MTT represents a specific indicator of the initial events underlying the mechanism of β-amyloid peptide toxicity,38,39 MTT test was first carried out exposing the three cell lines at increasing concentrations of Aβ(25−35) fragment (12.5, 25.0, 50.0, and 100.0 μM). Aβ(25−35) fragment was found to induce cytotoxicity in SK-N-BE-(2)-C cell line. The results of the MTT test carried out after exposure of SK-N-BE-(2)-C cell line to Aβ 25−35 increasing concentrations (12.5, 25.0, 50.0, and 100.0 μM) allowed us to identify the domain concentration to which it induced the highest cell viability inhibition. The effects of cotreatment of the cell line for 24 h with Aβ 25−35 100.0 μM and increasing extract dose levels (25.0, 50.0, and 100.0 μg/mL) were assessed through MTT test (Figure 4B). Pl-M extract was able to reduce 11963



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the blood-brain barrier.55 In this latter context, little and controversial information deals with the interaction of polyphenols or their circulating metabolites with the blood brain barrier (BBB). The layer of brain endothelial cells is sealed by complex tight junctions that possess few pinocytotic vesicles but express a number of specific uptake and efflux transport systems and metabolic enzymes. Together, these properties enable the BBB to restrict the passage of most small polar molecules and macromolecules from the cerebrovascular circulation to the brain, to exert tight control over transendothelial molecular traffic and to contribute to regulation of brain extracellular fluid composition. Youdim and co-workers56 investigated the potential BBB permeability of flavonoids and their physiologically relevant metabolites using the ECV304/ C6 coculture BBB model. Aglycones, due to their lipophilicity, were found to cross BBB by passive transcellular diffusion. Moreover, some evidence suggested that polyphenol constituents from Pistacia lentiscus leaves could be able to cross BBB. Epigallocatechin gallate (EGCG), for instance, was reported to enter the brain after a gastric administration of [3H]EGCG,57 whereas gallic acid was found to pass through BBB and protect or improve neuronal function in critical brain regions involving cognition after cerebral damage by ischemia.58 14C-labeled grape polyphenols was also demonstrated to reach the brain following oral administration.59 Importantly, in in vitro and in situ models of the blood brain barrier, it was demonstrated that quercetin was able to enter different regions of the brain as a substrate of P-glicoproteins, a BBB efflux transporter.60 Studies have indicated that the accumulation of flavonoids in the brain is not dependent on the brain region. Vauzour and coworkers,61 reviewing the neuroprotective potential of flavonoids, stated that these molecules traverse BBB and are able to localize in the brain, suggesting that they are candidates for direct neuroprotective and neuromodulatory actions. Indeed, although the uptake and distribution of dietary polyphenols within the brain are well documented, the question of the dose reaching the target tissues remains uncertain. Data from studies using exsanguinated and perfused animals or applying the recently published mathematical correction model suggest that polyphenols usually localize in the brain at levels below 1 nmol/g tissue.62 In conclusion, a host of products from plant species and derivatives have neuroprotectant effects in vitro and in vivo. Reported data are in accordance with the preparation of a polyphenol-rich alcoholic extract of Pistacia lentiscus (Pl-M) with strong antiradical and antioxidant activity. The extract, which was highly cytocompatible in neuronal and glial tested cell lines, at concentrations up to 100 μg/mL, showed protective effect against hydrogen peroxide-induced oxidative stress and marked antiamyloidogenic activity in neuronal cells. The positive and promising effects, probably the result of an appropriate mixture of ingredients, allow us to hypothesize that the enrichment of a food matrix with polyphenol bioactives from a broadly available and renewable source, as Pistacia lentiscus leaves, could represent a valuable method to counteract Alzheimer’s disease onset. In the present study, LC-MS analyses were performed to unravel the chemical complexity of this potential botanical dietary supplements, which represents, likely green tea and wine, an active and rich source of gallic acid esters and glycosides. The massive presence of some of these compounds, e.g. galloyl and digalloyl quinic acids, poorly available in human diet, addresses Pl-M extract bioavailability and preclinical

studies, able to define its active doses and to assess Pistacia lentiscus polyphenol extract safety and efficacy as nutraceutical in an AD mouse model and aged mice.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: severina.pacifi[email protected]. Phone: +39 0823 274572. Fax: +39 0823 274571. Notes

The authors declare no competing financial interest.

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REFERENCES

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