Cyanidin-3-O-galactoside in ripe pistachio (Pistachia vera L. variety Bronte) hulls: Identification and evaluation of its antioxidant and cytoprotective activities

Journal of Functional Foods 27 (2016) 376–385

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Cyanidin-3-O-galactoside in ripe pistachio (Pistachia vera L. variety Bronte) hulls: Identification and evaluation of its antioxidant and cytoprotective activities Ersilia Bellocco a, Davide Barreca a, Giuseppina Laganà a, Antonella Calderaro a, Zineb El Lekhlifi b, Salima Chebaibi b, Antonella Smeriglio a,*, Domenico Trombetta a a

Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale F. Stagno d’Alcontres 31, Messina, Italy b Faculty of Sciences, University Moulay Ismail Meknes, BP 11201 Zitoune, Mekne, Morocco

A R T I C L E

I N F O

A B S T R A C T

Article history:

Anthocyanidins, compounds with several health promoting properties, are able to influ-

Received 29 July 2016

ence a broad range of biological processes. In the present work we identified cyanidin-3-

Received in revised form 20

O-galactoside in ripe pistachio hulls, which represented the most abundant compound (>96%),

September 2016

and we performed a deep analysis of its antioxidant and cytoprotective activities against

Accepted 23 September 2016

tert-butyl hydroperoxide induced toxicity on human lymphocytes. Cyanidin-3-O-galactoside

Available online

showed strong antioxidant activity in ORAC, DPPH•, ABTS•+, FRAP and O2•− assays, clearly superior to synthetic antioxidants BHT, BHA and Trolox, while no activity was detected with

Keywords:

H2O2. Moreover, in the concentration range 0.1–10 µM, cyanidin-3-O-galactoside was found

Cyanidin-3-O-galactoside

able to counteract cytotoxic effects of tert-butyl hydroperoxide decreasing or completely avoid-

Antioxidant activity

ing cell death, LDH release, caspase 3 activation and DNA damages.

Cytoprotective activity

© 2016 Elsevier Ltd. All rights reserved.

Anthocyanins RP-HPLC-DAD

1.

Introduction

There is an increased interest of consumers towards a healthy lifestyle, with particular attention for diet and functional foods,

which can help reduce the risk of diseases and increase life span and well-being. Anthocyanins are natural pigments belonging to the flavonoid family identified in several plants, flowers and fruits (Wu & Prior, 2005). They have gained research attention due to their interesting and useful human

* Corresponding author. Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale F. Stagno d’Alcontres 31, Messina, Italy. E-mail address: [email protected] (A. Smeriglio). Chemical compounds: Cyanidin-3-O-galactoside (PubChem CID: 44256700); Trolox (PubChem CID: 40634); Butylated hydroxytoluene (PubChem CID: 31404); Butylated hydroxyanisole (PubChem CID: CID: 8456). http://dx.doi.org/10.1016/j.jff.2016.09.016 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 27 (2016) 376–385

health-promoting properties such as antioxidant, cytoprotective, anti-obesity and lipidaemic, antimicrobial and antitumour activities as well as their positive therapeutic effects in reducing the risk of age-related neurodegenerative disorders, cardiovascular diseases, diabetes and cancer (Hou, 2003; Martorana et al., 2013; Prior & Wu, 2006; Seeram, Bourquin, & Nair, 2001; Smeriglio, Barreca, Bellocco, & Trombetta, 2016; Tomaino et al., 2010; Yang et al., 2011). These observations are also supported by epidemiological evidences highlighting a possible direct correlation between anthocyanins intake and the onset of chronic and degenerative diseases (Smeriglio et al., 2016). Pistachio (Pistacia vera L.) is a nut tree which belongs to the Anacardiaceae family (Martorana et al., 2013) cultivated in Iran, Turkey, USA, Syria, Italy, Tunisia and Greece. In Italy the pistachio nut production industry is principally located in Sicily, where it is mainly cultivated on lava-rich soils in the eastern part around Etna Mount, in the region of Bronte (Martorana et al., 2013; Tomaino et al., 2010). Pistachio nuts are considered a rich source of many important biofunctional compounds (anthocyanins, flavan-3ols, proanthocyanidins, flavonols, isoflavones, flavanones, stilbenes and phenolic acids) useful for human diet and known for their various pharmacological characteristics such as antimicrobial, anti-inflammatory, insecticidal, anti-nociceptive activities (Giner-Larza et al., 2002; Orhan, Küpeli, Aslan, Kartal, & Yesilada, 2006; Özçelik, Aslan, Orhan, & Karaoglu, 2005; Pascual-Villalobos & Robledo, 1998) and especially the antioxidant activity due to the anthocyanin group (Tomaino et al., 2010). Recently, we have analysed and described the nutraceutical, antioxidant and cytoprotective activity of several phenol extracts isolated and identified in the hulls of ripe pistachio, by-products (35–45% of waste production) from the pistachio industries (Barreca et al., 2016). Up until a few years ago, despite the pistachio hull being a promising source of primary and secondary metabolites, little information was available in the literature regarding its use, bioactive compounds and biological properties (Chahed et al., 2007; Goli, Barzegar, & Sahari, 2005). The anti-microbial and antimutagenecity activities, as well as the total phenol content, were analysed (Goli et al., 2005; Rajaei, Barzegar, Mobarez, Sahari, & Esfahani, 2010), although only recently this matrix was characterized through an in-depth analysis of phenol composition, antioxidant and cytoprotective properties (Barreca et al., 2016). These studies have allowed to identify this matrix as a promising source of healthy compounds. The methanol extract was found best in terms of phenol, vanillin index, total flavonol and proanthocyanidin content. Furthermore, these parameters made it possible to calculate the polymerization index of crude extracts under study, highlighting the dominance of monomeric molecules (Barreca et al., 2016). RP-HPLC-DAD-FLU analysis allowed the identification and quantification of 20 compounds, of which the most abundant were gallic acid, 4-hydroxybenzoic acid, protocatechuic acid, naringin, eriodictyol7-O-glucoside, isorhamnetin-7-O-glucoside, quercetin-3-Orutinoside, isorhamnetin-3-O-glucoside and catechin (Barreca et al., 2016). Moreover, the results obtained highlighted antioxidant and cytoprotective properties directly correlated to the high total phenol content, in particular flavonols, phenolic acids and flavan-3-ols. However, despite this, the differences found in the two extracts, in terms of bio-activity, allow us to ascribe

377

them not only to the simple difference in the total amount of bioactive compounds but as a function of the structure/ activity relationships given the presence of different flavonoid skeleton/substitution in the components identified (Barreca et al., 2016). The richness in compounds with health promoting properties of this matrix and the possibility to easily utilize it to produce nutraceuticals lead us to take into account its anthocyanin composition. In particular, in the present work we performed a RP-HPLC-DAD analysis of the ripe pistachio hulls acidified extract and identified the cyanidin-3-O-galactoside as main component. The richness and purity of this compound in the hulls, joined with the limited data present in the literature regarding its health promoting properties, lead us to perform an in-depth analysis of its antioxidant and cytoprotective activities compared to the most widely used synthetic antioxidants.

2.

Materials and methods

2.1.

Chemicals

2,2-diphenyl-1-picrylhydrazyl (DPPH), nitroblue tetrazolium, 2,2′azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS •+ ), 6-hydroxy-2,5,7,8-tetramethylchromane-2carboxyl acid (Trolox), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenazine methosulphate, methanol, gallic acid, 2,2′-Azobis(2-methylpropionamidine)dihydrochloride (AAPH), fluorescein disodium salt, tert-butyl hydroperoxide (tBOOH), 2,4,6-tris(2-piridil)-s-triazina (TPTZ), iron sulphate heptahydrate, sodium phosphate dibasic, potassium phosphate monobasic, sodium acetate and potassium peroxydisulfate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile, glacial acetic acid and phosphoric acid were HPLCgrade and were purchased from Merck (Darmstadt, Germany). Cyanidin-3-O-galactoside and Cyanidin-3-O-glucoside were purchased from Extrasynthese (Genay, France). Other chemicals were of analytical grade.

2.2.

Extraction procedure

The hulls of ripe pistachio nuts (P. vera L., Bronte variety), harvested in summer 2015 by a local farmer in Bronte (Catania, Italy), were separated from the nuts and frozen at −20 °C. The frozen hulls were ground to a powder with a frozen mortar and 34 g were extracted at room temperature under continuous stirring for 12 h, with 150 ml of methanol/water/acetic acid (70/ 29.5/0.5, v/v/v) mixture. The sample was then immediately centrifuged at 3000 g for 15 min and the supernatant was separated. This procedure was repeated four times. The supernatants were collected and evaporated by rotary evaporator. The yield of the residue was 16.78%. The dried extract was dissolved in 4% phosphoric acid solution/acetonitrile (90:10 v/v), filtered by 0,22 µm PTFE syringe filter and then injected into HPLC system.

2.3.

Anthocyanins determination

The qualitative and quantitative determination of anthocyanins in ripe pistachio hull extract was carried out according

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to Tomaino et al. (2010), using an Agilent high performance liquid chromatography system (1100 series) equipped with an UV-Vis photodiode-array detector (DAD) (G1315), a control system (G1323), a LC pump (G1312) and an autoinjector (G1313). Briefly, the chromatographic separation was obtained by a Chromolith Performance RP18e column (100 mm × 4.6 mm; Merck) with solvent A (4% phosphoric acid solution) and solvent B (acetonitrile) as mobile phase. The elution gradient program started with 95% A to reach 85% A and 15% B at 60 min and equilibrated 10 min for a total run time of 70 min. The flow rate was 0.75 ml/min, injection volume was 20 µl and the column was thermostated at 35 °C. Detection was performed at 520 nm. UV–Vis spectra of anthocyanins were recorded from 200 to 600 nm. Peak identity was confirmed by comparing their retention times and absorption spectra with those of pure (≥99%) commercially available standards (concentration range 1–50 µg/ ml). Quantification was carried out by external standard calibration curves.

2.4.

DPPH• assay

The DPPH free radical scavenging activity was performed according to Rapisarda et al. (1999) with some modifications. 1.5 ml of freshly prepared DPPH• methanol solution (10−4 M) was mixed with 37.5 µl of several concentration of cyanidin-3-O-galactoside solubilized in methanol (0–400 µM), and the mixture was vortexed for 10 s at room temperature. The absorbance decrease at 517 nm, against a blank of pure methanol without DPPH, was measured after 20 min using an UV–Vis Spectrophotometer (Shimadzu UV-1601). Dose–response curves to study the DPPH radical-scavenging activity of cyanidin-3-O-galactoside with respect to reference compounds BHA, BHT and Trolox (0– 400 µM) were used.

2.5.

Trolox equivalent antioxidant capacity assay (TEAC)

The ABTS assay was performed as described by Morabito et al. (2010) with some modifications. Briefly, the reaction mixture was prepared by mixing 4.3 mM potassium persulfate and 1.7 mM ABTS solution 1:5 (v/v) followed by 12–16 h incubation in the dark at room temperature. Before use, the ABTS+• solution was diluted with phosphate buffer (pH 7.4) in order to obtain an absorbance of 0.7 ± 0.02 at 734 nm. Aliquots of cyanidin-3-O-galactoside (50 µl) solubilized in methanol at several concentrations (0–200 µM) were added to 1 mL of ABTS+• solution and incubated in the dark at room temperature for 6 min; the absorbance was then recorded at 734 nm using an UV–Vis Spectrophotometer (Shimadzu UV-1601). Dose–response curves to study the ABTS ·+ radical-scavenging activity of cyanidin-3-O-galactoside with respect to reference compounds BHA, BHT and Trolox (0–200 µM) were used.

2.6.

Ferric reducing antioxidant power (FRAP)

The FRAP assay was performed according to Benzie and Strain (1996) with some modifications. The fresh working FRAP reagent was prepared daily by mixing acetate buffer (300 mM, pH 3.6), 2,4,6-Tris(2-pyridyl)-S-triazina (TPTZ) solution (10 mM in 40 mM HCl) and FeCl3. 6H2O solution (20 mM) 10:1:1 v/v/v. The reagent was warmed to 37 °C and the initial absorbance was re-

corded. 50 µl of a methanol solution containing different concentrations of Cyanidin-3-O-galoctoside (0–200 µM) or of the vehicle (methanol) alone was added to 3.0 ml of the FRAP reagent, and the absorbance was measured at 593 nm (UV– Vis Spectrophotometer; Shimadzu UV-1601) after incubation at 20 °C for 4 min, using the FRAP reagent as a blank. Dose– response curves to study the reduction of iron compounds 2,4,6tripyridyl-s-triazine (Fe3+-TPTZ) in their coloured form (Fe2+TPTZ) by cyanidin-3-O-galactoside with respect to reference compounds BHA, BHT and Trolox (0–200 µM) were used.

2.7.

Oxygen radical absorbance capacity (ORAC assay)

Antioxidant activity against AAPH radical was examined by the ORAC method according to Dávalos, Miguel, Bartolomé, and López-Fandiño (2004) with some modifications. Briefly, several concentrations (0–10 µM) of methanol solution of cyanidin-3O-galactoside (20 µl) in 75 mM phosphate buffer solution (pH 7.4), were mixed with 120 µl of fresh daily 117 nM fluorescein solution. After a pre-incubation time of 15 min at 37 °C, 60 µl of freshly daily AAPH solution (40 mM) were rapidly added. The fluorescence was recorded every 30 s for 90 min (λex: 485; λem: 520) using a Fluorescence Plate Reader (FLUOstar Omega, BMG LABTECH) and the decrease in fluorescence was monitored. A blank using phosphate buffer instead of sample was also included in each assay. Dose–response curves to study the absorption capacity of oxygen free radicals of cyanidin-3-Ogalactoside with respect to reference compounds BHA, BHT and Trolox (0–10 µM) were used.

2.8.

Superoxide anion scavenging assay (O2•−)

Measurement of superoxide anion scavenging activity of extracts was done using the Nishimiki method (Nishikimi, Appaji, & Yagi, 1972). The reaction mix was composed of: 1.0 ml of nitroblue tetrazolium (NBT) solution (156 µM NBT in 100 mM phosphate buffer, pH 7.4), 1 ml NADH solution (468 µM in 100 mM phosphate buffer, pH 7.4) and different final concentration (0–80 µM) of cyanidin-O-galactoside. The reaction was started by adding 100 µl phenazine methosulfate (PMS) solution (60 µM PMS in 100 mM phosphate buffer, pH 7.4) to the mixture. The reaction mixture was incubated at 25 °C for 5 min, and absorbance at 560 nm was measured against blank samples with a Varian Cary 50 UV–Vis spectrophotometer. Dose– response curves to study the superoxide anion scavenging activity of cyanidin-3-O-galactoside with respect to reference compounds BHA, BHT and Trolox (0–80 µM) were used.

2.9.

Hydrogen peroxide scavenging activity

The hydrogen peroxide scavenging assay was carried out following the procedure of Gülçin, Elmastas¸, and Aboul-Enein (2007) utilizing as reference compounds BHA, BHT and Trolox (0–80 µM).

2.10.

Lymphocyte isolation

Lymphocytes were isolated from heparinized whole blood collected from healthy volunteers, who has provided written informed consent as described above. Blood samples were

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379

diluted with equal volumes of balanced salt solution, layered over Histopaque-1077 (Sigma-Aldrich) in centrifuge tubes and centrifuged at 400 g for 30–40 min at 25 °C. The peripheral blood mononuclear cell (PBMC) layer was removed with a pipette and washed by centrifugation. The PBMCs are passed through a Percoll gradient according to Repnik, Knezevic, and Jeras (2003) to enrich the fraction in lymphocytes (viability > 90%) which were counted on a haemocytometer and suspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin G and streptomycin. Cell concentration was adjusted to 106 cells/ml.

electrophoresis, the slides were rinsed with 0.4 M Tris (pH 7.5) and stained with ethidium bromide. Analyses were carried out and monitored by means of a Leica DMRE microscope equipped with a fluorescence attachment. The images were acquired with a camera connected to the microscope. Untreated and treated cells (~100) were randomly acquired and analysed using the comet analysis macro (available at http://www.predictive -toxicology.org/comet/) (Helma & Uhl, 2000). The considered parameters were tail length (TL) and tail moment (TM, product of tail length and percentage of DNA in the tail).

2.11.

Data are presented as means ± standard deviation (S.D.). Data were analysed by one-way analysis of variance (ANOVA). The significance of the difference from the respective controls for each experimental test condition was assayed by using Tukey’s for each paired experiment. A P < 0.05 was regarded as indicating a significant difference.

Cytotoxicity assays

For the cytotoxicity assay, cells (1 × 106/ml) were incubated in complete medium with or without 0.1, 0.25, 0.5, 1.0, 5.0 or 10 µM of cyanidin-3-O-galactoside for 24 h in the presence of 100 µM tert-butyl hydroperoxide. Parallel controls were performed without t-BOOH for all experiments. After incubation, cell viability was assessed by trypan blue staining. Briefly, an aliquot of the cell suspension was diluted 1:1 (v:v) with 0.4% trypan blue and the cells were counted using a haemocytometer. Results are expressed as the percentage of viable or dead cells (ratio of unstained or stained cells to the total number of cells, respectively). Cytotoxicity was also measured by lactate dehydrogenase (LDH) release from damaged cells into culture medium and expressed as a percentage of total cellular activity. LDH activity in the medium was determined using a commercially available kit from BioSystems S.A. Compounds, at the concentration utilized in the experiments, did not show any evidence of interference with the LDH assay. For caspase activity determination, lymphocytes were collected and washed three times (after treatments) with phosphate saline buffer (PBS) and resuspended in Hepes-buffer (100 mM HEPES pH 7.5, 20% glycerol, 5 mM DTT and 0.5 mM EDTA). The lysates were clarified by centrifugation at 25,000 g for 10 min at 4 °C and supernatants were passed through Microcon YM 30 (Nominal Molecular Weight Limit 30,000 Da) to obtain a partial purification of caspase 3. The activity of caspase-3 was carried out for 1 h at 37 °C spectrophotometrically following the release of p-nitroaniline at 405 nm from enzyme-specific colorimetric substrates (Barreca et al., 2009; Bellocco et al., 2009). Caspase 3 was expressed in arbitrary units as a function of untreated sample.

2.12.

Single-cell gel electrophoresis (Comet Assay)

DNA damage was analysed with single-cell gel electrophoresis technique according to Singh, Tice, Stephens, and Schneider (1991). Lymphocytes were incubated in complete medium with or without 0.1, 0.25, 0.5, 1.0, 5.0 or 10 µM of cyanidin-3-Ogalactoside for 24 h in the presence of 100 µM tert-butyl hydroperoxide. Parallel controls were performed without t-BOOH for all experiments. After treatment harvested cells were embedded in a thin agarose gel (1% low melting) on a microscope slide. Cell lysis was performed with a cold lysis solution (2.5 M NaCl, 0.1 M EDTA and 10 mM Tris, 1% Triton X-100 and 10% DMSO) at 4 °C overnight. DNA was treated with 300 mM NaOH pH > 13 and 1 mM EDTA for 30 min and then submitted to electrophoresis for 30 min applying 300 mA. Following

2.13.

3.

Statistical analysis

Results and discussion

The anthocyanin content of the ripe pistachio hull extract was analysed by RP-HPLC-DAD method; we discovered that the ripe pistachio hull is a rich source of cyanidin-3-O-galactoside that represents, as shown in Fig. 1, the most abundant anthocyanin in the extract under study (96.57%, 2.55 mg/100 g FW). We have also identified traces of cyanidin-3-O-glucoside (0.38%, 0.02 mg/100 g FW). This is the first time that these compounds have been identified and quantified in ripe pistachio hulls Bronte variety. Furthermore, the richness and purity of cyanidins-3-O-galactoside in ripe pistachio hulls made us hypothesize a potential use of this raw material for nutraceutical employments. Beyond their use as food dyes, anthocyanins have been investigated for their biological and pharmacological properties and literature data have described a range of interesting activities: anticancer, antioxidant, antimicrobial, anti-ageing, anti-inflammatory, anti-neurodegenerative and anti-diabetic (Smeriglio et al., 2016). In light of this, understand the mechanisms responsible for the activities of these compounds, for instance, their antioxidant and cytoprotective activities, analysing the possible benefits derived from their dietary assumption as well as their pharmacological and therapeutic potential, becomes important. Amongst anthocyanins, the cyanidin-3-O-galactoside is not much studied and data regarding its health promoting properties are still lacking. In light of this we decided to perform an in-depth analysis of its antioxidant and cytoprotective activities by performing a comparison with the most widely used synthetic antioxidants (BHA, BHT and Trolox). The analysis of antioxidant and free radical scavenging power of a pure compound as well as of a complex matrix requires the use of several antioxidant assays (hydrogen atom transfer and electron transfer-based methods) in order to check their behaviour under different reaction environments and mechanisms typology. In light of this, different in vitro methods namely ORAC, FRAP, TEAC, DPPH and superoxide anion scavenging assays were used. Fig. 2 shows the antioxidant and free

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Fig. 1 – Representative HPLC chromatogram of anthocyanins in ripe pistachio hull extract. Peak identification was performed by matching retention time and UV spectra against commercially available reference compounds: (1) cyanidin-3O-galactoside and (2) cyanidin-3-O-glucoside.

radical-scavenging potential of cyanidin-3-O-galactoside towards DPPH• (A), ABTS•+ (B), O2•− (C), H2O2 (D), Fe3+-TPTZ (E) and AAPH (F). Cyanidin-3-O-galactoside showed a radical scavenging activity against DPPH• clearly superior to Trolox, BHA and BHT (≥1.98, ≥2.91 and ≥3.14 fold respectively at each concentration tested) (Fig. 2A). Based on this preliminary evidence of potential antioxidant power, we analysed its superoxide anion and ABTS•+ radical scavenging activity (Fig. 2B and C). In both assays, we found that the cyanidin-3-O-galactoside outperformed Trolox, BHA and BHT in scavenging radicals, similarly to the DPPH assay. The presence of cyanidin-3-O-galactoside was found to scavenge ABTS•+ radicals more effectively than Trolox, BHA and BHT (≥2, ≥2.99 and ≥8 fold respectively at each concentration tested). A similar behaviour was observed regarding superoxide anion. In particular the tested anthocyanin, at 10 µM concentration, is ~ 6.2, 16.0 and 20.0 fold more efficient than Trolox, BHA and BHT in scavenging this former radical (Fig. 2C). This ability may have a fundamental role in living organism defence systems, where primary and relative weak antioxidants (such as O2•−) can be the precursor or combine with others bringing to the formation of more dangerous and reactive species such as hydrogen peroxide, peroxynitric radical, hydroxyl radical. Nevertheless the anthocyanin, in the range of concentration tested, did not show any significant effects on the elimination of hydrogen peroxide (Fig. 2D). Thus, the effects of cyanidin-3-O-galactoside were further analysed by FRAP and ORAC assays to measure its capacity to reduce iron and to scavenge peroxyl radicals (Fig. 2E and F). In both assays, the potentiality of cyanidin-3-O-galactoside was found to be by far superior to that of Trolox, BHA and BHT (≥2.10,

≥2.77 and ≥3.02 fold respectively at each concentration tested for FRAP assay and ≥12.64, ≥5.84 and ≥33.65 fold respectively at each concentration tested for ORAC assay). Expressing the results obtained into ORAC assay, as µmol of Trolox equivalent (TE)/µmol of pure compound, we can make an immediate comparison between what we observed and the literature data. Cyanidin-3-O-galactoside was found to possess the strong antioxidant activity with respect to BHA and BHT (13.64, 2.24 and 0.37 µmol of TE/µmol of pure compound, respectively). On the other hand, the activity of the tested compound was comparable to flavonoids quercetin and catechin that showed the highest ORAC values (10.5 and 14.9 µmol of TE/µmol of pure compound, respectively) compared with several phenolic acids including caffeic, protocatechuic, chlorogenic, ferulic and p-coumaric acids (6.63, 6.70, 5.70, 4.47 and 4.51 µmol of TE/µmol of pure compound, respectively) (Dávalos et al., 2004). Furthermore, it is known that amongst anthocyanins of the same hydroxylation pattern in the A and C rings, compounds with only one OH group in the B ring (4′-OH) including pelargonidin, malvidin, and peonidin were found to possess lower ORAC activity compared to cyanidin, characterized by a catechol structure (Smeriglio et al., 2016) as well as, the latter one, had a positive effect on TEAC, FRAP and DPPH assays (Csepregi, Neugart, Schreiner, & Hideg, 2016). It has been observed that the antioxidant activity of flavonoids in all the above mentioned assays was strictly related to the catechol structure; the 2,3 double bond in conjugation with a 4-oxo function in the C ring and the presence of a 3-hydroxyl group in the C ring only partially affected the antioxidant behaviour of this polyphenol class with an increase of FRAP and DPPH and TEAC

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Fig. 2 – Radical scavenging activity of cyanidin-3-O-galactoside, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and Trolox measured by (A) DPPH, (B) TEAC, (C) O2•−, (D) H2O2 (E), FRAP and (F) ORAC assays. Data represent average ± SD (n = 3) and were expressed as inhibition % for A, B and E, as residual absorbance % for C and D, and as area under the fluorescence decay curve (AUC) for F. (• cyanidin-3-O-galactoside; ○ BHA; ▼ BHT; Δ Trolox).

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Fig. 3 – Cytoprotective effects of cyanidin-3-O-galactoside on lymphocytes. Lymphocytes plus 100 µM of t-BOOH were incubated for 24 h in the absence (b) or in the presence of 10, 5.0, 1.0, 0.5, and 0.1 µM GAE (c–g). Lymphocytes incubated under the same experimental condition without t-BOOH but in the presence or the same amounts of acidified-methanol present in the samples (a). Cell vitality, integrity and apoptotic events were analysed by trypan blue staining (A), LDH release (B) and caspase 3 activation (C), respectively. Asterisks indicate significant differences (P < 0.05) with respect to lymphocytes treated in the presence of only t-BOOH.

and FRAP values, respectively. The results obtained revealed the potentiality of cyanidin-3-O-galactoside to be powerful scavenger of neutral, negative and positively charged radicals, with a primary antioxidant capacity which could be ascribed to the peroxyl radicals scavenging ability. According to our results, cyanidin glycosides have been found to possess higher antioxidant activity in a liposomal membrane system with respect to certain reference compounds, e.g. Trolox, BHA, BHT and tert-butyl hydroxyl quinone (Smeriglio et al., 2016). Different patterns of hydroxylation and glycosylation in anthocyanins appear to modulate their antioxidant properties (Smeriglio et al., 2016). The scavenging of peroxyl radicals is a critical point in the prevention of lipid peroxidation by breaking the chain of propagation of free radical reactions. Therefore, the evidence presented here suggests that nutraceutical employment of pistachio hull, as rich source of cyanidin-3-O-galactoside, may be useful in lowering the risk of certain pathophysiologies associated with freeradical mediated events. These promising results in antioxidant assays lead to check its potentiality to protect isolated lymphocytes from t-BOOH treatment. Incubation of lymphocytes for 24 h at 37 °C in the presence of the oxidant (100 µM) induced a decrease of cellular vitality up to ~40% (Fig. 3A). The presence of cyanidin-3-O-galactoside improved remarkable cell survival at all the tested concentrations (0.1–10 µM), with a decrease of cell death (following treatment with 100 µM t-BOOH) of ~4.0, 3.2, 2.1,

1.76 and 1.4 fold. These results are very interesting taking into account that other anthocyanins (such as cyanidin-3-O-glucoside, malvidin-3-O-glucoside, pelargonidin-3-O-glucoside or their aglycon) also showed cytoprotective activity against oxidative injuries, but required concentration between 25 and 150 µM, well higher than the one utilized in our experiments (Bognar et al., 2013; Nam, Hah, Nam, Kim, & Park, 2016; Paixão, Dinis, & Almeida, 2011, 2012). The activity of the tested compound is, on the other hand, comparable to the one of the strongest natural antioxidant quercetin, rutin and catechin that ranges from 0.15 to 2.65 µM (Zhang, Melton, Adaim, & Skinner, 2008; Zhang, Stanley, Adaim, Melton, & Skinner, 2006). Structure–activity relationship can help us shed some light on the potentiality of our tested compound. Recent studies have ascribed the biological activities of anthocyanins mainly to their antioxidant properties and, in particular, to the hydroxylation of B-ring and to the electron delocalization of the conjugated double bond system (Kähkönen & Heinonen, 2003; Prior & Wu, 2006). For this reason, cyanidin-3-O-galactoside is characterized by a high degree of hydroxylation of the B-ring (presence of catechol structure) associated to a hydroxyl group in the 3 position and conjugated double bond system. These elements, taken together, seem to confer a significant higher general antioxidant potential, if compared to the one characterized by phenol or methoxy substituted B-ring (i.e. pelargonidin and malvidin). In this latter case

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A

C

B

D 350

E

400

250 200 150

**

**

**

**

Tail moment

Tail length

300

100

300 200 100

**

**

**

**

50 0

0 a

b

c

d

e

f

g

a

b

c

d

e

f

g

Fig. 4 – Influences of cyanidin-3-O-galactoside on t-BOOH-induced DNA fragmentation in isolated lymphocytes. Representative images of control lymphocytes (A) or lymphocytes treated with 100 µM t-BOOH in the absence or in the presence of 10 µM cyanidin-3-O-galactoside. The images of the cell were digitalized and analysed in function of tail length (D) and moment (E). Asterisks indicate significant differences (P < 0.05) with respect to lymphocytes treated in the presence of only t-BOOH.

the activity is not ascribed to the presence of the substituents on its basic skeleton, but to a direct interaction of the molecule with biological targets, activation of signalling cascades and mechanisms of cellular defence systems. Therefore, the cytoprotective effects of cyanidin-3-O-galactoside has been further analysed taking into accounts the release of LDH from lymphocytes and stop of caspase 3 activation. As shown in Fig. 3B and C lymphocytes treatment with t-BOOH resulted in a significant increase in the release of LDH and in the activation of caspase 3. LDH is a soluble cytosolic enzyme, whose release in the culture medium is function of membrane integrity loss, resulting from cell- or compound-induced cytotoxicity. The presence of 10 and 5 µM of cyanidin-3-O-galactoside results in a statistical significant decrease of this release, with data almost completely superimposable with the one obtained in cells without treatment. The decrease in the LDH release supports the hypothesis that cyanidin-3-O-galactoside can directly act on t-BOOH by decreasing its strong oxidant activity, well evident at the level of cytoplasmatic fatty acids, and scavenging the reactive species originated at the membrane level and in the cytosol.This action is further confirmed by the process of caspase 3 activation, where the presence of reactive species is one of the main trigger for its activation. Lymphocytes treatment with t-BOOH induces the activation of caspase 3 from inactive zymogenic precursors, starting the apoptotic event that results finally also in the release of LDH, as shown in the above described results. Once again the presence of cyanidin-3-O-

galactoside results in a remarkable decrease of enzyme activation. These results support and supplement the results obtained by antioxidant assays, where cyanidin-3-O-galactoside results a better antioxidant in all the performed assays, able to scavenge, neutral, anion and cation radicals. In particular, as shown in our results, this compound is able to scavenge superoxide anion, one of the most active reactive species produced in our organism. This strong antioxidant activity is also supported by literature data, where similar molecules have also activity against hydroxyl radical (Garcia-Alonso et al., 2005). The cytoprotective activity of the molecule on t-BOOHinduced lymphocytes DNA damage was further analysed by COMET assay. Treatment of lymphocyte with this strong oxidant resulted in extensive DNA damage, as reflected by the comet tail length (2.8 fold) and tail moment (4.4 fold), compared with control cells (Fig. 4). As shown in the figure, in the presence of cyanidin-3-O-galactoside there was a reduction of both elements, with similar values with respect to controls as far as 1–10 µM concentrations are concerned.

4.

Conclusions

We have recently identified, for the first time, the ripe pistachio hull (P. vera L., Bronte variety), as an attractive source of health-promoting compounds potentially helpful in preventing

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the onset of various oxidative stress-related disorders. This further study, allowed us to investigate, amongst the polyphenol class, the presence of anthocyanins and to identify and quantify cyanidin-3-O-galactoside as the most abundant compound in ripe pistachio hull (P. vera L., Bronte variety). The richness and purity of this anthocyanin, which we have demonstrated to possess remarkable antioxidant, free radical scavenging and cytoprotective properties with respect to widely used synthetic antioxidants, make the ripe pistachio hull a valuable raw material for nutraceutic production characterized by well-defined and specific health promoting properties.

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