In vitro antioxidant and in vivo photoprotective effect of pistachio (Pistacia vera L., variety Bronte) seed and skin extracts

    In vitro antioxidant and in vivo photoprotective effect of pistachio (Pistacia vera L., variety Bronte) seed and skin extracts Maria Martorana, Teresita Arcoraci, Luisa Rizza, Mariateresa Cristani, Francesco Paolo Bonina, Antonina Saija, Domenico Trombetta, Antonio Tomaino PII: DOI: Reference:

S0367-326X(13)00002-6 doi: 10.1016/j.fitote.2012.12.032 FITOTE 2600

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

23 October 2012 20 December 2012 22 December 2012

Please cite this article as: Martorana Maria, Arcoraci Teresita, Rizza Luisa, Cristani Mariateresa, Bonina Francesco Paolo, Saija Antonina, Trombetta Domenico, Tomaino Antonio, In vitro antioxidant and in vivo photoprotective effect of pistachio (Pistacia vera L., variety Bronte) seed and skin extracts, Fitoterapia (2013), doi: 10.1016/j.fitote.2012.12.032

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ACCEPTED MANUSCRIPT In vitro antioxidant and in vivo photoprotective effect of pistachio (Pistacia vera L.,

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variety Bronte) seed and skin extracts

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Short title: Photoprotective effect of pistachio seed and skin extracts

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Maria Martoranaa, Teresita Arcoracia, Luisa Rizzab, Mariateresa Cristania, Francesco Paolo Boninab, Antonina Saijaa, Domenico Trombettaa, Antonio Tomaino*a,

Dept. Farmaco-Biologico, Faculty of Pharmacy, University of Messina

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a

Polo Annunziata, 98168 Messina, Italy

Dept. of Pharmaceutical Science, Faculty of Pharmacy, University of Catania,

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b

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Viale Andrea Doria, 95125 Catania

*Corresponding author: Antonio Tomaino, tel. +39 90 6766577;

fax +39 90 6766474;

e-mail: [email protected]; Dept. Farmaco-Biologico, Faculty of Pharmacy, University of Messina; Polo Annunziata 98168 Messina (Italy)

Abbreviations: AA, ascorbic acid; AP-1, activator protein; AUC, area under the curve; E.I., erythema index; HPLC, High Performance Liquid Chromatography; IC50, inhibiting concentration 50%; MDA, Malondialdehyde; MED, minimal erythema dose; NF-kB, nuclear factor kappa B; O/W, oil/water; PC, Phosphatidylcholine; PIE, 1

ACCEPTED MANUSCRIPT percentage inhibition skin erythema; ROS, Reactive Oxygen Species; SP, pistachio seeds; TOC, tocopheryl acetate; TP, pistachio skins; UV-IP, UV-induced peroxidation

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in liposomal membranes.

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ACCEPTED MANUSCRIPT Abstract Pistachio (Pistacia vera L.) nuts are a rich source of phenolic compounds, known for

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their high antioxidant activity, and contained not only in the seeds but also in the skin.

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A pistachio cultivar of high quality is typical of Bronte, Sicily, Italy. The purpose of our study was to investigate the chemical composition and antioxidant properties of two

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polyphenol-rich extracts from skins (TP) and decorticated seeds (SP) of Bronte pistachios, and to verify the potential use of these extracts for topical photoprotective products.

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Chemical analysis showed that the TP and SP extracts contain high levels of phenolic compounds, but the TP extract is about ten times richer in phenols than the SP extract,

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being anthocyanins the most abundant compounds found in the TP extract. Both these extracts, and especially the TP extract, possess good radical scavenger/antioxidant

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properties, as shown in a series of in vitro assays carried out using homogenous and

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non-homogenous chemical environment. Furthermore both the TP extract and, although at a lower degree, the SP extract reduce, when topically applied, UV-B-induced skin erythema in human volunteers. These findings suggest that extracts from Bronte TP and SP could be successfully employed as photoprotective ingredients in topical cosmetic and pharmaceutical formulations.

Keywords: pistachio skins; pistachio seeds; Pistacia vera L., var. Bronte; polyphenolic content; antioxidant activity; in vivo photoprotection

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ACCEPTED MANUSCRIPT 1. Introduction Pistachio (Pistacia vera L., belonging to the Anacardiaceae family) is native of aride

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zones of Central and West Asia and distributed throughout the Mediterranean basin. In

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Italy, a pistachio cultivar typical of Bronte, Sicily (an area around the Etna volcano, where the lava land and climate allow the production of a nut with intense green colour

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and aromatic taste), is very appreciated for its high quality in international markets. Pistachio nuts have recently been ranked among the first 50 food products highest in antioxidant potential [1] and are a rich source of phenolic compounds known for their

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high antioxidant activity. In particular, pistachios have been reported to be the only nuts containing anthocyanins, the pigments responsible for the colour of pistachio kernel [2]

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as well as of many vegetables and fruits. We have previously reported that Bronte pistachios are very rich of anthocyanins, at a concentration higher than that contained in

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pistachio of different other geographic origin [3]. One has to point out that not only

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pistachio seeds but also skins contain discrete amounts of bioactive compounds. This is a very interesting issue because, for most of their uses, pistachio nuts are used after removing skin; pistachio skins industrially removed from the nut constitute about 10% of the total shelled pistachio weight, and if not processed further, become waste and have potential to cause environmental pollution. Numerous studies have demonstrated the high capacity of several plant polyphenolic antioxidants (such as flavonoids and hydroxycinnamic acids) to protect living organism from alterations produced by reactive oxygen species (ROS) overproduction, including skin damages caused by exposure to ultraviolet (UV) radiation [4]. In particular there is evidence supporting that anthocyanins are endowed with strong antioxidant properties and, both as pure molecules or as anthocyanin-rich plant extracts, can efficiently protect skin cells against deleterious effects of UV light [5, 6]. 4

ACCEPTED MANUSCRIPT The use of sunscreens and the avoidment of intense sun exposure are recommended as expedient means of protection against UV light-induced skin damage, but also the

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employment of products containing herbal antioxidant ingredients may be a useful

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strategy [7]. Natural phenols have been shown able to perform photoprotective action, not only when taken with the diet but also when topically applied [8-11].

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The purpose of our study was to investigate, by means of in vitro and in vivo experiments, the properties of polyphenol-rich extracts from Bronte pistachio skins (TP) and decorticated seeds (SP) and to verify the potential use of these extracts as ingredient

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in topical photoprotective formulations. At this aim two methanolic/aqueous TP and SP extracts were characterized by HPLC for their polyphenolic content (phenolic acids and

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flavonoids). The in vitro radical scavenger/antioxidant properties of these extracts were studied by means of a series of different cell-free assays carried out in homogenous

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(Folin-Ciocalteau method; reducing power test) and non-homogenous (β-carotene

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bleaching assay; UVC light-induced peroxidation on phosphatidylcholine multilamellar vesicles) chemical environment. Thus, on the basis of the results obtained in in vitro experiments, the pistachio extracts were tested in vivo to assess the ability to reduce, when topically applied, UV-B-induced skin erythema (monitored by reflectance spectrophotometry) in healthy human volunteers. This test is regarded as one of the most suitable models for studying in vivo skin damage after acute UV exposure [12-14]. For this purpose, three formulations containing the pistachio extracts or tocopheryl acetate (TOC; used as reference compound) were applied on the skin immediately after exposure to UV-B radiation.

2. Material and methods 2.1. Chemicals 5

ACCEPTED MANUSCRIPT Chloroform, phosphoric acid, acetic acid, methanol and acetonitrile in HPLC-grade were purchased from VWR (Briare, France). Folin-Ciocalteau phenol reagent, sodium

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carbonate, methansulfonic acid 99,5%, N-methyl-2-phenylindole 99%, 1,1,3,3,-

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tetramethoxypropan, L-α-phosphatidylcholine from egg yolk type XI-e, L-ascorbic acid, Prussian Blue soluble, Tween 40, sodium phosphate dibasic (Na2HPO4), sodium

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phosphate monobasic monohydrate (NaH2PO4•H2O), β-carotene, linoleic acid, tocopheryl acetate, iron(III) chloride (FeCl3), potassium ferricyanide (K3Fe(CN)6) were purchased from Sigma–Aldrich (Steinheim, Germany). Methanol, hexane, 20%

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trichloroacetic acid (TCA) were purchased from Farmitalia Carlo Erba S.p.A. (Milan, Italy).

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To carry out the in vitro tests, the TP extract and the SP extract were reconstituted in the appropriate volume of methanol/water 2/1. For the in vivo experiments the extracts,

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reconstituted in ethanol, were employed as gel formulations (see the section

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“Preparation of topic formulations”).

2.2. Sample preparation

Pistachio (Pistacia vera L., variety Bronte) nuts, harvested in the summer of 2009, were obtained from Consorzio del Pistacchio di Bronte (Catania, Italy) and stored in the dark at 4 °C. Pistachios (100 g) were immersed for 20 s in liquid nitrogen and skins were manually separated. To obtain the TP extract, an aliquot (2 g) of crushed skins was extracted with 2 ml of a methanol/water (2/1) mixture by homogenization for 5 min and ultrasonication (Branson ultrasonic cleaner mod. 2200, 40 kHz frequency) at 25°C for 10 min; after centrifugation (at 16,770xg for 10 min) the supernatant was withdrawn and the same 6

ACCEPTED MANUSCRIPT procedure was repeated for three times. The supernatants were collected together and dried by a rotary evaporator.

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To obtain the SP extract, pistachio decorticated seeds were powdered in a mortar by

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means of a pestle. An aliquot (2 g) was extracted for three times with hexane (20 ml) by homogenization for 5 min and ultrasonication for 10 min; the supernatants separated by

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centrifugation (at 16,770xg for 10 min) were discarded. The residue was then extracted with a methanol/water (2/1) mixture following the same procedure described before. The dried TP and SP extracts obtained by this procedure weighed 576.25 mg and

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252.28 mg respectively.

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2.3. HPLC analysis of phenolic compounds

The quali/quantitative determination of phenolic acids, flavones, isoflavones,

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flavanones, flavonols and flavan-3-ols in TP and SP extracts was carried out using

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Shimadzu HPLC system equipped with an UV-Vis photodiode-array detector (DAD; Shimadzu) and a fluorescence detector (Hewlett Packard). The apparatus was controlled by a control system (SCL-10A VP), equipped with an LC pump (LC-10 AD VP) and with an auto-injector (SIL-10AD VP). Briefly, the chromatographic separation was obtained by a 5 µm ODS3 reversed-phase Prodigy column (250 mm x 4.6 mm; Phenomenex) with solvent A (water/acetic acid, 97/3, v/v) and solvent B (methanol) under the following gradient conditions: 0-3 min, 0% B; 3-9 min, 3% B; 9-24 min, 12% B; 24-30 min, 20% B; 30-33 min, 20% B; 33-43 min, 30% B; 43-63 min, 50% B; 63-66 min, 50% B; 66-76 min, 60% B; 76-81 min, 60% B; 81-86 min, 0% B. The flow rate was 1 ml/min, the injection volume was 50 µl, and the column was thermostated at 25°C [3]. UV spectra of phenolic acids, flavones, isoflavones, flavanones and flavonols were recorded from 240 to 400 nm. Concerning 7

ACCEPTED MANUSCRIPT quantitative analysis, the absorbance was measured at 260 nm for phenolic acids, flavones and isoflavones, at 292 nm for flavanones, and at 370 nm for flavonols. The

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analytical conditions for fluorescence detection of flavan-3-ols were: ex: 276 nm, em:

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316 nm.

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Anthocyanin determination in TP and SP extracts was carried out using a Varian HPLC system equipped with an UV-Vis detector (UV-Vis Prostar, Varian). The apparatus consisted of a model 240 pump and 410 autosampler and Galaxie Workstation software. The chromatographic separation was obtained by a Chromolith Performance RP18e

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(100 x 4.6 mm; Merck) with solvent A (4% aqueous phosphoric acid) and solvent B (acetonitrile) as mobile phase. The gradient program started with 95% A to reach 85%

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A and 15% B at 60 min. The flow rate was 0.75 ml/min, and the column was thermostatically-controlled at 35° C. Detection was performed at 520 nm [3]. Peak

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identity was confirmed by comparing their retention times with those of pure standards.

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All analyses were carried out in duplicate; identified phenols were quantified by comparison with curves constructed with solutions (1-30 µg/ml) of each pure commercial compound.

2.4. Evaluation of antioxidant activity 2.4.1. Folin-Ciocalteau method The antioxidant capacity of SP and TP extracts was determined by the Folin-Ciocalteau reagent [14]. Total phenol content was expressed as mg of gallic acid equivalents/g of extract. Each determination was performed in triplicate and repeated at least three times.

2.4.2. Reducing Power test The reducing power of the pistachio extracts was determined according to the method of 8

ACCEPTED MANUSCRIPT Oyaizu [15] with some modifications. In this assay, the yellow colour of the test solution turns to a green/blue colour whose intensity depends on the reducing power of

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the antioxidants present in the solution, that cause the reduction of the Fe3+/ferricyanide

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complex to the ferrous form; therefore, Fe2+ can be monitored by the measurement of the absorbance at 700 nm.

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Briefly, 200 µl of methanol/water (2/1) solutions containing pistachio extracts at a concentration able to give a final reading between 0.3 – 0.7 AU were mixed with 0.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 0.5 ml of 1% K3Fe(CN)6, and then

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incubated in a water bath at 50°C for 20 min. Then, 0.5 ml of 10% TCA were added to the mixture which was centrifuged at 8,300xg for 10 min. The supernatant (0.5 ml) was

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then mixed with 0.5 ml of distilled water and 0.1 ml of 0.1% ferric chloride solution. The intensity of the blue–green colour was measured at 700 nm. Each determination

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was performed in triplicate and repeated at least three times.

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Under the same conditions described above, different concentrations of ascorbic acid (AA) were tested in order to measure their reducing power. A calibration curve of Prussian Blue (concentration range 10-150 µM), dissolved in a mixture of water, phosphate buffer, TCA and methanol (71.48/13.37/13.37/1.78), was used to calculate the number of nmoles of Prussian Blue formed by the reaction between AA and Fe 3+. The number of Fe2+ nmoles was calculated as follow:

Equation 1

where 55.8 is the atomic weight of iron; AC is the absorbance at 700 nm of the reaction mixture containing AA; 0.0083 and 0.0053 are intercept and slope values of Prussian

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ACCEPTED MANUSCRIPT Blue equation, respectively; 329.196 is the molecular weight of Prussian Blue; 3.74 is the dilution factor.

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Thus, plotting the number of AA nmoles employed vs the number of formed Fe2+

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nmoles, a straight line is obtained, which allows to calculate the reducing power of the extracts under investigations. The results were expressed as mmoles of AA equivalents

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(AAE) per gram of extract.

2.4.3. β-Carotene bleaching test

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This assay was carried out according to the method of Wannes et al. [16] with some modifications. To prepare a stock solution of a β-carotene-linoleic acid mixture, 1 mg of

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β-carotene was dissolved in 10 ml of chloroform (HPLC grade), and then 5 ml of this solution were added to 40 μl of linoleic acid and 400 μl of Tween 40. Chloroform was

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removed using a rotary evaporator at 40◦C for 5 min and then 100 ml of distilled water

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were slowly added to the residue to form an emulsion. Five milliliters of the emulsion were added to 200 μl of methanol/water (2/1) solution containing the extracts to be studied at different concentrations (0.25 – 10 mg/ml); a same volume of the solvent alone (methanol/water, 2/1) was used in control samples. The absorbance was immediately measured (t = 0 min) at 470 nm against a blank, consisting of an emulsion without β-carotene. Then the samples were placed in a water bath at 50◦C and the oxidation of the emulsion was monitored by measuring absorbance at 470 nm 120 min after the beginning of the reaction. The percentage of inhibition respect to the control was calculated as follows: % of inibition = [(At-Ct)/(C0-Ct)]·100 where At and Ct are the absorbances measured for the sample to be tested and the control one, respectively, at t = 120 min, and C0 is the absorbance value for the control 10

ACCEPTED MANUSCRIPT measured at t = 0 min. Each determination was carried out in triplicate and repeated at least three times. Results were expressed as mg/ml of extract needed to inhibit -

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carotene bleaching by 50% (IC50).

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2.4.4. UV-induced peroxidation in liposomal membranes (UV-IP test) The protective effect of TP and SP extracts against UV-C-induced peroxidation was evaluated on phosphatidylcholine (PC) multilamellar vesicles [17]. Briefly, a volume (950 µl) of liposome suspension containing 10 mg/ml of PC (in a glass flask with a 3

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cm3 exposure surface area) was exposed to UV radiation from a 15 W Philips germicidal lamp (254 nm) for 1.5 h. Exposure was given at 10 cm from the lamp, and

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the experiment was carried out at 37 °C. Fifty µl of a methanol/water (2/1) mixture containing the extracts to be tested at different concentrations were added to the system;

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an equal volume (50 µl) of the vehicle alone was added to control tubes.

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Malondialdehyde (MDA) concentration formed in the mixtures following UV-C irradiation was measured using a colorimetric assay. In this assay, N-methyl-2phenylindole reacts with MDA to give a stable chromophore with a max at 586 nm. To calculate the amount of MDA formed in the samples, a calibration curve was prepared by submitting methanolic solutions of 1,1,3,3-tetramethoxypropan, at different concentrations, to the same procedures described above. All determinations were carried out in triplicate and repeated at least three times. The results were expressed as concentration of the extract needed to inhibit MDA formation by 50% (IC50) respect to the control.

2.5. In vivo study on photoprotective activity 11

ACCEPTED MANUSCRIPT 2.5.1. Preparation of topic formulations To in vivo evaluate the photoprotective effect of the extracts under investigations, we

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used a formulation containing 2% SP or TP extracts or 2% TOC (as a reference

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ingredient). The composition of each formulation is reported in Table 1. Each oil/water (O/W) emulsion was prepared by slowly adding the aqueous phase to the oily phase and

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to the blend of surfactants under continuous agitation, and maintaining the phases at 70°C. This mixture was stirred until cool, so forming the emulsion formulation.

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Moreover, a formulation without active ingredients was used in the study (Blank).

2.5.2. Instruments

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Skin erythema was induced by UV-B irradiation using a UVM-57 ultraviolet lamp (UVP, San Gabriel, CA, USA). This source emits radiation in the range 290-320 nm

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with an output peak at 302 nm. The flux rate measured at the skin surface was 0.80 mW

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cm-2. For each subject the minimal erythema dose (MED) was determined and an irradiation dose corresponding to double the MED was used throughout the study. UV-B-induced skin erythema was monitored by using a reflectance visible spectrophotomer X-Rite model 968 (X Rite Inc. Grandville, MI, USA), having 0° illumination and 45° viewing angle, calibrated and controlled as previously reported [18]. Reflectance spectra was obtained over the wavelength range 400-700 nm using illuminant C and 2° standard observer.

2.5.3. Subjects In vivo experiments were performed on twelve healthy volunteers of skin types II and III, aged 25-35 years. The volunteer subjects were recruited after medical screening including filling in a health questionnaire and physical examination of the application 12

ACCEPTED MANUSCRIPT sites. Subjects exhibiting features which might interfere with evaluation, such as sunburn, skin lesions or abnormal sensitivity to sunlight, or taking medication at the

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time of the study, were excluded from the study. The volunteers were fully informed

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about the nature of the study, and about the products and procedures involved, and gave their written consent. Each subject was rested for 15 min before the experiments and

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room conditions were set at 22 ± 2 °C and 40-50 % relative humidity. Two research assistants were responsible for recruitment and data collection.

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2.5.4. Protocol

For each subject recruited in the experiment, ten sites in the ventral surface of each

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forearm were defined using a circular template (1 cm2) and demarcated with permanent ink. Baseline skin assessment was performed by reflectance spectrophotometry in all

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sites. Each site was exposed to UV-B irradiation, then 200 mg of each formulation

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(containing SP, TP, TOC or the blank) were spread uniformly on the sites by means of a solid glass rod. For each forearm, two of the ten sites were used as control (no drug treatment). Afterwards, each skin site was occluded for 3 h, using Hill Top chambers (Hill Top Research, Inc, Cincinnati, OH, USA), to prevent any loss of the material from the skin surface. After the occlusion period the chambers were removed and the skin surfaces were washed to remove the formulation and allowed to dry for 15 min, after which the induced erythema was monitored for 58 h by reflectance spectrophotometry. Since erythema is due to an increment of blood supply in the subpapillary plexus of the skin, erythema index (E.I.) values were calculated by subtracting the logarithm of inverse reflectance (log 1/R) values of 510 nm and 610 nm (mainly due to melanin absorption), from the sum of log 1/R values of 540, 560 and 580 nm, which represent the wavelengths of haemoglobin absorption peaks (Equ. 2) [19]. 13

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Equ. 2

To evaluate the time course of skin erythema, E.I. baseline values (before UV-B

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irradiation) were subtracted from the E.I. values obtained after UV-B exposure at each

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time point, to calculate ∆.E.I. values. For each site, ∆.E.I. was plotted versus time and the area under the curve (AUC) was computed using the trapezoidal rule to obtain AUC0-58, a dimensionless index directly related to the degree of skin erythema.

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To better compare the efficacy of the tested formulations, the percentage inhibition of UV-B skin erythema (PIE) was calculated from formulation AUC0-58 values using the

% inhibition (PIE)

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Equ. 3

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equation (Equ. 3):

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Where AUC(C) is the area under the response-time curve of the sites no treated (control) and AUC(T) is the area under the response-time curve of the sites treated with the formulations under investigation. Statistical analysis of AUC values, expressed as means and standard deviations, was performed using unpaired Student’s t-test; p < 0.05 was considered to be significant.

2.6. Results and discussion In the present study, the phenolic composition, the in vitro antioxidant activity and the in vivo photoprotective effects of two extracts from decorticated seeds and skins of pistachios cultivated in Bronte were investigated. The phenolic profile of SP and TP extracts was characterized by HPLC analysis. The results of the HPLC quantitative analysis of the two TP and SP extracts are reported in 14

ACCEPTED MANUSCRIPT Table 2. Both extracts show high levels of phenolic compounds, but the TP extract is about ten times richer in phenols than the SP extract. Furthermore, significant

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differences between SP and TP extracts are evident concerning not only their

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quantitative chemical profile, but also the qualitative one. In fact, the anthocyanins cyanidin-3-O-galactoside and cyanidin-3-O-glucoside (the most abundant compounds

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contained in the TP extract), as well as epicatechin and the aglycones quercetin, naringenin, luteolin and kaempferol, were recovered only in the TP extract. Furthermore also the content of gallic acid (the only found phenolic acid) and catechin is very higher

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in the TP extracts than in the SP extract. Conversely, the isoflavones genistein, daidzein and genistein-3-O-glucoside were recovered only in the SP extract, representing the

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flavonoidic class more abundant in this extract, together with a small amount of apigenin. All these compounds are known to possess good antioxidant/radical scavenger

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properties [20]. Thus we have tested the antioxidant/radical scavenger power of the

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pistachio extracts under investigation. Concerning the tests carried out in homogenous chemical systems, we performed two electron-transfer based assays, the Folin-Ciocalteau method and the Reducing Power test, but the first assay is carried out in alkaline conditions and the second one in acidic conditions. In fact pH has an important effect on the reducing capacity of phenolic antioxidants, because its may be suppressed in acidic conditions, due to protonation, and enhanced in alkaline conditions, due to proton dissociation [7]. Besides these methods, we used two tests both performed in heterogeneous systems (the

-carotene bleaching test and the UV-IP test); in fact the presence of two phases, one hydrophilic and one hydrophobic, might limit the antioxidant power of compounds unable to reach the organic phase (where lipoperoxidation occurs) and/or to interact with lipidic mycelles/biomembranes. However these two assays employ different pro15

ACCEPTED MANUSCRIPT oxidant agents, that are heating and UV-C light respectively. The -carotene bleaching test is a proton-transfer based assay, given that the scavenging of the lipoperoxyl

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radicals by an antioxidant is thought to be a proton-transfer based reaction, and thus it

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allows us to evaluate the capacity of a compound to act as a chain-breaking antioxidant

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in the process of lipid peroxidation [21].

As expected on the basis of their different qualitative/quantitative phenolic profiles, the TP extract possesses an higher antioxidant activity than the SP extract in all tests employed (Table 3).

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The difference between the two extracts was more evident in the Folin-Ciocalteau assay (TP: 320.42 ± 25.19 mg GAE/g; SP: 19.49 ± 1.24 mg GAE/g) than in the Reducing

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Power test (TP: 2.69 ± 0.18 mmoles AAE/g; SP: 1.13 ± 0.07 mmoles AAE/g). One may

Power test.

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suppose that these results are influenced by the acidic conditions used in the Reducing

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A similar trend was evident also observing the results obtained in the -carotene bleaching test and in the UV-IP test. In fact in the -carotene bleaching test, IC50 values were 0.26 ± 0.02 mg/ml and 2.06 ± 0.18 mg/ml, for TP extract and SP extract respectively; but in the UV-IP test, IC50 values were 2.49 ± 0.18 mg/ml and 4.05 ± 0.36 mg/ml for TP extract and SP extract respectively. The antioxidant compounds present in the SP and TP extracts are clearly able to reach and interact with the lipidic phase, protecting it from peroxidative reactions, but differences in their water/oil solubility, stability when exposed to the oxidant agents used in these assays (heating and UV-C light) or capability to act as UV screens can influence their behaviour and contribute to explain the present findings. The results obtained in these tests make the SP and TP extracts good candidates as topical photoprotective agents and thus they were tested in in vivo experiments for their 16

ACCEPTED MANUSCRIPT capability to ameliorate skin erythema elicited by acute UV-B exposure in human volunteers.

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The time course of erythema for skin sites treated with SP, TP and TOC formulations

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after UV-B irradiation is shown in Figure 1 and AUC0-58 values, for each volunteer, are reported in Table 4. Both the formulations containing SP and TP extracts, as well as

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that containing TOC, were able to protect the skin against UV-B-induced erythema, and appeared active in comparison with the blank formulation. No difference was observed between the intensity of erythema developed in skin sites treated with blank formulation

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(only vehicle) and that monitored in control skin sites (no treatment). Interestingly, AUC0-58 values calculated for skin sites treated with the TP were lower

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than those obtained using the SP formulation or the TOC formulation (p<0.05). Moreover, no significant difference was observed between the AUC0-58 values of SP and

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TOC formulations (p>0.05). These data were expressed also as PIE values, which were

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66.81%, 33.18% and 22.60% for TP, SP and TOC gel formulations, respectively (Figure 2). Consistently with these findings, several polyphenol-rich herbal products have proven to be able, when topically employed, to protect skin from UV light-induced damage. For example, extracts of Culcitium reflexum H.B.K. leaves [22], Anthurium versicolor leaves [23], and wine from Jaquez grapes [24] inhibit the development of skin erythema following UV-B-irradiation in humans, very likely due to their content in flavonoids and phenolic acids. Taken together these results clearly demonstrate the protective action of SP and, in particular, TP extracts against photooxidative damage. A part from the high content of phenolic compounds, the in vivo photoprotective activity of SP and TP extracts may be probably related to their particular phenolic profile. Interestingly, the TP extract is very rich in cyanidin-3-O-galactoside, which (as demonstrated by a radical scavenging 17

ACCEPTED MANUSCRIPT chemical assay carried out by means of thin-layer chromatography) is, partially at least, responsible for the antioxidant activity of pistachio skins, together with gallic acid,

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catechin, eriodictyol-7-O-glucoside and epicatechin [3]. There is much evidence

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supporting that anthocyanins, accumulated in plant skin during acclimation to strong sunlight, can serve as an efficient UV-B screen [25]; in fact, they play an important role

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in the resistance of the plant photosynthetic apparatus to the UV-B component of sun radiation. Similar data are reported also for other flavonoids, such as quercetin and kaempferol [26-28]. On the other hand we previously reported that quercetin is stable

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when exposed to UV-C irradiation [29], and Tarozzi et al. [6] demonstrated the photostability of cyanidin-3-glucoside exposed to UV-A and UV-B irradiation. Thus the

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capability of the bioactive components contained in SP and TP extracts to act also as

study.

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UV-absorbing screens could contribute to their photoprotective effect observed in our

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However also other mechanisms are very likely involved in the in vivo photoprotective effects of pistachio extracts, in particular the capability of their active components to interact with cellular biochemical pathways. For example, in in vitro studies on different cell lines anthocyanins have been shown to modulate cell molecular events (increase of the translocation of transcription factors NF-kB and AP-1, overexpression of the proinflammatory cytokine IL-8, cleavage of procaspase-3, and DNA fragmentation) responsible of skin cell damage following UV exposure [7, 30-32]. On the basis of these data one can hypothesize that the studied pistachio extracts do not work only as antioxidants or sunscreens in our experimental conditions. In addition to their antioxidant properties, flavonoids possess an interesting antiinflammatory profile, related to their capability to interfere with a variety of molecular (cyclooxygenase, lipoxygenase) and cellular targets (macrophages, lymphocytes, 18

ACCEPTED MANUSCRIPT epithelial cells, endothelium) [33]. These properties might contribute to the in vivo protective effect of the SP and TP extracts against UV-B light-induced skin

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inflammation; in fact, lipid peroxidation products exert acute inflammatory effects in

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mammalian skin [34]. Furthermore several papers support the usefulness of the UV-B erythema test employed in our study for evaluation of steroidal and non-steroidal anti-

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inflammatory agents [35].

Conclusions

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The present findings demonstrate that SP extract and especially TP extract are endowed with good in vitro antioxidant activity and in vivo skin photoprotective properties, very

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likely due to the polyphenols contained in them. This is particularly interestingly as to the extract from TP; in fact products from pistachio skin, a significant by-product of

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pistachio industrial processing, might be employed as a low cost ingredient with high

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added value in the cosmetic and pharmaceutical field for photoprotective applications.

References 19

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[2] M.L. Dreher Pistachio nuts: composition and potential health benefits. Nutr Rev 70 (2012) 234-240.

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[3] A. Tomaino, M. Martorana, T. Arcoraci, D. Monteleone, C. Giovinazzo, A. Saija, Antioxidant activity and phenolic profile of pistacchio (Pistacia vera L., variety Bronte) seeds and skins. Biochimie 92 (2010) 1115-1122. [4] V.M. Adhami, D.N. Syed, N. Khan, F. Afaq, Phytochemicals for prevention of solar ultraviolet radiation-induced damages. Photochem Photobiol 84 (2008) 489-500.

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[7] D. Huang, B. Ou, R.L. Prior, The chemistry behind antioxidant capacity assays. J Agric Food Chem 53 (2005) 1841-1856.

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[8] A. Saija, A. Tomaino, D. Trombetta, M. Giacchi, A. De Pasquale, F. Bonina, Influence of different penetration enhancers on in vitro skin permeation and in vivo photoprotective effect of flavonoids. Int J Pharm 175 (1998) 185-199. [9] F. Bonina, C. Puglia, D. Ventura, R. Aquino, S. Tortora, A. Sacchi, A. Saija, A. Tomaino, M.L. Pellegrino, P. de Caprariis, In vitro antioxidant and in vivo photoprotective effects of a lyophilized extract of Capparis spinosa L buds. J Cosmet Sci 53 (2002) 321-335. [10] A. Saija, A. Tomaino, R. Lo Cascio, P. Rapisarda, J.C. Dederen, In vitro antioxidant activity and in vivo photoprotective effect of a red orange extract. Int J Cosmet Sci 20 (1998) 331-342. [11] F. Bonina, M. Lanza, L. Montenegro, C. Puglisi, A .Tomaino, D. Trombetta, F. Castelli, A. Saija, Flavonoids as potential protective agents against photo-oxidative skin damage. Int J Pharm 145 (1996) 87-94. [12] F. Bonina, L. Montenegro, In vivo evaluation of radical scavenger compounds in cosmetic formulations by means of skin reflectance spectrophotometry. SÖFW-J 122 (1996) 1-3. [13] L. Montenegro, F. Bonina, J.C. Dederen, In vivo protective effect of bis(carboxyethyl)germanium sesquioxide. J Soc Cosmet Chem 47 (1997) 307-313. 20

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[14] G. Mandalari, A. Tomaino, T. Arcoraci, M. Martorana, V. Lo Turco, F. Cacciola, G.T. Rich, C. Bisignano, A. Saija, P. Dugo, K.L. Cross, M.L. Parker, K.W. Waldron, M.S.J. Wickham, Characterization of polyphenols, lipids and dietary fibre from almond skins (Amygdalus communis L.). J Food Comp Analysis 23 (2010) 166-174.

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[15] M. Oyaizu, Studies on product of browning reaction prepared from glucose amine. Japan J Nutr 44 (1986) 307-315.

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[16] W.A. Wannes, B. Mhamdi, J. Sriti, M.B. Jemia, O. Ouchikh, G. Hamdaoui, M.E. Kchouk, B. Marzouk, Antioxidant activities of the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf, stem and flower. Food Chem Toxicol 48 (2010) 1362-1370.

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[17] A. Saija, A. Tomaino, D. Trombetta, M.L. Pellegrino, B. Tita, C. Messina, F.P. Bonina, C. Rocco, G. Nicolosi, F. Castelli, “In vitro” antioxidant and photoprotective properties and interaction with model membranes of three new quercetin esters. Eur J Pharm Biopharm 56 (2003) 167-174.

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[18] F. Bonina, C. Puglia, D. Ventura, R. Aquino, S. Tortora, A. Sacchi, A. Saija, A. Tomaino, M.L. Pellegrino, P. de Caprariis, In vitro antioxidant and in vivo photoprotective effects of a lyophilized extract of Capparis spinosa L buds. J Cosmet Sci 53 (2002) 321-335.

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[19] A. Saija, A. Tomaino, D. Trombetta, M.L. Pellegrino, B. Tita, S. Caruso, F. Castelli, Interaction of melatonin with model membranes and possible implications in its photoprotective activity. Eur J Pharm Biopharm 53 (2002) 209-215.

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[20] R. Tsao, Chemistry and biochemistry of dietary polyphenols. Nutrients 2 (2010) 1231-1246. [21] M. G. Traber, J.F. Stevens Vitamins C and E: Beneficial effects from a mechanistic perspective. Free Radic Biol Med 51 (2011) 1000-1013. [22] R. Aquino, S. Morelli, A. Tomaino, M.L. Pellegrino, A. Saija, L. Grumetto, C. Puglia, D. Ventura, F. Bonina, Antioxidant and photoprotective activity of a crude extract of Culcitium reflexum H.B.K. leaves and their major flavonoids. J Ethnopharmacol 79 (2002)183-191. [23] R. Aquino, S. Morelli, M.R. Lauro, S. Abdo, A. Saija, A. Tomaino, Phenolic constituents and antioxidant activity of an extract of Anthurium versicolor leaves. J Nat Prod 64 (2001) 1019-1023. [24] G. Spagna, A. Tomaino, F. Cimino, R.N. Barbagallo, D. Ventura, F. Bonina, A. Saija, Chemical analysis and photoprotective effect of an extract of wine from Jacquez grapes. J Sci Food Agric 82 (2002) 1867-1874. [25] J.B. Harborne, C.A. Williams, Advances in flavonoid research since 1992. Phytochem 55 (2000) 481-504. [26] M. Josuttis, H. Dietrich, D. Treutter, F. Will, L. Linnemannstöns, E. Krüger, Solar UVB response of bioactives in strawberry (Fragaria × ananassa Duch. L.): a 21

ACCEPTED MANUSCRIPT comparison of protected and open-field cultivation. J Agric Food Chem 58 (2010) 12692-12702.

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[27] A. Fini, C. Brunetti, M. Di Ferdinando, F. Ferrini, M. Tattini, Stress-induced flavonoid biosynthesis and the antioxidant machinery of plants. Plant Signal Behav 6 (2011) 709-711.

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[28] A. Fini, L. Guidi, F. Ferrini, C. Brunetti, M. Di Ferdinando, S. Biricolti, S. Pollastri, L. Calamai, M. Tattini, Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: an excess light stress affair? J Plant Physiol 169 (2012) 929-939. [29] F. Bonina, M. Lanza, L. Montenegro, C. Puglisi, A .Tomaino, D. Trombetta, F. Castelli, A. Saija, Flavonoids as potential protective agents against photo-oxidative skin damage. Int J Pharm 145 (1996) 87-94.

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[30] [45] F. Cimino, M. Cristani, A. Saija, F.P. Bonina, F. Virgili, Protective effects of a red orange extract on UVB-induced damage in human keratinocytes. Biofactors 30 (2007) 129-138.

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[31] C. Huang, D. Zhang, J. Li, Q. Tong, G.D. Stoner, Differential inhibition of UVinduced activation of NF kappa B and AP-1 by extracts from black raspberries, strawberries, and blueberries. Nutr Cancer 58 (2007) 205-212.

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[32] J.Y. Bae, S.S. Lim, S.J. Kim, J.S. Choi, J. Park, S.M. Ju, S.J. Han, I.J. Kang, Y.H. Kang, Bog blueberry anthocyanins alleviate photoaging in ultraviolet-B irradiationinduced human dermal fibroblasts. Mol Nutr Food Res 53 (2009) 726-38. [33] R. González, I. Ballester, R. López-Posadas, M.D. Suárez, A. Zarzuelo, O. Martínez-Augustin, F. Sánchez de Medina, Effects of flavonoids and other polyphenols on inflammation. Crit Rev Food Sci Nutr 51 (2011) 331-362. [34] M.S. Matsui, A. Hsia, J.D. Miller, K. Hanneman, H. Scull, K.D. Cooper, E. Baron, Non-sunscreen photoprotection: antioxidants add value to a sunscreen. J Investig Dermatol Symp Proc 14 (2009) 56-59. [35] P. Bjerring, Comparison of the bioactivity of mometasone furoate 0.1% fatty cream, betamethasone dipropionate 0.05% cream and betamethasone valerate 0.1% cream in humans. Inhibition of UV-B-induced inflammation monitored by laser Doppler blood flowmetry. Skin Pharmacol 6 (1993) 187-192.

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Figure 1

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Figure 2

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Graphical Abstract

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ACCEPTED MANUSCRIPT Figure legends

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Figure 1: Typical trend of erythema index variations (ΔEI) versus time for one subject. Formulations containing 2% of the active ingredients under study (pistachio seed, SP, extract, or pistachio skin, TP, extract, or tocopheryl acetate, TOC) or containing no active ingredient (Blank) were applied to the skin after UV-B exposure. No treated skin sites were used as control.

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Figure 2. Percentage inhibition of erythema (PIE) values obtained after skin application of formulations containing 2% pistachio seeds extract (SP), pistachio skins extract (TP) or tocopheryl acetate (TOC), calculated vs blank samples.

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SP

TP

PPG-15 stearyl ether (7 g); isohexadecane\PPG-15 stearyl ether (3 g); tocopheryl acetate (2 g)

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PPG-15 stearyl ether (8 g); isohexadecane\PPG-15 stearyl ether (4 g)

Distilled water (76.7 g)

Distilled water (76.7 g)

Steareth 2 (3.5 g); steareth 21 (2.5 g); stearic acid (2.5 g); cetylstearylic acid (2.1 g); xanthan gum (0.3 g); undebenzophenon (0.4 g)

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TOC

Surfactants and structurizing agents Steareth 2 (3.5 g); steareth 21 (2.5 g); stearic acid (2.5 g); cetylstearylic acid (2.1 g); xanthan gum (0.3 g); undebenzophenon (0.4 g)

Aqueous phase

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Blank

Oil phase

PPG-15 stearyl ether (7 g); isohexadecane\PPG-15 stearyl ether (3 g); pistachio seed extract (2 g)

Distilled water (76.7 g)

Steareth 2 (3.5 g); steareth 21 (2.5 g); stearic acid (2.5 g); cetylstearylic acid (2.1 g); xanthan gum (0.3 g); undebenzophenon (0.4 g)

PPG-15 stearyl ether (8 g); isohexadecane\PPG-15 stearyl ether (4 g)

Distilled water (74.7 g) pistachio skin extract (2 g)

Steareth 2 (3.5 g); steareth 21 (2.5 g); stearic acid (2.5 g); cetylstearylic acid (2.1 g); xanthan gum (0.3 g); undebenzophenon (0.4 g)

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Table 1

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Formulation

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TP (µg/g)

Phenolic acids gallic acid

113.28 ± 4.99 113.28 ± 4.99

5703.22 ± 382.06* 5703.22 ± 382.06*

- glycosides cyanidin-3-O-galactoside cyanidin-3-O-glucoside

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Flavan-3-ols - aglycones catechin epicatechin

Isoflavones

- glycosides genistein-7-O-glucoside

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Flavanones

Flavonols

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- glycosides eriodictyol-7-O-glucoside naringenin-7-O-neohesperidoside

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- aglycones quercetin kaempferol

- glycosides quercetin-3-O-rutinoside

16606.64 ± 1036.88 16439.32 ± 1023.55 167.32 ± 13.33

24.33 ± 1.74 24.33 ± 1.74 N.D.

1932.42 ± 131.05* 1390.40 ± 102.59 542.02 ± 28.46

876.72 ± 58.90 520.93 ± 30.60 355.79 ± 28.30

N.D.

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- aglycones genistein daidzein

- aglycones eriodictyol naringenin

N.D.

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Anthocyanins

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SP (µg/g)

Phenols

416.12 ± 24.33 416.12 ± 24.33

N.D.

112.26 ± 3.65 112.26 ± 3.65 N.D.

302.50 ± 18.15* 244.51 ± 14.89 57.99 ± 3.26

636.51 ± 52.48 274.85 ± 20.14 361.66 ± 32.34

1449.34 ± 107.07* 1120.66 ± 83.71 328.68 ± 23.36

N.D.

77.19 ± 3.96 71.15 ± 3.68 6.04 ± 0.28

874.11 ± 44.55 874.11 ± 44.55

28.53 ± 4.96* 28.53 ± 4.96

8.01 ± 0.08

85.14 ± 4.93* 85.14 ± 4.93

Flavones

- aglycones luteolin apigenin

8.01 ± 0.08

Table 2

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TP

mg GAE/g

19.49 ± 1.24

320.42 ± 25.19*

mmoles AAE/ga

1.13 ± 0.07

2.69 ± 0.18*

-carotene bleaching test

IC50b (mg/ml)

2.06 ± 0.18

0.26 ± 0.02*

UV-IP test

IC50c (mg/ml)

4.05 ± 0.36

2.49 ± 0.18*

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Reducing Power test

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Folin-Ciocalteau assay

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*p < 0.01 vs SP extract. a mmoles of Ascorbic Acid Equivalent/g of extract b mg/ml of extract needed to inhibit by 50% -carotene bleaching c mg/ml of extract needed to inhibit by 50% MDA production

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Table 3

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PIE (%)

948.61 ± 64.93 1205.08 ± 75.76 975.33 ± 71.64 1020.71 ± 70.84 1109.43 ± 74.12 984.37 ± 68.29 1098.27 ± 55.51 1247.09 ± 47.26 996.20 ± 69.81 1058.38 ± 56.37 1108.34 ± 70.15 1007.44 ± 68.94

1373.86 117.33

1345.57 137.13

-----

p<0.05 vs TOC;

----b

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920.35 ± 79.07 912.08 ± 61.62 705.37 ± 41.94 1012.84 ± 75.08 1091.07 ± 79.07 820.74 ± 52.74 815.59 ± 55.37 936.46 ± 69.16 878.37 ± 70.39 998.09 ± 64.38 1114.77 ± 59.67 810.15 ± 46.34

TP 418.61 ± 46.54 305.44 ± 34.75 527.90 ± 63.16 439.66 ± 56.69 691.28 ± 69.17 398.01 ± 40.01 359.57 ± 46.30 466.08 ± 48.25 591.48 ± 39.15 546.26 ± 42.17 387.81 ± 40.85 338.57 ± 37.72

1063.27 93.34b

917.99 121.66a,b

455.89 113.84a, b

22.60

33.18

66.81

p < 0.05 vs Blank

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a

Blank 1285.70 ± 70.77 1584.93 ± 70.96 1375.16 ± 65.86 1306.94 ± 65.14 1230.5 ± 64.66 1108.87 ± 59.48 1496.24 ± 76.27 1288.54 ± 55.65 1437.47 ± 78.53 1184.87 ± 45.34 1393.01 ± 69.06 1454.56 ± 57.00

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Mean ± S.D.

Control 1394.37 ± 73.71 1492.18 ± 77.13 1175.33 ± 66.95 1278.05 ± 69.00 1398.81 ± 70.37 1305.87 ± 36.17 1337.55 ± 49.36 1529.07 ± 68.12 1581.82 ± 58.77 1372.63 ± 60.25 1253.61 ± 55.91 1367.08 ± 43.07

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Subject A B C D E F G H I L M N

AUC0-58 TOC

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Table 4

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Table 1 Composition of topical formulations containing pistachio seed (SP) extract, pistachio skin (TP) extract or tocopheryl acetate as reference substance (TOC), or without active ingredients (Blank)

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Table 2: Quantitative analysis of phenolics in SP and TP extracts. Results are expressed as mean ± S.D. of three independent samples and were analyzed by Student’s t test for unpaired data. *p < 0.01 vs SP extract.

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Table 3: Antioxidant activity of pistachio seed (SP) and skin (TP) extracts measured by means of different in vitro test. Data are expressed as mean ± SD of three experiments and were analyzed by Student’s t test for unpaired data.

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Table 4. AUC0-58 values obtained by applying the pistachio seed extract (SP), pistachio skin extract (TP), tocopheryl acetate (TOC) or blank formulations to UV-B-exposed skin sites. Data are expressed as mean ± SD and analyzed by Student’s t test for unpaired data.

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