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Article

Identification of Phenolic Compounds in Red and Green Pistachio (Pistacia vera L.) Hulls (Exo- and Mesocarp) by HPLC-DAD-ESI-(HR)-MS

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Sevcan Er#an, Özlem Güçlü Üstünda#, Reinhold Carle, and Ralf M. Schweiggert J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01745 • Publication Date (Web): 11 Jun 2016 Downloaded from http://pubs.acs.org on June 12, 2016

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Journal of Agricultural and Food Chemistry

Identification of Phenolic Compounds in Red and Green Pistachio (Pistacia vera L.) Hulls (Exo- and Mesocarp) by HPLC-DAD-ESI-(HR)-MSn

Sevcan Erşan,†, ‡ Özlem Güçlü Üstündağ,‡ Reinhold Carle,†,§ and Ralf M. Schweiggert†*

†

Chair of Plant Foodstuff Technology and Analysis, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 25, 70599 Stuttgart, Germany ‡

§

Department of Food Engineering, Faculty of Engineering, Yeditepe University, 26 Ağustos Yerleşimi, Kayışdağı Cad., 34755, Istanbul, Turkey

Biological Science Department, King Abdulaziz University, P. O. Box 80257, Jeddah 21589, Saudi Arabia

*

To whom correspondence should be addressed: Phone +49 711 459 22995, Fax: +49 711 459 24110, Email: [email protected]

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ABSTRACT

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Phenolic constituents of the non-lignified red and green pistachio hulls (exo- and

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mesocarp) were assessed by HPLC-DAD-ESI-MSn as well as by HR-MS. A total of 66

4

compounds was identified in the respective aqueous methanolic extracts. Among them,

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gallic acid, monogalloyl glucoside, monogalloyl quinic acid, penta-O-galloyl-β-D-

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glucose, hexagalloyl hexose, quercetin 3-O-galactoside, quercetin 3-O-glucoside,

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quercetin 3-O-glucuronide, and (17:1)-, (13:0)-, (13:1)-anacardic acids were detected at

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highest signal intensity. The main difference between red and green hulls was the

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presence of anthocyanins in the former ones. Differently galloylated hydrolyzable

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tannins, anthocyanins, and minor anacardic acids were identified for the first time.

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Pistachio hulls were thus shown to be a source of structurally diverse and potentially

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bioactive phenolic compounds. They therefore represent a valuable by-product of

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pistachio processing having potential for further utilization as raw material for the

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recovery of pharmaceutical, nutraceutical, and chemical products.

15 16

Keywords: Pistacia vera L., waste, by-product, gallotannin, flavonoid, quercetin

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glycoside, anthocyanin, anacardic acid, mass spectrometry, high resolution mass

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spectrometry, bioactive.

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INTRODUCTION

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Pistachio (Pistacia vera L.) seeds are an important commercial crop, whose annual

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production has doubled during the past decade to reach a worldwide production of

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approx. 1 million tons in 2013. Top producing countries are Iran followed by the USA

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and Turkey.1 Although the fruit of the pistachio tree is botanically considered a drupe,

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pistachio seeds are often regarded as “nuts”, being most commonly consumed as roasted

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and salted snack food. In addition, a certain amount of pistachio seeds is used as food

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ingredient, e.g., in pastry, ice-cream, chocolate, confectionary production, and

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mortadella. Pistachio drupes consist of an edible seed characterized by light-green

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cotyledons (kernels), a mauvish seed coat (testa) covered by a creamy lignified shell

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(endocarp), and a green to yellow-red colored outer hull (exo- and mesocarp) depending

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on the degree of ripeness.2 According to their final use, they are processed to separate

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either the non-lignified hull for snack pistachios or the entire hull, shell, and seed coat to

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obtain the isolated kernels.3,4 In both cases, significant amounts of waste accrue having

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no or low commercial value, which need to be disposed at the processor’s expense.

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Therefore, valorization of pistachio by-products is of great interest, so far being

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hampered by missing identification of valuable target compounds which merit utilization.

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Extracts of pistachio outer hull, the main by-product of pistachio processing, have been

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shown to exert antioxidant,5–8 antimicrobial,8–10 antimutagenic8 and cytoprotective6 as

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well as potential antitumor11 and anticancer activities.12 Despite such potent bioactivities,

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the phytochemicals in the pistachio hull remain to be comprehensively characterized.

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Only a limited number of previous studies has aimed at elucidating the phenolic profiles 3 ACS Paragon Plus Environment


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of pistachio hulls (either ripe exocarp or green exo- and mesocarp), suggesting phenolic

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acids (gallic, protocatechuic, hydroxybenzoic, p-coumaric, and vanillic acids) and

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flavonoids (quercetin glycosides, isorhamnetin glycosides, naringenin, eriodictyol, and

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catechin) represent the main phenolic constituents, as only being identified by HPLC-

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DAD-FLD or HPLC-DAD-MS,5,6 together with (13:0)-, (13:1)-, (15:0)-, (15:1)-, and

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(17:1)-anacardic acids.13 Since pistachio belongs to the Anacardiaceae, a wide array of

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phenolic compounds is to be expected in pistachio hull.14 For instance, flavonol

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glycosides, gallotannins, and specific phenolic lipids have been reported in other

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members of the Anacardiaceae such as mango (Mangifera indica L.),15–17 Brazilian

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pepper (Schinus terebinthifolius Raddi),18 and cashew (Anacardium occidentale L.).19

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Thus, we sought to provide a comprehensive analysis of the phenolic constituents present

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in pistachio hull using highly sensitive tools such as HR-MSn, particularly, aiming at the

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further utilization of pistachio hull as a source of potent phenolic bioactives.

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Pistachio drupes are harvested at different maturity stages based on their desired

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properties and final use. A high portion of pistachio drupes are harvested red colored at

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full maturity, and preferentially used for the production of snack food, because their fully

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developed taste and high shell splitting ratio are important quality traits. However, green

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drupes, i.e. early-harvested drupes, are desired to produce intensely green colored kernels

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for pastry and confectionary industry of high market value. Thus, both green and red

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pistachio hulls, either in fresh or dried form depending of agricultural practices of the

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country, can be obtained in high amounts as by-products of pistachio processing.

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Particularly, dried pistachio hulls accrue in large amounts from numerous processors,

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because drying allows the off-season processing of the otherwise highly perishable


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pistachio drupes. Hitherto, no attention has been given to their compositional differences,

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and a lack of unambiguous sample descriptions in previous studies often hampers their

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comparative consideration.5–8 Therefore, in this study, we additionally sought to compare

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the phenolic profiles of red and green pistachio hull as a further reference for their

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utilization as a phenolic source.

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For the above mentioned purposes, dried red and green hulls were obtained from a

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commercial pistachio processor to be extracted with aqueous methanol yielding phenolic-

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rich samples. Subsequently, these were screened for bioactive phenolic compounds by

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HPLC-DAD-ESI-MSn and HPLC-HR-MS.

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MATERIALS AND METHODS

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Reagents

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Gallic acid, protocatechuic acid, penta-O-galloyl-β-D-glucose, and (15:0)-anacardic acid

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were obtained from Sigma–Aldrich Chemie (Steinheim, Germany). β-Glucogallin (1-O-

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galloyl β-D-glucopyranose) was from PhytoLab (Vestenbergsgreuth, Germany), quercetin

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3-O-glucuronide, quercetin 3-O-glucoside, quercetin 3-O-galactoside, myricetin 3-O-

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galactoside and cyanidin 3-O-β-D-galactopyranoside were from Extrasynthèse (Genay

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Cedex, France). HPLC grade methanol from VWR (Darmstadt, Germany), analytical

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grade formic acid from Merck (Darmstadt, Germany), and deionized water were used

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throughout the study.

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Samples and sample preparation



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Dried red and green pistachio drupes cv. ‘Uzun’ (Figure 1) were obtained from a

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pistachio processor (Gaziantep, Turkey). Both red and green pistachio drupes were

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harvested between August and September in 2013 in Gaziantep region of Turkey,

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traditionally sun-dried to decrease their moisture contents to 4.6% ± 0.2% as determined

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according to AOAC Official Method 934.0120 and stored for a year by the producer as

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usual in Turkey. Commonly, moisture content of pistachio drupes during commercial

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storage was reported to range from 40-50% in fresh form to 3-5% in dried form21.

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Average air temperatures were between 3.4 °C and 28.6 °C for this region during the year

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the drupes were stored with a relative humidity of 29.0-78.9%.22

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Pistachio drupes were sampled in triplicate (each 500.0 g ± 0.1 g). Hulls and seeds were

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separated manually and weighted. Proportions of hulls and seeds of the whole pistachio

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drupe were approx. 20.5% and 37.4% on dry weight basis, respectively. Pistachio hulls

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were subsequently ground using an A11 laboratory mill (IKA, Staufen, Germany).

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Ground samples were stored at -20 °C until analyses.

99 100

Extraction of phenolics

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Ground red and green pistachio hulls (1.00 g ± 0.01 g) were combined with 5 mL of

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acidified (0.1% HCl, v/v) aqueous methanol (80%, v/v) and subjected to probe sonication

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at 70% amplitude for 30 s. After centrifugation (1233 x g, 3 min), the supernatant was

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collected and the solid residues were re-extracted four times as described above. The

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combined methanolic extracts were evaporated to dryness in vacuo at 30 °C. Then, the

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dried extract was dissolved in 1 mL of 50% aqueous methanol containing 1% (v/v)


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formic acid and membrane-filtered (0.45 µm, regenerated cellulose) into amber vials

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prior to HPLC analyses.

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HPLC-DAD-ESI-MSn analyses

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An 1100 series HPLC system with G1322A degasser, a G1312A pump module, a

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G1313A autosampler, a G1316A column thermostat and a G1315A diode array detector

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(Agilent, Waldbronn, Germany) was equipped with a 250 mm × 4.6 mm i.d., 5 µm

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particle size, 100 Å pore size Kinetex C18 core-shell reversed-phase column fitted with

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4.6 mm x 2 mm i.d. SecurityGuard Ultra C18 guard column both from Phenomenex

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(Aschaffenburg, Germany). Mobile phases were water for eluent A and methanol for

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eluent B both containing 1% (v/v) formic acid. Chromatographic separation was achieved

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at 35 °C column temperature, 1 mL/min flow rate, and using the following gradient

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profile: isocratic at 2% B for 10 min, from 2-37% B in 27 min, isocratic at 37% B for 5

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min, from 37-40% B in 18 min, from 40-60% B in 10 min, from 60-100% B in 20 min,

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isocratic at 100% B for 14 min, from 100-2% B in 1 min followed by isocratic

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conditioning at 2% B for 7 min prior to the next run. Total run time was 112 min and

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injection volume was 5 µL.

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For multi-stage mass spectrometry, the above described LC system was interfaced with

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an Esquire 3000+ ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany),

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which had been fitted with an ESI source operated in positive ion mode for anthocyanins

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and negative ion mode for all other analyses. Ion scan rate was in the range of m/z 100-

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2000 at a scan speed of m/z 13,000/s. Nitrogen was used both as drying and nebulizing

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gas at a flow rate of 11 mL/min and at pressure of 60 psi, respectively. Nebulizer 7 ACS Paragon Plus Environment


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temperature was 365 °C. The potential on the capillary was set at ±2287 V for both

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negative and positive ion modes. Helium at a pressure of 4 x 10-6 mbar was used for

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collision induced dissociation (CID) at a fragmentation amplitude of 1.2 V. Ion

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chromatograms were analyzed using Esquire Control software.

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HPLC-ESI-HR-MS analyses

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HPLC-HR-MS analyses were performed using micrOTOF-Q mass spectrometer (Bruker

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Daltonics, Bremen, Germany) coupled to an Agilent 1200 series HPLC system operated

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according to the above mentioned parameters. HR-MS was operated in negative ESI

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mode with +2200 V capillary voltages. Ion scan rate was in the range of m/z 250-3000 at

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a scan speed of m/z 12,900/s (at m/z 996.8). Nitrogen was used both as nebulizing and

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drying gas at a pressure of 3.0 bar and a flow rate of 8 L/min, respectively. Drying gas

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was heated to 300 °C. Instrument calibration was carried out according to the

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manufacturer’s instructions using sodium formate. DataAnalysis 3.4 software was used to

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generate molecular formulas based on accurate mass measurements.

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RESULTS AND DISCUSSION

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Analysis of phenolic compounds by HPLC-DAD-ESI-MSn and HPLC-ESI-HR-MS

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A total of 66 phenolic compounds was detected in pistachio hull extracts, representative

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structures are presented in Figure 2. Monitoring was performed at different wavelengths

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as shown in Figure 3. All analytical data obtained for the examined compounds are listed

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in Table 1, including their retention times, UV absorption maxima, ESI-MSn


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fragmentation pattern, and the respective high resolution mass-to-charge (m/z) signals.

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The identification of the examined 66 compounds led to their allocation into three

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structurally related groups, i.e., gallotannins, flavonoids, and anacardic acids

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Gallotannins

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Gallotannins represent a subgroup of hydrolyzable tannins, more specifically being esters

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of at least one gallic acid molecule with polyols such as sugars, shikimic acids or quinic

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acids.23 While gallic acid, 2, was tentatively identified by comparing the obtained

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retention time, UV absorption and mass spectra to those of an authentic standard, a total

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of 30 related gallic acid derivatives and gallotannins was identified in pistachio hull

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extracts, mainly based on the formation of characteristic product ions at m/z 169 ([gallic

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acid-H]-) and 125 ([gallic acid-CO2-H]-) as well as due to the specific neutral loss of a

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dehydrated galloyl moiety (152 Da). The identification was corroborated by their UV

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absorption spectra and high resolution MS data.

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Galloyl hexoses

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Compound 1 with a parent ion [M-H]- at m/z 331 revealed a daughter ion [M-H-162]- at

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m/z 169 upon CID fragmentation, indicating the loss of a hexose moiety. It was identified

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as 1-O-galloyl β-D-glucopyranose (β-glucogallin) after comparing its retention time, UV

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absorption and mass spectra with those of an authentic standard. Glucogallin has already

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been reported in many plants including other members of Anacardiaceae, such as



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Brazilian pepper18 and mango,24 and is considered a primary metabolite and galloyl donor

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for gallotannin biosynthesis.25,26

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The parent ion [M-H]- at m/z 493 of compound 8 formed daughter ions [M-H-162]- at m/z

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313 and [M-H-162-162]- at m/z 169. In agreement with its UV and high resolution MS

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data, compound 8 was tentatively identified as a galloyl dihexose. Similar galloyl

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dihexoses have been previously found in plant parts of other Anacardiaceae, namely in

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sumac (Rhus coriaria L.).27 Compounds 6 and 7 exhibited both UV absorption spectra

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and high resolution mass signals identical to those of compound 8 (Table 1). However,

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their different fragmentation patterns indicated that they might represent distinct isomers

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of galloyl dihexoses as reported previously.28 For instance, while predominant CID

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daughter ions [M-H-180]- at m/z 313 and [M-H-324]- at m/z 169 were observed for

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compounds 6 and 8, those of compound 7 were observed at m/z 271 ([M-H-162-60]-), at

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m/z 211 ([M-H-162-120]-) and at m/z 313 ([M-H-180]-) (Table 1).24

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Two further minor compounds, 14 and 16, were tentatively assigned as digalloyl hexoses

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(m/z 483) due to loss of a dehydrated galloyl (152 Da) and a hexose (162 Da) moiety,

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ultimately yielding gallic acid (m/z 169) as daughter ion. The loss of dehydrated galloyl

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units (152 Da) may indicate depsidically linked gallic acids due to previously reported

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predominance of galloyl fission in these types of linkages.29

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The pseudo-molecular ions [M-H]- of three compounds, 31, 37 and 39, exhibited

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sequential losses of galloyl moieties (152 Da) from their parent ions at m/z 787, 939 and

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1091, respectively. These compounds were tentatively identified as tetra- , penta- , and

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hexagalloyl hexose, respectively, based on their retention order, UV absorption maxima,

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high resolution MS data, and MSn fragmentation pattern as compared with literature.30,31 10 ACS Paragon Plus Environment

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The identity of compound 37 was further confirmed as penta-O-galloyl-β-D-glucose after

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comparing its analytical data with that of the corresponding authentic standard.

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Hexagalloyl hexose, 39, was the gallotannin having the highest degree of galloylation

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that was detected as a separate peak, although several unresolved peaks eluting after 40

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min (Figure 3) may be attributed to the presence of higher degrees of galloylated tannins.

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The chromatographic separation of such highly galloylated gallotannins was previously

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shown to be most intricate due to the increased number of possible gallotannin isomers

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with the increase in the number of galloyl units.32 In agreement, our extracted ion

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chromatograms containing the respective traces of penta-, hexa-, hepta-, octa-, and

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nonagalloyl tannins are shown in Figure 4 to illustrate the increasing complexity of these

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compounds and their related mass signals. In addition, characteristic doubly charged

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pseudo-molecular ions [M-2H]2− and corresponding fragment ions [M-n × 152-2H]2−

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with (n = 1–4) were observed in the region of highly galloylated gallotannins.18,33

208 209

Galloyl quinic acids

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By analogy to differently galloylated hexoses, quinic acid was found to be galloylated to

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different degrees. Compounds 4, 19, and 28 were tentatively identified as mono-, di- and

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trigalloyl quinic acids due to sequential losses of galloyl moieties (152 Da) from their

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parent ions at m/z 343, 495 and 647, respectively, and the formation of a final product ion

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at m/z 191 (deprotonated quinic acid).34,35 HR-MS measured exact molecular masses

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were also in good agreement with calculated masses of the respective galloyl quinic

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acids, corroborating their identification (Table 1).



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An additional quinic acid derivative (compound 29) was also tentatively identified due to

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the formation of a putative product ion at m/z 191 (deprotonated quinic acid) upon CID

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experiments. Compounds with different degrees of galloyl quinic acids were also

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reported in Pistacia lentiscus L.,34 Myrtus communis L.,36 green tea (Camellia sinensis

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L.), and tara (Caesalpinia spinosa (Molina) Kuntze).35

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Galloyl shikimic acids

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Three compounds, 9, 10 and 12, with identical UV absorption spectra, identical high

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resolution MS data, and identical pseudo-molecular ions [M-H]- at m/z 325 were

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characterized by the loss of 156 Da, yielding a daughter ion at m/z 169. In agreement with

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our analytical data, these compounds were identified as monogalloyl shikimic acids. Two

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compounds, 23 and 24, with pseudo-molecular ions [M-H]- at m/z 477 produced daughter

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ions at m/z 325 due to the neutral loss [M-H-152]- of a putative galloyl moiety. Further

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fragmentation of m/z 325 yielded spectra similar to the above mentioned monogalloyl

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shikimic acids, 9, 10, and 12. In agreement with their chemical formula (Table 1),

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compounds 23 and 24 were tentatively identified as the isomers of digalloyl shikimic

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acids. Despite their uncommon presence in plants, differently galloylated shikimic acids

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were previously reported in other plants,37–40 including Brazilian pepper from the

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Anacardiaceae.18 As shikimic acid has been reported as a precursor of gallate synthesis,41

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galloylated shikimic acids may represent intermediates for the biosynthesis of higher

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molecular weight gallotannins.

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The presence of gallotannins has previously been reported in other Anacardiaceae such as

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mango30 and sumac.27 However, this is the first detailed report on pistachio hull 12 ACS Paragon Plus Environment

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gallotannins. Behgar et al.42 previously reported the total “tannin content” of pistachio

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hull as determined by an unspecific protein precipitation based radial diffusion assay.

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Other gallic acid derivatives

243 244

Two peaks, 13 and 20, exhibited parent ions [M-H]- at m/z 321. Their CID fragmentations

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resulted in product ions at m/z 169 and 125 characteristic of gallic acid. Thus, these

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compounds were tentatively identified as digallic acids, although their differences on

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linkages between the two galloyl moieties remain unknown despite their slightly different

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UV absorption maxima (Table 1). The pseudo-molecular ion [M-H]- of compound 32 at

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m/z 473 tentatively indicated the presence of a trigallic acid due to sequential loss of two

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galloyl moieties, yielding product ions specific for gallic acid. We were unable to provide

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evidence that these three compounds, 13, 20, and 32, represented depsides, although

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depsidically linked gallic acids were previously found in tanoak acorns (Notholithocarpus

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densiflorus (Hook. & Arn. ) Manos, Cannon & S. H. Oh)28 and Anacardiaceae such as

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sumac,27 mango peel31 as well as in Rhus chinensis Mill. leaves, a traditional Chinese

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herb.43

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Compound 15 exhibited a parent ion [M-H]- at m/z 183 with a corresponding

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demethylated product ion [M-H-15]- at m/z 168, thus being tentatively assigned as methyl

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gallate in agreement with previous reports.31 Similarly, compound 36 with a parent ion at

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m/z 335 was identified as methyl digallate due to loss of a digalloyl moiety during CID.

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In addition, a product ion [M-H-152]- at m/z 183 (methyl gallate) was observed in the

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MSn spectra. Noteworthy, methyl gallate, 15, and methyl digallate, 36, may represent

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potential artefacts, resulting from methanolysis of depsidically linked gallotannins in the 13 ACS Paragon Plus Environment


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course of extraction and analysis.29,35 In agreement, an increase in both peak area and

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height of compound 15 and 36 was observed upon re-analysis of methanolic extracts kept

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at room temperature for 24 h (data not shown).

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Compound 26 produced a parent ion [M-H]- at m/z 319 and its HR-MS measured exact

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molecular mass revealed a good fit to luteic acid, a digallic acid with an additional C-C

268

bond between its benzene rings (Table 1). Although compound 17, 26 and 44 had

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common MSn product ions at m/z 139, which may indicate the formation methyl

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pyrogallol fragments upon CID, the identities of compounds 17 and 44 yet remain

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unknown. Luteic acid, a molecule present in the structure of myrobalanitannin, has been

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reported in the fruits of Terminalia chebula Renz. as an intermediate of ellagic acid

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biosynthesis.44

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Five compounds, 3, 11, 18, 21, and 22, were tentatively identified as gallic acid

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derivatives due to the formation of a deprotonated gallic acid at m/z 169 as MSn daughter

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ion, although their further characterization remains pending. The yet unidentified gallic

277

acid derivative, compound 3, may contain nitrogen due to its even-numbered mass-to-

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charge ratio at m/z 296 (Table 1).

279 280

Flavonoids

281 282

Flavonols

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A total of 17 flavonols was tentatively identified in red pistachio hull extracts, including

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quercetin, myricetin and kaempferol derivatives. Quercetin derivatives, 38, 40-43, 45-47,


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50 and 51, were the major flavonol constituents of the hulls under investigation,

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according to their highly characteristic UV absorption maxima at ca. 350 nm, common

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fragment ions at m/z 301 (deprotonated quercetin), and a characteristic fragment of the

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quercetin aglycone at m/z 179. The major quercetin derivatives, quercetin 3-O-

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galactoside, 40, quercetin 3-O-glucuronide, 41, and quercetin 3-O-glucoside, 42, were

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identified by comparing their retention times, UV absorption and MS data to those of

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authentic standards (Figure 3). In addition to quercetin hexosides, a quercetin pentoside,

292

47, was tentatively identified. Moreover, several types of galloylated quercetin glycosides

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were tentatively identified according to their analytical data shown in Table 1, such as

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quercetin galloyl hexosides, 38, 43, and 45, quercetin galloyl deoxyhexose, 46, quercetin

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galloyl hexuronide, 50, and quercetin galloyl pentoside, 51.

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Four myricetin derivatives, 30, 33-35, were tentatively assigned based on their

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characteristic fragment ion at m/z 317 (myricetin aglycone) and their characteristic

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secondary fragments at m/z 299 and 271. Compound 33 was tentatively identified as

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myricetin hexuronide due to its parent ion [M-H]- at m/z 493 and the derived, previously

300

reported29 predominant daughter ion at m/z 317, indicating the loss of an uronic acid

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moiety in agreement with its high resolution MS data. Compounds 34 and 35 were

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tentatively identified as myricetin hexosides based on the analytical data presented in

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Table 1. Compound 34 was further identified as myricetin 3-O-galactoside after

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comparison of its analytical data with that of an authentic standard. The parent ion [M-H]-

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of compound 30 at m/z 631 was characterized by the sequential loss of 162 Da (hexose)

306

and 152 Da (galloyl moiety), thus being identified as myricetin galloyl hexoside in

307

agreement with its high resolution MS data. Moreover, three kaempferol derivatives,



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308

namely two hexosides and one pentoside, 49, 52 and 53, were identified in trace amounts.

309

HR-ESI-MS accurate mass measurements were in agreement with all proposed flavonols

310

(Table 1).

311

Flavonols of pistachio hull have been recently investigated using HPLC-DAD.5

312

According to this study, quercetin rutinoside represented the major flavonol accompanied

313

by lower amounts of quercetin, quercetin galactoside, quercetin glucoside, kaempferol

314

and isorhamnetin glycosides. The presence of these flavonol derivatives was confirmed

315

by our study, except for quercetin rutinoside and isorhamnetin derivatives which were not

316

detected in our samples. In further contrast, in our samples, quercetin galactoside,

317

quercetin glucuronide and quercetin glucoside were the major flavonols accompanied by

318

low amounts of myricetin and kaempferol derivatives (Table 1).

319 320

Anthocyanins

321

Compound 26 was the main peak observed in the chromatogram recorded at 520 nm

322

(Figure 3), whose parent ion [M]+ at m/z 449 exhibited a characteristic daughter ion [M-

323

162]+ at m/z 287, the cyanidin aglycone. After comparing its retention time, UV/Vis

324

absorption and mass spectra with those of an authentic standard, compound 25 was

325

tentatively identified as cyanidin 3-O-β-D-galactopyranoside. Minor amounts of a

326

putative cyanidin pentoside, 27, were also tentatively identified based on the formation of

327

a cyanidin aglycone at m/z 287 after the loss of a pentose (132 Da) upon CID

328

fragmentation. The presence of cyanidin 3-O-galactoside in P. vera seed coat (named as

329

“skin” in the study)45,46 and cyanidin 3-O-glucoside in leaves of P. lentiscus34 has

330

previously been reported. However, this is the first report on the occurrence and 16 ACS Paragon Plus Environment

Page 17 of 35


331

identification of cyanidin derivatives in red pistachio hull, although a previous study

332

reported

333

spectrophotometrically.47 Interestingly, 7-O-methylated anthocyanins have not been

334

observed in our study, although their presence in mango,48 cashew apple,49 Brazilian

335

pepper,18 and sumac27 has been proposed to be a chemotaxonomic marker of the

336

Anacardiaceae. Noteworthy, our samples underwent drying and storage prior to analyses,

337

and thus, further studies on freshly collected pistachio fruits should be done. On the other

338

hand, it is worth mentioning that P. vera has occasionally been classified in its own

339

family Pistaciaceae rather than in the Anacardiaceae, which may be supported by the

340

aforementioned lack of 7-O-methylated anthocyanins.

total

anthocyanin

contents

of

pistachio

hulls

as

determined

341 342

Anacardic acids

343

A total of 11 anacardic acids 54-60, 62-64, 66, with different lengths of alkyl chains

344

(C13, C15 and C17) and saturation degrees (fully saturated or mono-, di-, or

345

triunsaturated) were identified in pistachio hull extracts (Table 1). They eluted late at 88-

346

94 min (Figure 3) due to their lipophilic alk(en)yl side chain. In the following, the

347

compounds are named according to the length of their side chain and the number of

348

double bonds in the side chain (Figure 2). All of them produced similar UV absorption

349

spectra with maximum absorbance at 250 and 311 nm. Their CID mass spectra had a

350

product ion [M-44]- in common, indicating a CO2 loss from the phenolic carboxyl group.

351

Furthermore, characteristic product ions at m/z 106 or m/z 107 have been reported to

352

occur due to the elimination of the phenol group, while product ions at m/z 119 and 149

353

were described to result from fragmentation at the allyl position of unsaturated anacardic 17 ACS Paragon Plus Environment


Page 18 of 35

354

acids.50,51 Compound 63 was assigned as (15:0)-anacardic acid after comparing its

355

retention time, UV absorption and mass spectra including the characteristic loss of CO2

356

(44 Da), and the product ion at m/z 106 with those of an authentic standard. The

357

identification of further anacardic acids was based on UV and mass spectra including

358

accurate mass measurements and the corresponding molecular formulas that were

359

consistent with previously published data of Jerz et al.50 As shown in Figure 3, the most

360

abundant representatives were (13:0)-, 59, (13:1)-, 58, (15:0)-, 63, (15:1)-, 60, and (17:1)-

361

anacardic acids, 64, being in agreement with an earlier study on phenolic acids of P.

362

vera.13 Their elution occurred later with increasing chain length and decreasing degree of

363

saturation as previously shown.50 Based on the oxidative degradation of these major

364

compounds, Yalpani et al.13 determined the localization of the double bond of the

365

unsaturated (13:1)-, (15:1)-, and (17:1)-anacardic acids to be at the 8 position of the alkyl

366

chain for monounsaturated anacardic acids from green pistachio hull (named as “outer

367

green shell” in their study). We assume that this allocation of the double bonds may also

368

be valid for our results. Besides confirming these major compounds, our study is the first

369

report of the occurrence of six minor anacardic acids, namely (16:1)-, (13:2)-, (11:0)-,

370

(15:3)-, (17:2)-, and (17:0)-anacardic acids. In contrast to pistachio kernels,51 cardanols,

371

decarboxylated derivatives of anacardic acids, were not detected in pistachio hulls.

372

Interestingly, anacardic acids are currently considered to be chemotaxonomic markers of

373

the Anacardiaceae,14 consistently occurring in cashew nuts and shells,50 as well as in

374

mango.52 These findings might be of interest for the above mentioned discussion on the

375

assignment of the genus Pistacia.

376


Page 19 of 35

377


Minor compound

378

Compound 5 with a parent ion [M-H]- at m/z 153 was tentatively identified as

379

protocatechuic acid after comparison of its analytical data with those of an authentic

380

standard. Protocatechuic acid has been reported in pistachio hull before.5,6

381 382

Comparison of red and green hulls

383

When red and green type pistachio hulls were compared (Figure 3), virtually identical

384

phenolic profiles were observed, except for the anthocyanins that only occurred in red

385

hulls. These findings indicate that green and red pistachio hulls may be utilized without

386

separation as a source of phenolic compounds. However, further research on the quantity

387

and contribution of each class of phenolic compounds to the biological activity of red and

388

green pistachio hull extracts should be performed to determine technologically optimal

389

recovery strategies for phenolics from different types of pistachio hulls.

390 391

In conclusion, the complex phenolic profiles of dried red and green P. vera hulls were

392

characterized in this study to provide basic knowledge for their future utilization. Apart

393

from anthocyanins that are characteristic of red hulls, phenolic constituents of pistachio

394

hulls may largely be grouped in three major phenolic classes: gallotannins, flavonoids

395

and anacardic acids. The identity of the gallotannins of pistachio hull was elucidated for

396

the first time, revealing the presence of galloyl hexoses with up to nine galloyl units, and

397

galloyl quinic and shikimic acids with up to three galloyl units. Pistachio hulls also

398

contained glycosides of flavonoids such as quercetin glycosides and a cyanidin 3-O-β-D19 ACS Paragon Plus Environment


Page 20 of 35

399

galactopyranoside. Furthermore, anacardic acids, a distinct class of polar phenolic lipids,

400

were identified and may be useful as chemotaxonomic markers. In brief, a wide range of

401

phenolic compounds was present in pistachio hulls, ranging from simple (gallic acid) to

402

very complex ones (gallotannins) and from polar/mid-polar (gallotannins and flavonoids)

403

to amphiphilic (anacardic acids) ones. Thus, pistachio hull represents an interesting

404

source for the production of multifunctional phenolic extracts. Further research on the

405

extraction and isolation of these compounds and the determination of their relation with

406

attributed biological functions should be encouraged.

407

ACKNOWLEDGEMENTS

408

We thank Joachim Trinkner (University of Stuttgart, Germany) for conducting the

409

HPLC-ESI-HR-MS analyses.

410

Funding

411

TUBITAK 2211/C and 2214/A Programs are acknowledged for financial support of one

412

of the authors (S.E.).

413

Notes

414

The authors declare no competing financial interest.

415

ABBREVIATIONS

416

(11:0)-anacardic acid, undecylsalicylic acid; (13:0)-anacardic acid, tridecylsalicylic acid;

417

(13:1)-anacardic

acid,

tridecenylsalicylic

acid;

(13:2)-anacardic

acid, 20


Page 21 of 35


418

tridecadienylsalicylic acid; (15:0)-anacardic acid, pentadecylsalicylic acid; (15:1)-

419

anacardic

420

pentadecatrienylsalicylic acid; (16:1)-anacardic acid, hexadecenylsalicylic acid; (17:0)-

421

anacardic acid, heptadecylsalicylic acid; (17:1)-anacardic acid, heptadecenylsalicylic

422

acid; (17:2)-anacardic acid, heptadecadienylsalicylic acid.

acid,

pentadecenylsalicylic

acid;

(15:3)-anacardic

acid,



423

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Page 26 of 35

FIGURE CAPTIONS Figure 1.

Photograph of dried (A) green and (B) red Pistacia vera L. drupes. Background was cut off without further manipulation. Note that the green color has been lost upon drying and storage due to its instability.

Figure 2.

Representative structures of phenolic compounds detected at high signal intensity in aqueous methanolic extracts of Pistacia vera L. hulls

Figure 3.

HPLC separation of phenolic compounds from (A) red and (B) green Pistacia vera L. hulls at 280 nm. (C) Chromatogram of phenolic compounds from red hull monitored at different wavelengths. Peak assignments are shown in Table 1.

Figure 4.

Extracted ion chromatograms indicating the putative presence of (A) penta- (m/z 939), (B) hexa- (m/z 1091), (C) hepta- (m/z 1243), (D) octa(m/z 1395), and (E) nona- (m/z 1547) galloyl hexoses.


Page 27 of 35


Table 1. HPLC Retention Times, UV/Vis Spectra, and MS Data of Pistachio (Pistacia vera L.) Hull Phenolics a

HPLCUV/Vis abs. max (nm) 274

HR-ESI(-)-MS [M-H]m/z exp. (theo.)

molecular formula

HPLC-ESI-MSn experiment m/z

331.0684 (331.0671)

C13H16O10

[331]: 169(100), 271(56), 193(31), 211(31), 183(25), 315(10)

gallic acidb gallic acid derivative (1)

272 276

nac 296.0788 (296.0789)

na na

11.2 13.4 14.5

galloyl quinic acid protocatechuic acidb galloyl dihexose (1)

275 259 274

343.0689 (343,0671) na 493.1195 (493.1194)

C14H16O10 na C19H26O15

7

16.5

galloyl dihexose (2)

274

493.1209 (493.1194)

C19H26O15

8

17.2

galloyl dihexose (3)

274

493.1205 (493.1199)

C19H26O15

9

18.0

galloyl shikimic acid (1)

274

325.0571 (325.0565)

C14H14O9

10

19.6


274

325.0568 (325.0565)

C14H14O9

11

20.2

gallic acid derivative (2)

276

na

na

12

20.8


274

325.0576 (325.0565)

C14H14O9

13

21.1

digallic acid (1)

280

321.0261 (321.0252)

C14H10O9

14

21.5

digalloyl hexose (1)

280

483.0805 (483.0780)

C20H20O14

15

22.3

methyl gallate

272

na

na

16

23.8

digalloyl hexose (2)

288

483.0794 (483.0780)

C20H20O14

17

25.0


274

509.0972 (509.0996)

C15H26O19

compound no.

tR (min)

compound identity

1

6.0

1-O-galloyl β-Dglucopyranose (βglucogallin)b

2 3

6.8 10.0

4 5 6

[331→169]:125(100) [169]: 125(100) [296]:169(100), 125(15), 195(5), 107(5), 171(2) [296→169]:125(100) [343]: 191(100), 169(17), 125(12) [153]: 109(100) [493]: 313(100), 169(39), 283(29), 331(10) [493→313]: 283(100), 169(44), 223(43), 135(39), 241(15) [493]: 271(100), 313(25), 211(20), 331(20), 169(7) [493→271]: 211(100), 125(4) [493]: 313(100) [493→313]: 169(100), 125(92), 189(30), 242(18) [325]: 169(100), 125(22), 139(9), 193(8) [325→169]: 125(100) [325]: 169(100), 125(15), 281(7), 111(4), 173(3) [325→169]: 124(100), 125(28) [571]: 285(100), 169(7) [571→285]: 133(100), 169(49), 170(10) [325]: 169(100), 125(15), 137(3), 173(3) [325→169]: 125(100) [321]: 169(100) [321→169]: 125(100) [483]: 331(100), 271[(16), 169(15) [483→331]: 169(100), 313(60), 271(51), 193(25), 123(15), 241(12), 195(12), 211(10) [183]: 168(100), 124(10) [183→168]: 124(100) [483]: 331(100), 169(27), 332(17), 271(11) [483→331]: 169(100), 241(53), 125(15) [509]: 267(100), 429(11) [509→267]: 139(100)



18

26.0


274

423.0931 (423.0933)

C19H20O11

19

26.4

digalloyl quinic acid

274

495.0776 (495.0780)

C21H20O14

20

27.3

digallic acid (2)

275

321.0246 (321.0252)

C14H10O9

21

28.1


268

403.1254 (403.1246)

C17H24O11

22

29.5


274

467.1197 (467.1195)

C21H24O12

23

30.6

digalloyl shikimic acid (1)

275

477.0688 (477.0675)

C21H18O13

24

31.0

digalloyl shikimic acid (2)

276

477.0703 (477.0675)

C21H18O13

25

31.6

cyanidin 3-O-β-Dgalactopyranosideb

278, 517

na

na

26

33.2

luteic acid

278

319.0133 (319.0096)

C14H8O9

27 28

33.7 33.8

cyanidin pentoside trigalloyl quinic acid

520 273

na 647.0873 (647.0890)

na C28H24O18

29

34.5

quinic acid derivative (1)

276

523.1455 (523.1157)

C24H28O13

30

35.3

myricetin galloyl hexoside

270, 354

631.0934 (631.0941)

C28H24O17

31

35.8

tetragalloyl hexose

278

787.1010 (787.0999)

C34H28O22

32

36.7

trigallic acid

274

473.0373 (473.0362)

C21H14O13

33

36.9

myricetin hexuronide

357, 252

493.0640 (493.0624)

C21H18O14

34

37.1

myricetin 3-O-galactoside

359, 252

479.0848 (479.0831)

C21H20O13

35

37.4

myricetin hexoside

357, 252

479.0884 (479.0831)

C21H20O13

36

37.9

methyl-digallate

274

335.0414 (335.0409)

C15H12O9

37

38.3

penta-O-galloyl-β-D

280

939.1130 (939.1109)

C41H32O26

Page 28 of 35

[423]: 313(100), 169(70), 125(36), 211(24) [423→313]: 169(100), 313(39), 295(17) [495]: 343(100), 191(29), 344(16), 271(4) 169(3) [495→343]: 191(100) [321]: 169(100), 125(16) [321→169]: 125(100) [403]: 169(100), 151(55), 313(37), 125(36), 271(21), 211(18), 179(15), [403→169]: 124(100), 125(34), 107(34) [467]: 313(100), 169(17), 295(7) [467→313]: 169(100), 153(10), 191(5) [477]: 325(100), 169(47), 326(33) [477→325]: 169(100) [477]: 325(100), 169(47) [477→325]: 169(100), 125(11), 281(10) d [449]: 287(100) [449→287]: 137(100) [319]: 239(100), 139(15), 240(14) [319→239]: 139(100), 124(12) d [419]: 287(100) [647]: 495(100), 343(22), 191(2) [647→495]: 343(100), 191(46) [523]: 209(100), 371(45), 505(30), 313(19), 169(14), 191(14) [523→209]: 191(100), 165(21), 151(18), 123(2) [631]: 479(100), 317(10) [631→479]: 316(100), 317(69), 325(15), 271(11) [787]: 617(100) [787→617]: 465(100), 589(55), 221(41), 277(34), 449(30), 600(26), 296(20), 235(18), 466(12) [473]: 321(100), 169(25) [473→321]: 169(100), 125(6) [493]: 317(100), 299(4), 151(3), 137(2) [493→317]: 227(100), 151(23), 137(18) [479]: 317(100), 271(20), 179(19), 287(15) [479→317]: 215(100), 271(99), 164(24), 270(22), 287(22), 242(17) [479]: 316(100), 317(57), 271(12) [479→316]: 271(100), 179(40), 317(35), 180(29), 255(26), 137(23) [335]: 183(100), 253(5) [335→183]: 168(100) [939]: 769(100), 617(18), 787(12)


Page 29 of 35


glucoseb [939→769]: 617(100), 387(43), 601(40), 465(28), 323(21), 259(19), 403(19), 725(17), 245(12), 573(12), 386(11) [615]: 463(100), 301(32)

38

38.9

quercetin galloyl hexoside (1)

257, 354

615.1031 (615.0992)

C28H24O16

39

40.6

hexagalloyl hexose

263, 353

1091.1423 (1091.1213)

C48H36O30

40

41.0

quercetin 3-O-galactosideb

252, 352

463.0922 (463.0882)

C21H20O12

41

41.2

quercetin 3-Oglucuronideb

255, 355

477.0715 (477.0675)

C21H18O13

42

41.6

quercetin 3-O-glucosideb

260, 355

463.0915 (463.0877)

C21H20O12

43

42.1


254, 358

615.1031 (615.0986)

C28H24O16

44

42.7


279

347.0409 (347.0406)

C16H12O9

45

42.9


276, 358

615.1098 (615.0986)

C28H24O16

[615→301]: 179(100), 151(99) [347]: 267(100), 139(5), 249(4), 269(4), 124(2) [347→267]: 139(100), 249(28), 83(16), 140(10), 205(8) [615]: 301(100), 179(5), 463(3)

46

43.3

quercetin galloyl deoxyhexose

274, 355

599.1075 (599.1037)

C28H24O15

[615→301]: 151(100), 170(32), 107(21), 179(20), [599]: 463(100), 301(36)

47

43.7

quercetin pentoside

254, 355

433.0810 (433.0771)

C20H18O11

48

44.1

unknown (1)

275, 357

na

na

49

45.0

kaempferol hexoside

272, 350

447.0963 (447.0927)

C21H20O11

50

45.4

quercetin galloyl hexuronide

276, 352

629.0852 (629.0779)

C28H22O17

[599→463]: 301(100), 255(7) [433]: 301(100), 255(6), 242(4), 193(4) [433→301]: 271(100), 179(42), 151(41), 255(11), 243(9), [629]: 327(100), 459(15), 477(15), 328(11) [629→327]: 113(100), 169(49), 283(40), 175(38), 170(37), 177(35) [447]: 285(100), 255(28), 327(19) [447→285]: 255(100), 223(6), 256(5) [629]: 477(100), 301(26)

51

46.3

quercetin galloyl pentoside

277, 352

585.0910 (585.0881)

C27H22O15

[629→477]: 301(100), 323(5), 175(3) [585]: 301(100)

[615→463]: 301(100), 179(8), 229(7), 253(6), 272(6), 151(2) [1091]: 939(100), 769(16) [1091→939]: 769(100), 770(13), 617(10) [463]: 301(100), 179(8) [463→301]: 151(100), 229(58), 271(43), 343(30), 254(26), 258(24), 181(16) [477]: 301(100), 179(7), 151(2) [477→301]: 179(100), 152(20), 180(11), 121(8), 256(7), 273(6), 229(6) [463]: 301(100) [463→301]: 179(100), 271(52), 152(39), 272(32), 255(31), 151(27), 203(21) [615]: 301(100), 313(15), 315(8), 273(6), 463(3)

[585→301]: 151(100), 179(45), 165(44), 121(9)



52

46.9

kaempferol hexoside

270, 350

447.0952 (447.0927)

C21H20O11

53

48.9

kaempferol pentoside

270, 350

417.0907 (417.0822)

C20H18O10

54

88.4

(16:1)-anacardic acid

238, 310

359.2521 (359.2586)

C23H36O3

55

88.5


238, 310

315.1968 (315.1966)

C20H28O3

56

88.9


238, 308

291.1960 (291.1966)

C18H28O3

57

89.1


238, 310

341.2112 (341.2122)

C22H30O3

58

89.6


246, 310

317.2115 (317.2117)

C20H30O3

59

90.7


248, 312

319.2289 (319.2279)

C20H32O3

60

91.0


240, 310

345.2429 (345.2434)

C22H34O3

61

91.2

unknown (2)

260

455.3510 (455.3405)

C30H48O3

62

91.4


238, 310

371.2599 (371.2592)

C24H36O3

63

92.0

(15:0)-anacardic acidb

242, 311

347.2623 (347.2592)

C22H36O3

64

92.2


248, 312

373.2743 (373.2743)

C24H38O3

65

92.4

unknown (3)

260

373.2748 (373.2748)

C24H38O3

66

93.1


238, 312

375.2951 (375.2977)

C24H40O3

Page 30 of 35

[447]: 285(100), 284(92), 255(63), 163(18), 151(17) [447→285]: 255(100), 197(13), 240(10), 198(8), 213(7), 227(6), 169(5) [417]: 284(100), 255(37) [417→284]: 255(100), 160(5), 165(4), 227(4), 195(3), 151(3) [359]: 315(100), 341(43), 161(21), 315(17), 107(13), 343(13), 317(11) [359→315]: 108(100) [315]: 271(100), 107(6) [315→271]: 107(100) [291]: 247(100) [291→247]: 106(100) [341]: 297(100) [341→297]:149(100) [317]: 273(100) [317→273]: 107(100) [319]: 275(100) [319→275]: 106(100) [345]: 301(100), 119(4) [345→301]: 119(100) [455]: 418(100) [455→418]: 434(100), 395(66), 375(60), 399(15) [371]: 327(100) [371→327]: 327(100), 119(18), 107(10) [347]: 303(100) [373→303]: 106(100) [373]: 329(100) [373→329]: 106(100) [373]: 329(100) [373→329]: 119(100) [375]: 331(100) [375→331]: 106(100)

a

tR: retention time. Verified by reference standards. c na: not available. d Positive ionization mode was used for the identification of anthocyanins. e All compounds were detected in both red and green pistachio hulls, except for the anthocyanins, 25 and 27, which were only found in red hulls. b


Page 31 of 35


Figure 1 79x74mm (600 x 600 DPI)



Figure 2 117x80mm (600 x 600 DPI)


Page 32 of 35

Page 33 of 35


Figure 3 156x136mm (600 x 600 DPI)



Figure 4 120x86mm (600 x 600 DPI)


Page 34 of 35

Page 35 of 35


TOC Graphical Abstract 44x23mm (600 x 600 DPI)


Pistacia vera L. - ACS Publications - American Chemical Society

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