Nutrition 29 (2013) 338344

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Nutrition
journal homepage: www.nutritionjrnl.com

Basic nutritional investigation

Bioaccessibility of pistachio polyphenols, xanthophylls, and tocopherols during

simulated human digestion
Giuseppina Mandalari Ph.D. a, b, *, Carlo Bisignano Ph.D. b, Angela Filocamo Ph.D. b,
 M.Sc. c, Germana Torre M.Sc. c,
Simona Chessa Ph.D. a, Mariagiovanna Saro
a
Richard M. Faulks M.Sc. , Paola Dugo Ph.D. c
a
b
c

The Model Gut, Institute of Food Research, Norwich, United Kingdom

Pharmaco-Biological Department, University of Messina, Messina, Italy
Pharmaco-Chemistry Department, University of Messina, Messina, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 11 April 2012
Accepted 21 August 2012

Objective: The bioaccessibility of bioactives from pistachios has not been previously evaluated. In
the present study we quantied the release of polyphenols, xanthophylls (lutein), and tocopherols
from pistachios (raw pistachios, roasted salted pistachios, and mufns made with raw pistachios)
during simulated human digestion.
Methods: A dynamic gastric model of digestion that provides a realistic and predictive simulation of
the physical and chemical processing and accurately mimics the residence time and the luminal
environment within the human stomach was used for the digestion studies.
Results: More than 90% of the polyphenols were released in the gastric compartment, with virtually
total release in the duodenal phase. No signicant differences were observed between raw shelled
and roasted salted pistachio. The presence of a food matrix (mufn) decreased the bioaccessibility
of protocatechuic acid (78%) and luteolin (36%). Almost 100% bioaccessibility of lutein and
tocopherols was found after duodenal digestion, with no difference among the three samples.
Conclusion: The rapid release of the assayed bioactives in the stomach maximizes the potential for
absorption in the duodenum and contributes to the benecial relation between pistachio
consumption and health-related outcomes.
2013 Elsevier Inc. All rights reserved.

Keywords:
Pistachios
Bioactives
Simulated digestion
Food matrix
Bioaccessibility

Introduction
Epidemiologic and clinical studies have demonstrated that
nut consumption decreases the risk of cardiovascular disease:
when subjects consumed test diets including mixed nuts, a 25%
greater cholesterol-lowering response was found and this effect
was attributed to the large proportion of unsaturated fatty acids
present in nuts [1,2]. The results of three almond (50100 g/d),
two peanut (3568 g/d), one pecan nut (72 g/d), and four
walnut (4084 g/d) studies have demonstrated a decrease in
total cholesterol (216%) and low-density lipoprotein (LDL)
cholesterol (219%) compared with control subjects [3]. For
pistachios in particular, recent publications have shown benecial effects on cardiovascular disease risk factors, lipid
This work was funded by the American Pistachio Growers (Fresno, CA, USA).
* Corresponding author. Tel.: 44-1603-251405; fax: 44-1603-507723.
E-mail address: giusy.mandalari@ifr.ac.uk (G. Mandalari).
0899-9007/$ - see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nut.2012.08.004

parameters, endothelial function, inammation, and oxidative

status [4,5]. In a randomized cross-over controlled feeding
study, the inclusion of pistachios decreased total cholesterol,
LDL cholesterol, nonhigh-density lipoprotein cholesterol, and
plasma stearoyl-coenzyme A desaturase activity in a dosedependent manner [4]. When pistachios were given to 32
normolipidemic healthy young men for 4 wk, signicant
decreases in blood glucose, total cholesterol, and serum
interleukin-6 were observed, with improved endothelium
vasodilation and total antioxidant status [5]. The consumption
of pistachio nuts has been shown to signicantly decrease
oxidative stress, improving total cholesterol and its LDL levels
in healthy volunteers [6]. Li et al. [7] showed pistachio
consumption decreased plasma triacylglycerols and body
weight when compared with a carbohydrate snack in obese
subjects. Compared with other tree nuts, pistachios are very
rich in phytosterols, potassium, vitamin B6, carotenoids, and
tocopherols [8,9] and have been ranked among the 50 foods

G. Mandalari et al. / Nutrition 29 (2013) 338344

highest in antioxidants [10]. Extensive in vivo and in vitro

experiments on the effect of phenolic compounds have shown
benecial health activities as protective agents against cancer
and cardiovascular, inammatory, and aging disorders, and
human pathogens [1113]. Catechins have been shown to be
particularly effective in cardiovascular disease prevention and
in decreasing the oxidation of LDL [14].
A major challenge in evaluating the role of individual healthpromoting components in the pistachio is the lack of information on their behavior in the gastrointestinal (GI) tract and in
particular on factors that inuence their bioavailability. A
component of bioavailability is bioaccessibility, which is
dened as the relative amounts of nutrients or phytochemicals
released from a complex food matrix in the lumen of the GI
tract, and therefore could potentially be available for absorption
into the body [15]. This process is highly dependent on the
physical and chemical properties of the food matrix and the
way these change during digestion [16]. The choice of food
matrix is therefore crucial for testing the bioaccessibility of
several compounds under simulated GI digestion. We recently
showed that cell walls play a crucial role in regulating nutrient
bioaccessibility from almonds and that polyphenols from
almond skin are potentially available for absorption during
digestion [15,17].
In this study, we characterized the polyphenols, carotenoids,
and tocopherols in raw pistachios and roasted, salted pistachios
and investigated their release during simulated human digestion
in the gut. A dynamic gastric model of digestion that provides
a realistic and predictive simulation of the physical and chemical
processing and accurately mimics the transit time and the
luminal environment within the human stomach was used for
the digestion studies [18,19], together with a duodenal simulated
digestion.
To assess the effect of the food matrix, mufns containing raw
pistachios were used to investigate the bioaccessibility of polyphenols, xanthophylls, and tocopherols.

339

and centrifuged. The pellets were extracted four more times. All methanol fractions were combined and evaporated, after which the residues were dissolved in
distilled water (40 mL) and extracted ve times with ethyl acetate (40 mL). The
organic phases were combined, dried with Na2SO4 for 20 min, and evaporated
under vacuum.
For HPLC separations, an Ascentis Express C18 column (150  4.6 mm,
2.7 mm; Ascentis Express, Supelco, Bellefonte, PA, USA) was used. The mobile
phase was water/formic acid (99.9:0.1, v/v; solvent A) and acetonitrile/formic
acid (99.9:0.1, v/v; solvent B); the linear gradient prole was as follows: 0 min 0%
B, 60 min 100% B, 70 min 100% B, and 71 min 0% B. The data were acquired using
a photodiode array detector in the range of 190 to 400 nm and chromatograms
were extracted at 270 nm and by mass spectrometry. The mass spectrometric
acquisition was performed using electrospray ionisation in negative mode.
The results were expressed as milligrams per 100 g.
Carotenoid analysis
Milled NPs, RPs, or pistachio mufns (10 g) were extracted ve times in the
dark with n-hexane (100 mL) under magnetic stirring for 2 h at room temperature. All extracts were combined together and subjected to rotary evaporation to
remove the solvent.
For the b-carotene determination, the ve hexane portions were combined,
ltered, concentrated, and made up to a known volume of dichloromethane to
measure the b-carotene spectrophotometrically using a previously prepared
calibration curve. The b-carotene was quantied using a Shimadzu UV-2410PC
ultraviolet-visible spectrophotometer (Shimadzu Corp., Kyoto, Japan) at 450 nm.
For xanthophyll determinations, the oil obtained (5 g) was re-extracted by
dimethylformamide (30 mL) and subsequently treated with ve portions (10 mL)
of n-hexane in a separating funnel. All chlorophylls, chlorophyll derivates, and
xanthophylls were retained in the dimethylformamide phase, whereas lipids
and carotenes were retained in the n-hexane phase. The dimethylformamide
phase was treated with a 2% Na2SO4 solution at 0 C and transferred to a mixture
(100 mL) of n-hexane/ethyl ether (1:1, v/v). The aqueous phase was discarded to
remove polyphenols and other water-soluble compounds.
The organic phase was evaporated to dryness at 30 C. The dry residue was
then dissolved in a mixture of methanol/methyl-tert-butyl ether (1:1, v/v) and
analyzed by HPLC [21].
For HPLC separations, a YMC 30 (YMC Europe, Schermbeck, Germany)
analytical column (250  2.1 mm inner diameter, 5-mm particle size) was used.
The mobile phase consisted of a binary gradient of methanol (solvent A) and
methyl-tert-butyl ether (solvent B), with the following gradients: 0 min 5% B,
30 min 45% B, 50 min 95% B, 50 to 55 min 5% B, and 55 to 60 min 5% B. Ultravioletvisible spectra were acquired in the range of 325 to 750 nm and chromatograms
acquired at 450 nm for the xanthophylls and 660 nm for the chlorophylls.
The results were expressed as milligrams per 100 g.

Materials and methods

Tocopherol analysis
Pistachios
Natural, raw, shelled pistachios (NPs; Pistacia vera L.) and roasted salted
pistachio (RP) kernels (P. vera L.) from California were kindly provided by the
American Pistachio Growers (Fresno, CA, USA). Home-made mufns containing
NPs were prepared using the following ingredients: sugar (sucrose 178 g), butter
at room temperature (118 g), eggs (two standard eggs), full-fat plain yogurt (240
g), white our (470 g), baking soda (5 g), salt (1.2 g), and NPs (112 g). Each cooked
mufn weighed 70 g and contained 12 g of NPs (whole nuts). Control mufns
without pistachios were also prepared using the same ingredients.

Weighed and milled NPs, RPs, or pistachio mufns (10 g) were extracted ve
times with n-hexane (100 mL) in the dark under magnetic stirring for 2 h at room
temperature. All aliquots were combined and subjected to rotary evaporation to
remove the solvent.
For HPLC separations, a microsilica column (Ascentis Supelco SI; 250  1.0
mm, 5-mm particle size) was used. The mobile phase consisted of n-hexane/isopropanol (99:1), the ow rate was 50 mL/min, and the injection volume was 2 mL.
The method was validated according to the Eurachem guidelines for each
component, namely a-tocopherol, g-tocopherol, and d-tocopherol [22].
The results were expressed as milligrams per 100 g.

Chemicals and enzymes

Simulated human digestion
Egg L-a-phosphatidylcholine (lecithin grade 1, 99% purity) was obtained from
Lipid Products (South Nuteld, Surrey, UK). Porcine gastric mucosa pepsin,
bovine a-chymotrypsin, pancreatic a-amylase, porcine trypsin, porcine colipase,
porcine pancreatic lipase, and bile salts were obtained from Sigma (Poole, Dorset,
UK). Lipase for the gastric phase of digestion was a gastric lipase analogue of
fungal origin from Amano Enzyme, Inc. (Nagoya, Japan).
Lutein and b-carotene standards were purchased from Extrasynthese (Genay,
France). Polyphenol standards were purchased from Sigma-Aldrich (Milan, Italy).
All the solvents used were high-performance liquid chromatographic (HPLC)
grade and purchased from Sigma-Aldrich.
Determination of polyphenols
Pistachio polyphenolic extracts were prepared as previously reported [20].
NPs, RPs, or pistachio mufns (10 g) were rst homogenized with an Ultra-Turrax
(IKA Works, Inc., Wilmington, DE, USA) and then extracted ve times with
n-hexane (100 mL) under constant agitation (2 h) to remove lipids. After ltration, the residues were mixed with 100 mL of methanol/HCl 0.1% (v/v), extracted,

Oral digestion
The aim of this procedure was to simulate the chewing of the pistachio meals
in the mouth. This is the initial step in the digestion process and was designed to
simulate the salivary amylase activity and mechanical breakdown of the food. The
NPs or RPs (50 g) were minced three times using a mincer (Lexen, Grove City, OH,
USA) to simulate the mechanical oral breakdown. Simulated salivary uid (25 mL)
at pH 6.9 (0.15 M NaCl, 3 mM urea) and human salivary amylase (900 U) dissolved
in simulated salivary uid (1 mL) were added to the minced pistachios and mixed
for 20s. This produced a paste consisting of equal amounts of the solid and
aqueous phases as calculated by human chewing (Institute of Food Research,
unpublished data). The simulated oral processing of pistachio mufns was performed by mixing each minced mufn (70 g) with simulated salivary uid (45 mL)
and human salivary amylase (1260 U) to produce a paste that could be swallowed.
Gastric digestion
The dynamic gastric model of digestion incorporates the inhomogeneous
gastric mixing, antral shearing, and rate of delivery to the duodenum with

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G. Mandalari et al. / Nutrition 29 (2013) 338344

acidication and addition of gastric enzymes in the normal physiologic range. A

suspension of single-shelled lecithin liposomes, prepared as previously described
[15], was added to the gastric enzyme solution at a nal concentration of 0.127
mM. Gastric digestions were carried out immediately after oral digestion on the
chewed pistachio samples (50 g for NPs and RPs, 70 g for pistachio mufns)
together with a representative drink of water (175 mL) in the presence of the
priming acid (20 mL). Six samples (61 g for NPs and RPs, 48 g for pistachio
mufns) were removed from the dynamic gastric model of digestion every
10 min. The amounts of acid secretions added during gastric digestion were
42.2  3, 24.2  1, and 31.6  2 mL for the NPs, RPs, and pistachio mufns,
respectively. The amounts of gastric enzymes added during gastric digestion
were 37.7  2, 34.1  1 and 28.1  1 mL for the NPs, RPs, and pistachio mufns,
respectively. Each gastric sample was weighed, and its pH was recorded and
adjusted to 7.0 with NaOH (1 M) to inhibit gastric enzyme activity.

The pancreatic enzyme solution contained NaCl (125.0 mM), CaCl2 (0.6 mM),
MgCl2 (0.3 mM), and ZnSO4$7H2O (4.1 mM). Porcine pancreatic lipase (590 U/mL),
porcine colipase (3.2 mg/mL), porcine trypsin (11 U/mL), bovine a-chymotrypsin
(24 U/mL), and porcine a-amylase (300 U/mL) were added to the pancreatic juice.
Each gastric sample and the pooled duodenal sample were centrifuged at
3700 rpm for 15 min (7 C) to separate the soluble fraction from the residue. All
samples were immediately frozen and retained for analyses.
Statistical analysis
Differences among polyphenol, lutein, and tocopherol releases were assessed
by analysis of variance followed by the Tukey pairwise comparison. Two-sample
t tests (two-tailed) were used to examine differences between NPs and RPs. The
regression values were considered statistically signicant at P < 0.05.

Duodenal digestion
A pooled sample (30 g) obtained from an aliquot (5 g) of each gastric sample
was transferred to a plastic tube (Sterilin, Ltd., ThermoFisher Scientic, Newpoet,
UK) for duodenal digestion with the addition of the simulated bile solution (7 mL)
and the pancreatic enzyme solution (20 mL) and incubated at 37 C under shaking
(170 rpm) for 2 h. The simulated bile was prepared fresh daily. It contained
lecithin (6.5 mM), cholesterol (4 mM), sodium taurocholate (12.5 mM), and
sodium glycodeoxycholate (12.5 mM) in a solution containing NaCl (146.0 mM),
CaCl2 (2.6 mM), and KCl (4.8 mM).

5.5

Results
Flavonoids and phenolic acids in pistachios and pistachio mufns
Typical chromatograms of the polyphenols detected in the
NPs and RPs are shown in Figure 1. Eleven avonoids (avanols,
avonols, and avanones) and phenolic acids were identied

mAU(x10)
270nm,4nm (1.00)

5.0

4.5

4.0
3.5
3.0
4

2.5
2

2.0

7
1.5

1.0
5

0.5

10 11

0.0
0.0

8.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

20.0

22.5

min

mAU (x10)
270nm,4nm (1.00)

7.0

6.0
5.0
2
4.0
3.0
8

2.0

10

7
34

1.0

11
5

0.0
0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

min

Fig. 1. Typical high-performance liquid chromatograms of avonoids and phenolic compounds extracted at 270 nm in (A) natural, raw, shelled pistachios and (B) roasted,
salted pistachios. Peaks are marked with the same numbers reported in Table 1. Peak identication was performed by matching the information from ultraviolet and mass
spectrometric data to reference materials.

G. Mandalari et al. / Nutrition 29 (2013) 338344

341

Table 1
Identied polyphenols in natural, raw, shelled and roasted, salted pistachios
No.

Common name

Systematic name

Compound type

lmax (nm)

Formula [M]

[M-H]

1
2
3

gallic acid
protocatechuic acid
chlorogenic acid

benzoic acid derivate

benzoic acid derivate
cinnamic acid derivate

128; 270
258; 293
325

C7H6O5
C7H6O4
C16H18O9

169; 125
153
353; 191

catechin

avan-3-ol derivate

278

C15H14O6

289; 245; 205; 161; 137

epicatechin

avan-3-ol derivate

278

C15H14O6

289; 245; 205; 161; 137

eriodictyol7-O-glucoside

avanone-glycoside
derivate

280; 325sh

C21H22O11

451; 287

rutin

3,4,5-trihydroxybenzoic acid
3,4-dihydroxybenzoic acid
(1R,3R,4S,5R)-3-[(E)-3-(3,4dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5trihydroxy-cyclohexane-1-carboxylic acid
(2S,3R)-2-(3,4-dihydroxyphenyl)-3,4dihydro-1-(2H)-benzopyran-3,5,7-triol
(2R,3R)-2-(3,4-dihydroxyphenyl)-3,4dihydro-1-(2H)-benzopyran-3,5,7-triol
2-(3,4-dihydroxyphenyl)-5-hydroxy-7[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6(hydroxymethyl)oxan-2-yl]oxy-chroman4-one
quercetin-3-O-rutinoside

205,254, 354

C27H30O16

609; 301

isoquercetin

avonol-glycoside
derivate
avonol derivate

287

C21H20O12

463

daidzein

isoavone derivate

210; 260sh; 301

C15H10O4

253

10

eriodictyol

avanone derivate

287

C15H12O6

287

11

luteolin

avone derivate

210; 265sh; 348

C15H10O6

285

3-[(2S,3R,4R,5R)-5-[(1R)-1,2dihydroxyethyl]-3,4-dihydroxyoxolan-2yl]oxy-2-(3,4-dihydroxyphenyl)-5,7dihydroxychromen-4-one
7-hydroxy-3-(4-hydroxyphenyl) chromen4-one
2S-2-(3,4-dihydroxyphenyl)-5,7dihydroxy-4-chromanone
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4chromenone

lmax, maximum absorbance for compound identication in ultraviolet visibility; [M], molecular formula; [M-H], pseudomolecular ion; sh, shoulder

(Table 1). The ultraviolet spectra of the different compounds

were recorded from 200 to 380 nm and quantied at 270 nm.
As previously described [23], the major compounds identied
in the pistachio samples were gallic acid, ()-catechin, and isoquercetin. Hydroxybenzoic acids (as gallic and protocatechuic
acids) and chlorogenic acid, avan-3-ols (as []-catechin and
[]-epicatechin), avonols (as glycosides; quercetin-3-Orutinoside and quercetin-3-O-glucoside), avones (as luteolin),
isoavones (as daidzein), and avanones (as aglycones; eriodictyol and glycosides; eryodictiol-7-O-glucoside) were identied. The most abundant forms of avonol glycosides were
glucosides, mainly quercetin-3-O-rutinoside.
Overall, the total amount of avonoids and phenolic acids was
slightly higher in NPs compared with RPs (Table 2), probably
owing to the effect of roasting, as reported by other investigators
[24]. However, the amounts of hydroxybenzoic acids and luteolin
increased in the RPs, and chlorogenic acid and daidzein were

Table 2
Flavonoids and phenolic acids in NPs, RPs, control mufns, and pistachio mufns
Peak no.

Compound

NPs

RPs

Control
mufns

Pistachio
mufns*

1
2
3
4
5
6
7
8
9
10
11

gallic acid
protocatechuic acid
chlorogenic acid
catechin
epicatechin
eriodictyol-7-O-glucoside
quercetin-3-O-rutinoside
isoquercetin
daidzein
eriodictyol
luteolin
Total phenols

1.22
0.83
d
2.11
0.23
0.02
0.56
1.48
d
0.08
0.19
6.72

1.98
0.92
0.14
0.98
0.11
0.04
0.55
0.83
0.17
0.09
0.21
6.02

d
d
d
d
d
d
d
0.08
d
d
d
0.08

0.51
0.34
d
d
d
d
0.15
0.14
d
0.02
0.10
1.26

d, trace of detected compound; NPs, natural, raw, shelled pistachios; RPs,

roasted, salted pistachios
Values are expressed as milligrams per 100 g and represent the average of
triplicate measurements. The SD was always lower than 5%.
* On average, 100 g of pistachio mufns contained 17.1 g of raw pistachios.

detected only in RPs. Of the 11 avonoids and phenolic acids

identied in the pistachio samples, only six compounds were
detected in signicant amounts in the mufns containing NPs,
with trace amounts of the other compounds (Table 2). This may
be due to the smaller amount of pistachios in mufns, e.g.,
a 100-g mufn contained on average 17.1 g of pistachios. Gallic
acid and protocatechuic acid were the main compounds identied in the pistachio mufns. Only traces of all polyphenols were
detected in the control mufns.
Carotenoids and tocopherols in pistachios and pistachio mufns
The b-carotene content ranged from 0.3 to 0.4 mg/100 g of
pistachios, with no signicant differences between the NP and
RP samples (results not shown).
The lutein and tocopherol contents in the NPs, RPs, and
control and pistachio mufns are reported in Table 3. Although
acetone provided a better extraction of chlorophyll pigments,
n-hexane was more appropriate for polar carotenoids, including
xanthophylls (lutein). The lutein content was 34% higher in the
NP compared with RP samples; this effect could have been
caused by roasting. As expected, the lutein content was six times
Table 3
Lutein and tocopherols in NPs, RPs, control mufns, and pistachio mufns
Compound
Xanthophylls
Lutein
Tocopherols
a-Tocopherol
d-Tocopherol
g-Tocopherol
Total

NPs

RPs

Control
mufns

Pistachio
mufns*

1.66

1.10

0.05

0.32

1.11
0.58
21.00
22.69

1.54
3.08
21.94
26.56

1.96
d
d
1.96

2.53
0.46
14.21
17.20

d, trace of detected compound; NPs, natural, raw, shelled pistachios; RPs,

roasted, salted pistachios
Values are expressed as milligrams per 100 g and represent the mean of triplicate
measurements. The SD was always lower than 5%.
* On average, 100 g of pistachio mufns contained 17.1 g of raw pistachios.

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G. Mandalari et al. / Nutrition 29 (2013) 338344

Fig. 2. Release of avonoids and phenolic acids from natural, raw, shelled (black bars) and salted, roasted (white bars) pistachios. Values are presented as percentages of gallic
acid (A), protocatechuic acid (B), isoquercetin (C), eriodyctiol (D), and luteolin (E) released during in vitro gastric (60 min) and gastric plus duodenal (180 min) digestion.
Values represent the average  SD (always <10%) of triplicate measurements.

in the pistachio mufns compared with the control mufns

(Table 3).
The g-tocopherol was most abundant in the NPs, RPs, and
pistachio mufns, followed by a -and d-tocopherols (Table 3).

Slightly larger tocopherol amounts were observed in RPs

compared with NPs, probably resulting from the higher lipid
content in the RPs (50.4%) compared with the NPs (49.5%).
Only a-tocopherol was detected in the control mufns,

G. Mandalari et al. / Nutrition 29 (2013) 338344

343

shown in the pistachio mufns, with total solubilization at the

end of G digestion.
The total tocopherols of the NPs and RPs ranged from 23 to 27
mg/100g, of which 83% to 92% was represented by g-tocopherol,
4.9% to 5.8% by a-tocopherol, and the remainder by d-tocopherol.
The tocopherol releases (percentages) from in vitro digested
samples are listed in Table 4. More than 90% of a-tocopherol was
released in the gastric compartment from the two pistachio
samples, and nearly 100% become available for absorption in the
duodenum. A slightly different trend was shown for d-tocopherol
and g-tocopherol, where 88% and 89% were released from NPs
after G digestion, respectively, and a nearly complete release of
the two compounds was detected from RPs in the stomach. No
signicant differences in tocopherol releases were observed with
pistachio mufns compared with NPs and RPs (Table 4).
Discussion
Fig. 3. Release of lutein from natural, raw, shelled pistachios (black bars), salted and
roasted pistachios (white bars), and pistachio mufns (gradient ll). Values are
presented as percentages of lutein released during in vitro gastric (60 min) and
gastric plus duodenal (180 min) digestion. Values represent the average  SD
(always <10%) of triplicate measurements.

whereas g-tocopherol was the most abundant in the pistachio

mufns.
Release of polyphenols, lutein, and tocopherols from pistachios
during in vitro digestion
Figure 2 shows the release of avonoids and phenolic acids
from the NPs and RPs after in vitro gastric (G) and gastric plus
duodenal (G D) digestion. More than 90% of the pistachio
polyphenols were released during simulated digestion over 3 h.
No signicant differences were observed between the NPs and
RPs, with an initial slower release for the hydroxybenzoic acids
(Fig. 2A, B) compared with isoquercetin, eriodictyol, and luteolin
(Fig. 2CE). Similar trends were observed with pistachio mufns
for most of the identied polyphenols (results not shown), with
the exception of protocatechuic acid and luteolin, whereas
a slower release was observed in the gastric and small intestinal
environments as a result of the food matrix. Releases of 64.7%
and 78.3% were detected for protocatechuic acid in pistachio
mufns after G and G D digestion, respectively, whereas lower
releases were found for luteolin, 27.0% and 35.8%, after G and
G D digestion, respectively.
The release of lutein as a percentage of the original amount
present for the pistachio samples and the pistachio mufns after
G and G D digestion is shown in Figure 3. As observed for the
polyphenol releases, no signicant differences were shown
between NPs and RPs. For all samples, most of the lutein was
released in the gastric environment, with G D digestion
producing only a slight increase in lutein release over that
observed in the stomach. No differences in lutein release were

The results presented in this work demonstrate that polyphenols, tocopherols, and lutein in pistachios are bioaccessible
during simulated human gastric digestion and therefore available for absorption in the upper GI tract. Phenolic acids such as
gallic and chlorogenic acids are generally absorbed in the upper
GI tract within 1 to 2 h after their intake [25].
Polyphenols have been shown to have higher antioxidant
capacity in vitro compared with vitamins and carotenoids and
they represent the main dietary antioxidant [26]. The interactions with other food components and the potential synergy or
antagonism among different compounds are relevant factors in
the bioaccessibility and subsequent benets associated with
polyphenol intake [27]. It has been shown that absorption of
avonols is affected by the attached sugars and the presence of
fat as emulsiers, whereas epimerization reactions occurring
during processing could affect the absorption of avanols such as
catechins [28].
In the present study, more than 90% of the pistachio polyphenols were released in the gastric compartment, with little or
no increase in the duodenal phase (Fig. 2). A similar trend has
been observed with nutrients and polyphenols from almonds
[15,17]. Although the effects of the food matrix on the polyphenol
bioaccessibility have not been examined in much detail, some
studies have shown that the fat content in cocoa samples
increase the release of some phenolic compounds during
duodenal digestion [29]. The absorption of phenolic acids seems
to be affected by the attached sugar, which can covalently link
these compounds to the food matrix [28]. Therefore, the rate of
absorption of polyphenols in the small intestine may be signicantly affected by the food matrix. In the present study, we have
shown that the release of protocatechuic acid and luteolin was
affected by the presence of the mufn, and this may be due to the
matrix composition and the processing. However, roasting had
no effect on polyphenol bioaccessibility (Fig. 2). It has been
shown that roasting signicantly increases the antioxidant

Table 4
Tocopherol release from pistachios due to in vitro gastric and gastric plus duodenal digestion
Pistachio meal

NPs
RPs
Mufns

In vitro gastric duodenal digestion

In vitro gastric digestion

a-Tocopherol (%)

d-Tocopherol (%)

g-Tocopherol (%)

a-Tocopherol (%)

d-Tocopherol (%)

g-Tocopherol (%)

94.0  5.1
99.8  3.6
93.4  2.7

88.1  3.0
98.4  2.8
96.9  4.1

88.8  4.8
99.7  3.8
97.2  3.7

98.9  4.0
98.5  3.4
98.9  2.1

97.9  3.6
99.8  3.7
98.3  2.7

98.7  3.8
99.9  2.7
99.3  2.5

NPs, natural, raw, shelled pistachios; RPs, roasted, salted pistachios.

Values are presented as mean  SD (n 3).

344

G. Mandalari et al. / Nutrition 29 (2013) 338344

capacity of nut extracts, such as almond skins, compared with

blanching and oven drying because of the degradation of polymerized polyphenols and the hydrolysis of glycosides [30]. In
this study, we found a slight decrease of total polyphenols in RPs
compared with NPs (Table 1), although some compounds were
found only after roasting; however, their antioxidant capacity
has not yet been investigated.
The lutein content found is in agreement with previous data
[31]. The marked antioxidant activity of lutein has been shown to
protect the eye against age-related macular degeneration by
preventing damage to the retina [32]. In this report, we have
shown that most of the lutein in NPs, RPs, and pistachio mufns
was released during simulated digestion (Fig. 3). In comparison,
only 37% of lutein in spinach was recovered in micelles in vitro
when using a simulated digestion system [33]. However, this
may be dependent on the fat content of spinach, which is lower
than in pistachios. The bioaccessibility of g-tocopherol also has
ranged from 6.5% (apple) to 59% (cheese). This variable degree of
bioaccessibility of carotenoids and tocopherols across the food
sources conrms that the food matrix has an effect on highly
lipophilic food microconstituents. It has been shown that processing has a benecial effect on carotenoid bioaccessibility [34].
In the present work, almost 100% of pistachio tocopherols were
released in the upper GI tract, and these data may be correlated
with lipid absorption, which has been shown to decrease the
metabolizable energy from pistachios by 5% [35].
Conclusion
We have demonstrated that bioactives from pistachios
become rapidly accessible in the stomach, maximizing the
possibility of absorption in the upper small intestine, which
would contribute to the benecial relation between pistachio
consumption and health-related outcomes. Further human
clinical studies are needed to validate the in vitro ndings on the
release of bioactives from pistachios.
Acknowledgments
The authors acknowledge the American Pistachio Growers
and Setton Farms for providing the samples.
References
[1] Kris-Etherton PM, Yu-Poth S, Sabate J, Ratcliffe HE, Zhao G, Etherton TD.
Nuts and their bioactive constituents: effects on serum lipids and other
factors that affect disease risk. Am J Clin Nutr 1999;70:504S11S.
[2] Kris-Etherton PM, Zhao G, Binkoski AE, Coval SM, Etherton TD. The effects
of nuts on coronary heart disease risk. Nutr Rev 2001;59:10311.
[3] Mukkudem-Petersen K, Oosthuizen W, Jerling JC. A systematic review
of the effects of nuts on blood lipid proles in humans. J Nutr 2005;
135:20829.
[4] Gebauer SK, West SG, Kay CD, Alaupovic P, Bagshaw D, Kris-Etherton PM.
Effects of pistachios on cardiovascular disease risk factors and potential
mechanisms of action: a doseresponse study. Am J Clin Nutr 2008;88:6519.
[5] Sari I, Baltaci Y, Bagci C, Davutogen V, Erel O, Celik H, et al. Effect of
pistachio diet on lipid parameters, endothelial function, inammation, and
oxidative status: a prospective study. Nutr 2010;26:399404.
[6] Kocyigit A, Koylu AA, Keles H. Effects of pistachio nuts consumption on
plasma lipid prole and oxidative status in healthy volunteers. Nutr Metab
Cardiovasc Dis 2006;16:2029.
[7] Li Z, Song R, Nguyen C, Zerlin A, Karp H, Naowamondhol K, et al. Pistachio
nuts reduce triglycerides and body weight by comparison to rened
carbohydrate snack in obese subjects on a 12-week weight loss program. J
Am Coll Nutr 2010;29:198203.
[8] US Department of Agriculture, Agricultural Research Service. USDA
nutrient database for standard reference, release 24. Nutrient data laboratory. Available at: http://www.ars.usda.gov/nutrientdata. Accessed
November 8, 2011.

[9] Philips KM, Ruggio DM, Ashraf-Khorassani M. Phytosterol composition of

nuts and seeds commonly consumed in the United States. J Agric Food
Chem 2005;53:943645.
[10] Halvorsen BL, Carlsen KM, Philips KM, Bohn SK, Holte K, Jacobs DR Jr, et al.
Content of redox-active compounds (ie antioxidants) in food consumed in
the United States. Am J Clin Nutr 2006;84:95135.
[11] Lau FC, Shukitt-Hale B, Joseph JA. The benecial effects of fruit poplyphenols on juice on cognitive and motor decits in aging. Nutrition
2006;22:295302.
[12] Zafra-Stone S, Yasmin T, Bagchi M, Chatterjee A, Vinson JA, Bagchi D. Berry
anthocyanins as novel antioxidants in human health and disease prevention. Mol Nutr Food Res 2007;51:67583.
[13] Puupponen-Pimia R, Nohynek L, Alakomi H-L, Oksman-Caldentey KM.
Bioactive berry compoundsdnovel tools against human pathogens. Appl
Microbiol Biotechnol 2005;67:818.
[14] Velayutham P, Babu A, Liu D. Green tea catechins and cardiovascular
health: an update. Curr Med Chem 2008;15:184050.
[15] Mandalari G, Faulks RM, Rich GT, Lo Turco V, Picout DR, Lo Curto RB, et al.
Release of protein, lipid, and vitamin E from almond seeds during digestion. J Agric Food Chem 2008;56:340916.
[16] Cilla A, Alegria A, de Ancos B, Sanchez-Moreno C, Cano MP, Plaza L, et al.
Bioaccessibility of tocopherols, carotenoids, and ascorbic acid from milkand sot-based fruit beverages: inuence of food matrix and processing. J
Agric Food Chem 2012;60:728290.
[17] Mandalari G, Tomaino A, Rich GT, Lo Curto R, Arcoraci T, Martorana M, et al.
Polyphenol and nutrient release from skin of almonds during simulated
human digestion. Food Chem 2010;122:10838.
[18] Vardakou M, Mercuri A, Barker SA, Craig DQ, Faulks RM, Wickham MS.
Achieving antral grinding forces in biorelevant in vitro models: comparing
the USP dissolution apparatus II and the dynamic gastric model with
human in vivo data. AAPS PharmSciTech 2011;12:6206.
[19] Pitino I, Randazzo CL, Mandalari G, Lo Curto A, Faulks RM, LeMarc Y, et al.
Survival of Lactobacillus rhamnosus strains in the upper gastrointestinal
tract: static and dynamic digestion systems. Food Microbiol 2010;27:
11217.
[20] Mandalari G, Tomaino A, Arcoraci T, Martorana M, Lo Turco V,
Cacciola F, et al. Characterization of polyphenols, lipids and dietary bre
from skins of almonds (Amygdalus communis L.). J Food Comp Anal
2010;23:16674.
[21] Giuffrida D, Saitta M, La Torre L, Bombaci L, Dugo G. Carotenoid, chlorophyll
and chlorophyll-derived compounds in pistachio kernels (Pistacia vera L.)
from Sicily. Ital J Food Sci 2006;18:30916.
[22] Fanali C, Dugo L, Cacciola F, Beccaria M, Grasso S, Dacha M, et al. Chemical
characterization of sacha inchi (Plukenetia volubilis L.) oil. J Agric Food
Chem 2011;59:130439.
[23] Tomaino A, Martorana M, Arcoraci T, Monteleone D, Giovinazzo C, Saija A.
Antioxidant activity and phenolic prole of pistachio (Pistacia vera L.,
variety Bronte) seeds and skins. Biochimie 2010;92:111522.
[24] Ballistreri G, Arena E, Fallico B. Inuence of ripeness and drying process on
the polyphenols and tocopherols of Pistacia vera L. Molecules 1999;
30:435869.
[25] Lafay S, Gil-Izquierdo A. Bioavailability of phenolic acids. Phytochem Rev
2008;7:30111.
[26] Richelle M, Tavazzi I, Offord E. Comparison of the antioxidant activity of
commonly consumed polyphenolic beverages (coffee, cocoa and tea)
prepared per cup serving. J Agric Food Chem 2001;49:343842.
[27] Lambert JD, Hong J, Yang GY, Liao J, Yang CS. Inhibition of carcinogenesis by
polyphenols: evidence from laboratory investigations. Am J Clin Nutr 2005;
81:284S91S.
[28] Scholz S, Williamson G. Interactions affecting the bioavailability of dietary
polyphenols in vivo. Int J Vitam Nutr Res 2007;77:22435.
[29] Ortega N, Reguant J, Romero M-P, Macia A, Motilva MJ. Effect of fat content
on the digestibility and bioaccessibility of cocoa polyphenol by an in vitro
digestion model. J Agric Food Chem 2009;57:57439.
 mez-Cordove
s C, Bartolome B. Polyphenols and
[30] Garrido I, Monagas M, Go
antioxidant properties of almond skins: inuence of industrial processing. J
Food Sci 2008;73:C10615.
[31] Kornsteiner M, Wagner K-H, Elmadfa I. Tocopherols and total phenolics in
10 different nut types. Food Chem 2006;98:3817.
[32] Seddon JM, Ajani UA, Sperduto RD, Hiller H, Blair N, Burton TC, et al.
Dietary carotenoids, vitamins A, C, and E, and advanced age-related
macular degeneration. Eye Disease CaseControl Study Group. JAMA
1994;272:141320.
[33] Reboul E, Richelle M, Perrot E, Desmoulins-Mazelet C, Pirisi V, Borel P.
Bioaccessibility of carotenoids and vitamin E from their main dietary
sources. J Agric Food Chem 2006;54:874955.
[34] Van het Hot KH, de Boer BC, Tijburg LB, Lucius BR, Zijp I, West CE, et al.
Carotenoid bioavailability in humans from tomato processed in different
ways determined from the carotenoid response in triglyceride-rich lipoprotein fraction of plasma after a single consumption and in plasma after 4
days of consumption. J Nutr 2000;130:118996.
[35] Bauer DJ, Gebauer SK, Novotny JA. Measured energy value of pistachios in
the human diet. Br J Nutr 2011;107:1205.