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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
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.
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.
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
340
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.
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
128; 270
258; 293
325
C7H6O5
C7H6O4
C16H18O9
169; 125
153
353; 191
catechin
avan-3-ol derivate
278
C15H14O6
epicatechin
avan-3-ol derivate
278
C15H14O6
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
C15H10O4
253
10
eriodictyol
avanone derivate
287
C15H12O6
287
11
luteolin
avone derivate
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 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
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
342
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.
343
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
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
344