Antioxidant and antiproliferative activities of common edible nut seeds

Jun Yang
*
,1
, Rui Hai Liu, Linna Halim
Department of Food Science, Stocking Hall, Cornell University, Ithaca, NY 14853, United States
a r t i c l e i n f o
Article history:
Received 15 April 2008
Received in revised form 15 July 2008
Accepted 15 July 2008
Keywords:
Nuts
Phytochemicals
Phenolics
Antioxidant
Antiproliferative activity
a b s t r a c t
Frequent consumption of nuts has been linked to a lowered risk of cardiovascular disease.
Phytochemicals, especially phenolics, in nuts may be considered to be the major bioactive compounds for
health benets. Nine types of tree nuts and peanuts commonly available in the United States were
evaluated for total phenolic and avonoid contents, antioxidant, and antiproliferative activities. The
proles of total phenolics and avonoids, including both soluble free and bound forms, were investigated
by utilizing solvent extraction, base digestion, and solid-phase extraction methods. Walnuts had the
highest total phenolic and avonoid contents (1580.5 58.0 mg/100 g, 744.8 93.3 mg/100 g, respec-
tively). Walnuts also possessed the highest total antioxidant activity (458.1 14.0 mmol of vitamin C
equiv/g of nut). Both soluble phenolic and avonoid contents were positively correlated with total
antioxidant activity (R
2
0.9901, p <0.05; and R
2
0.9749, p <0.05, respectively). The proliferation of
HepG
2
and Caco-2 cells was signicantly inhibited in a dose-dependent pattern after exposure to the
extracts of nuts, with walnuts and pecans exhibiting the highest antiproliferative activity. These results
provide new knowledge about health functions of nuts and may inuence consumers toward purchasing
nuts exhibiting greater potential health benets.
2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Consumption of nuts has been linked to a lowered risk of
cardiovascular heart disease (CHD) (Albert, Gaziano, Willett,
Manson, & Hennekens, 2002; Fraser, Sabate , Beeson, & Strahan,
1992; Hu, Stampfer, & Manson, 1998; Kris-Etherton, Pearson, Wan,
& Hargrove, 1999; Prineas, Kushi, Folsom, Bostick, & Wu, 1993;
Sabate et al., 1993). Several large prospective cohort studies have
reported a signicant inverse correlation between frequent nut
consumption and CHD. The Adventist Health Study with a 6-year
follow-up was the rst epidemiological study on the relationship
between nut consumption and risk of CHD (Fraser et al., 1992). In
this study, subjects who consumed nuts ve or more times per
week had signicantly lowered risks of both fatal CHD and nonfatal
myocardial infarction. Prineas et al. (1993) found that women who
consumed nuts two to four times per week reduced risks of fatal
CHD. More than 80,000 women who consumed nuts about ve
times per week lowered their risk of heart disease by 35% and type
2 diabetes by 27%, reported in the Harvard Nurses Health Study (Hu
et al., 1998). The Iowa Womens Health Study has recorded that
frequent nut consumption may offer postmenopausal women
modest protection against the risk of death fromall causes and CHD
(Ellsworth, Kushi, & Folsom, 2001). In the Physicians Health Study,
followed up for an average of 17-years, US male physicians who
consumed nuts two or more times per week had a 47% lower risk of
sudden cardiac death and a 30% lower risk of total CHD death
compared to men who rarely or never consumed nuts (Albert et al.,
2002). In the Seventh-Day Adventists study, one to four intakes of
nuts per week were associated with a 33% decrease in the risk of
colorectal cancer compared to the non-consumers (Singh & Fraser,
1998). Additionally, in both the Adventist Health Study and the
Harvard Nurses Health Study, persons who consumed more nuts
tended to weigh less. The EPIC cohort study conducted in 10
European countries showed that women consuming more than
6.2 g of nuts and seeds daily reduced their risk of colon cancer by
31% (Jenab, Ferrari, & Slimani, 2004). However, epidemiological
evidence in the effect of nut consumption on the risk of cancer is
insufcient.
Nut consumption lowers the risk of CHD, which may be partly
explained by the cholesterol-lowering effect. The favorable fatty
acid composition and lipid lowering effect of nuts have been
demonstrated in experimental studies with almonds (Hyson,
Schneeman, & Davis, 2002), macadamia nuts (Curb, Wergowske,
Dobbs, Abbott, & Huang, 2000), peanuts (Alper & Mattes, 2003),
pecans (Morgan & Clayshulte, 2000), pistachios (Edwards, Kwaw,
Matud, & Kurtz, 1999), and walnuts (Ros, 2000). A diet high in nuts
(walnuts, peanuts, or almonds) reduces LDL cholesterol and
decreases the ratio of total to HDL cholesterol (Kris-Etherton et al.,
* Corresponding author. 7701 Legacy Drive, R&D, FritoLay North America Inc.,
Plano, TX 75024, United States. Tel.: 1 (972) 334 4863; fax: 1 (972) 334 2329.
E-mail address: junyang97@gmail.com (J. Yang).
1
Note: This work has been done when Dr. Jun Yang was a graduate student in the
Department of Food Science at Cornell University.
Contents lists available at ScienceDirect
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j ournal homepage: www. el sevi er. com/ l ocat e/ l wt
0023-6438/$34.00 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2008.07.007
LWT - Food Science and Technology 42 (2009) 18
1999). Almonds used as snacks in the diets of hyperlipidemic
subjects signicantly decreased CHD risk factors (Jenkins et al.,
2002). Almond consumption may reduce colon cancer risk after
the investigation of whole almond-, almond meal-, or almond
oil-containing diet effects on aberrant crypt foci (ACF) in
azoxymethane-treated F344 male rats. Almonds also showed to be
more effective than wheat bran in inhibiting colon cancer cells
(Davis & Iwahashi, 2001). The macadamia nut-based diet had
potentially benecial effects on cholesterol and LDL cholesterol
levels when compared with a typical American diet (Curb et al.,
2000). Regular peanut consumption lowers serum triacylglycerol
(TAG) and increases dietary folate intake, thereby lowering plasma
homocysteine concentration (Alper &Mattes, 2003). Higher nut and
peanut butter consumption was associated with a decreased risk of
type 2 diabetes inwomen (Jiang et al., 2002). In addition to lowering
serum cholesterol, pecan-enriched diets also increased HDL
cholesterol and lowered TGA concentrations, leading to favorably
altering the lipid prole (Rajaram, Burke, Connell, Myint, & Sabate,
2001). After three weeks of eating pistachios, moderate hypercho-
lesterolemia subjects experienced a decrease in total cholesterol, an
increase in HDL, a decrease in the total cholesterol/HDL ratio, and
a reduction in the LDL/HDL ratio (Edwards et al., 1999). Walnut diets
improved endothelium-dependent vasodilation and decreased
levels of vascular cell adhesion molecule-1 in a randomized,
crossover feeding trial by replacing a walnut-enriched diet for
a Mediterranean-type diet (Ros, 2000). Whole nuts containa variety
of constituents that may have cardio-protective and anticancer
effects by a number of different mechanisms. The proposed mech-
anisms of action for the health benets of nuts may include
a favorable fatty acid prole, improved blood lipoprotein prole,
antiatherogenic effect through the arginineNO pathway (Cooke
et al., 1993), antiarrhythmic properties with a higher intake of
a-linolenic acid (Kang & Leaf, 1996), benets for glucose and insulin
homeostasis via MUFA and PUFA (Jiang et al., 2002), antioxidant
activity (Wu et al., 2004), reduction of tumour initiation or promo-
tion, regulation of cell differentiation and proliferation, repair of
DNA damage, regulation of immunological activity and inamma-
tory response, induction of phase II metabolic enzymes, regulation
of hormones by acting as phytoestrogens (Kris-Etherton, Hecker, &
Bonanome, 2002), and a high content of nutrients such as magne-
sium, selenium, folic acid, potassium, ber, and vitamin E.
Nuts are rich sources of unsaturated fatty acids, protein, ber,
micronutrients, vitamins, and phytochemicals (Rainey & Nyquist,
1997) although the levels vary among nuts. Walnuts are good
sources of both antioxidants and n-3 fatty acids, in particular high
amounts of a-linolenic acid (6.3 g/100 g), whereas other nuts such
as almonds, pecans, and pistachios possess much smaller amounts
(0.40.7 g/100 g). Almonds are especially high in vitamin E and
magnesium. Brazil nuts are particularly rich in the antioxidant
compound selenium, while pecans are rich in bone-building
manganese. Cashews, macadamia nuts and Brazil nuts contain
slightly more saturated fatty acids than other nuts. Nuts are also
a source of dietary ber (1.75 g soluble ber per 100 g nuts).
Peanuts are good sources of folate and resveratrol. Nuts are a good
source of phytochemicals, including phenolics (tannins, ellagic acid,
and curcumin), avonoids (luteolin, quercetin, myricetin, kaemp-
ferol, and resveratrol), isoavones (genistein and daidzein),
terpenes, organosulfuric compounds, and vitamin E (Bravo, 1998;
Kris-Etherton et al., 2002). For example, avonoids, including
catechins, avonols, and avonones in their aglycone and glycoside
forms, are found in abundance in almonds (Sang et al., 2002).
Walnuts contain a wide variety of phenolics and tocopherols; non-
avonoids such as ellagitannins are also found in walnuts
(Anderson et al., 2001). Peanuts have several avonoids and are rich
in resveratrol (Lou, Yuan, Yamazaki, Sasaki, & Oka, 2001), while
alkyl phenols exist in abundance in cashews (Trevisan et al., 2006).
The potential mechanisms of action of components in nuts that
may intervene in the prevention of cancer have not been
completely understood. To date, nut research has been mostly
focused on the lipid prole. Little research has been concentrated
on the phytochemical prole of nuts, and even less attention has
been paid to the associated antioxidant and antiproliferative
activities in nuts. To more fully characterize the antioxidant prole
and possible associated health benets of different nuts and
peanuts, it is worth quantifying these bioactive compounds.
Therefore, the objectives for this study were (1) to determine the
prole of total phenolics, including both soluble free and bound
forms in nuts, (2) to measure the total antioxidant activities, (3) to
evaluate the antiproliferative activities of nuts on human liver and
colon cancer cell growth, and (4) to examine correlations between
antioxidant or antiproliferative activity and total phenolic or
avonoid content.
2. Materials and methods
2.1. Materials
()-Catechin, sodium nitrite, FolinCiocalteu reagent (FCR),
hydrochloric acid, glucagon, hydrocortisone, insulin, and a-keto-
g-methiolbutyric acid (KMBA) were purchased from Sigma Chem-
ical Co. (St. Louis, MO). Aluminum chloride, sodium hydroxide,
methanol, and acetone were purchased from Fisher Scientic
(Pittsburgh, PA). Gallic acid was purchased from ICN Biomedical
Inc. (Costa Mesa, CA). 2,2
0
-azobis-amidinopropane (ABAP) was
purchased from Wako Chemicals (Richmond, VA).
Nine commonly consumed tree nuts (almonds, Brazil nuts,
cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios,
and walnuts) and peanuts available in the United States were
purchased from the local market (Table 1). The peanut or
groundnut (Arachis hypogaea) is a species of the legume family
Fabaceae. All data collected for each sample were reported as
means SD for at least three replications.
2.2. Methods
2.1.1. Extraction of soluble free phytochemical compounds
Total phenolic extraction of nuts is shown in the owchart of
Fig. 1. Soluble free phenolics of samples were extracted using the
method reported previously in our laboratory (Yang, Meyers, van
der Heide, & Liu, 2004). Briey, 25 g of nuts were blended for 5 min
in 200 g of chilled 80% acetone (1:8, w/w) using a Waring blender.
The mixture was then homogenized in a Virtis High Speed
Homogenizer for 5 min and ltered under vacuum. Water in the
ltrate was evaporated using a rotary evaporator at 45

C until the
weight of the evaporated ltrate was less than 10% of the weight of
the original ltrate. After measurement of the residue volume, 100%
methanol was added to a nal methanol concentration of 50%
methanol/water mixture. The methanol/water mixture was
Table 1
Description of tree nuts and peanuts
Nut Variety Process
Almonds California Shelled meat, whole, dry roasted
Brazil nuts Unknown Shelled meat, whole, dry roasted
Cashews Venguria Shelled meat, whole, dry roasted
Hazelnuts Ennis Shelled meat, whole, dry roasted
Macadamia nuts Ikaika Shelled meat, whole, dry roasted
Peanuts Runner Shelled meat, whole, dry roasted
Pecans Stuart Shelled meat, whole, dry roasted
Pine nuts Korean pine Shelled meat, whole, dry roasted
Pistachios California Shelled meat, whole, dry roasted
Walnuts Chandler Shelled meat, whole, dry roasted
J. Yang et al. / LWT - Food Science and Technology 42 (2009) 18 2
washed using 50 mL hexane by centrifugation at 2500g for 10 min.
The hexane fraction was removed and the extract was washed
twice. Finally, the methanol/water mixture was evaporated using
a rotary evaporator at 45

C until the weight of the evaporated
ltrate was less than 10% of the weight of the original ltrate. The
nal extract was diluted to 10 mL with MilliQ water, aliquoted into
2 mL per tube, and saved as solublefree phenolics. The extracts
were stored at 40

C until use.
2.1.2. Extraction of bound phytochemical compounds
Bound phytochemicals of nuts were extracted by the method
reported previously (Krygier, Sosulski, & Hogge, 1982) and modied
in our laboratory (Adom, Sorrells, & Liu, 2003). Briey, 10 g of the
solid residues from the soluble-free extraction were collected,
ushed with nitrogen gas, sealed, and hydrolyzed directly with
40 mL of 4 mol/L NaOH at room temperature for 1 h with shaking.
The mixture was then neutralized with concentrated hydrochloric
acid to pH 7.0. Ten milliliters of the solution was applied to
a column packed with 20 g of mufed Celite (ratio 1:2, v/w).
Approximately 200 mL of 20% methanol/ethyl acetate was used as
a mobile phase to wash the phytochemicals out of the column. The
washout was evaporated at 45

C to dryness. Bound phenolics were
reconstituted in 10 mL of MilliQwater, aliquoted into 2 mL per tube,
and saved as bound phenolics. The extracts were stored at 40

C
until use.
2.1.3. Total phenolic content measurement
The total phenolic content in nut extracts was determined using
the FolinCiocalteu colorimetric method (Singleton, Orthofer, &
Lamuela-Raventos, 1999), which was modied in our laboratory
(Yang et al., 2004). Briey, all extracts were diluted in the ratio 1:10
with distilled water to obtain readings within the standard curve
range of 0.0800.0 mg of gallic acid/mL. The nut extracts were
oxidized by the FolinCiocalteu reagent, and the reaction was
neutralized with sodium carbonate. The absorbance was measured
at 760 nm after 90 min at room temperature by an MRX II Dynex
plate reader (Dynex Technologies, Inc., Chanilly, VA). The absor-
bance values were then compared with those of standards with
known gallic acid concentrations. All values were expressed as the
mean (milligrams of gallic acid equivalents per 100 g of nut) SD
for three replications.
2.1.4. Total avonoid content measurement
The total avonoid content of the nut extracts was quantied
using a modied colorimetric method (Jia, Tang, & Wu, 1999; Yang
et al., 2004). Briey, 0.25 mL of 1:10 diluted nut extract was mixed
with 1.25 mL of distilled water and subsequently with 0.075 mL of
5% sodium nitrite solution and allowed to react for 5 min. Then
0.15 mL of 10% aluminum chloride was added and allowed to
further react for 6 min before 0.5 mL of 1 mol/L sodium hydroxide
was added. Distilled water was added to bring the nal volume of
the mixture to 3 mL. The absorbance of the mixture was immedi-
ately measured at a wavelength of 510 nm against a prepared blank
using an MRX II DYNEX spectrophotometer. The avonoid content
was determined by a catechin standard curve and stated as the
mean (milligrams of catechin equivalents per 100 g of nut) SD for
the triplicate extracts.
2.1.5. Determination of total antioxidant capacity
The total antioxidant capacity of the nut extracts was measured
using a total oxyradical scavenging capacity (TOSC) assay (Winston,
Regoli, Duga, Fong, & Blanchard, 1998) as modied in our laboratory
(Yang et al., 2004). In this assay, peroxy radicals produced from2,2
0
-
azobis-amidinopropane (ABAP) oxidized a-keto-g-methiolbutyric
acid (KMBA) to form ethylene gas, which was measured by a gas
chromatographic headspace analysis. Briey, antioxidant activity
was quantied after 15, 30, 45, and 60 min for four different extract
concentrations plus a control. The amount of ethylene generated by
the reaction was expressed as peak area. The TOSC value corre-
sponding to each extract concentration was calculated by inte-
grating the area under the kinetic curve and assessed by the
following equation:TOSC 100
R
SA=
R
CA 100, where !SA is
the integrated area from the sample reaction, and !CA are the
integrated areas from the control reaction. The median effective
dose (EC
50
) was determined for each nut sample from the dose
response curve of nut concentration versus the TOSC value. The
TOSC value is expressed as mmole of vitamin C equivalents per gram
of nut. All values were presented as the mean SD for three
replications.
2.1.6. Measurement of inhibition of HepG
2
and Caco-2 cell
proliferation by nut extracts
The antiproliferative activity of different nut extracts was
assessed by the measurement of the inhibition of HepG
2
and Caco-2
(The AmericanType Culture Collection, ATCC, Rockville, MD) human
cancer cell proliferation. Antiproliferative activities were deter-
mined by the MTS colorimetric assay (MTS-based cell titer 96
nonradioactivity cell proliferation assay; Promega, Madison, WI)
reported previously (Yang et al., 2004). HepG
2
human liver cancer
cells were cultured in Williams medium E (WME), containing
10 mMHepes, 5 mg/mL insulin, 0.05 mg/mL hydrocortisone, 2 mg/mL
glucagon, 5% fetal bovine serum (Gibco, Life Technologies, Grand
Island, NY), 50 units/mL penicillin, 50 mg/mL streptomycin, and
100 mg/mL gentamicin. Caco-2 human colon cancer cells were
maintainedinDMEM, containing 10 mMHepes, 5%FBS, 50 units/mL
penicillin, 50 mg/mL streptomycin, and 100 mg/mL gentamicin.
HepG
2
and Caco-2 cells were maintained in a 5% CO
2
/37

C incu-
bator. A total of 2.5 10
4
HepG
2
or Caco-2 cells in growth media
were placed in each well of a 96-well at-bottom plate. Cell prolif-
eration was measured by the ability of viable cells to reduce 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)
-2H-tetrazolium (MTS) to formazan. After 4 h of incubation, the
growth medium was removed and media containing various
concentrations (1, 2, 5, 7.5, 10, 12.5, 15, 20, 30, 40, 50, 75, 100, 125,
Phytochemicals in Nuts
Edible Part
Homogenization
Base Digestion
Solid Extraction
Bound Form Soluble Free
Soluble Insoluble
Hexane Washing
80 Acetone Extraction
Fig. 1. Flowchart of phytochemical extraction of tree nuts and peanuts.
J. Yang et al. / LWT - Food Science and Technology 42 (2009) 18 3
150, and 200 mg/mL) of nut extracts were added to the cells. Control
cultures received the extractionsolutionminus the nut extracts, and
blank wells contained 100 mL of growth mediumwithout cells. Cell
proliferation (percent) was determined at 96 h from the MTS
absorbance (490 nm) reading for each concentration compared to
the control, using at least three replications for each sample. The
effective median dose (EC
50
) was determined and expressed as
milligrams of nut extract per milliliter SD.
2.1.7. Statistical analysis
Statistical analysis was performed using Minitab Student
Release 12 (Minitab Inc., State College, PA) and SigmaStat Version
8.0 (Jandel Corp., San Raphael, CA). Results were subjected to
ANOVA, and differences between means were located using Tukeys
multiple comparison test. Correlations between various parameters
were also investigated. Signicance was determined at p <0.05. All
data were reported as the mean SD for three replications.
3. Results and discussion
3.1. Phenolic content of common nuts
Phenolic contents of the nine selected common tree nuts and
peanuts are shown in Table 2. Among all the samples analyzed,
walnuts contained the highest soluble-free phenolic content
(p <0.05) followed by pecans, peanuts, pistachios, cashews,
almonds, Brazil nuts, pine nuts, and macadamia nuts. Hazelnuts
had the lowest free phenolic content. The free phenolic content of
walnuts and pecans was signicantly higher than those of other
nuts (p <0.05). Signicant differences were found in free phenolic
content in comparisons between walnuts, pecans, peanuts, and
cashews (p <0.05); however, no signicant differences in free
phenolic content were found between almond and cashews, or
among Brazil nuts, macadamia nuts, and pine nuts (p >0.05). There
was an approximately 59-fold difference in free phenolic content
between the highest and lowest ranked nuts, walnuts and hazel-
nuts (p <0.05). The distribution pattern of bound phenolics in the
samples was different from the free soluble phenolics. Macadamia
nuts had the highest bound phenolics (p <0.05) followed by
peanuts, hazelnuts, walnuts, pecans, pistachios, cashews, almonds,
Brazil nuts, and pine nuts. Signicant differences in bound
phenolics were found between macadamia nuts, peanuts, walnuts,
and almonds (p <0.05); however, signicant differences were not
found in bound phenolics in comparisons between hazelnuts and
peanuts, among walnuts, pecans, and pistachios, or among
almonds, Brazil nuts, and pine nuts (p >0.05). There was a 4-fold
difference in bound phenolic content between the highest and
lowest ranked nuts, macadamia nuts and pine nuts (p <0.05). Data
shown here indicated that free and bound phenolic distribution
in nuts is different. The free phenolic contents in walnuts,
pecans, peanuts, and pistachios were 5.2-, 5.2-, 1.2-, and 1.5-fold,
respectively, higher than the bound phenolic content in each;
Conversely, bound phenolic contents in macadamia nuts, hazelnuts,
cashews, almonds, Brazil nuts, and pine nuts were 12.8-, 13.0-, 2.6-,
1.6-, 2.7-, and 2.9-fold, respectively, higher than the free phenolic
content in each. The total phenolic content was the lowest in pine
nuts and the highest inwalnuts, showing an over 10-fold difference.
Therewas a signicant difference (p <0.05) intotal phenolic content
between walnuts, pecans, peanuts, cashews, macadamia nuts, pine
nuts, and hazelnuts; however, a signicant difference in total
phenolic content was not found between peanuts and pistachios,
pistachios and macadamia nuts, or among cashews, almonds, and
Brazil nuts (p >0.05). Basedonthe FolinCiocalteumethod, the total
phenolic content (mg of GAE/g) of the 10 nuts reported by Wu et al.
(2004) was in the order pecans >pistachios >walnuts >hazelnuts
>almonds >peanuts >Brazil nuts >cashews >macadamias >
pine nuts, which shows a different pattern fromour data. The main
reason may be that both agronomic and environmental factors play
important roles in the phenolic composition (Tomas-Barberan &
Espin, 2001).
3.2. Flavonoid content of common nuts
Flavonoid contents of nine selected tree nuts and peanuts are
also shown in Table 2. Among all the samples tested, pecans
contained the highest soluble free avonoid content (p <0.05)
followed by walnuts, peanuts, pistachios, cashews, almonds, Brazil
nuts, hazelnuts, pine nuts, and macadamia nuts. The free avonoid
content of pecans and walnuts was signicantly higher than those
of other nuts (p <0.05). Signicant differences in free avonoid
content were found in comparisons between pecans, walnuts,
peanuts, and pistachios (p <0.05); however, signicant differences
in free avonoid content were not found among hazelnuts, pine
nuts, and macadamia nuts (p >0.05). The free avonoid content of
cashews was similar to levels in almonds and Brazil nuts (p >0.05).
There was a 68-fold difference in free avonoid content between
the highest and lowest ranked nuts, pecans and macadamia nuts
(p <0.05). The distribution pattern of bound avonoids in nuts is
different from free soluble avonoids. Walnuts had the highest
bound avonoid content (p <0.05) followed by macadamia nuts,
hazelnuts, Brazil nuts, pecans, pistachios, almonds, peanuts, pine
nuts, and cashews. Signicant differences in bound avonoid
content were found between walnuts and pecans, Brazil nuts, and
cashews (p <0.05); however, no signicant differences were found
in bound avonoid content in comparisons among peanuts, pista-
chios, cashews, almonds, and pecans (p >0.05). There was a 9.7-
fold difference in bound avonoid content between the highest and
lowest ranked nuts, walnuts and cashews (p <0.05). The results
showthat the free avonoid content was an approximately 1.69.8-
fold higher than the bound avonoid content in walnuts, pecans,
peanuts, and pistachios. The bound avonoid content was roughly
1.313.7-fold higher than the free avonoid content in cashews,
Table 2
Total phenolic and avonoid contents of nine tree nuts and peanuts (mean SD, n 3)
Edible nut seeds Phenolics (mg/100 g) Flavonoids (mg/100 g)
Free form Bound form Total Free form Bound form Total
Almonds 83.0 1.3 129.9 13 212.9 12.3 39.8 2.0 53.7 11.9 93.5 10.8
Brazil nuts 46.2 5.7 123.1 18.4 169.2 14.6 29.2 7.2 78.6 9.2 107.8 6.0
Cashews 86.7 8.1 229.7 15.1 316.4 7.0 42.1 3.8 21.6 5.2 63.7 2.1
Hazelnuts 22.5 1.1 292.2 48.4 314.8 47.3 13.9 2.3 99.8 28.5 113.7 30.2
Macadamia nuts 36.2 2.6 461.7 51.2 497.8 52.6 9.4 0.7 128.5 9.3 137.9 9.9
Peanuts 352.8 22.2 293.1 25.0 645.9 47.0 145.5 10.0 44.2 5.2 189.8 13.1
Pecans 1227.3 8.4 236.6 28.1 1463.9 32.3 639.3 17.0 65.4 12.7 704.7 29.5
Pine nuts 39.1 0.6 113.8 14.3 152.9 14.1 13.0 1.5 32.0 6.8 45.0 5.4
Pistachios 339.6 15.1 232.2 13.3 571.8 12.5 87.4 14.0 55.9 13.6 143.3 18.7
Walnuts 1325.1 37.4 255.4 25.0 1580.5 58.0 535.4 71.5 209.4 22.1 744.8 93.3
J. Yang et al. / LWT - Food Science and Technology 42 (2009) 18 4
almonds, Brazil nuts, pine nuts, macadamia nuts, and hazelnuts.
The total avonoid content was highest in walnuts and pecans, and
these levels were signicantly different from the other nuts. The
total avonoid content was lowest in pine nuts and highest in
walnuts, presenting around a 16-fold difference. Signicant differ-
ences were found in the total avonoid content in comparisons
between pecans, peanuts, and pine nuts (p <0.05); however,
signicant differences in the total avonoid content were not
shown between pecans and walnuts, cashews and pine nuts, or
among peanuts, pistachios, almonds, Brazil nuts, hazelnuts, and
macadamia nuts (p >0.05). The signicance of bound phyto-
chemicals in nuts to human health is not clear. Bound phyto-
chemicals, such as b-glycosides, cannot be digested by human
enzymes and could survive stomach and small intestine digestion
to reach the colon (Sosulski, Krygier, & Hogge, 1982). It may be
hypothesized that nuts with bound phytochemicals can be digested
and absorbed at different sites of the gastrointestinal tract and play
their unique health benets.
3.3. Total antioxidant activity
The total antioxidant activities of nut- and peanut-free soluble
extracts, expressed as micromoles of vitamin C equivalents per
gram of sample, are summarized in Fig. 2. Among the 10 samples
tested, walnuts had the greatest antioxidant activity (458.1
14.0 mmol/g, p <0.05) followed by pecans (427.0 21.6), peanuts
(81.3 3.2), pistachios (75.9 1.2), cashews (29.5 2.7), almonds
(25.4 2.0), Brazil nuts (16.0 1.2), pine nuts (14.6 1.1), macad-
amia nuts (13.4 0.4), and hazelnuts (7.1 0.9). A statistically
signicant difference (p <0.05) in antioxidant activity was found
between walnuts and pecans, pecans and peanuts, and pistachios
and cashews. There was no signicant difference (p >0.05) in
antioxidant activity between peanuts and pistachios, or among
cashews, almonds, Brazil nuts, pine nuts, macadamia nuts, and
hazelnuts. The phytochemical extracts of the samples exhibited
potent antioxidant activities. Nut variety, process methods, in-shell
or without shell, cultivation conditions, and/or storage conditions
markedly affect the content of total antioxidants. It was reported
that nuts without the pellicle contained less than 50% of the total
antioxidants compared to nuts with the pellicle (Blomhoff, Carlsen,
Andersen, & Jacobs, 2006). For instance, in walnuts, less than 10% of
total antioxidant activity is retained after removing the skin. In our
study, the total antioxidant activity of 100 g of walnuts was
equivalent to that of 8067 mg of vitamin C, followed by pecans
(7520 mg/100 g), peanuts (1431 mg/100 g), pistachios (1336 mg/
100 g), cashews (520 mg/100 g), almonds (447 mg/100 g), Brazil
nuts (282 mg/100 g), pine nuts (257 mg/100 g), macadamia nuts
(236 mg/100 g), and hazelnuts (125 mg/100 g). Using both hydro-
philic and lipophilic ORAC assays, the total antioxidant capacities
(mmole of TE) of the 10 nuts reported by Wu et al (2004) were in
the order pecans >walnuts >hazelnuts >pistachios >almonds >
peanuts >cashews >macadamias >Brazil nuts >pine nuts. Based
on FRAP, TRAP, and TEAC assays, the contribution of bound
phytochemicals from six nuts, extracted by methanol and alkaline,
to the total antioxidant capacity (TAC) was evaluated by Pellegrini
et al. (2006). In all three assays, walnuts had the highest TAC values
followed by pistachios. Pine nuts ranked the lowest of the TAC
values in the FRAP and TRAP assays. There exists a wide range of
both free and bound TAC values measured for all six nuts and for all
three assays. A positive correlation between phenolics and total
antioxidant activity has been previously demonstrated for a variety
of fruits and vegetables both in our laboratory and by others
(Velioglu, Mazza, Gao, & Oomah, 1998; Yang et al., 2004). This study
suggests that phenolics and avonoids in nuts and peanuts
comprise a generous portion of the total antioxidant activity
(R
2
0.9901, p <0.05; and R
2
0.9749, p <0.05, respectively, Table 3),
suggesting that the combination of phytochemicals and synergistic
mechanisms in the nut matrix may be responsible for their potent
antioxidant activities.
3.4. Inhibition of human cancer cell proliferation
The antiproliferative activities of 10 soluble free extracts toward
the growth of HepG
2
human liver and Caco-2 human colon cancer
cells in vitro are presented in Figs. 3 and 4, respectively. Among the
10 nuts, the soluble free extracts of walnuts, pecans, peanuts, and
almonds showed relatively high antiproliferative activities toward
both HepG
2
and Caco-2 cells in a dose-dependent manner. The
antiproliferative activities of nuts are expressed as the median
effective dose (EC
50
), with a lower EC
50
value signifying a higher
antiproliferative activity (Fig. 5). The phytochemical extracts of
walnuts (7.5 0.1 mg/mL) and pecans (9.7 0.5 mg/mL) contained
the highest antiproliferative activities toward HepG
2
cells with
the lowest EC
50
values (p <0.05) followed by peanuts (26.2
0.5 mg/mL), and almonds (122.9 3.5 mg/mL). The phytochemical
extracts of cashews and macadamia nuts exhibited a weak anti-
proliferative activity toward HepG
2
cells at higher doses, with EC
50
values of 183.4 19.9 and 191.7 2.6 mg/mL, respectively. The EC
50
values toward HepG
2
cells of the pine nuts, pistachios, Brazil nuts,
and hazelnuts could not be determined at the doses of soluble free
extracts tested in this experiment. The phytochemical extracts of
walnuts and pecans exhibited the highest antiproliferative effect
(p <0.05) toward Caco-2 cells with the lowest EC
50
values (1.8 0.1
and 2.5 0.1 mg/mL, respectively) followed by peanuts (13.5 1.2)
and pine nuts (57.8 4.5). The phytochemical extracts of almonds
and macadamia nuts showed weak antiproliferative activities at
higher doses with EC
50
values of 72.9 2.8, and 86.9 2.9 mg/mL,
respectively (Fig. 5). However, the EC
50
values toward Caco-2 cells
W
a
ln
u
ts
P
e
c
a
n
P
e
a
n
u
ts
P
is
ta
c
h
io
s
C
a
s
h
e
w
A
lm
o
n
d
B
r
a
z
il N
u
t
P
in
e
n
u
t
M
a
c
a
d
a
m
ic
N
u
t
H
a
z
e
ln
u
t
0
100
200
300
400
500
a
b
c
c
d d
d d d
d
T
o
t
a
l

A
n
t
i
o
x
i
d
a
n
t

A
c
t
i
v
i
t
y

(



m
o
l

v
i
t
a
m
i
n

C

e
q
u
.
/
g

s
a
m
p
l
e
)
Fig. 2. Total antioxidant activity of phytochemical extracts of nine tree nuts and
peanuts (mean SD, n 3). Bars with on letters in common are signicantly different
(p <0.05).
Table 3
Correlation analysis of phytochemical content, antioxidant activity, and
antiproliferative activity
Total antioxidant activity
a
Antiproliferative activity
HepG
2
cells Caco-2 cells
Phenolics 0.9901*
,b
0.6997* 0.7396*
Flavonoids 0.9749* 0.6732* 0.7146*
Total antioxidant activity 0.6362 0.6778*
The signicant values are shown with asterisk.
a
Correlation coefcient R
2
.
b
Signicantly different p <0.05.
J. Yang et al. / LWT - Food Science and Technology 42 (2009) 18 5
of the cashews, pistachios, Brazil nuts, and hazelnuts could not be
determined at the doses of soluble free extracts used in this
experiment.
The inhibition of cell proliferation was observed in a dose-
dependent manner after exposure to the free soluble extracts of
walnuts, pecans, and peanuts; these nuts demonstrated greater
antiproliferative activity than the pine nuts, almonds, cashews,
macadamia nuts, Brazil nuts, and pistachios. The antiproliferative
activities of 10 samples differed in the HepG
2
and Caco-2 cell lines.
Overall, the 10 samples had a greater ability to inhibit Caco-2 colon
cancer cell proliferation than HepG
2
liver cancer cell proliferation.
The inhibition of cancer cell proliferation by nut extracts can be
partially explained by the total phenolic contents in the nuts tested,
suggesting that a specic phenolic compound or a class of phenolics
in nuts was responsible for their antiproliferative activities. Alter-
natively, particular phenolic compounds may act additively,
synergistically, and/or antagonistically with other compounds to
play a role in antiproliferative activity. For instance, it was reported
that resveratrol inhibits the expression of the enzymes which
metabolize aryl hydrocarbons to genotoxic metabolites in human
HepG
2
cells (Ciolino, Daschner, & Yeh, 1998). The antiproliferative
activities of resveratrol is proposed by the direct inhibition of
ribonucleotide reductase, which supplies proliferating cells with
deoxyribonucleotides required for DNA synthesis (Fontecave,
Lepoivre, Elleingand, Gerez, & Guittet, 1998). In addition, it is
hypothesized that bound phytochemicals reach the colon in an
undigested formwhich could be metabolized by the microora. For
example, diferulic acids from cereal brans are ester-linked to cell
wall polysaccharides and cannot be absorbed in this form. It has
been demonstrated that esterase from human and rat colonic
microora are able to release the dietary diferulates. Further, free
diferulic acids can be absorbed and enter the circulatory system
(Andreasen, Kroon, Williamson, & Garcia-Conesa, 2001).
3.5. Relationship between total phenolic, antioxidant, and
antiproliferative activity
The correlations between free soluble phytochemical content
and total antioxidant and antiproliferative activities are summa-
rized in Table 3. There was a direct relationship between phenolic
content and total antioxidant activity (R
2
0.9901, p <0.05) and
between avonoid content and total antioxidant activity
(R
2
0.9749, p <0.05) in soluble free phytochemical extracts of
different nuts. The positive correlation indicates that the higher
phenolic/avonoid contents resulted in a higher antioxidant
activity. The relationship between soluble free phenolic/avonoid
contents and the median effective dose (EC
50
) toward the inhibition
of HepG
2
and Caco-2 cell proliferation was also investigated. There
was a weak correlation between the inhibition of HepG
2
cell
proliferation and the soluble free phenolic content (R
2
0.6997,
p <0.05) and the soluble free avonoid content (R
2
0.6732,
p <0.05). However, there was no signicant linear relationship
between total antioxidant activity and antiproliferative activity of
HepG
2
cells in the nuts tested (R
2
0.6362, p >0.05). Similarly,
a weak correlation was observed between the inhibition of Caco-2
cell proliferation and the soluble free phenolic content
(R
2
0.7396, p <0.05), the soluble free avonoid content
(R
2
0.7146, p <0.05), and the total antioxidant activity
(R
2
0.6778, p <0.05).
Tree nuts and peanuts possess numerous phytochemicals that
may offer benecial effects and lower the risk of chronic diseases.
Consumption of avonoids was shown to be inversely associated
with morbidity and mortality resulting from CHD. Flavonoids have
many biological activities such as antioxidant activity, the inhibi-
tion of plasma platelet aggregation and cyclooxygenase activity, the
suppression of histamine release and SRS-A biosynthesis in vitro;
potent nitric oxide radical scavenging activity, and exhibiting
W
a
ln
u
ts
P
e
c
a
n
s
P
e
a
n
u
ts
P
in
e
n
u
ts
A
lm
o
n
d
s
M
a
c
a
d
a
m
ia
n
u
ts
C
a
s
h
e
w
s
E
C
5
0

o
f

A
n
t
i
p
r
o
l
i
f
e
r
a
t
i
v
e

A
c
t
i
v
i
t
y

(
m
g
/
m
L
)
0
50
100
150
200
250
Fig. 5. EC
50
values of antiproliferative activity of phytochemical extracts of six tree
nuts and peanuts (mean SD, n 3). ( ) Caco-2; ( ) HepG2.
Concentration of nut extracts (mg/mL)
0 50 100 150 200
0
20
40
60
80
100
120
C
e
l
l

P
r
o
l
i
f
e
r
a
t
i
o
n

(




)
Fig. 4. Percent inhibition of Caco-2 cell proliferation by nine tree nuts and peanuts
extracts (mean SD, n 3). () control; (B) pistachios; (;) Brazil nuts; (6) hazelnuts;
(-) pine nuts; (,) macadamia nuts; (A) cashews; (>) almonds; (6) peanuts;
(P) pecans; ( ) walnuts.
Concentration of nut extracts (mg/mL)
0 50 100 150 200
0
20
40
60
80
100
120
C
e
l
l

P
r
o
l
i
f
e
r
a
t
i
o
n

(




)
Fig. 3. Percent inhibition of HepG
2
cell proliferation by nine tree nuts and peanuts
extracts (mean SD, n 3). () control; (B) pistachios; (;) Brazil nuts; (6) hazelnuts;
(-) pine nuts; (,) macadamia nuts; (A) cashews; (>) almonds; (6) peanuts; (P)
pecans; ( ) walnuts.
J. Yang et al. / LWT - Food Science and Technology 42 (2009) 18 6
antibacterial, antiviral, anti-inammatory, and antiallergenic
effects (Cook & Samman, 1996). Generally, phytochemicals present
in nuts include phenolics, carotenoids, and phytosterols. Phenolics
in nuts contain phenolic acids, avonoids, and stilbenes. Walnuts
are a good source of phenolics with 1625 mg GAE equivalents/100 g
(Chen & Blumberg, 2008). In our study, the total phenolic concen-
tration in walnuts is 1580.5 58.0 mg/100 g, being similar to this
reported one. Pecans, almonds, and pistachios have 34, 15, and
12 mg avonoids/100 g of nuts, respectively, from the USDA Data-
base for the Flavonoid Content of Selected Foods, which showed
a higher level of avonoids than those in this study. Resveratrol is
reported in peanuts and pistachios at 84 and 115 mg/100 g,
respectively. The level of proanthocyanidins in almonds, cashews,
hazelnuts, pecans, pistachios, peanuts, and walnuts ranges from 9
to 494 mg/100 g (Chen & Blumberg, 2008). The above bioactive
compounds in nuts play an important role in the prevention of
chronic diseases. This study mainly focuses on the relationship
between soluble free phenolics/avonoids and antioxidant/anti-
proliferative activities in nuts, not bound forms. The main reason
would be that phenolics/avonoids are absorbed mostly in free
forms rather than bound forms in GI tract. The bound forms will be
broken down by microbes in the large intestine before absorption.
The study of antioxidant and antiproliferative activities in free
forms has more signicance than in bound forms.
4. Conclusions
Little information has been documented from the literature in
phenolics of nuts. Furthermore, the phenolic content and its anti-
oxidant activities in nuts were underestimated in the literature,
because bound phenolics were not included. This study established
a more complete prole of total phenolic contents in nuts by
further digesting and extracting the bound phytochemicals.
Phenolics in nuts are present in both free and bound forms. The
contribution of total avonoid content comes from both free and
soluble-conjugated avonoids. The soluble antioxidant activity was
quantied. It was inferred that the higher the total phenolic nut
content, the higher was the antioxidant activity observed. The
phytochemicals present in the nine tree nuts and peanuts also
contributed to their antiproliferative activity. It is proposed here
that the additive and synergistic actions of phytochemicals in nuts
are responsible for these potent antioxidant and anticancer activi-
ties, and that the benet of a diet rich in nuts is ascribed to the
interaction of phytochemicals present in whole nuts (Liu, 2003).
These results provide newknowledge about the health functions of
nuts and may inuence consumers toward purchasing nuts
exhibiting greater potential health benets. While many of these
bioactive constituents remain to be fully identied and character-
ized, further identication of specic phytochemicals for their
antioxidant and antiproliferative activities is worthy of
investigation.
Acknowledgements
The authors thank Sharon Johnston for her technical assistance.
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