Isolation and Identification of Strawberry Phenolics
with Antioxidant and Human Cancer Cell
YANJUN ZHANG, NAVINDRA P. SEERAM,* RUPO LEE, LYDIA FENG, AND
Center for Human Nutrition, David Geffen School of Medicine, University of California,
Los Angeles, California 90095
Studies suggest that consumption of berry fruits, including strawberries (Fragaria × ananassa Duch.),
may have beneficial effects against oxidative stress mediated diseases such as cancer. Berries contain
multiple phenolic compounds, which are thought to contribute to their biological properties.
Comprehensive profiling of phenolics from strawberries was previously reported using high-
performance liquid chromatography with mass spectrometry (HPLC-MS) detection. The current study
reports the isolation and structural characterization of 10 phenolic compounds from strawberry extracts
using a combination of Amberlite XAD16-resin and C18 columns, HPLC-UV, and nuclear magnetic
resonance (NMR) spectroscopy methods. The phenolics were cyanidin-3-glucoside (1), pelargonidin
(2), pelargonidin-3-glucoside (3), pelargonidin-3-rutinoside (4), kaempferol (5), quercetin (6),
kaempferol-3-(6′-coumaroyl)glucoside) (7), 3,4,5-trihydroxyphenyl-acrylic acid (8), glucose ester of
(E)-p-coumaric acid (9), and ellagic acid (10). Strawberry crude extracts and purified compounds
1–10 were evaluated for antioxidant and human cancer cell antiproliferative activities by the Trolox
equivalent antioxidant capacity (TEAC) and luminescent ATP cell viability assays, respectively. Among
the pure compounds, the anthocyanins 1 (7156 µM Trolox/mg), 2 (4922 µM Trolox/mg), and 4 (5514
µM Trolox/mg) were the most potent antioxidants. Crude extracts (250 µg/mL) and pure compounds
(100 µg/mL) inhibited the growth of human oral (CAL-27, KB), colon (HT29, HCT-116), and prostate
(LNCaP, DU145) cancer cells with different sensitivities observed between cell lines. This study adds
to the growing body of data supporting the bioactivities of berry fruit phenolics and their potential
impact on human health.
KEYWORDS: Strawberries; polyphenols; antioxidant; antiproliferative
Oxidative damage is thought to be one of the major
mechanisms involved in chronic human diseases such as cancer
and heart disease, the leading causes of mortality in the United
States. An overwhelming and large body of studies suggests
that a phytochemical-rich diet, which includes colorful fruits
and vegetables, may reduce the risk of chronic human diseases.
Phenolic compounds (commonly referred to as “flavonoids” or
“polyphenols”) are ubiquitous phytochemicals present in plant
foods with numerous biological activities including antioxidant
properties. Phenolics exert antioxidant properties through various
mechanisms of actions including the scavenging of free radicals
and inhibition of the generation of reactive species during the
course of normal cell metabolism, thereby preventing damage
to lipids, proteins, and nucleic acids and eventually cell damage
and death (1). It has been shown that antioxidant-rich diets can
reduce oxidative damage to DNA, thus preventing a critical step
in the onset of carcinogenesis (2), and the impact of antioxidants
Strawberry (Fragaria × ananassa Duch.) fruits are popularly
consumed in fresh forms, as processed food products, and as
botanical extracts for dietary supplements. Strawberries have
high antioxidant activity, which has been linked to their content
of phenolic compounds (5–7). Strawberry juice extracts exhib-
ited high levels of antioxidant capacity against superoxide
radicals, hydrogen peroxide, hydroxyl radicals, and singlet
oxygen free radicals (8). The contents of phenolics in strawber-
ries have been associated with the total antioxidant capacity
for low-density lipoproteins of the fruit extracts (5).
Our group is currently investigating the pharmacokinetics and
bioavailability of strawberry phenolics in animals and healthy
human volunteers, and chemical standards are needed for
evaluations of plasma and urinary metabolites. In addition, we
are interested in evaluating the biological actions of phenolics
* Present address: Department of Biomedical and Pharmaceutical
Sciences, College of Pharmacy, University of Rhode Island, Kingston,
RI 02881 (e-mail email@example.com).
J. Agric. Food Chem. 2008, 56, 670–675
10.1021/jf071989c CCC: $40.75
2008 American Chemical Society
Published on Web 01/23/2008
as singly purified compounds compared to their combined “food-
matrices” forms (9, 10). Our previous work reported the
comprehensive profiling of phenolics from strawberries using
liquid chromatography electrospray ionization mass spectrom-
etry (LC-ESI-MS) methods (11). Strawberries were found to
contain a wide variety of phenolics such as ellagic acid, ellagic
acid glycosides, ellagitannins, gallotannins, flavonols (quercetin
and kaempferol glucuronides and glycosides), anthocyanins
(pelargonidin and cyanidin glycosides), flavanols, and coumaroyl
glycosides (11). The objectives of the current study were (1) to
isolate and structurally characterize individual phenolic con-
stituents of strawberry fruits and (2) to evaluate the strawberry
extracts generated, and their purified phenolic compounds, for
biological activities in vitro. Although extracts of strawberries
have previously been evaluated for antioxidant and human
cancer cell antiproliferative properties (12–14), this is the first
report of the isolation and biological investigation of these
purified phenolic constituents from strawberry fruits.
MATERIALS AND METHODS
Reagents. All solvents were of high-performance liquid chroma-
tography (HPLC) grade and purchased from Fisher Scientific Co.
(Tustin, CA). Amberlite XAD-16 resin was purchased from Sigma-
Aldrich Co. (St. Louis, MO). C18silica gel was obtained from DyChrom
General Experimental Procedures. The nuclear magnetic resonance
(NMR) spectra were recorded on Bruker instruments operating at 400
MHz for1H and at 100 MHz for13C, respectively. Chemical shifts for
compounds 1, 3, 4, and 7 were recorded in CD3OD/CF3COOD (19:1,
v/v), and chemical shifts for compound 9 were recorded in DMSO-d6.
Chemical shifts are presented in δ (parts per million) and the coupling
constants (J) in hertz.
Generation and Composition of Strawberry Crude Extracts. A
freeze-dried whole strawberry fruit powder (SFP), provided by the
California Strawberry Commission (Watsonville, CA), has been previ-
ously described (11). The SFP was subjected to an extraction protocol
as outlined in Figure 1. Briefly, a portion of SFP (845 g) was extracted
by cold percolation with methanol (6 L) under room temperature for
12 h to yield a methanol extract (624 g, 73.8% yield). The methanol
extract was then subjected to a sequence of solvent-solvent partitioning
to yield a chloroform extract (CHCl3-X, 16.4 g; enriched in sterols
and fatty acids), an ethyl acetate extract (EtOAc-X, 6.4 g; enriched in
flavonoids and phenolic acids), and an aqueous portion. The aqueous
portion was further adsorbed on an XAD-16 (Amberlite Resin, Sigma,
St. Louis, MO) column and eluted with water followed by acidic
methanol (adjusted with 6 N hydrochloric acid, HCl, to a pH of 3) as
previously described (11) to yield a water extract (H2O-X; enriched in
natural fruit sugars and organic acids) and an acidic methanol fraction
(25 g, polyphenol-enriched fraction), respectively. The acidic methanol
fraction was further purified by suspending the extract in 200 mL of
distilled water and filtering to yield a water-soluble fraction (MeOH-
1, 14 g, dark red powder; enriched with anthocyanins) and a water-
insoluble fraction (MeOH-2, 10.9 g; enriched in ellagitannins, ellagic
acid, and other polymeric molecules). Crude extracts and their relative
composition used in our experiments were (1) CHCl3-X (sterols, fatty
acids), (2) EtOAc-X (flavonols, small phenolic compounds), (3) H2O-X
(natural fruit sugars and acids), (4) Anth-X (anthocyanin enriched),
and (5) ellagitannin-X (ellagitannin enriched).
Purification and Structural Elucidation of Strawberry Phenolics.
Pure compounds 1–10 were obtained as follows. Briefly, medium-
pressure liquid chromatography (MPLC) C18 columns were used to
separate the anthocyanins, and silica gel MPLC and preparative-scale
thin layer chromatography (TLC) were employed to purify the other
The Anth-X (anthocyanin enriched, 5 g) fraction was subjected to a
C18column and eluted with acidic methanol/water (pH 3 adjusted with
HCl) gradient (1:9 v/v, 500 mL; 2:8 v/v, 500 mL; 3:7 v/v, 100 mL;
5:5 v/v, 500 mL; and 100% methanol, 500 mL). The color bands were
collected; otherwise, 200 mL aliquot fractions were collected. A total
of four colored fractions (AnthX1-X4), noncolored fractions (AnthX0),
and a pure methanol eluted fraction (AnthX5) were obtained. The
colored fractions were further purified by preparative HPLC with UV
detector by using a C18column and elution with methanol/water (7:3,
v/v). Single peaks detected at 520 nm (i.e., the characteristic absorption
wavelength of anthocyanins) were collected as compounds 1 (21 mg),
2 (15.3 mg), 3 (296 mg), and 4 (16.5 mg), respectively, according to
retention times. A pale solid that precipitated from fraction AnthX5
yielded compound 10 (140 mg), which was further purified after
filtration and recrystallization from methanol.
A portion of the EtOAc-soluble fraction (EtOAc-X, 2 g) was
subjected to a silica gel MPLC column and eluted with a chloroform/
acetone (C/A) solvent system under gradient conditions (C/A 9:1 v/v,
500 mL; C/A 4:1 v/v, 500 mL; C/A 3:1 v/v, 500 mL; C/A 2:1 v/v, 500
mL; C/A 1:1 v/v, 500 mL; C/A 1:2 v/v, 500 mL; acetone, 250 mL;
methanol, 250 mL), affording 15 fractions (200 mL each, A1-A15).
Fractions A4-A6 were further purified by preparative-scale TLC by
collecting a band at a retention factor (Rf) of 0.45 (hexane/acetone,
8:2, v/v) to yield compound 6 (6.6 mg). Fraction A7 was precipitated
with acetone to yield pure compound 5 (17 mg) as a pale yellow
precipitate. Pale yellow needles, which precipitated from fraction A5,
were filtered and recrystallized from acetone to yield pure compound
7 (44 mg).
A portion of the EtOAc-soluble fraction (EtOAc-X, 2 g) was
subjected to a C18MPLC column eluting with methanol/water (M/W)
solvent system under gradient conditions (M/W 9:1 v/v, 500 mL; M/W
4:1 v/v, 500 mL; M/W 7:3 v/v, 500 mL; M/W 2:1 v/v, 500 mL; M/W
1:1 v/v, 500 mL; methanol, 500 mL), affording 12 fractions (M1-M12,
200 mL each, plus a methanol-eluted fraction M13). Compound 8 (11
mg) was precipitated as a needle form M4 fraction. Compound 9 was
precipitated from fraction M2 (10 mg) as a white needle precipitate.
Compound 7 (kaempferol-3-(6′-coumaroyl)glucoside):
(CD3OD) δ 7.98 (2H, dd, J ) 9.0, 2.1 Hz, H-2′, 6′), 7.39 (1H, d, J )
16.0 Hz, H-8′′′), 7.29 (2H, dd, J ) 8.6, 1.84, H-2′′′, 6′′′), 6.81 (2H,
dd, J ) 9.0, 2.1 Hz, H-3′, 5′), 6.78 (2H, dd, J ) 8.6, 1.84 Hz, H-3′′′,
5′′′), 6.29 (1H, d, J ) 2.12 Hz, H-8), 6.12 (1H, d, 2.12 Hz, H-6), 6.07
(1H, d, J ) 16.0 Hz, H-7′′′), 5.24 (1H, d, J ) 7.68 Hz, H-1′), 4.31
(1H, dd, J ) 11.80, 2.20 Hz, HR-6′), 4.19 (1H, dd, J ) 11.80, 8.30
Hz, H?-6′), 3.50 (1H, m, H-5′), 3.47 (2H, m, H-2′, 4′), 3.35 (1H, m,
H-3′);13CNMR δ 179.5 (C-4), 168.9 (C-9′′′), 166.0 (C-7), 163.1 (C-
5), 161.6(C-4′), 161.3 (C-4′′′), 159.4 (C-2), 158.5(C-9), 146.7 (C-7′′′),
Figure 1. Generation of crude strawberry crude extracts for bioassays.
CHCl3, chloroform; EtOAC, ethyl acetate; MeOH, methanol.
Berry Health Benefits SymposiumJ. Agric. Food Chem., Vol. 56, No. 3, 2008
135.4 (C-3), 132.4 (C-2′, 6′), 131.3 (C-2′′′, 6′′′), 127.2 (C-1′′′), 122.8
(C-1′), 116.9 (C-3′, 5′), 116.2 (C-3′′′, 5′′′), 114.9 (C-8′′′), 105.7 (C-
10), 104.1 (C-1′′), 100.1 (C-6), 95.0 (C-8), 78.1 (C-3′′), 75.9 (C-2′′,
C-5′′), 71.8 (C-4′′), 64.5 (C-6′′).
Compound 9 [glucose ester of (E)-p-coumaric acid]:1H NMR (400
MHz, DMSO-d6) δ 10.07 (s, 1H, -OH), 7.64 (1H, d, J ) 15.9 Hz,
H-7), 6.40 (1H, d, J ) 15.9 Hz, H-8), 7.58 (2H, d, J ) 8.6 Hz, H-2,
6), 6.80 (2H, d, J ) 8.6 Hz, H-3, 5), 5.46 (1H, d, J ) 7.96 Hz, H-1′),
3.65 (1H, dd, J ) 12.00, 5.6 Hz, H-6′), 3.44 (1H, dd, J ) 12.0, 5.6,
H-6′). 3.37–3.48 (4H, m, H-2′, H-3′, H-4′, H-5′).
Trolox Equivalent Antioxidative Capacity (TEAC). The TEAC
assay was performed as previously reported (15). Briefly, 2′,2′-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)
radical cations were prepared by adding solid manganese dioxide (80
mg) to a 5 mM aqueous stock solution of ABTS•+(20 mL using a 75
mM Na/K buffer of pH 7). Trolox (6-hydroxy-2,5,7,8-tetramethylchro-
man-2-carboxylic acid), a water-soluble analogue of Vvitamin E, was
used as an antioxidant standard. A standard calibration curve was
constructed for Trolox at 0, 50, 100, 150, 200, 250, 300, and 350 µM
concentrations. Samples were extracted in methanol/water (1:1, v/v)
(10 mg/mL concentrations) by vortexing for 30 min, sonicating for 5
min, and centrifuging for 10 min at 2000g. Samples were diluted
appropriately according to antioxidant activity in Na/K buffer pH 7.
Diluted samples were mixed with 200 µL of ABTS•+radical cation
solution in 96-well plates, and absorbance was read (at 750 nm) after
5 min in a ThermoMax microplate reader (Molecular Devices,
Sunnyvale, CA). Samples were assayed in six replicates. TEAC values
were calculated from the Trolox standard curve and expressed as Trolox
equivalents (TE, in micromolar per milligram).
Cell Culture Materials. All cell lines were obtained from American
Type Culture Collection (ATCC, Rockville, MD). KB oral cancer cells
and MCF-7 breast cancer cells were grown in minimum essential
medium (MEM); CAL-27 oral cancer cells were grown in Dulbecco’s
minimum essential medium (DMEM); LNCaP and DU145 prostate
cancer cells were grown in RPMI 1640; HT-29 and HCT116 colon
cancer cells were grown in McCoy’s 5A medium, modified. All media
contained 10% fetal bovine serum (FBS) in the presence of 100 units/
mL penicillin and 0.1 g/L streptomycin. Cells were incubated at 37 °C
with 95% air and 5% CO2. All cells were maintained below passage
20 and used in experiments during the linear phase of growth.
Cell Proliferation Assay. Proliferation was measured utilizing the
CellTiter-Glo Luminescent Cell Viability Assay (Technical Bulletin
288, Promega Corp., Madison, WI). When added to cells, the assay
reagent produces luminescence in the presence of ATP from viable
cells. Cells were plated in 96-well plates at a density of 5000 cells/
well and incubated for 24 h. Test samples were solubilized in deionized
water by sonication, filter sterilized, and diluted with media to the
desired treatment concentration. Cells were treated with 100 µL of
media or test samples and incubated for 48 h drug exposure duration.
Extracts were tested at 250 µg/mL, and pure compounds were tested
at 100 µg/mL concentrations. At the end of 48 h, plates were
equilibrated at room temperature for 30 min, 100 µL of the assay reagent
was added to each well, and cell lysis was induced on an orbital shaker
for 2 min. Plates were incubated at room temperature for 10 min to
stabilize the luminescence signal, and results were read on an Orion
Microplate Luminometer (Bertholds Detection Systems, Pforzheim,
Germany). All plates had control wells containing medium without cells
to obtain a value for background luminescence that was subtracted from
the test sample readings. Data are expressed as percentage of untreated
cells, mean ( SE, for three replications.
Statistical Analysis. Cell proliferation data were analyzed by either
Student’s t test or one-way ANOVA followed by Dunnett’s multiple-
range test (R ) 0.05) with Graph Pad Prism 3.0 (Graph Pad Software
Inc.) as appropriate. TEAC values were determined in six replicates,
and the mean values ( SD are reported.
RESULTS AND DISCUSSION
Isolation and Identification of Strawberry Phenolics. The
compounds were identified by comparison of their nuclear
magnetic resonance (NMR) data with published literature values
and by comparison of their HPLC-UV retention times to those
of authentic standards when available. Figure 2 shows the
chemical structures of the phenolics isolated and identified from
strawberries: cyanidin-3-glucoside (1), pelargonidin (2), pelar-
gonidin-3-glucoside (3), pelargonidin-3-rutinoside (4), kaempfer-
ol (5), quercetin (6), kaempferol-3-(6′-coumaroyl)glucoside (7),
3,4,5-trihydroxyphenyl-acrylic acid (8), glucose ester of (E)-p-
coumaric acid (9), and ellagic acid (10).
A brief description of the structural elucidation of the
compounds 1, 3, 4, 7, and 9 follows. Compound 1 was identified
as cyanidin-3-glucoside, and its NMR data corresponded to
previously published values (16). Similarly, the NMR data for
compounds 3 and 4 corresponded to the reported data for
pelargonidin-3-glucoside and pelargonidin-3-rutinoside, respec-
tively (17–19). The1H NMR data of compounds 1, 3, and 4
are summarized in Table 1.
signals for a kaempferol moiety [δ 7.98 (2H, dd, J ) 9.0, 2.1
Hz), 6.81 (2H, dd, J ) 9.0, 2.1 Hz), 6.29 (1H, d, J ) 2.1 Hz),
6.12 (1H, d, J ) 2.1 Hz)] and a trans coumaroyl moiety [δ
7.39 (1H, d, J ) 16.0 Hz), 7.29 (2H, d, J ) 8.6 Hz), 6.78 (2H,
d, J ) 8.6 Hz), 6.07 (1H, d, J ) 16.0 Hz)] and signals for an
anomeric proton of a sugar at δ 5.24, d, J ) 7.7 Hz. The
coupling constant of J ) 7.7 Hz indicated a ?-configuration
for the glucose moiety. Because the H-6′′ proton signal of the
glucose moiety was shifted downfield at δ 4.31 (1H, dd, J )
11.8, 2.2 Hz), 4.19 (1H, dd, J ) 11.8, 8.3 Hz), it was concluded
that the coumaroyl moiety was attached to the C-6 of glucose
in the trans configuration. The structure of compound 7 was
1H NMR spectrum of compound 7 exhibited typical
Figure 2. Chemical structures of phenolic compounds 1–4, 7, and 9,
J. Agric. Food Chem., Vol. 56, No. 3, 2008Zhang et al.
confirmed as kaempferol-3-(6′-coumaroyl)glucoside by com-
parison with published NMR data (20).
The LC-ESI-MS of compound 9 gave an [M - H]-ion at
m/z 325 and indicated that the molecular formula of compound
9 was C15H18O8. The characteristic signals for 1,4-disubstituted
benzene protons at δ 7.58 (2H, d, J ) 8.6 Hz) and 6.80 (2H, br
d, J ) 8.6 Hz) and the pair of trans-olefinic proton signals at δ
7.64 (1H, d, J ) 16.0 Hz) and 6.40 (1H, d, J ) 16.0 Hz), which
were conjugated with a carbonyl group, clearly indicated that
there is a trans-coumaroyl moiety in the structure. The anomeric
proton signal at δ 5.46 (1H, d, J ) 8.0 Hz) of the sugar unit
demonstrated a ?-configuration. Therefore, compound 9 was
identified as 1-[3-(4-hydroxyphenyl)-2-propenoate]-?-D-glu-
copyranoside that corresponded with published NMR data (21).
Compounds 2, 5, 6, 8, and 10 were identified as pelargonidin,
kaempferol, quercetin 3,4,5-trihydroxyphenyl-acrylic acid, and
ellagic acid, respectively, first by comparison with correspon-
dence standards on HPLC and were further confirmed by
Antioxidant Activities. The antioxidant activity using the
TEAC assay reached 7156 µM Trolox/mg for the pure com-
pounds as shown in Figure 3. Among the purified phenolics,
the anthocyanins 1 (7156 µM Trolox/mg), 2 (4922 µM Trolox/
mg), and 4 (5514 µM Trolox/mg) were the most potent
antioxidants. Anthocyanins have been reported to have potent
antioxidant properties (10), which corroborate the results
reported here. Structure–activity-related (SAR) studies have been
conducted for anthocyanins, with respect to their antioxidant
activities (22, 23), and these structural differences may explain
the TEAC values observed for the anthocyanins in this
Antiproliferative Activities. Strawberry crude extracts were
evaluated for their ability to inhibit the growth of human colon
(HT-29 and HCT-116, Figure 4A), prostate (LNCaP and
DU145, Figure 4B), and oral (KB and CAL-27, Figure 4C)
tumor cell lines. Pure compounds 1–10 were also evaluated for
their ability to inhibit the growth of human colon (HT-29 and
HCT-116, Figure 5A), prostate (LNCaP and DU145, Figure
Table 1.1H Nuclear Magnetic Resonance Data for Compounds 1, 3, and 4 (in MeOH-d4Containing 1%TFA-d)a
aδ in parts per million; J in hertz.
Figure3. TEACresultsof thepurecompoundsisolatedfromstrawberry
Figure 4. Inhibition of proliferation of human tumor cell lines by crude
exposed to extracts at 250 µg/mL for 48 h.
Berry Health Benefits SymposiumJ. Agric. Food Chem., Vol. 56, No. 3, 2008
5B), and oral (KB and CAL-27, Figure 5C) tumor cell lines. It
is noteworthy that the anticancer effects of individual phy-
tochemical constituents of strawberries, as well as whole
strawberry extracts, have been previously demonstrated (re-
viewed in ref 6). In agreement with previously published studies
(6), these results confirm that strawberry extracts and their
purified compounds inhibit human cancer cell growth in a dose-
dependent manner with various degrees of potency.
In conclusion, the current study contributes to the growing
body of literature which demonstrates that strawberry extracts,
and their purified phenolic compounds, show antioxidant and
human tumor cell antiproliferative activities in vitro. Other berry
fruit extracts, similar to the strawberry, have also been reported
to inhibit the growth of human tumor cell lines in vitro (24). It
is noteworthy that the in vitro doses used for the antioxidant
and antiproliferative assays in this study are not physiologically
relevant. Nevertheless, the main objective of this study was to
isolate and identify phenolic compounds from the strawberry
fruit to serve as chemical standards for planned animal and
human studies. There is an urgent need for continued and future
in vivo studies before the impact of strawberry consumption
on human health and disease can be thoroughly evaluated (25).
We thank Dr. Jane Strouse for assistance in acquiring NMR
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Received for review July 3, 2007. Revised manuscript received October
4, 2007. Accepted November 27, 2007. Funding for this project was
provided by the Califronia Strawberry Commission (Watsonville, CA).
NMR data were obtained from equipment supported by National
Science Foundation Equipment Grant CHE-0116853 in the Department
of Chemistry and Biochemistry, UCLA.
Berry Health Benefits SymposiumJ. Agric. Food Chem., Vol. 56, No. 3, 2008