Content uploaded by Navindra P Seeram
Author content
All content in this area was uploaded by Navindra P Seeram on Jan 20, 2015
Content may be subject to copyright.
1
Strawberry Phytochemicals and Human Health: A Review
Navindra P. Seeram
UCLA Center for Human Nutrition, David Geffen School of Medicine, University of California,
Los Angeles, CA 90095, USA
*Corresponding author. Tel: (310) 825-6150; fax: (310) 206-5264;
E-mail address: nseeram@mednet.ucla.edu (N. P. Seeram)
2
Abstract
A growing body of data suggests that consumption of a phytochemical-rich diet reduces the risk
of certain chronic human illnesses such as cancer, heart and neurodegenerative diseases.
Strawberry (Fragaria x ananassa Duch.) fruits are a rich source of phytochemicals (plant-
chemicals) of which phenolic compounds predominate. Research conducted by our group and
others have shown that phenolic compounds have potent antioxidant, anticancer, anti-
atherosclerotic and anti-neurodegenerative properties in both in vitro and in vivo studies.
Strawberry phenolics consist of large polymeric compounds (ellagitannins and gallotannins), as
well as monomeric molecules (ellagic acid and ellagic acid glycosides, anthocyanins, flavonols,
catechins and coumaroyl glycosides). Ellagitannins are hydrolyzable tannins also found in other
popularly consumed foods such as pomegranates, red and black raspberries, blackberries and
some nuts. Anthocyanins are pigments that impart the attractive colors to berry fruits, red grapes
(and therefore red wine) and many vegetables. Flavonols (such as quercetin found in onions,
apples and other berries) and catechins (antioxidants present in green tea) are also found in the
strawberry fruit. This paper reviews the chemistry and biological activities of preventive
phytochemicals found in strawberries in relation to their potential impact on human health.
Ongoing and completed animal and human studies conducted by our group and others, which
investigate the potential health benefits that may result from strawberry consumption, are the
focus of this review article.
Keywords: Strawberries; Phytochemicals; Phenolics; Health; Diseases
3
Introduction
Consumption of phytochemical-rich foods such as fruits, vegetables, spices and some
beverages (red wine, green and black tea etc.) are associated with a reduced risk of diseases
mediated by oxidative stress and inflammation such as certain cancers, atherosclerosis and
neurodegenerative diseases (Halliwell, 1994). Among colorful fruits, strawberries (Fragaria x
ananassa) are popularly consumed in human diet in fresh and processed forms such as
beverages, yogurts, jellies and jams. In addition, there has been recent growing popularity in the
use of strawberry extracts as ingredients in functional foods and botanical supplements for their
potential human health benefits.
Many berry fruits contain micronutrients such as minerals, vitamin C and folic acid,
which are essential for health. However berries may provide additional health benefits because
they also contain high levels of a diverse range of phytochemicals/phytonutrients consisting
predominantly of phenolic type molecules. Phenolic compounds contain aromatic ring (s)
bearing hydroxyl group (s) and can range from simple monomeric molecules to very large
oligomers (Figure 1). They frequently occur naturally in berry fruits in their glycosylated forms,
which make them more water-soluble although the higher molecular weight oligomers are more
insoluble (Bravo, 1998). Berry fruits are reported to contain a wide variety of phenolics
including hydroxybenzoic and hydroxycinnamic acid derivatives, anthocyanins, flavonols,
flavanols, condensed tannins (proanthocyanidins) and hydrolyzable tannins (Machiex et al.,
1990).
Studies conducted in vitro (‘in laboratory assays’) indicate that berry phenolics have a
wide range of biological properties such as anti-cancer, antioxidant, anti-inflammatory, and cell
regulatory effects (reviewed in Seeram, 2006a; Seeram and Heber, 2006b). However the in vitro
4
biological properties of berry phytochemicals does not necessarily translate to biological
activities borne out in vivo (‘in the living system’). Therefore there have been renewed research
interests by many laboratories in conducting studies to evaluate how berry phytochemicals exert
their beneficial effects in animal models and human subjects.
Among commonly and popularly consumed small and soft berry fruits, strawberries are
widely known for their potential health benefits due to their high fiber, potassium, vitamin C and
folate contents. In addition, strawberries are abundant in phenolics, phytonutrients that have
been the subject of much investigation by numerous research laboratories. Specifically, our
laboratory is interested in investigating the biological activities, bioavailability, metabolism, and
tissue distribution of these molecules. Our group and others have been investigating the
biological properties of strawberry phenolics on biomarkers of oxidative stress and inflammation
in animals and human subjects. While some of these studies in many investigators’ laboratories
are still ongoing, the current review focuses on the phytochemicals present in strawberry fruits in
relation to the impact of strawberry consumption on human health.
This review focuses specifically on recent in vivo studies that have been conducted with
strawberry fruits and strawberry-related materials and updates the literature since the last
published review on strawberry health benefits (Hannum, 2004). It is noteworthy that recent and
significant advances have been made in understanding the bioavailability and metabolism of
ellagitannins and ellagic acid (major phenolics present in strawberries), which are discussed
herein. Studies of the absorption, metabolism, tissue distribution and in vivo biological effects
and mechanisms of action of strawberry phenolics are necessary to evaluate their impact on
human health and diseases. In fact, the chemistry and biology of phenolic molecules are
important when taken in context of their biological effects exerted in the human body. Therefore
5
this review surveys the in vitro and in vivo studies that have been conducted with strawberries
with a specific focus on the latter studies in order to evaluate their potential impact on human
health and diseases.
B. Strawberry Phytochemicals
Because of the reported biological properties associated with strawberry fruits and its
related materials (Chung et al., 2002; Cao et al., 1998; Carlton, et al., 2001; Stoner et al., 1999;
Joseph, et al, 1998; Seeram et al., 2001; Xue et al., 2001; Meyers et al., 2003), the identification
of strawberry phenolics is necessary for the evaluation of the impact of strawberry consumption
on human health. This is because phenolic compounds occur naturally as glycosylated and
conjugated forms and therefore the nature, size, structure, solubility, degree and position of
glycosylation, and conjugation with other compounds can influence their bioavailability,
absorption, distribution, metabolism and excretion in humans (Aherne and O’Brien, 2002;
Hollman, 2001).
Our laboratory has recently conducted a detailed phytochemical investigation to identify
the phenolics present in strawberries using liquid chromatography electrospray ionization mass
spectroscopy (LC-ESI-MS) methods (Seeram et al., 2006c). These compounds are grouped as
hydrolyzable tannins [ellagitannins (ETs), gallotannins (GTs), and ellagic acid (EA)],
anthocyanins, flavonols, hydroxycinnamic acid derivatives and their esters, and flavanols
(catechins) and are described in detail below
Hydrolyzable tannins. These are polymeric polyphenolic compounds consisting of ETs
and GTs. ETs are esters of hexahydroxydiphenic acid (commonly referred to as HHDP) and a
polyol usually glucose or quinic acid which hydrolyze to release the bislactone, EA. Therefore
6
EA can be considered to be present in bound forms (as EA-glycosides or as ETs) and its
detection in free forms in strawberry fruits and related food-materials is usually as a result of its
release from ET hydrolysis. GTs can also be transformed to ETs by oxidative C-C coupling
between spatially adjacent galloyl groups to form HHDP groups. ETs are abundant in other
berry fruits such as pomegranates, red and black raspberries, blackberries and some nuts and
after hydrolytic conversion, are commonly detected and quantified in the form of EA (Amakura,
et al., 2000). We have identified sanguiin H-6, polymeric molecules containing galloyl-bis-
HHDP-glucose moieties, and EA-glycosides among the major hydrolysable tannin constituents
present in strawberries (Seeram et al., 2006c).
Anthocyanins. Anthocyanins, water-soluble pigments, which belong to the general
class of plant secondary metabolites known as flavonoids, are found naturally occurring as the
glycosides of anthocyanidins (i.e. the aglycon forms). These brightly colored pigments impart
the attractive red-purple and blue colors to berry fruits and many vegetables. The six most
common anthocyanidins present in fruits and vegetables are cyanidin (Cy), delphinidin (Dp),
malvidin (Mv), pelargonidin (Plg), peonidin (Peo) and petunidin (Pt); among these aglycons, Cy
is the most ubiquitous. Our group has used LC-ESI-MS methods and shown that the major
anthocyanins present in strawberries are based on Plg and Cy aglycons (Figure 1) and
specifically are Plg-diglucoside, Cy-glucoside, Plg-glucoside, and Plg-rutinoside (Seeram et al.,
2006c).
Flavonols. These phenolics also belong to the general class of flavonoids and are found
in strawberries as the glucosides and glucuronides of quercetin and kaempferol aglycons (Figure
1). The flavonols present in strawberries are quercetin-rutinoside, quercetin-glucoside, quercetin-
glucuronide and kaempferol-glucuronide ((Seeram et al., 2006c). The occurrence of kaempferol
7
in strawberry fruits is quite uncommon and this trait is shared with other berries such as artic
bramble, and gooseberries (Hakkinen et al., 1999; Maata-Riihinen, et al., 2004). It is also
noteworthy that these two aglycons occur in strawberries as glucuronides, which has also been
observed in the raspberry (Ryan and Coffin, 1971; Maata-Riihinen, et al., 2004), although
Maata-Riihinen (2004), only reported kaempferol-glucuronide in strawberries.
Human bioavailability of flavonoids differs significantly depending on the type of
glycosides that these molecules possess. It follows that the occurrence of flavonols as their
glucuronide forms in strawberry fruits is interesting since numerous studies are focused on the
biotransformation of these dietary phenolics in vivo (Manach and Donovan, 2004). Polyphenol
bioavailability is generally thought to be poor based on low blood levels observed after ingestion
of a polyphenol-rich meal. However on absorption, phase-II enzymes conjugate these
polyphenols and/or their aglycons into glucuronidated, sulfated and methylated analogs, among
others, and enterohepatic circulation may result in substantial levels of these conjugated
metabolites observed in circulation. Whether these conjugates are bioactive in vivo and whether
they accumulate in target tissues and exert their biological effects therein are areas of our group’s
research interest and remains to be fully explored.
Other Compounds: Flavanols and Hydroxycinnamic Acid derivatives. Catechin, but not
its isomer, epicatechin, has been found in strawberries (Seeram et al., 2006c). We also identified
isomeric forms of p-coumaroyl-glucoside and a p-coumaroyl sugar ester in strawberries (Seeram
et al., 2006c). Strawberries are reported to contain p-coumaric acid as their common
hydroxycinnamic acid aglycon by other groups (Maata-Riihinen et al., 2004).
Unidentified Compounds. During our phytochemical examination of the strawberry fruit
(Seeram et al., 2006c), several phenolic compounds remained un-identified. Therefore, as further
8
phytochemical investigations are conducted into the strawberry fruit, it is possible that additional
compounds will be added to the diverse list of phenolics that have already been identified in the
fruit.
Strawberry Health Benefits
Anticancer Effects. The anticancer effects of individual phytochemical constituents of
strawberries, as well as whole strawberry extracts, have been demonstrated (reviewed in Seeram,
2006a). These anticancer effects are exerted through multi-mechanistic means of action
including the antioxidant actions of the berry’s phenolic constituents by protecting DNA from
damage, and also through effects exerted beyond antioxidation (reviewed in Seeram and Heber,
2006b). The biological activities of strawberry phytochemicals include the regulation of phase-II
enzymes and the modulation of gene expression and sub-cellular signaling pathways of cell
proliferation, angiogenesis and apoptosis (programmed cell death). Although there have been
many published reports on the anticancer effects of individual phenolics known to be present in
the strawberry fruit, the following discussion focuses on studies conducted specifically with
whole strawberry fruit freeze-dried powder and extracts per se.
Our laboratory recently reported on the dose-dependent anti-proliferative effects of a
characterized phenolic-enriched strawberry extract on a panel of human oral, breast, colon and
prostate tumor cells lines in vitro (Seeram et al., 2006d). In addition, we showed that the
strawberry extract stimulated apoptosis of HT-29 colon tumor cells, a cyclooxygenase-2 (COX2)
expressing tumor cell line.
The effects of whole strawberry fruit extract on the viability and apoptosis of human
hepatoma HepG2 cells has been reported (Ramos et al., 2005). Cell viability was inhibited in a
9
dose-dependent manner by the strawberry fruit extract. Similarly, the test materials showed pro-
apoptotic effects on the HepG2 cells and the extract arrested the G1 phase in cell-cycle
progression experiments, prior to apoptosis.
Wang and co-workers (2005) showed the inhibitory effects of strawberries on the
transcription factors, activating protein-1 (AP-1) and nuclear factor kappa B (NFκB), thereby
suppressing cancer cell proliferation and transformation. The study also showed that strawberry
extracts inhibited the proliferation of human A549 lung epithelial cancer cells and decreased
tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of mouse epidermal
cells. In addition, pretreatment of the mouse epidermal cells with strawberry extracts resulted in
the inhibition of both ultraviolet-B (UVB-) and TPA-induced AP-1 and NFκB transactivation.
Furthermore, the strawberry extracts also blocked TPA-induced phosphorylation of extracellular
signal-regulated kinases (ERKs) and UVB-induced phosphorylation of ERKs and JNK kinase in
the mouse epidermal cell culture. The authors suggested that the ability of strawberries to block
UVB- and TPA-induced AP-1 and NFκB activation might be due to their antioxidant properties
and their ability to reduce oxidative stress.
In another study, a freeze-dried strawberry extract was analyzed for anti-transformation
and chemopreventive activity using the Syrian hamster embryo (SHE) cell model (Xue et al.,
2001). The extract did not produce an increase in morphological transformation. However, in
the experiments to evaluate chemopreventive properties, SHE cells were treated with test
samples and benzopyrene for 7 days. In the latter experiments purified fractions from
strawberries were observed to produce a dose-dependent decrease in transformation compared
with the benzopyrene treatment only. Therefore the authors concluded that a possible
mechanism by which the purified fractions inhibited cell transformation could involve
10
interference of uptake, activation, detoxification of benzopyrene and/or intervention of DNA
binding and DNA repair (Xue et al., 2001).
The anti-angiogenic properties of a ‘synergistic’ combination of berry extracts that
include strawberries have been shown by a group of investigators (Roy et al., 2002; Bagchi et
al., 2004). These studies reported that the total berry extract inhibited both hydrogen peroxide
and TNF-α-induced VEGF (Vascular Endothelial Growth Factor) expression by human
keratinocytes (Roy et al., 2002; Bagchi et al., 2004). VEGF is a key regulator of tumor
angiogenesis.
Strawberry extracts have also been evaluated for their ability to inhibit mutation by the
direct-acting mutagen methyl methanesulfonate, and the metabolically activated carcinogen,
benzopyrene (Hope et al., 2004). The berry significantly inhibited mutagenesis caused by both
carcinogens. Ethanol extracts from freeze-dried fruits of several strawberry cultivars were also
evaluated and hydrolyzable tannin-containing fractions from strawberries were found to be most
effective at inhibiting mutations (Hope et al., 2004).
Induction of Antioxidant Enzymes. Oxidative stress and the resulting unrepaired
oxidative damage have been suggested to play a role in many chronic diseases, including cancer
(Ames et al., 1993). Normal endogenous metabolic process and exogenous factors, such as
ionizing radiation, diet and xenobiotics, produces reactive oxygen species (ROS) (Halliwell and
Gutteridge, 1989; Davies, 1987). ROS overproduction coupled with the deficiencies of
antioxidant defense or repair mechanisms, results in oxidative stress, which causes irreversible
damage to critical cellular macromolecules such as DNA (Davies, 1987). Therefore organisms
constantly battle the adverse effects of ROS by increasing the production of biochemical
antioxidants or by inducing endogenous antioxidant enzymes. These scavenging antioxidant
11
molecules and the endogenous antioxidant enzymes attenuate the ROS concentration to maintain
an intracellular reduction and oxidation (redox) balance.
The antioxidant capacity of strawberry extracts against ROS and the activity of
antioxidant enzymes in strawberries have been shown (Wang and Lin, 2000; Wang and Zheng,
2001). The activities of antioxidant enzymes in strawberries were shown to be positively
correlated to their antioxidant capacity (Wang et al., 2005). Studies have been designed to
investigate correlations between antioxidative potential and anti-proliferative activities of berries.
For example, Meyers and co-workers (2003) investigated eight strawberry cultivars to find out if
their antioxidant capacities, by the total oxyradical scavenging capacity (TOSC) assay, can be
correlated to their antiproliferative activities. Overall, although the proliferation of HepG2
human liver cancer cells was significantly inhibited in a dose-dependent manner after exposure
to all strawberry cultivar extracts, these workers found no relationship between antiproliferative
activity and antioxidant content.
Inhibitors of Phase-II Detoxification Enzymes. Phase-II metabolizing enzymes play an
important role in the detoxification and biotransformation of carcinogens and xenobiotics in the
human body. Kansanen and co-workers (1996) have investigated the in vitro effects of
strawberry extracts on CYP1A1 isozyme, a key phase-II enzyme, and have shown that the berry
extract was an effective inhibitor of CYP1A1.
Inhibitors of Cyclo-oxygenase Enzymes. The cyclooxygenase (COX) enzymes are
involved in the conversion of arachidonic acid to various eicosanoids involved in inflammation,
which has now been implicated as a common mechanism of many chronic human diseases.
Research conducted by the authors’s group has shown that strawberry extracts were moderately
effective in inhibiting the activity of COX-1 enzyme (a constitutive ‘house-keeping’ enzyme) but
12
more effective against the COX-2 enzyme (an inducible enzyme that is upregulated in many
cancers) (Seeram et al., 2001).
Anti-Neurodegenerative Properties. Research has shown that rodent diets supplemented
with strawberries have the ability to retard and even reverse age-related deficits in behavior and
signal transduction in rats (Joseph et al., 1998; Shukitt-Hale et al., 1999). A recent study
evaluated the efficacy of berry diets on irradiation-induced deficits by maintaining rats on these
diets or a control diet for 8 weeks prior to being exposed to whole-body irradiation (Shukitt-Hale
et al., 2006). Irradiation impaired performance in the Morriswater maze and measures of
dopamine release 1 month following radiation but these deficits were protected by the berry
diets. The strawberry diet offered better protection against spatial deficits in the maze and the
authors observed that the strawberry-fed animals were better able to retain place information (a
hippocampally mediated behavior) compared to controls. The authors suggested that irradiation
caused deficits in behavior and signaling in rats which were ameliorated by antioxidant diets and
that the polyphenols in berry fruits might be acting in different brain regions. Therefore the
studies that have been pioneered by this group of investigators suggest that phytochemicals
present in antioxidant-rich foods such as strawberries may have benefits in retarding functional
age-related Central Nervous System and cognitive behavioral deficits thereby positively
impacting neurodegenerative disease.
Metabolism of Strawberry Phenolics in Human Subjects
The biological activities attributed to berry fruits have been related to their high content
of antioxidant phenolic compounds and therefore in vivo bioavailability and metabolism studies
of these constituents present in their natural food matrix are necessary. In addition, the tissue
13
distribution and accumulation of potentially active metabolites of these compounds are also
important in order to understand their role in health promotion. Although investigators have
conducted studies to investigate the bioavailability of single phenolics compounds found in
strawberries such as quercetin (reviewed in Hannum, 2004), the focus of this section is on human
studies conducted with whole strawberries. A literature survey has revealed there are few
published human studies that have investigated the metabolism and excretion of strawberry
phenolics after ingestion of whole strawberry fruits, which are described below.
A study conducted by Rosher and co-workers (1997), involved urine collection for
monitoring the excretion of 2,5-dimethyl-4-hydroxy-3[2H]furanone (Furaneol, DMHF), an
important flavor constituent of strawberry fruits, after administration of fresh strawberries to six
healthy volunteers (four males and two females). Male and female volunteers excreted 59-69%
and 81-94%, respectively, of the DMHF dose (total of free and glycosidically bound DMHF in
strawberries) as DMHF-glucuronide in urine within 24 hours. The amount of DMHF excretion
was independent of the dose size and the ratio of free to glycosidically bound forms of DMHF in
strawberry fruit. However it is noteworthy that DMHF, DMHF-glucoside and its 6'-O-malonyl
derivative, naturally occurring in strawberries, were not detected in human urine.
Another human study also involved the collection of urine samples after six healthy
volunteers (three males and three females) consumed 200 g strawberries (providing 179 μmol
pelargonidin-3-glucoside) (Felgines et al., 2003). Intact pelargonidin-3-glucoside, its aglycon, as
well as three monoglucuronides and a sulfoconjugate of pelargonidin were detected as urinary
metabolites. Total urinary excretion of strawberry anthocyanin metabolites corresponded to 1.80
+/- 0.29% (mean +/- SEM, n = 6) of pelargonidin-3-glucoside ingested. More than 80% of this
excretion was related to a monoglucuronide. The authors observed that more than two-thirds of
14
anthocyanin metabolites were excreted at four hours after the ingestion but urinary excretion
continued until the end of the 24 h experiment. The study concluded that anthocyanins were
glucuro- and sulfo-conjugated in humans and that the main metabolite of strawberry
anthocyanins in human urine was pelargonidin-monoglucuronide.
Mazur and co-workers (2000) conducted a study to determine the pharmacokinetics
and urinary excretion pattern of the mammalian lignan, enterolactone derived from plant lignans.
Five healthy women and two men consumed a single strawberry-meal containing known
amounts of the plant lignans, secoisolariciresinol and matairesinol. Basal and post-meal blood
and urine samples were collected at short intervals and analyzed using time-resolved
fluoroimmunoassay of enterolactone. The meal increased plasma concentration of enterolactone
after 8-24 h and in urine in the 13-24 h and 25-36 h urine collections. Enterolactone excreted in
the urine collected throughout the 48 h post-meal yielded on average 114% of the plant lignans
consumed. The author observed high individual variability in metabolic response and concluded
that berries containing relatively high concentrations of plant lignans which contribute to plasma
and urinary levels of mammalian enterolactone in human subjects.
Cerda and co-workers (2005) conducted a study which was designed to investigate the
metabolism of ETs using different ET-rich foods. Forty healthy volunteers were distributed in
four groups who consumed, in a single dose, a different ET-containing foodstuff: strawberries
(250 g), red raspberries (225 g), walnuts (35 g), and oak-aged red wine (300 mL). After
ingestion, urine collections were monitored for ETs and EA-metabolites. Although neither ETs
nor EA were detected in urine after LC-ESI-MS/MS analyses, colonic microbial metabolites i.e.
3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one (‘urolithin A’ derivatives) were detected
independent of the consumed foodstuff. The mean percentage of metabolite excretion ranged
15
from 2.8% (strawberries) to 16.6% (walnuts) regarding the ingested ETs. Similar to the
previously discussed Mazur (2000) study, considerable interindividual differences were noted,
and "high and low metabolite excreters" were identified in each group, which supported the
involvement of the colonic microflora in ET-metabolism. The study results indicated that
urolithin A is a biomarker of human exposure to dietary ETs and may be useful in intervention
studies with ET-containing products. Therefore the authors concluded that the biological effects
of dietary ETs and EA should be considered in the gastrointestinal tract whereas the study of
potential systemic activities should be focused on the bioavailable urolithin A derivatives.
Recent work conducted by our group has established the pharmacokinetic parameters of
EA after ingestion of pomegranate juice, an ET-rich food (Seeram et al., 2004; Seeram et al.
2006e). We detected EA in plasma of all subjects with a maximum concentration (Cmax) of 0.06
+/- 0.01 micromol/L, area under concentration time curve (AUC) of 0.17 +/- 0.02 (micromole x
h) x L(-1), time of maximum concentration (Tmax) of 0.98 +/- 0.06 h, and elimination half-life
(T1/2E) of 0.71 +/- 0.08 h. EA-metabolites, including dimethylellagic acid glucuronide and
urolithin derivatives, were also detected in plasma and urine in conjugated and free form which
was in agreement with the Cerda (2005) study. Therefore urolithins, formed by intestinal
bacteria, may contribute to the biological effects of ET-rich foods, such as strawberries, as they
may persist in plasma and accumulate in tissues thereby exerting their biological properties and
positively impacting human health benefits. It is noteworthy that genetic polymorphisms in EA-
metabolizing enzymes e.g. catechol-O-methyl transferase (‘methylating’ enzyme), glucuronosyl
transferase (‘glucuronidating’ enzyme), and sulfotransferases (‘sulfating’ enzymes), would be
related to variations in response to ET-rich foods which remains to be established. In addition,
further research is warranted to determine the stability of the urolithin-producer phenotype and
16
the nature of the interindividual differences in bacteria that are responsible for production of
urolithins (Cerda et al., 2006; Seeram et al., 2006e).
Our group at the University of California Los Angeles (UCLA) Center for Human
Nutrition was recently funded (2007; Principal Investigator: Seeram N.P.) by the California
Strawberry Commission (CSC, Watsonville, CA, USA) to conduct a pharmacokinetic study with
whole strawberries in twelve healthy volunteers. Whilst these studies are yet to be conducted
and reported, funding of these types of experiments signifies the positive research direction and
focus of the CSC to evaluate and understand the impact of strawberry consumption in humans.
Ongoing & Current California Strawberry Commission (CSC) Funded Studies
The following section describes a brief update of ongoing projects currently funded by
the CSC, which was presented at the NASS/ NAGSA proceedings by the author. Some of these
studies have been published as abstracts, some are papers in press and some are unpublished.
I. Strawberries and Endothelial Dependent Relaxation in Rabbit Aorta. A recent study
conducted by Edirisinghe and co-workers (2007) showed that strawberry extract causes
endothelial dependent relaxation in rabbit aorta. Because polyphenolic compounds have
vasodilatory properties and may help to lower blood pressure, the investigators hypothesized that
a crude strawberry extract (1.15 mmol/gallic acid equivalents in 1 mg/ml crude solution) would
cause endothelium dependent relaxation (EDR) in rings of rabbit aorta. The authors concluded
that the strawberry extract potently stimulated vasorelaxation and that the mechanism of
relaxation was endothelium dependent.
II. Improving palatability of an effective cholesterol-lowering combination diet
(dietary portfolio), with Strawberries. Nguyen and co-workers (2007) conducted a study where
17
oat bran bread was exchanged for strawberries in a portfolio diet to evaluate improvement in
palatability. Twenty-eight hyperlipidemic subjects who had taken the dietary portfolio
consisting of soy products, viscous fibers, plant sterols and almonds for mean duration of 1.5
year took additional oat bran bread (65 g/d, 112 kcal, ≈ 2g β-glucan) or strawberries (454 g/d,
112 kcal) for one month in random order with a 2-week washout. On a scale of 1 (unpalatable)
to 10 (highly palatable) the strawberries had a palatability score of 8.8 ± 0.3 versus oat bran 6.2 ±
0.4 at the end of the respective phase (P<0.001). At week 4 on the strawberry phase the LDL-C
reduction from baseline (1.5 years pre-study) was 13.3 ± 2.1% (P<0.001) and the total:HDL-C
was 15.7 ± 1.7% (P<0.001). Similar reductions were observed at week 4 on the oat bran of 13.9
± 2.3% (P<0.001) and 14.6 ± 2.1% (P<0.001), respectively. The authors concluded that
strawberries enhanced the palatability of a cholesterol-lowering diet while maintaining the serum
lipid reductions of the dietary portfolio.
III. Strawberries and Heart Health in Human Subjects. Two human studies were
conducted at the Health Research and Study Center (Los Altos, CA, USA; Principal Investigator:
Spiller, G.A.; personal communication). The studies were aimed at investigating whether
consumption of strawberries would increase plasma folate levels and decrease plasma
homocysteine (HCY) and C-reactive protein (CRP) levels in healthy human subjects. HCY is an
amino acid currently under investigation for its relationship to heart disease due to the finding
that individuals with elevated serum HCY (homocysteinemia) are at increased risk for
cardiovascular disease. Strawberries are a good source of folate (30 µg/6-oz serving) and HCY
levels are known to decrease with supplemental folate. CRP, a general marker of inflammation,
has recently become the focus of much research since it appears to be associated with risk of
myocardial infarction, stroke, peripheral vascular disease and cardiovascular death. In addition,
18
in these studies, Spiller and co-workers measured serum triglycerides (TG) and lipoproteins, total
cholesterol (TC), low-density-lipoprotein (LDL) and high-density-lipoprotein (HDL) cholesterol
as secondary objectives, given their established relationship to cardiovascular disease. Blood
pressure was also measured to monitor any potential changes in the subjects.
In the first study (Study 1), the intervention consisted of the addition of daily strawberries
to the participants’ usual diet. The participants were asked to consume at least one serving
(about 6-7 oz) of California strawberries 6 days a week for 8 weeks. Participants were asked to
not change their typical diet or their supplement routine. After 8 weeks of daily strawberry
consumption, there was a statistically significant increase of 22% in plasma folate levels
(P<0.001). However, no significant changes were observed in plasma HCY and CRP. When
only the participants with baseline HCY ≥ 9 µmol/L were analyzed, HCY levels were observed
to have decreased in 7 of the 12 participants, with a mean reduction of 0.8%. It is noteworthy
however, that the results did not reach statistical significance. At the end of this study there was a
statistically significant reduction (4%, P<0.001) in systolic blood pressure, whereas diastolic
blood pressure did not change over the course of the study. No significant changes were
observed in serum lipids or lipoproteins at the end of the study.
In the second study (Study 2), in order to maximize the effect of strawberry consumption
on the parameters evaluated, participants were asked to follow a diet low in fruits and vegetables
and whole grains but were instructed to consume strawberries (~12 oz) daily. At the end of the
study, diet diaries were reviewed by the study investigator for compliance with study diet
guidelines. Participants complied with a low fruit, vegetable and whole grains diet. The
participants’ weight remained unchanged from the end of the baseline phase to the end of the
Strawberry Diet Phase (80 ± 17 kg; BMI changed 28 ± 4 kg/m2). However, there was a
19
significant increase (P<0.05) in folate (14%) but no significant changes in HCY or CRP. CRP
levels increased by 53% but the results did not reach statistical significance. When only the
subset of participants with baseline HCY ≥ 9 µmol/L was considered, HCY levels dropped in
each individual, with a mean statistically significant reduction of 1% (P<0.002). Changes in
systolic or diastolic pressure were not observed after 8 weeks of strawberry consumption and no
significant changes were observed in serum lipids or lipoproteins at the end of the study.
In conclusion, in Study 1, daily consumption of 6-7 oz of strawberries for 8 weeks
resulted in a statistically significant but modest (22%) increase in plasma folate levels as
predicted by the addition of a high-folate fruit to daily dietary habits. It also brought a significant
but small reduction in systolic blood pressure (4%), which may be explained by the participants’
higher intake of fruits and dairy products at the end of the study compared to baseline. Plasma
levels of homocysteine and CRP remained unchanged. When data were analyzed for participants
with baseline homocysteine levels higher than 9 µmol/L, however, most participants (58%)
experienced a drop of HCY levels, with a mean reduction of 0.8% was observed, although the
reduction was not statistically significant.
Similarly, in Study 2 a slightly larger serving of strawberries (~12 oz/day) incurred a
significant (14%) increase in plasma folate but no changes in HCY levels. An increase in CRP
was seen for which factors other than the study intervention might be responsible. Whereas in
Study 1 there was only a trend toward a reduction in HCY in participants with baseline HCY
levels higher than 9 µmol/L, a significant decrease in HCY was observed in this group in Study
2, confirming the hypothesis that plasma HCY may be susceptible to dietary changes when
higher than a certain threshold. The amount of dietary folate added by strawberry consumption
in these two studies (30-60 µg/6-12 oz of strawberries) was much lower than that obtainable with
20
supplementation. The authors concluded that a modest benefit for HCY levels could be seen
with daily consumption of strawberries (6-12 oz/day) in individuals with considerably high HCY
levels.
IV. Ongoing In vivo Studies. The following is a list of ongoing human and animal
studies funded by the CSC: 1) Cholesterol lowering effects of fresh strawberries (University of
Toronto; Principal Investigator: D. Jenkins); 2) Effects of fresh strawberries on chronic disease
risk in overweight subjects (University of California at Davis; Principal Investigator: B. Burton-
Freeman); 3) Anti-inflammatory effects of strawberries in obese individuals (USDA Western
Human Nutrition Research Center; Principal Investigator: S. Zunino); 4) Bioavailability and
bioactivity of strawberry phytonutrients in twenty healthy volunteers (University of California at
Los Angeles; Principal Investigators: Z. Li, S. Henning, N. Seeram); 5) Effects of strawberries
on diabetes and hypertension in women (Harvard Medical School; Principal Investigator: H.
Sesso); 6) Effects of freeze-dried strawberries on oral cancer prevention (Ohio State University;
Principal Investigator: B. Casto); 7) Effects of freeze-dried strawberries on esophageal cancer
(Ohio State University/China; Principal Investigator: T. Chen); 8) Mechanistic studies of
strawberries on cognitive and neuronal communication in aging in rats (Tufts University;
Principal Investigator: J. Joseph); 9) Effects of strawberry consumption on serum lipid oxidation
in hypercholesterolemic subjects and its pharmacokinetics in healthy subjects (University of
California at Los Angeles; Principal Investigator: N. Seeram); 10) Effects of strawberry
consumption on blood pressure in pre-hypertension patients (University of California at Davis;
Principal Investigator: B. Burton-Freeman).
Epidemiological Studies
21
It has been shown that healthy older women who consumed strawberry (240 g) have an
increase in serum antioxidant capacity of up to 2 hours after consumption (Cao et al., 1998).
Because strawberries contain several key nutrients, including fiber, folate, potassium, vitamin C,
and various phenolics, then its consumption may play a role in cardiovascular disease (CVD)
prevention. Sesso and co-workers (2007) recently studied the effects of strawberry intake on
lipids, CRP, and the risk of CVD in the Women’s Health Study. These researchers examined
strawberry intake for both its prospective association with CVD risk in 38,176 women and its
cross-sectional association with lipids and CRP in a subset of 26,966 women. Strawberry intake
was assessed from a baseline semi-quantitative food frequency questionnaire, along with other
self-reported lifestyle, clinical and dietary factors. Participants returned baseline bloods, which
were assayed for lipids and CRP and the relative risks for a total of 1,004 CVD case occurring
during 10.9 years of follow-up was computed. The study found a lack of association for
individual CVD endpoints and comparing mean levels of lipids and CRP by category of
strawberry intake. However, women consuming ≥2 servings of strawberries per week had a
borderline but significant, multivariate 14% lower likelihood of an elevated CRP (≥3 mg/L)
compared to women who did not consume strawberries. The authors concluded that the intake of
strawberries was not associated with the risk of incident CVD, lipids, or CRP in middle-aged and
older women. However higher strawberry intake may slightly reduce the likelihood of having
elevated CRP levels although the authors cautioned that additional epidemiological data are
needed to clarify the role of strawberries in CVD prevention.
22
Conclusions and Future Directions
In conclusion, a number of cell culture, animal and human studies suggest that
strawberries may have a positive impact on human health and diseases. The phytochemical
constituents (predominantly phenolics) within the whole strawberry fruit matrix may act
individually, additively and/or synergistically to exert their biological properties and this needs
to be further examined in well-designed experiments. It should be cautioned that results
obtained from in vitro and animal studies cannot necessarily be translated into biological
endpoints that will be observed in humans. Therefore future studies should be designed to
investigate the potential of stawberries for the prevention and treatment of specific diseases in
human subjects. In addition, details on absorption, distribution, metabolism and mechanisms of
action of strawberry phytochemicals in humans should aid in determining effective dietary
portions of the berry. Whether the biological properties of strawberries are enhanced by the
combination of phytochemicals obtained from other fruits and vegetables should also be
investigated. Finally, given the number of ongoing human studies currently being conducted
with strawberries, another review will be warranted in a few years.
Acknowledgements
The literature review was supported by the California Strawberry Commission (Watsonville,
CA, USA).
23
Literature Cited
Ames, B.N., M.K. Shigena, and T.M. Hagen. 1993. Oxidants, antioxidants and the
degenerative diseases of aging. Proc. Natl. Acad. Sci. U.S.A. 90:7915-7922.
Aherne, S.A. and N.M O’Brien. 2002. Dietary flavanols: chemistry, food content and
metabolism. Nutrition 18:75-81.
Amakura, Y., M. Okada, S. Tsuji, and Y. Tonogai. 2000. High performance liquid
chromatographic determination with photodiode array detection of ellagic acid in fresh
and processed fruits. J. Chromatogr. A 896:87-93.
Bagchi, D., C.K. Sen, M. Bagchi, and M. Atalay. 2004. Anti-angiogenic, Antioxidant,
and Anti carcinogenic Properties of a Novel Anthocyanin-Rich Berry Extract
Formula. Biochem. (Trans. of Biokhimiya) 69:75-80.
Bravo, L. 1998. Polyphenols: chemistry, dietary sources, metabolism and nutritional
significance. Nutr. Reviews 56:317-333.
Cao, G., R.M. Russell, N. Lischner, and R.L. Prior. 1998. Serum antioxidant capacity is
increased by consumption of strawberries, spinach, red wine, or vitamin C in elderly
women. J. Nutr 128:2383-2390.
Carlton, P.S., L.A. Kresty, J.C. Siglin, M.A. Morse, J. Lu, C. Morgan, and G.D. Stoner. 2001.
Inhibition of N-nitrosomethylbenzylamine-induced tumorigenesis in the rat esophagus by
dietary freeze-dried strawberries. Carcinogenesis 22:441-446.
Cerda B., F.A. Tomas-Barberan, and J.C. Espin. 2005. Metabolism of antioxidant and
chemopreventive ellagitannins from strawberries,raspberries, walnuts, and oak aged wine
in humans: identification of biomarkers and individual variability. J
Agric Food Chem. 53(2): 227-35.
24
Chung, M.J., S.H. Lee, and N.J. Sung. 2002. Inhibitory effect of whole strawberries, garlic juice
or kale juice on endogenous formation of N-nitrosodimethylamine in
humans. Cancer Letters 182:1-10.
Davies, K.J. 1987. Protein damage and degradation by oxygen radicals. I. General aspects. J.
Biol. Chem. 262:9895-9901.
Edirisinghe, I., B. Burton-Freeman, and T. Kappagoda. 2007. Strawberry extract causes
endothelial dependent relaxation in rabbit aorta. Abstract # 8087, Experimental
Biology. April 28-May 2, Washington DC, USA.
Felgines C., S. Talavera, M.P. Gonthier, O. Texier, A. Scalbert, J.L. Lamaison, and
C. Remesy. 2003. Strawberry anthocyanins are recovered in urine as glucuro- and
sulfoconjugates in humans. J. Nutr. 133: 1296-301.
Halliwell, B. 1994. Free radicals, antioxidants and human disease: Curiosity, cause or
consequence? The Lancet 344:721-724.
Halliwell, B., and J.M.C. Gutteridge. 1989. Free Radicals in Biology and Medicine, 2
nd ed.
Clarendon Press, Oxford, U.K.
Hakkinen, S.H., S.O. Karenlampi, M. Heinonen, H.M. Mykkanen, and A.R. Torronen.
1999. Content of the flavanols quercetin, myricetin and kaempferol in 25 edible berries.
J. Agric. Food Chem. 47:2274-2279.
Hannum, S.M. 2004. Potential impact of strawberries on human health. Crit. Rev. Food Sci.
Nutr. 44:1-17.
Hollman, P.C. 2001. Evidence for health benefits of plant phenols: Local or systemic effects? J.
Sci. Food Agric. 81:842-852.
25
Hope, S.S., P.L. Tate, G. Huang, J.B. Magee, K.M. Meepagala, D.E. Wedge, and L.L. Larcom.
2004. Antimutagenic activity of berry extracts. J. Med. Food 7:450-455.
Joseph, J.A., B. Shukitt-Hale, N.A. Denisova, R.L. Prior, G. Cao, A. Martin, G. Taglialatela, and
P.C. Bickford. 1998. Long-term dietary strawberry, spinach, or vitamin E
supplementation retards the onset of age-related neuronal signal-transduction and
cognitive behavioral deficits. J. Neurosci. 18:8047-8055.
Kansanen, L., H. Mykkanen, and R. Torronen. 1996. Flavonoids and extracts of strawberry and
black currant are inhibitors of the carcinogen-activating enzyme CYP1A1 in vitro.
Natural Antioxidants and Food Quality in Atherosclerosis and Cancer Prevention
181:386-388.
Maatta-Riihinen, K.R., A. Kamal-Elin, and A.R. Torronen. 2004. Identification and
quantification of phenolic compounds in berries of Fragaria and Rubus species (Family
Rosaceae). J. Agric. Food Chem. 52:6178-6187.
Machiex, J.J., Fleuriet, A., and Billot, J. 1990. Fruit phenolics. CRC Press, Boca Raton,
FL.
Manach, C. and J.L. Donovan. 2004. Pharmacokinetics and metabolism of dietary flavanoids in
humans. Free Radical Research, 38:771-785.
Mazur W.M., M. Uehara, K. Wahala, H. and Adlercreutz. 2000. Phyto-oestrogen content of
berries, and plasma concentrations and urinary excretion of enterolactone after a single
strawberry-meal in human subjects. Br. J. Nutr. 83: 381-7.
Meyers, K.J., C.B. Watkins, M.P. Pritts, and R.H. Liu. 2003. Antioxidant and
antiproliferative activities of strawberries. J. Agric. Food Chem. 51:6887-6892.
Nguyen, T.H., C.W.C. Kendall, D.A. Faulkner, C. Ireland, K. Galbraith, A. Marchie,
26
C. Christian, and D.J.A. Jenkins. 2007. Strawberries to improve palatability of a
cholesterol lowering diet, Abstract # 6923, Experimental Biology. April 28- May 2,
Washington DC, USA.
Ramos, S., M. Alia, L. Bravo, and L. Goya. 2005. Comparative effects of food-derived
polyphenols on the viability and apoptosis of a human hepatoma cell line HepG2. J.
Agric. Food Chem. 53:1271-1280.
Roy, S., S. Khanna, H.M. Alessio, J. Vider, D. Bagchi, M. Bagchi, and C.K. Sen.
2002. Anti angiogenic property of edible berries. Free Rad. Res. 36:1023-1031.
Roscher R., H. Koch, M. Herderich, P. Schreier, and W. Schwab. 1997. Identification of 2,5
dimethyl-4 hydroxy-3[2H] furanone beta-D-glucuronide as the major metabolite of a
strawberry flavour constituent in humans. Food Chem Toxicol. 35(8): 777-82.
Ryan, J.J., and D.E. Coffin. 1971. Flavanol glucuronides from red raspberry (Rubus
Idaeus) ‘Rosaceae’. Phytochemistry 10:1675-1677.
Seeram, N. P., R.A. Momin, L.D. Bourquin, and M.G. Nair. 2001. Cyclooxygenase inhibitory
and antioxidant cyanidin glycosides in cherries and berries. Phytomedicine 8:362-369.
Seeram, N.P., R. Lee, and D. Heber. 2004. Bioavailability of ellagic acid in human plasma after
the consumption of ellagitannins from pomegranate (Punica
granatum L.) juice. Clin. Chim. Acta. 348:63-68.
Seeram, N.P. 2006a. Berries, p. 615-625. In: D. Heber et al. (eds). Nutritional Oncology, 2nd ed.
Academic Press, London, UK.
Seeram, N.P., and D. Heber. 2006b. Impact of berry phytochemicals on human health: Effects
beyond antioxidation. In: C.T. Ho et al. (eds). Lipid Oxidation and Antioxidants:
27
Chemistry, Methodologies and Health Effects. ACS Symposium Series. Oxford
University Press.
Seeram, N.P., R. Lee, H.S. Scheuller, and D. Heber. 2006c. Identification of phenolic
compounds in strawberries by liquid chromatography electrospray ionization mass
spectroscopy. Food Chem. 97:1-11.
Seeram, N.P., L.S. Adams, Y. Zhang, R. Lee, D. Sand, H.S. Scheuller, and D. Heber. 2006d.
Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts
inhibit growth and stimulate apoptosis of human cancer cells in vitro. J. Agric. Food
Chem. 54:9329-39.
Seeram, N.P., S.M. Henning, Y. Zhang, M. Suchard, Z. Li, and D. Heber. 2006e. Pomegranate
juice ellagitannin metabolites are present in human plasma and some persist in urine for
up to 48 hours. J. Nutr. 136:2481-5.
Sesso, H.D., J.M. Jenkins, D.J. Gaziano, and J.E. Buring. 2007. Strawberry intake, lipids, C-
reactive protein, and the risk of cardiovascular disease in women. J. Am. Coll Nutr. In
press.
Shukitt-Hale, B., A.N. Carey, D. Jenkins, B.M. Rabin, and J.A. Joseph. 2006. Beneficial effects
of fruit extracts on neuronal function and behavior in a rodent model of accelerated
aging. Neurobiol Aging. In press.
Shukitt-Hale, B., D.E. Smith, M. Meydani, and J.A. Joseph. 1999. The effects of dietary
antioxidants on psychomotor performance in aged mice. Exp Gerontol. 34(6):797-808.
Stoner, G. D., L.A. Kresty, P.S. Carlton, J.C. Siglin, and M.A. Morse. 1999. Isothiocyanates and
freeze-dried strawberries as inhibitors of esophageal cancer. Toxicol. Sci.
52(supplement):95-100.
28
Wang, S.Y., and H.S. Lin. 2000. Antioxidant activity in fruit and leaves of blackberry,
raspberry, and strawberry is affected by cultivar and maturity. J. Agric. Food
Chem. 48:140-146.
Wang, S.Y., and W. Zheng. 2001. Effect of plant growth temperature on antioxidant
capacity in strawberry. J. Agric. Food Chem. 49:4977-4982
Wang, S.Y., R. Feng, Y. Lu, L. Bowman, and M. Ding. 2005. Inhibitory Effect on
Activator Protein-1, Nuclear Factor-Kappa B, and Cell Transformation
by Extracts of Strawberries (Fragaria X ananassa Duch). J. Agric. Food
Chem. 53:4187-4193.
Xue, H., R.M. Aziz, N. Sun, J.M. Cassady, L.M. Kamendulis, Y. Xu, G.D. Stoner, and J. E.
Klaunig. 2001. Inhibition of cellular transformation by berry extracts. Carcinogenesis
22:351-356.
29
Figure 1: Chemical structures of phenolics found in strawberries
O
O
HO
OH
OH
R
OH
Flavonols
Quercetin R = OH
Kaempferol R = H
O
R2
HO
OH
OH
Flavanols
(+)-Catechin (2R, 3S) R1 = OH, R2 = H
OHO
OH
OH
Anthocyanidins
Cyanidin R = OH
Pelargonidin R = H
OH
R
+
R1
OH
O
OH
Hydroxycinnamic acid
p-coumaric acid R = H
HO
R
30
O
O
OH
OH
O
O
HO
HO
Ellagitannin
Ellagic acid
OH
HO
HO
HO
HO
OH
C
C
O
O
O
O
O
O
OCO
HO
HO
OH OH OH
OH
O
O
OH
OH
O
HO
HO
HO
HO
HO
OH
C
C
O
O
O
O
O
O
OCO
HO
HO
OH OH OH
OH
O
O
OH
OH
OH
Ellagitannin
Sanguiin H-6
OH
HO
HO
HO
HO
OH
C
C
O
O
O
O
O
O
OCO
HO
HO
OH OH OH
OH
O
O
OH
OH
OH
Ellagitannin
Galloyl-bis-HHDP-glucoside
