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Functional properties of spinach (Spinacia oleracea L.)

Cite this: Food Funct., 2016, 7, 3337
phytochemicals and bioactives
Joseph L. Roberts*a,b and Régis Moreaua

Overwhelming evidence indicates that diets rich in fruits and vegetables are protective against common
chronic diseases, such as cancer, obesity and cardiovascular disease. Leafy green vegetables, in particular,
Published on 22 June 2016. Downloaded on 6/8/2019 12:11:56 AM.

Received 13th January 2016,
Accepted 22nd June 2016
DOI: 10.1039/c6fo00051g
www.rsc.org/foodfunction
are recognized as having substantial health-promoting activities that are attributed to the functional pro-
perties of their nutrients and non-essential chemical compounds. Spinach (Spinacia oleracea L.) is widely
regarded as a functional food due to its diverse nutritional composition, which includes vitamins and min-
erals, and to its phytochemicals and bioactives that promote health beyond basic nutrition. Spinach-
derived phytochemicals and bioactives are able to (i) scavenge reactive oxygen species and prevent
macromolecular oxidative damage, (ii) modulate expression and activity of genes involved in metabolism,
proliferation, inflammation, and antioxidant defence, and (iii) curb food intake by inducing secretion of
satiety hormones. These biological activities contribute to the anti-cancer, anti-obesity, hypoglycemic,
and hypolipidemic properties of spinach. Despite these valuable attributes, spinach consumption remains
low in comparison to other leafy green vegetables. This review examines the functional properties of
spinach in cell culture, animals and humans with a focus on the molecular mechanisms by which
spinach-derived non-essential phytochemicals and bioactives, such as glycolipids and thylakoids, impart
their health benefits.

Introduction
a
Department of Nutrition & Health Sciences, University of Nebraska–Lincoln, Spinach (Spinacia oleracea L.), a dark green leafy vegetable, is a
Lincoln, NE 68583, USA
b member of the family Amaranthaceae that includes beets and
Mailstop 1518-002-7BB (SPH: Global Health), Nutrition and Health Sciences
Program, Emory University, Atlanta, GA 30322, USA. chard.1 Believed to have originated in Persia (Iran), the vege-
E-mail: joseph.roberts@emory.edu; Tel: +1-252-915-9723 table spread through other parts of Asia and Europe through

Joseph Roberts, MS, is a PhD Régis Moreau, PhD, is Assistant

Joseph L. Roberts
student in nutrition and health
sciences at Emory University. He
graduated from North Carolina
State University (2013), and
earned a Master of Science in
nutrition and health sciences
from the University of Nebraska–
Lincoln (2015). His master’s
project focused on the lipid-
lowering properties of alpha-
lipoic acid and phenylbutyric
acid. His current research
focuses on the molecular mecha-
nisms by which dietary bioactive
compounds promote musculo-
skeletal health.
Professor at the University of
Nebraska–Lincoln. He teaches
advanced human nutrition to
undergraduate and graduate stu-
dents. His research program is
focused on understanding the
mechanisms of action of natural
food organosulfur and phenolic
compounds and evaluating their
therapeutic potential in chronic
metabolic disorders and associ-
Régis Moreau ated enteric, hepatic and cardio-
vascular dysfunction.

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trading routes.2 The earliest written record of spinach is by
the Chinese in 647 A.D., where it was referred to as the herb
of Persia.1,2 Spinach is broadly classified into three types
according to the leaf texture: (i) smooth-leaf; (ii) savoy; and (iii)
semi-savoy. Smooth-leaf spinach is the preferred leaf type for
processing and salads; whereas savoy and semi-savoy are used
for cooking. Approximately 82 unique varieties of spinach exist
including, but not limited to, Amsterdam Giant, Bloomsdale,
Gaundry, Victoria, Viroflay, Dark Green Bloomsdale, Long
Standing Bloomsdale, Hollandia, King of Denmark, Juliana,
Nobel, Virginia Savoy, Canner King, Darkie, Del Monte,
Domino, Old Dominion, Presto, Viking, Virginia Savoy Wilt
Resistant Winter Giant, Samish, Ozarka II, and Winter King.
Hybrid species, first introduced in the 1950’s, are the major
type of spinach cultivated today. Spinach is widely cultivated
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around the world, and worldwide production has increased by
418.7% since 1970.3 China, the largest producer of spinach,
produced 21.1 million tons in 2013 amounting to about 90%
of the world spinach production.3 The United States produced
approximately 1.4% or 0.34 million tons of spinach in
2013 making it the second largest producer.3 Other major pro-
ducers include Japan (1.1%), Turkey (1%), Indonesia (0.6%),
and France (0.5%). Although the U.S. is the second largest pro-
ducer of spinach in the world, Americans consume spinach
infrequently when compared to other commonly consumed
green leafy vegetables, such as lettuce (Lactuca sativa), cabbage
(Brassica oleracea var. capitata), and broccoli (Brassica oleracea
L. var. italic) (Fig. 1). In 2012, Americans consumed on average
approximately 0.91 g per day of fresh spinach per capita
and 0.49 g per day of processed spinach (e.g., frozen, canned
spinach).4 Though consumption has remained low, spinach
availability in the U.S. increased in the last 42 years from
0.81 kg per person in 1970 to 1.17 kg per person in 2012.
Similarly, using the European Food Safety Authority Compre-
hensive Database, the mean consumption of fresh spinach
in 19 European countries is approximately 1.6 g per day per
person.
Fig. 1 Consumption trends for select green, leafy vegetables in the
United States.4
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Nutritional and chemical composition
of spinach
Spinach is a versatile plant that is consumed in both raw (e.g.,
salads, smoothies) and cooked forms (e.g., steamed, casser-
oles, soups). Spinach is primarily composed of water (91.4%),
and contains small amounts of protein (2.9%), carbohydrate
(3.6%), and fat (0.4%). The lipid fraction is mainly composed
of mono- and poly-unsaturated fatty acids (e.g., alpha-linolenic
acid, linoleic acid, oleic acid) and trace levels of saturated fatty
acids (e.g., capric acid, myristic acid, stearic acid).5–7 Spinach
(100 g serving) contains 2.2 g fiber, meeting 8.8% of the rec-
ommended dietary allowance (RDA) for a 2000 kcal diet. The
vitamin and mineral composition is considerably diverse com-
pared to other commonly consumed leafy green vegetables
(Fig. 2A–D). For this review, comparisons were made with broc-
coli, cabbage, and lettuce due to similar culinary use and the
availability of data on their chemical composition. For
instance, a 100 g serving of spinach contains high levels of
magnesium, potassium, and iron that meet 20%, 16%, and
15%, respectively of their RDA (Fig. 2A).6 That is substantially
more that broccoli, cabbage and lettuce (Fig. 2B–D). A 100 g
serving of spinach contains enough of several vitamins to par-
tially meet or exceed their respective RDA, including vitamin K
(604%), vitamin A (188%), folate (49%), and vitamin C (47%)
(Fig. 2A).6
The variety, cultivation method, leaf size, and storage
duration of spinach can influence the nutrient composition,
including vitamin C, vitamin E, folate, phylloquinone
(vitamin K1), and pro-vitamin A β-carotene.8,9 There was
marked variation in average vitamin C (total ascorbic and
dehydroascorbic acid) concentration in 27 varieties of
spinach, ranging from 24.57 to 48.49 mg per 100 g FW in the
Spalding and the advanced breeding line 618, respectively.9
Similarly, the concentrations of γ- and α-tocopherol were sig-
nificantly higher in the cultivar Lazio than Samish.8 Spinach
grown during the summer solstice in the subartic (Nova
Scotia, Canada) had significantly higher folate and tocopherol
compared to spinach grown during the winter solstice in the
subtropics (Texas, USA).8 The mineral composition of spinach
is also affected by growing season. Spinach grown in Fall
(October and November) contained higher levels of nitrogen,
potassium, calcium, magnesium, copper, zinc, and manga-
nese in their leaves than spinach grown in winter (December–
February).10 Production method further impacts nutrient com-
position as spinach cultivated using organic methods had
approximately 31% more vitamin C than conventionally
grown spinach.9 Post-harvest factors (e.g., cooking) influence
the micronutrient content in spinach as well. For example,
boiling or steaming fresh spinach decreased folate (−47% and
−3%, respectively) and vitamin C (−75% and −34%, respec-
tively) concentrations.11 Collectively, these studies suggest that
the growing season, cultivar, cultivation and cooking methods
should be taken into account when examining the nutritional
value of spinach.
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Fig. 2 Vitamin, mineral, and phytochemical composition of select green, leafy vegetables per 100 g serving. Data are presented as % of daily value
(DV) for a 2000 kcal diet (A–D) or average concentration (E–H). Foods providing 20% or more of the DV are considered good sources of the nutrient.
(A) Spinach meets 100% of the DV for vitamin K and vitamin A, and over 40% of the DV for folate and vitamin C. (B) Broccoli exceeds the DV for
vitamin K and vitamin C. (C) White cabbage meets 95% and 61% of the DV for vitamin K and vitamin C, respectively. (D) Lettuce satisfies 30% of the
DV for vitamin K.6 (E) Spinach contains more total phenolic compounds (mg gallic acid equivalents (GAE)) than lettuce and cabbage, and similar
levels as broccoli.20 (F) Flavonoids are highest in lettuce, followed by broccoli, spinach, and cabbage. Total flavonoid content is expressed in mg
catechin equivalent (CE).21 (G) Broccoli has the highest total phenolic acid content, followed by spinach, lettuce, and cabbage.18 (H) Broccoli has the
highest total lignan content, followed by cabbage, spinach, and lettuce.26

Phytochemicals, such as carotenoids and phenolic com-
pounds, are non-essential nutrients ubiquitously present
throughout the plant kingdom, many of which are found in
spinach. Carotenoids are naturally occurring pigments that
serve as components of photosynthetic light-harvesting com-
plexes ( photosystem II), where they quench chloroplast triplet
states, stabilize protein–lipid structures, scavenge reactive
oxygen species (ROS), and dissipate excess radiant energy not
required for photosynthesis.12 Spinach is considered to be
one of the richest plant sources of the carotenoid, lutein,13
which is implicated in the prevention of age-related macular
degeneration and cataracts.14,15 Spinach also contains notable
amounts of phenolic compounds that are produced as sec-
ondary metabolites in response to various stressors such as
ultraviolet radiation, low temperatures, pathogens, and nutri-
ent deficiency.16 Phenolic compounds function as anti-
oxidants, signaling molecules, antimicrobial, antiviral, and
antifungal agents.17–19 Among commonly consumed leafy
green vegetables, spinach contains ∼40% and ∼70% more
phenols than cabbage and lettuce, respectively (Fig. 2E).20
Despite being an excellent source of phenolics, spinach only
contributes approximately 0.8 mg gallic acid equivalents
(GAE) per day per person due to low daily intake (2.5 g
spinach per day).21
Polyphenols are classified according to their chemical struc-
tures into one of four groups: flavonoids, phenolic acids, stil-
benes, and lignans.22,23 In comparison to other leafy green
vegetables, spinach contains slightly more flavonoids than
white cabbage, but less total flavonoids than head lettuce or
broccoli (Fig. 2F).21 Spinach contains negligible amounts of
common flavonoids (e.g., quercetin, kaempferol), but contains
at least 17 distinct flavonoid derivatives of patuletin, spinace-
tin, spinatoside, jaceidin, and flavone (Fig. 3A).9,24 Phenolic
acids are divided into two classes; hydroxycinnamic acid (e.g.,
p-coumaric, ferulic) and hydroxybenzoic acid (e.g., p-hydroxy-
benzoic, vanillic acids).18,23 Spinach, along with broccoli, have
the highest total phenolic acid content among leafy green veg-
etables (Fig. 2G). The predominant phenolic acids are ferulic
acid and p-coumaric acid.18 Spinach contains fewer lignans
than cabbage or broccoli (Fig. 2H). Lignans are diphenolic
compounds; they are metabolized by the gut microflora into
biologically active phytoestrogens, enterolactone and entero-
diol.25 The main lignans found in spinach are lariciresinol,
secoisolariciresinol, and pinoresinol.26

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Fig. 3 Chemical structures of common spinach phytochemicals and bioactive compounds. (A) Structures of the predominant flavonoids in spinach
and IUPAC names of identified flavonoids.9 (B) Structure of lutein, a highly abundant carotenoid in spinach. (C) Structures of identified phytochem-
icals in spinach NAO, including p-coumaric acid derivatives and flavonoid derivatives.35 (D) Spinach glycolipids and major fatty acids associated with
each glycolipid. Abbreviations: MGDG, monogalactosyl diacylglycerol; DGDG, digalactosyl diacylglycerol; SQDG, sulfoquinovosyl diacylglycerol.75

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Importantly, the phytochemical composition of spinach
varies in response to a number of agricultural and post-harvest
factors; consequently, the reported phenolic contents can
greatly fluctuate (Fig. 4A and B). A 2-fold variation in total fla-
vonoids was observed in five spinach cultivars, suggesting that
genotype strongly affects the levels of total phenolics and flavo-
noids.27 Advanced breeding lines (e.g., F-380, 88–120, 88–354)
have higher levels of total phenolics and flavonoids than com-
mercial cultivars (e.g., Samish, Fallgreen, Wintergreen).27,28
Furthermore, cultivars bred for disease resistance (e.g., 88–354,
Wintergreen, F380, Fallgreen, Ozarka II) synthesize high levels
of phenols.28 Leaf maturity should also be taken into account
when considering the phytochemical composition of spinach.
It has been shown that midmature leaves contained more fla-
vonoids and total phenolic compounds than immature (baby
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in September and harvested in December (four-month
growing season) had only half the phenolic content of
cultivars grown over six months in winter (October–March),
suggesting that biotic and abiotic stressors associated with a
long growing season spanning winter induce the production
of phenolics.28
Post-harvest factors also influence the phytochemical com-
position of spinach. Simulated retail lighting (e.g., 24 h fluo-
rescent lighting) was beneficial in maintaining the carotenoids
lutein, zeaxanthin, and β-carotene vs. dark storage conditions.8
With regards to the cooking method, microwaving frozen
spinach in distilled water increased the concentration of leaf
trans- and cis-lutein by 91% and 39%, respectively, when com-
pared to uncooked spinach.30 This increase is likely a result of
water loss during cooking and suggests that microwave

spinach) and mature spinach leaves.29 The cultivation method
and growing season also greatly influence the phytochemical
profile of spinach. Organically grown spinach contains signifi-
cantly more flavonoids than conventionally grown spinach; a
difference that may be attributed to the elevated level of stress
(e.g., pests) that vegetables grown organically experience.9 With
regards to the growing season, spinach cultivars planted
cooking is an effective method to increase lutein bioavailability
of spinach.30 In contrast, cooking fresh spinach can decrease
the flavonoid and lignan contents by releasing the phenolic
compounds from the leaves. Gil et al.31 reported that boiling
spinach for 10 minutes at 90 °C lowered the flavonoid content
by approximately 41% and that of total lignans by 67% (laricir-
esinol, −85%; pinoresinol, −21%).25

Fig. 4 Factors influencing the nutritional and phytochemical composition of spinach. (A) The nutritional and phytochemical composition of
spinach is affected by genetics, agricultural, and post-harvest factors. (B) Summary of the effects of agricultural and post-harvest influences on
flavonoid and phenolic composition of spinach. Advanced breeding lines contain more flavonoids than commercial cultivars.28 Leaf maturity affects
flavonoid content, with midmature having the most effect, followed by immature and mature leaves.29 Organically grown spinach has higher levels
of flavonoids than conventionally grown spinach.9 Additionally, spinach grown over-winter contains more total phenolics.28 Finally, cooking spinach
decreases the flavonoid content.31 Data represent mean ± SD. Abbreviations: ABL, advanced breeding lines; CC, commercial cultivars; GAE, gallic
acid equivalents; CAE, chlorogenic acid equivalents; FW, fresh weight.

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Functional properties of spinach in vitro based on the scavenging of 2,2′-azino-bis(3-ethylbenzo-

Worldwide, the incidence of chronic diseases such as cancer,
diabetes, and cardiovascular disease is on the rise.32 A clear
relationship between diet and these physiopathologies has
fueled broad interest in functional foods as cost-effective
means of prevention and treatment.33 Among the functional
properties of spinach, the antioxidant (Table 1), anti-inflam-
matory (Table 2), antiproliferative (Table 3), anti-obesity
(Table 4), hypoglycemic (Table 5), and lipid-lowering (Table 6)
activities of spinach leaves and spinach-derived extracts are
thought to be central to the protection against chronic dis-
eases. The following sections review the aforementioned func-
tions of spinach based on studies carried out in cells, animals,
and humans.
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thiazoline-6-sulfonic acid; ABTS) radical, superoxide anion
(O2•−), Fe2+, peroxyl radical, hydroxyl radical, and peroxy-
nitrite.20,34 Compared to lettuce and cabbage extracts, the
total oxyradical scavenging capacity (TOSC) value of spinach
extracts was higher by 39.5% and by 24.2%, respectively, but
was not different from that of broccoli.20 A water-soluble
natural antioxidant (NAO) mixture extracted from spinach
also demonstrated considerable antioxidant activity. NAO is
comprised primarily of aromatic polyphenols, including
p-coumaric acid derivatives, flavonoids, and other hydrophilic
molecules (Fig. 3C). It has been shown that spinach NAO
prevented peroxide and malondialdehyde (MDA) formation
in vitro by ∼35%.35 Since individually isolated p-coumaric
acid, flavonoid and hydrophilic compounds demonstrated

noticeably less antioxidant potency ( peroxide and MDA

Antioxidant properties
Diets rich in fruits and vegetables protect against chronic
diseases in part through the antioxidant capacity of the
phytochemicals, vitamin C, and vitamin E they contain.
The antioxidant capacity of spinach extract was established
formation was decreased by ∼5–7%), it was suggested that
the phenolics and other hydrophilic compounds in NAO act
synergistically.35 Spinach and spinach extracts also contain
glucuronide derivatives of the antioxidant flavonoids, patule-
tin and spinacetin.31 In particular, patuletin-3-O-β-D-gluco-
pyranosyl(1→6)-[β-D-apiofuranosyl-(1→2)]-β-D-glucopyranoside,
patuletin-3-O-β-D-(2″-feruloylglucopyranosyl)-(1→6)-[β-D-apiofurano-

Table 1 Antioxidant effects of spinach

Model Dosage and duration Key effects Ref.

Cell culture
PC3 and DU145, human
prostate adenocarcinoma
Animal models
Sprague-Dawley rats fed with a
high fat-cholesterol diet
Fischer rats
UV-irradiated mice
TRAMP mice
Human studies
7–8 healthy female adults
23 healthy males
9 healthy females
48 healthy males and females
Spinach NAO (1.6 mg mL−1) for 1 h. 1. Significant decrease in ROS levels. 39
Diets supplemented with 5% freeze-dried spinach 1. Significantly lower liver thiobarbituric acid 36
powder for 6 weeks. reactive substances.
2. Significantly lower leukocyte DNA damage.
Diets supplemented with 0.64% spinach extract for 1. Significant decrease in 2′,7′-dichloro- 37
8 months. fluorescin production in the striatum and
cerebellum.
Topically applied spinach NAO (1% suspension). 1. Prevented formation of lipid peroxidation 38
product, MDA.
2. Significant decrease in lipooxygenase
activity in the epidermis.
Oral administration of spinach NAO (200 mg per kg 1. Significant decrease in plasma lipid 39
per day) for 13 weeks. peroxides/H2O2.
2. No effect on plasma SOD activity.
Subjects consumed a single meal of spinach (294 g), 1. Significantly higher serum oxygen radical 42
serum analyzed 4 h postprandial. absorbance capacity.
2. Significantly higher serum ferric reducing
ability.
3. Significantly higher serum Trolox
equivalent antioxidant capacity.
Subjects consumed freeze-dried spinach powder 1. Decreased DNA strand breaks in blood 43
(10 g per day) for 2 weeks. lymphocytes.
2. No significant reduction of oxidized
pyrimidine bases.
Subjects consumed spinach (150 g per day) for 3 weeks. 1. Significant decrease in lymphocyte DNA 46
damage.
Subjects consumed spinach (20 g per day) as either 1. Significant increase in erythrocyte 47
whole-leaf, minced, or liquefied (via enzymatic glutathione reductase activity.
digestion) for 3 weeks.

Abbreviations: MDA, malondialdehyde; NAO, water-soluble natural antioxidant; ROS, reactive oxygen species; SOD, superoxide dismutase.

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Table 2 Anti-inflammatory properties of spinach

Model Dosage and duration Key effects Ref.

Animal models
Wistar rats challenged with Animals injected with spinach NAO (10 mg per kg 1. Reduced frequency and reduced severity of 49
LPS injection per day) for 8 days. LPS-induced hepatic lesions.
2. Attenuated COX-2-mediated activation of
inflammatory cells.
New Zealand rabbits Animals injected with spinach NAO (10 mg per kg 1. Reduced LPS-related lesions in the liver, 50
challenged with LPS injection per day) for 8 days. thymus, spleen, eyes, and adrenal gland.
Fischer 344 rats Animals were fed diets containing freeze-dried 1. Significant decrease in TNF-α and TNF-β in 57
spinach (0.02% w/w) for 6 weeks. the cerebellum.

Human studies
25 healthy males and females
20 overweight females
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Subjects consumed freeze-dried spinach 1. No significant reduction in C-reactive protein 61
(10.4 g per day) for 8 weeks. levels.
Subjects consumed a high carbohydrate test meal 1. No effect on plasma TNF-α. 88
containing spinach thylakoid (3.7 g or 7.4 g).

Abbreviations: COX-2, cyclooxygenase 2; LPS, lipopolysaccharide; NAO, water-soluble natural antioxidant; TNF-α, tumor necrosis factor-alpha;
TNF-β, tumor necrosis factor-beta.

syl-(1→2)]-β-D-glucopyranoside, and the glucuronated flavonoid
5,3′,4′-trihydroxy-3-methoxy-6:7-methylendioxyflavone-4′-β-D-glucur-
onide demonstrated the highest antioxidant activity in vitro
(Fig. 3A).27,31
The antioxidant properties of spinach and spinach extracts
have been examined in animals. Hyperlipidemic Sprague-
Dawley rats fed a high fat-cholesterol diet supplemented with
5% freeze-dried spinach powder exhibited significantly lower
liver thiobarbituric acid reactive substances (a measure of lipid
peroxidation) and leukocyte DNA damage than the high fat-
cholesterol diet controls.36 Diets supplemented with 0.64%
spinach extract for eight months led to a significant decrease
in 2′,7′-dichlorofluorescin production (a broad marker of oxi-
dative stress) in the striatum and cerebellum when compared
to Fischer rats fed the control diet.37 In a separate study,
topically applied spinach NAO (1% suspension) was more
effective than vitamin E (5% suspension) at preventing MDA
formation and decreasing lipooxygenase activity in the epider-
mis of UV irradiated mice; lipooxygenase catalyzes the for-
mation of reactive oxygen species and pro-inflammatory
leukotrienes from arachidonic acid.38 NAO (200 mg per kg per
day; 13 weeks) also demonstrated in vivo oxidant scavenging
properties when administered orally in TRAMP (Transgenic
Adenocarcinoma of the Mouse Prostate) mice.39 In these
animals, NAO led to a significant decrease (−15%) in plasma
lipid peroxides and H2O2 levels compared to the control
animals.
It should also be noted that spinach NAO is rich in glycosy-
lated derivatives of p-coumaric acid and flavonoids, and the
carbohydrate moieties either interact with epithelial glucose
transporters or undergo β-glucosidase-mediated deglycosyla-
tion prior to absorption.40,41 As could be expected, multiple
factors affect the bioavailability of these polyphenolic com-
pounds, including β-glucosidase activity, diet composition,
and consumption patterns, all of which have been reviewed in
detail elsewhere.40,41 Nonetheless, research examining the
absorption, pharmacokinetics, and biotransformation of
spinach-derived phytochemicals in humans is only beginning.
Future studies should emphasize the postprandial fate of
phytochemicals found in whole spinach and their combined
antioxidant value.
Human intervention studies have also provided some
insight into the antioxidant potential of spinach. Elderly
women (mean age = 66.9 year; n = 7–8) who consumed a
single meal of spinach (294 g) had significantly higher
oxygen radical absorbance capacity (ORAC, +25%), ferric
reducing ability of plasma (FRAP, +24%), and Trolox equi-
valent antioxidant capacity (TEAC, +21%) four hours after the
meal than control subjects not consuming spinach.42 This
antioxidant potential likely stems from the action of non-
protein antioxidants (e.g., polyphenols), as proteins were pre-
cipitated from serum prior to the assay. This notion was con-
firmed in a study by Pool-Zobel et al.43 in which daily
consumption of 10 g spinach powder (dissolved in milk or
water) by healthy male subjects for two weeks decreased DNA
strand breaks in lymphocytes. In this study, the consumption
of spinach powder was associated with a 2-fold increase in
blood plasma lutein levels. As a scavenger of superoxide and
hydroxyl radicals, and inducer of antioxidant enzymes (cata-
lase, superoxide dismutase, glutathione reductase) and gluta-
thione, spinach-derived lutein may protect against oxidants
in blood.44,45 A study conducted in 9 healthy females con-
suming a diet enriched in spinach (150 g per day) for 3
weeks revealed that spinach intake significantly decreased
lymphocyte DNA damage and concomitantly increased serum
lutein by 37%.46 In a larger study of 48 healthy men
and women, spinach (20 g per day for 3 weeks) consumption
as either whole-leaf, minced, or liquefied (via enzymatic
digestion) increased plasma lutein and erythrocyte gluta-
thione reductase activity above that of control group.47
Collectively, these human studies support the notion that
spinach, through lutein, improves oxidative stress via a
mechanism that may involve the upregulation of glutathione
reductase.

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Table 3 Anti-cancer effects of spinach
Model
Cell culture
PC3, human prostate
adenocarcinoma
DU145, human prostate
carcinoma
HepG2, human hepatocellular
carcinoma
HT-29, human colon carcinoma
NUGC-3, human gastric
adenocarcinoma
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HL-60, human promyleocytic
leukemia
Molt-4, human T-Cell acute
lymphoblastic leukemia
Colon-26, mouse colon
adenocarcinoma
A549, human lung epithelium
adenocarcinoma
HeLa, human cervix
adenocarcinoma
BaLL-1, human B-cell acute
lymphoblastoid leukemia
Animal models
TRAMP mice
BALB/c mice with
subcutaneously implanted
Colon-26 carcinoma cells
BALB/c mice with
subcutaneously implanted
S-180 sarcoma cells
BALB/c mice with
subcutaneously implanted
Colon-26 carcinoma cells
BALB/c mice with
subcutaneously implanted
Colon-26 carcinoma cells
Wistar rats fed diets
supplemented with heme
Abbreviations: NAO, water-soluble natural antioxidant; PCNA, proliferating cell nuclear antigen.
Anti-inflammatory properties
Inflammation is a natural immune response to trauma or infec-
tion, that if unabated contributes to the development of serious
chronic diseases including cancer, cardiovascular disease, and
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Dosage and duration
Spinach NAO (0.8, 1.6, and 3.2 mg mL−1)
for 24, 48, and 72 h.
Spinach NAO (0.8, 1.6, and 3.2 mg mL−1)
for 24 or 72 h.
Acetone extracted soluble phenolics from
spinach (1, 5, 10, 20, 30, 40, and 50 mg
mL−1) for 96 h.
Lyophilized spinach leaves (0.5 g FW
mL−1) for 0, 12, and 24 h.
Spinach-derived glycolipid (100 µg mL−1)
or DGDG, SQDG, and MGDG (0, 20, 40, 60,
80, 100 µg mL−1) for 48 h.
Spinach-derived glycolipid fractions
(100 µg mL−1) for 48 h.
Spinach-derived glycolipid fraction (0, 20,
40, 60, 80, 100 µg mL−1) for 24 h.
Spinach-derived MGDG (0, 10, 20, 30, 40 or
50 µg mL−1) for 24 h.
Spinach-derived glycolipid fraction (0, 20,
40, 60, 80, 100 µg mL−1) for 24 h.
Spinach-derived glycolipid fraction (0, 20,
40, 60, 80, 100 µg mL−1) for 24 h.
Spinach-derived glycolipid fraction (0, 20,
40, 60, 80, 100 µg mL−1) for 24 h.
Oral administration of spinach NAO
(200 mg per kg per day) for 13 weeks.
Oral administration of spinach glycolipids
(4 mg kg−1 or 20 mg per kg per day) for
26 days.
Oral administration of spinach glycolipids
(20 mg per kg per day) for 2 weeks.
Oral administration of spinach glycolipids
(20 mg per kg per day) for 2 weeks prior to
colon cell implantation.
Oral administration of spinach glycolipids
(10 or 20 mg per kg per day) for 2 weeks.
Animals were fed diets supplemented with
freeze-dried spinach powder (82 g kg−1
food) for 2 weeks.
type-2 diabetes.48 Current pharmacological therapies for inflam-
mation, while effective, are associated with adverse side effects
(e.g., vomiting, nausea, constipation, dizziness), and thus func-
tional foods are appealing alternatives in the treatment and pre-
vention of inflammation and inflammation-mediated diseases.
View Article Online
Food & Function
Key effects Ref.
1. Dose dependent inhibition of cell proliferation, with 39
IC50 of 2.5 mg mL−1.
2. Halted the cell cycle in the G1 phase.
1. Dose dependent inhibition of cell proliferation, with 39
IC50 of 2.2 mg mL−1.
2. Halted the cell cycle in the G1 phase.
1. Dose-dependent inhibition of cell proliferation, with 20
EC50 of 42.51 mg mL−1.
1. Significant inhibition of cell proliferation. 69
1. MGDG significantly inhibited cell growth in dose- 72,75
dependent manner, with LD50 of 49 g mL−1, halted cell
cycle at the G1 phase, and induced apoptosis.
2. DGDG and SQDG had no effect on cell viability.
1. Significant inhibition of cell proliferation, with LD50 72
of 66.8–70.1 µg mL−1.
1. Significantly inhibited cell growth in dose-dependent 73
manner, with IC50 of 58.5 µg mL−1.
1. Inhibited cell proliferation in dose-dependent manner 74
with LD50 of 25 g mL−1.
2. Halted the cell cycle at the G1 phase.
1. Significantly inhibited cell growth in dose-dependent 73
manner, with IC50 of 81.7 µg mL−1.
1. Significantly inhibited cell growth in dose-dependent 73
manner, with IC50 of 57.2 µg mL−1.
1. Significantly inhibited cell growth in dose-dependent 73
manner, with IC50 of 59 µg mL−1.
1. Improved severity of hyperplasia in the pancreas 39
dorsal and lateral lobes.
2. Significant decrease in blood lipid
hydroperoxides/H2O2.
1. Dose-dependent decrease in tumor growth. 74
2. Significant decrease in number of mitotic cells and
expression of tumor cell proliferation makers, PCNA.
3. Significant increase in tumor cell apoptosis.
1. Decrease in sarcoma tumor growth. 76
1. Decrease in colon adenocarcinoma tumor growth. 76
2. Significant decrease in PCNA expression.
1. Dose-dependent decrease in Colon-26 tumor growth. 73
2. Significant decrease in number of mitotic cells and
expression of tumor cell proliferation makers Ki-67,
PCNA, and Cyclin E.
3. Significant increase in tumor microvessel density.
1. Significant inhibition of heme-induced colon cell 67
hyperproliferation.
2. Significant increase in heme excretion in feces.
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Table 4 Anti-obesity effects of spinach

Model Dosage and duration Key effects Ref.

Animal models
Sprague-Dawley rats Animals were fed diets containing spinach thylakoid (2 mg 1. Decreased food consumption. 82
fed a high fat diet of chlorophyll per g of food) for 13 days.
apoE-deficient mice Animals were fed diets containing spinach thylakoid (6 mg 1. Decreased food consumption. 86
fed a high fat diet of chlorophyll per g food) for 100 days. 2. Decreased body fat and body weight.
Cross-bred pigs fed a Oral administration of spinach thylakoid (0.5 g kg−1) then 1. Significantly higher CCK at 30 min post-OGTT. 95
high fat diet subjected to OGTT. 2. Significant attenuation of ghrelin at 120 min
post-OGTT.
Human studies
11 healthy males and Subjects consumed a high fat meal containing 25 g or 50 g 1. Dose-dependent elevation of CCK at 6 h. 83
females of spinach thylakoid and serum was analyzed 2 and 6 h after 2. Significant increase in leptin at 6 h.
test meal. 3. Significant attenuation of ghrelin at 2 h.
38 overweight females Subjects consumed spinach thylakoid (5 g per day) for 90 1. Significant increase in GLP-1. 85
days. 2. Decreased urge for sweets and chocolate.
3. No effect on total calories consumed, and urge
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for high carbohydrate snacks or salt.

20 overweight females Subjects consumed a high carbohydrate test meal
containing spinach thylakoid (3.7 g or 7.4 g).
60 overweight and Subjects consumed spinach thylakoid (5 g) prior to
obese males and consuming standardized breakfast with serum and food
females consumption measured at 2 h and 4 h after thylakoid
consumption, respectively.
4. Significant reduction in body weight.
5. No effect on fat-free mass, percent body fat,
and waist circumference.
1. Decreased postprandial subjective ratings of 88
hunger, thoughts of food, and urge to eat.
2. Increased plasma CCK at 180 min post-
intervention.
1. Significant reduction in ratings of hunger, 89
longing for food, prospective intake, desire for
salty foods, and thirst.
2. No effect on ratings of fullness, satisfaction,
desire for savory and sweet foods, or food intake
at the next meal.

Abbreviations: CCK, cholecystokinin; GLP-1, glucagon-like peptide-1; OGTT, oral glucose tolerance test.

Table 5 Hypoglycemic effects of spinach

Model Dosage and duration Key effects Ref.

Cell culture
3T3-L1 pre-adipocytes Whole juice and ethanol spinach extracts. 1. 3T3-L1 cell differentiation in the absence of 93
insulin and heightened differentiation in presence of
insulin.
Animal models
Wistar rats with Oral administration of 70% spinach ethanolic extract 1. Significant decrease in plasma glucose levels. 94
alloxan-induced (100 mg per kg per day) for 12 days.
diabetes
Sprague-Dawley Animals were fed diets containing spinach thylakoid 1. Significantly lower plasma insulin levels in a 2 h 84
rats (6 mg of chlorophyll per g of food) for 10 days. OGTT.
2. No effect on plasma glucose levels in 2 h OGTT.
Cross-bred pigs Oral administration of spinach thylakoid (0.5 g kg−1) then 1. Significant decrease in plasma glucose post-OGTT. 95
fed a high fat diet subjected to OGTT. 2. No effect on plasma insulin post-OGTT.

Human studies
11 healthy males and Subjects consumed a high fat meal containing 25 g or 1. Significant reduction in insulin levels. 83
females 50 g of spinach thylakoid and serum was analyzed 2 and
6 h after test meal.
60 overweight and Subjects consumed spinach thylakoid (5 g) prior to 1. Significant increase in 2 h postprandial plasma 89
obese males and consuming standardized breakfast with serum and food glucose concentrations.
females consumption measured at 2 h and 4 h after thylakoid
consumption, respectively.
38 overweight Subjects consumed spinach thylakoid (5 g per day) 5 min 1. Significantly lower postprandial plasma glucose 85
females prior to test meal, and prolonged supplementation for and insulin concentrations at 15 min
12 weeks. 2. No difference in plasma glucose or insulin levels
between the treated and placebo groups after 12
weeks of supplementation.
14 normal weight Subjects consumed boiled spinach (75 g) with a fat-rich 1. No effect on postprandial plasma glucose. 102
and 10 obese men meal.

Abbreviations: OGTT, oral glucose tolerance test.

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Table 6 Lipid-lowering properties of spinach

Model Dosage and duration Key effects Ref.

Animal models
Sprague-Dawley rats fed a
high fat diet
apoE-deficient mice fed a
high fat diet
Wistar rats with alloxan-
induced diabetes
Sprague-Dawley rats fed with
a high fat-cholesterol diet
Animals were fed diets containing spinach thylakoid 1. Significantly lower serum triglycerides. 82
(2 mg of chlorophyll per g of food) for 13 days.
Animals were fed diets containing spinach thylakoid 1. Significantly lower serum triglycerides 86
(6 mg of chlorophyll per g food) for 100 days. and free fatty acids.
Oral administration of 70% spinach ethanolic extract 1. Significant reduction in serum 94
(100 mg per kg per day) for 12 days. triglycerides, and normalized triglycerides to
levels in non-diabetic rats.
Diets supplemented with 5% freeze-dried spinach 1. No difference in serum triglycerides. 36
powder for 6 weeks.

Human studies
11 healthy males and
females
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Subjects consumed a high fat meal containing 25 g or 1. 50 g thylakoids led to significantly lower 83
50 g of spinach thylakoid and serum was analyzed postprandial serum free fatty acids.
2 and 6 h after test meal.

60 overweight and obese
males and females
38 overweight females
14 normal weight and
10 obese men
Subjects consumed spinach thylakoid (5 g) prior to 1. No effect on serum triglycerides or free 89
consuming standardized breakfast with serum measured fatty acids.
at 2 h after thylakoid consumption, respectively.
Subjects consumed spinach thylakoid (5 g per day) for 1. No effect on serum triglycerides. 85
90 days.
Subjects consumed boiled spinach (75 g) with a fat-rich 1. No effect on postprandial serum 102
meal. triglycerides.

The anti-inflammatory properties of spinach have been
evaluated in several animal models. Male Wistar rats that
received a daily dose of spinach-derived NAO (10 mg kg−1; i.p.)
for 8 days and then challenged with a single dose of lipopoly-
saccharide (LPS; 10 mg kg−1; i.p.) displayed (i) reduced fre-
quency and reduced severity of LPS-induced hepatic lesions,
and (ii) attenuated cyclooxygenase-2 (COX-2)-mediated acti-
vation of inflammatory cells (e.g., monocytes, polymorpho-
nuclear leukocytes).49 Similarly, New Zealand rabbits challenged
with LPS (10 mg kg−1, i.p.) after receiving NAO (10 mg kg−1,
i.p.) for 8 consecutive days had reduced LPS-related lesions in
the liver, thymus, spleen, eyes, and adrenal gland.50 It is well
accepted that LPS promotes inflammation through the toll-like
receptor-4 (TLR-4) signaling pathway and downstream acti-
vation of nuclear factor kappa B (NF-κB), which resides quies-
cent in the cytoplasm bound to IκB inhibitory protein (IκB).51
In this process, the binding of LPS to TLR-4 induces the phos-
phorylation and activation of IκB kinase (IKK), which phos-
phorylates IκB. IκB undergoes proteosomal degradation
thereby allowing NF-κB to dimerize and translocate to the
nucleus where NF-κB induces the expression of pro-inflamma-
tory cytokines (interleukin-1, IL-1; tumor necrosis factor-α,
TNF-α), and ROS generating enzymes (COX-2; inducible nitric
oxide synthase, iNOS).52–54 Mechanistically, spinach-derived
NAO may afford protection by inhibiting the cytosolic acti-
vation or DNA-binding activity of NF-κB.55 The aromatic phe-
nolics (including p-coumaric acid) in NAO likely contribute to
this reported anti-inflammatory properties35 In arthritic Wistar
rats, p-coumaric acid (100 mg per kg per day; i.p.) significantly
reduced the expression of TNF-α in synovial tissue.56 In prac-
tice, spinach leaves rather than fractionated parts are con-
sumed, thus it is most appropriate to examine the anti-
inflammatory effects of whole spinach. In so doing, Cartford
et al.57 found that the expression of NF-κB target genes, TNF-α
and TNF-β, were suppressed in the cerebellum of Fischer 344
rats fed a diet containing 0.02% (w/w) dry spinach for 6 weeks.
To date no human studies have evaluated the anti-inflamma-
tory properties of NAO or other spinach-extracts, thus repre-
senting an area for further study.
The anti-inflammatory properties of lutein (Fig. 3B), a caro-
tenoid with high bioavailability, have been investigated in
animal models and in humans. In animals, the administration
of lutein (10 mg kg−1; i.p.) to BALB/c mice subsequently chal-
lenged with LPS revealed that the plasma levels of prostaglan-
din E2 (PGE2), TNF-α, and IL-1β, and the liver abundance of
iNOS and COX-2 enzymes were decreased.58 Studies conducted
in LPS-primed peritoneal macrophages treated with 20 μM
lutein showed that lutein decreased the levels of PGE2, TNF-α,
and IL-1β.58 The authors concluded that, by scavenging ROS,
lutein decreased the availability of H2O2 to induce IKK-depen-
dent activation of NF-κB, and as a result decreased NF-κB
target gene expression. Moreover, in LPS-treated RAW 264.7
cells, lutein attenuated the activation of several NF-κB signal-
ing mediators including Akt, NIK (NF-κB inducing kinase),
IKKα, and IKKβ.58 Spinach also contains notable levels of
β-carotene that is thought to have anti-inflammatory properties
via a mechanism similar to lutein. In gastric epithelial AGS
cells, β-carotene inhibited the H2O2-induced activation of NF-
κB and subsequent expression of the pro-inflammatory chemo-
kine, IL-8.59 Importantly, the bioavailability of β-carotene from
whole leaf, minced, or liquefied spinach is much lower than
lutein and the two carotenoids negatively interact with each
other during intestinal absorption.47,60 As the concentration of
lutein in spinach is much higher than β-carotene, the anti-

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Food & Function
inflammatory mechanism is likely mediated by lutein;
however, it should be further investigated in human interven-
tion studies. Overall, the evidence from animal and cell
studies indicated that lutein ameliorates the inflammatory
response by decreasing the activation of NF-κB, and suggested
that inclusion of lutein-rich foods, like spinach, could sup-
press inflammation.
In humans, a randomized, double-blind, placebo-controlled
trial tested the effects of dried spinach (10.4 g per day) on
inflammation in twenty-five healthy men and women for 8
weeks. Subjects who had consumed dried spinach exhibited a
significant increase in serum lutein; however, the C-reactive
protein (CRP) levels, a marker of inflammation, were
unchanged.61 A link with NF-κB could not be made in that
study because proteins of the NF-κB pathway were not
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determined.
Further research in humans is therefore warranted to ascer-
tain whether the consumption of spinach NAO- or lutein-rich
foods, which attenuates inflammation in animals through
modulation of the NF-κB pathway, is effective clinically particu-
larly in individuals with chronic inflammation or inflam-
mation-mediated diseases.
Anti-cancer properties
Cancer is one of the leading causes of death worldwide with
an estimated 14 million new cases of cancer and 8.2 million
cancer-related deaths in 2012.62 Current projections suggest
the incidence rate will increase by 70% over the next two
decades, further highlighting the critical need to identify safe
and effective strategies to prevent and treat cancer.62 Among
the many factors associated with carcinogenesis, the influence
of diet on hyperplasia is well established. Diets rich in veg-
etables, in particular dark green vegetables, are associated
with a reduced risk for many cancers. Epidemiological studies
have demonstrated a protective role of spinach consumption
in breast, colon, and esophageal cancers. In a case-control
study composed of 6888 cases and 9428 controls, females with
higher intake of raw spinach (>52 servings per year) had a 45%
decreased risk of breast cancer.63 The carotenoid, lutein, was
specifically associated with a decreased risk of breast cancer.64
In a pooled analysis of 14 prospective cohort studies from
North America and Europe, consumption of one serving of
spinach (73 g) per week to one-half serving per day was associ-
ated with an 11% decreased risk of colon cancer.65 Spinach
was the only vegetable, among the 12 vegetables examined in
this study, to demonstrate a significant reduction in colon
cancer risk.65 In a retrospective cohort study of 490 802 partici-
pants recruited for the National Institutes of Health-AARP Diet
and Health Study, the highest tertile of spinach (raw or
cooked) consumption was associated with a significant
reduction in esophageal adenocarcinoma (hazard ratio = 0.66;
95% CI = 0.46–0.95).66
The chemoprotective mechanisms of action of spinach have
been studied in both animal and cancer cell models. From
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this research, we note that in male TRAMP mice, the oral
administration of spinach-derived NAO (200 mg per kg body
weight per day by gavage) improved the severity of hyperplasia
in the pancreas dorsal and lateral lobes.39 The antiproliferative
properties of whole spinach were further addressed in Wistar
rats fed diets supplemented with heme, a hyperproliferative
compound found in red meat. Spinach (82 g kg−1 food;
2 weeks) consumption significantly inhibited heme-induced
proliferation of colon epithelial cells, likely by interfering with
heme metabolism.67 These protective effects were attributed to
the high chlorophyll content of spinach, as chlorophyll alone
was protective against heme-induced hyperproliferation at
levels comparable to that of spinach.67 The regular inclusion
of spinach in the diet may thus be chemopreventive in
humans who frequently consume red meat; however, further
studies are needed to elucidate the mechanism of protection.
Some important insights into the cancer protective mecha-
nisms of spinach have come from cell culture studies. Spinach
extracts elicited dose- and time-dependent antiproliferative
properties in cells lines derived from human prostatic cancer
cells (PC3 and DU145), hepatocellular carcinoma cells
(HepG2), and human colon carcinoma cells (HT-29).20,68,69
Spinach soluble free phytochemicals, extracted using the
organic solvent acetone, proved the most antiproliferative out
of ten vegetable extracts tested in HepG2 cells.20 Spinach
extracts did not induce cellular apoptosis but blocked the G1/S
transition.39,70 Mechanistically, spinach NAO blocked the
association of Cyclin-A with cyclin dependent kinase-2 (CDK-2)
during the G1/S transition by increasing the expression of
cyclin-dependent kinase inhibitor (CDKI) p21cip1 and decreas-
ing the levels of Cylcin-A and CDK-2.70,71 The NAO-induced
suppression of Cyclin A/CDK-2 complex formation, in turn,
decreased the phosphorylation of retinoblastoma (Rb) pro-
teins, which when hypophosphorylated, sequester the tran-
scription factor E2F-1 (required for cell cycle progression) into
repressive complexes.70
Spinach leaves contain large quantities of glycolipids when
compared to green onion, chive, sweet pepper, garlic, or
carrot. These glycolipids include monogalactosyl diacylglycerol
(MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovo-
syl diacylglycerol (SQDG) (Fig. 3D).72 Spinach glycolipids
exhibited antiproliferation activity in multiple cancer cell lines
derived from various tissues including NUGC-3 (gastric
cancer), HL-60 (leukemia), A549 (lung cancer), HeLa (cervix
cancer), BALL-1 (B-cell acute lymphoblastoid leukemia), Molt-4
(T-cell acute lymphoblastic leukemia), and Colon-26 (colon
cancer) cells.72,73 In these cells, glycolipids decreased the
expression of tumor cell proliferation markers Ki-67, PCNA,
and Cyclin E, and halted the cell cycle at the G1 phase causing
apoptosis.73–75 SQDG dense fractions were anti-proliferative by
blocking the replicative activity of purified DNA polymerase-α
in vitro (IC50 = 41.0 μg ml−1).72 Purified MGDG also demon-
strated a marked ability to inhibit replicative DNA poly-
merases, without affecting the repair activity of the enzymes.75
In animals, BALB/c mice subcutaneously implanted with
Colon-26 carcinoma or S-180 sarcoma cells and subsequently
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administered spinach glycolipids orally (20 mg per kg body
weight) exhibited a 56.4% decrease in colon adenocarcinoma
tumor growth or a 90% decrease in sarcoma tumor volume,
respectively, compared to vehicle control mice.73,76 Animals
that were pretreated orally with glycolipids (20 mg per kg per
day) for 2 weeks and subcutaneously implanted with Colon-
26 cells showed a 48.9% reduction in tumor growth when
compared to vehicle controls.76 Some uncertainties remain
regarding the anticancer properties of dietary glycolipids in
light of reports indicating that glycolipids are degraded in
the GI tract and thus would not be absorbed as intact
glycolipids.77–79 Further studies examining the absorption of
spinach glycolipids are needed to determine if intact glyco-
lipids, their digestion products, or a combination of both are
responsible for the anticancer properties in the GI tract and
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extra-gastrointestinal tissues. Taken together, there is some
evidence that spinach-derived glycolipids have anti-prolifera-
tive properties in established tumor cells by blocking DNA
replication. With regards to spinach phytochemicals and
NAO, the data support the notion that they prevent cancers
through antioxidant mechanisms and disruption of mito-
genic signaling pathways.
While most studies that examined the protective effects of
spinach on cancers were conducted in rodent and cell models,
the epidemiologic data available suggest that spinach
decreases the risk of esophageal, colon, and breast cancers in
humans. However, there is a critical need for human interven-
tion studies aimed at delineating the anticancer properties of
spinach and its constituents.
Anti-obesity properties
While obesity statistics are staggering – approximately
80 million American adults and over 300 million adults world-
wide are obese (BMI ≥ 30 kg m−2) – short-term projections are
expected to remain high despite numerous public health
initiatives to curb the epidemic.80,81 Opposing the epidemic,
lifestyle modifications, which include diet, exercise and behav-
ioral therapy, are the cornerstone approaches implemented by
the medical community to control obesity. On the margins of
this arena, there is growing interest in the use of nutraceuticals
to induce satiety and suppress orexigenic signals as a means to
decrease calorie intake. A patented extract of spinach contain-
ing mostly thylakoids has been shown to induce satiety in
humans and lower food intake in animals.82–84 One hundred
grams of spinach thylakoid reportedly contain 23.5 g protein,
11.9 g fat, 41.7 g carbohydrate, 3.5 g salt, 3000 mg chlorophyll,
27.9 mg lutein, 730 µg zeaxanthin, 4760 µg β-carotene, 21 µg
vitamin A, 1313 µg vitamin K, 6.07 mg vitamin E and 166 µg
folic acid.85 Female Sprague-Dawley rats fed a high-fat diet sup-
plemented with thylakoids consumed significantly less food
(ca. −30 kcal per day) over the 13-day study, and had signifi-
cantly lower body weights (−18%) when compared to the
control rats.82 In a longer trial (100 days), thylakoid sup-
plementation lowered food consumption, body fat (−33%) and
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body weight (−27%) in female apoE-deficient mice.86 Mechan-
istically, it is suggested that thylakoids slow down the diges-
tion and absorption of dietary fat through the physical
interaction of the hydrophobic trans-membrane helices of thy-
lakoid membranes with the lipase/colipase complex, and in so
promote satiety because post-prandial high-fat content in the
small intestinal lumen lessens hunger and stimulates satiety
signals.87
In healthy humans, Köhnke et al.83 demonstrated that con-
sumption of a meal containing 0 g, 25 g, or 50 g thylakoids
caused a dose-dependent elevation of the gut-derived satiety
hormone cholecystokinin (CCK) 6 hours after the test meal.
The release of CCK from duodenal enteroendocrine cells was
likely increased in response to the thylakoid membrane
content of the chyme.83 In a separate study, levels of the enter-
oendocrine-derived satiety hormone glucagon-like pepetide-1
(GLP-1) was significantly elevated (+44%) postprandially on
day 90 in thylakoid-treated (5 g per day) overweight women.85
Similarly to CCK and GLP-1, the adipose-derived satiety
hormone leptin was significantly increased 6 hours following
the consumption of a meal containing 25 g or 50 g thylakoid.83
Serum ghrelin, a stomach-derived hunger hormone, was sig-
nificantly lower in humans 2 hours after the consumption of a
thylakoid-enriched meal and returned to baseline at 6 hours.83
The heightened secretion of satiety hormones led to signifi-
cant decreases in subjective measures of appetite. In this
regard, the inclusion of thylakoids (3.7 g or 7.4 g) in a meal
significantly decreased (i) postprandial ratings of hunger, (ii)
thoughts of food, and (iii) urge to eat in healthy overweight
women.88 When consumed for 12-weeks, thylakoids (5 g per
day) resulted in lower urge for sweets and chocolate in over-
weight women (n = 38) at study completion; however, total cal-
ories consumed and urge for high carbohydrate snacks or salt
did not differ between groups.85 In contrast, the satiety action
of spinach thylakoids proved to be weak in a larger, double-
blind, placebo-controlled randomized cross-over intervention
study with 60 overweight or obese participants. In this study,
food intake at the next meal (i.e., 4 hours after the first meal)
did not differ between the placebo and thylakoid (5 g)
groups.89
The effects of thylakoid-supplementation on anthropo-
metric parameters of body composition were examined in over-
weight women (n = 38) in a 12 week single-blinded,
randomized, placebo-controlled intervention study.85 Subjects
that consumed 5 g thylakoids per day experienced a 6.3% sig-
nificant reduction in body weight compared to the placebo
group.85 However, changes in fat-free mass, percent body fat,
and waist circumference did not differ between the two
groups.85
Overall, there is evidence to suggest that dietary supplemen-
tation of spinach-derived thylakoids is useful to control
interim hunger, calorie intake, and weight gain in healthy
individuals through the modulation of CCK, GLP-1, and
ghrelin secretions, but the relevance of these effects in longer
clinical trials, focusing on the obese, should be matter of
further investigation.
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Hypoglycemic activity
As one of the clinical manifestations of metabolic syndrome
and a risk factor for developing type-2 diabetes, hyperglycemia
(elevated blood glucose levels) is highly prevalent worldwide
and in the U.S. population.90 In 2014, 422 million adults
worldwide91 and 29 million Americans had diabetes (≥126 mg
fasting plasma glucose per dl).92 An additional, 86 million
Americans suffer from impaired fasting blood glucose
(100–125 mg fasting plasma glucose per dl) and thus are con-
sidered pre-diabetic.92 These alarming statistics highlight the
need to find new dietary and therapeutic strategies to better
control glycemia and lessen insulin resistance.90 In this
regard, spinach and spinach-derived compounds were shown
to improve insulin sensitivity and in turn plasma glucose.
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Evidence from cell culture experiments support the insulin-
like and insulin-sensitizing actions of spinach extracts. Park
et al.93 investigated the effects of fresh spinach either pro-
cessed as juice or extracted with ethanol on the differentiation
of 3T3-L1 pre-adipocytes. Adipocyte differentiation was chosen
to illustrate the critical role of insulin-mediated glucose
uptake via GLUT4 in this process. Park et al.93 found that both
spinach juice and ethanol extract induced 3T3-L1 cell differen-
tiation in the absence of insulin, and even more so in the pres-
ence of insulin. Moreover, spinach, as whole juice or ethanol
extract, was inhibitory towards the digestion of disaccharides
(−19.6%) by intestinal α-glucosidase in vitro;93 an effect that
may help improve glycemia by lowering the uptake of diet-
derived glucose.
The hypoglycemic action of spinach-derived compounds
has been examined in multiple animal studies. Using Wistar
rats dosed with alloxan monohydrate (to permanently destroy
pancreatic β-cells and cause insulin deficiency) and sub-
sequently administered orally a daily dose of 70% spinach
ethanolic extract (100 mg per kg body weight) for 12 days,
Kumar and Loganathan94 observed a significant lowering of
plasma glucose levels (152 mg dl−1) in treated rats when com-
pared to the control rats (250 mg dl−1). In crossbred pigs fed a
high-fat diet, Montelius et al.95 found that a single dose of
spinach thylakoid (0.5 g per kg body weight) was sufficient to
suppress blood glucose following an oral glucose tolerance test
(OGTT); however no differences in insulin levels were
observed. In a longer study, Sprague-Dawley rats fed spinach
thylakoid for 10 days had significantly lower plasma insulin
levels in a 2-hour OGTT when compared to the control diet
devoid of thylakoid.84
In the clinical setting, thylakoids were effective at modulat-
ing the postprandial insulin response in healthy men and
women.83 In this study, subjects who consumed a meal con-
taining 25 g or 50 g of thylakoids had significantly reduced
insulin levels even though the test meal contained more
protein and carbohydrate than the control meal.83 Attenuation
of the insulin response likely stems from decreased glucose
uptake, as thylakoids were shown to decrease transport of
methyl-glucose across the brush border of rat intestinal seg-
ments in vitro by binding to intestinal mucosa and decreasing
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intestinal permeability.96 In overweight and obese men and
women, consumption of a meal containing 5 g of thylakoids
led to significantly higher 2 h postprandial plasma glucose
concentrations than the placebo-containing meal.89 The un-
expected heightened plasma glucose concentration might have
resulted from attenuated insulin secretion, however, the post-
prandial insulin response was not measured in this study and
will require further investigation. In a separate study, over-
weight women who consumed 5 g of thylakoids five minutes
prior to consuming a test meal exhibited a significantly lower
postprandial plasma glucose and insulin concentrations at
15 minutes, and four hours later insulin concentrations con-
tinued to trend lower.85 If daily thylakoid supplementation was
prolonged for 12 weeks in the same individuals, results
showed that plasma glucose or insulin levels did not differ
between the treated and placebo groups.85 Collectively, these
studies suggest that thylakoids may be beneficial in controlling
postprandial glycemia and insulinemia by interfering with
glucose absorption and attenuating insulin secretion, but the
effects may be short lived.
Lipid-lowering properties
High blood triglyceride (also termed hypertriglyceridemia) is
clinically defined as fasting blood triglyceride levels above
150 mg dl−1 (>1.69 mM), and considered a significant risk
factor for pancreatitis, liver disease, and cardiovascular
disease.97 Hypertriglyceridemia is highly prevalent in develo-
ped countries, including the United Kingdom (27.5%),100
France (27%),98 South Korea (25%),99 and the United States
(24%),90 but it is also a public health concern in low-and
middle-income countries such as India (47%).101 Weight loss
and exercise, dietary supplementation with fish oil or niacin,
and drug intervention are conventional approaches to correct
hypertriglyceridemia. Spinach, in particular spinach thylakoids
and phytochemicals, may offer an alternative to these
strategies.
Thylakoid membranes isolated from spinach leaves have
been shown to lower blood lipids. Men and women consuming
a meal containing 50 g thylakoids had significantly lower
serum free fatty acids postprandially (ca. −25%) when com-
pared to the subjects not consuming spinach thylakoid.83
Similar results were observed in two animal models, Sprague-
Dawley rats and apoE-deficient mice, in which thylakoid-
enriched high-fat diets significantly lowered serum triglycer-
ides (−39%;82 ca. −25%86) and free fatty acids (ca. −15%86).
The lipid lowering properties of spinach phytochemicals were
examined in a rodent study, in which alloxan-induced type-1
diabetic Wistar rats were treated with a daily dose of 70% etha-
nolic spinach extract (100 mg per kg body weight, oral). In this
study, the rats treated with spinach extract exhibited a 62.3%
reduction in serum triglycerides when compared to control
diabetic rats.94 Spinach extract normalized plasma triglycer-
ides in diabetic rats to the levels observed in non-diabetic rats
(83 mg dl−1).94 The efficacy of whole spinach supplement on
Food Funct., 2016, 7, 3337–3353 | 3349

Review
serum lipids was also examined using Sprague-Dawley rats fed
a high fat-cholesterol diet (9.5% fat) supplemented with 5%
freeze-dried spinach powder for 6 weeks.36 Animals in the
spinach group exhibited an 11% reduction in serum triglycer-
ides compared to the control, however the decrease did not
reach statistical significance.36
Although animal studies revealed the potential of spinach-
derived thylakoids and phytochemicals to lower blood trigly-
cerides, the demonstration of potency in humans has not been
forthcoming. In humans, one-time supplementation of thyla-
koids (5 g) did not alter serum free fatty acids89 or triglycer-
ides.85,89 Similarly, consumption of boiled spinach (75 g
spinach per meal) did not significantly change postprandial
blood triglycerides in healthy weight or obese subjects who
consumed a fat-rich meal (25 g butter and 170 g white
Published on 22 June 2016. Downloaded on 6/8/2019 12:11:56 AM.
bread).102 However, no study has evaluated the effects of
boiled spinach vs. raw spinach on postprandial blood triglycer-
ides. Since cooking by means of boiling alters the composition
of spinach, it is possible that boiling altered the content
and/or activity of the lipid-lowering compounds in the leaves.
Given the scarcity of data, the effects of spinach and its con-
stituents on blood lipids should be matter of further
examination.
Conclusions
Spinach is a widely available green leafy vegetable, a relevant
source of vitamins and minerals, and also phytochemicals and
bioactive compounds that as of yet have poorly defined health-
promoting properties. Most prevalent chronic diseases have an
etiologic basis in oxidative stress and inflammation, and in
this context spinach has the documented potential to play a
role in the remediation of these conditions by (i) quenching
ROS, (ii) lowering oxidative damage, and (iii) inhibiting NF-κB
activation and ensuing proinflammatory markers. Epidemiolo-
gical studies revealed the chemoprotective and chemopreven-
tive effects of spinach consumption. Studies in cells and
animals uncovered some aspects of the chemoprotective
action of spinach extracts through cell cycle arrest and DNA
polymerase inhibition. Human and animal studies also
support the potential of spinach-derived compounds to
improve several components of the metabolic syndrome, i.e.,
obesity, hyperglycemia, and hypertriglyceridemia. From a
mechanistic viewpoint, spinach constituents would act
through induction of satiety hormones, insulin sensitizing
activity, and interaction with lipid and carbohydrate digestive
enzymes. Importantly, spinach-derived NAO, glycolipids, and
thylakoids were not associated with adverse side effects and
were deemed safe at doses utilized in the reviewed studies;
reinforcing the potential utility of spinach-derived compounds
in the prevention and treatment of chronic diseases. In spite
of its reputation as a health promoting food, worldwide
spinach consumption remains low especially in comparison to
other green, leafy vegetables. Although the 42-year trend
(1971–2013) suggests that the inclusion of spinach in the
3350 | Food Funct., 2016, 7, 3337–3353
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Food & Function
American diet will continue to increase, the overall consump-
tion will most likely remain low compared to lettuce. Yet,
adding spinach in everyday cooking and salads or partly sub-
stituting spinach for lettuce would increase exposure to the
phytochemicals and bioactives reviewed herein, and likely lead
to positive health outcomes.
Further research is encouraged to address certain gaps in
knowledge, with particular focus on the health benefits of
spinach leaves. While multiple studies reviewed here examined
some of the isolated constituents of spinach (e.g., NAO, glyco-
lipids, thylakoids), few studies have focused on the therapeutic
potential of spinach leaves. This raises questions about the
bioavailability of spinach phytochemicals and bioactives, and
the effective doses that can be obtained from whole leaves.
Metabolomic fingerprinting offers a unique opportunity to
study the systemic effects following spinach consumption, and
would provide a comprehensive picture of the biological pro-
perties of spinach. Additionally, a majority of the studies pre-
sented in this review utilized cell and animal models.
While these studies are highly informative, few studies still
have investigated spinach consumption and disease pro-
gression in humans. Future intervention studies should
address the functional properties reviewed above in subjects
with inflammation, cancer, obesity, hyperglycemia, or hypertri-
glyceridemia to inform the clinical effectiveness of spinach
and it’s bioactives. This area of research is critical because it
would inform on the role and place spinach should have in
the contemporary human diet.
Acknowledgements
Support was received from the University of Nebraska–Lincoln
Hatch Act, the Tobacco Settlement Funds, and the Layman
Seed Award.
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