Review pubs.acs.org/JAFC
Berries: Anti-inflammatory Effects in Humans Shama V. Joseph,† Indika Edirisinghe,† and Britt M. Burton-Freeman*,†,§ †
Center for Nutrition Research, Institute for Food Safety and Health, Illinois Institute of Technology, Bedford Park, Illinois 60501, United States § Department of Nutrition, University of California, Davis, California 95616, United States ABSTRACT: A sustained pro-inflammatory state is a major contributing factor in chronic disease development, progression, and complication, including the most commonly known diseases: cardiovascular disease, Alzheimer’s, and type 2 diabetes. Fruits, such as berries, contain polyphenol compounds purported to have anti-inflammatory activity in humans. Among the most notable polyphenols in berries are anthocyanins, responsible for their distinctive colors of red, blue, and purple. Berries have been studied widely for their antioxidant properties; however, preclinical data suggest important effects on inflammatory pathways. Correspondingly, the effects of berries, including extracts and purified anthocyanins, have been the subject of a number of human trials. This review aims to evaluate the current state of the human science on berry (products) as a source of dietary polyphenols, particularly anthocyanins, to modulate inflammatory status. Identifying dietary strategies that manage the modern-day inflammatory burden has important implications for chronic disease risk reduction and informing dietary guidelines aimed at achieving and maintaining health. KEYWORDS: berries, polyphenols, anthocyanins, inflammation, postprandial, clinical trials
■
Over the past decade or more, the “antioxidant” hypothesis has been the prevailing wisdom of how polyphenols/ flavonoids/anthocyanins impart their health benefits; however, this view has been under considerable scrutiny in recent years as research has revealed numerous other biological activities of these important plant components, among them antiinflammatory activity.40−44 In recent years several human studies investigating the effects of berries have been published, including a variety of reviews; however, none have reviewed the literature of berry intake by humans and inflammation. With this background, the goal of the present review was to identify and discuss published human clinical research investigating the effects of berry intake and berry extract supplementation on endpoints of inflammation. Clinical investigations were identified in Medline with PubMed searches on the following keywords: “berries” (including names of specific berries, such as “blueberry”, “strawberry”, “chokeberry”), “polyphenols”, “anthocyanins”, and “flavonoids”, in association with “inflammation”, “humans”, “clinical trial”, “IL-6”, “TNF-α”, “CRP”, “adhesion molecules”, “chronic disease”, “cardiovascular disease”, “diabetes”, and “postprandial”. On the basis of search criteria, 34 published studies investigating berries and inflammatory-associated endpoints were identified as of August 15, 2013 (including online-only publications), 3 of which had either multiple berry products or design (i.e., acute and chronic) investigations within a single publication and were discussed and counted separately. Only studies that used single berries or combinations of berries or were controlled to assess the “berry”
INTRODUCTION
Inflammation is a major contributing factor in the development of noncommunicable diseases (NCD) such as cardiovascular diseases (CVD), type 2 diabetes, cancer, and Alzheimer’s disease.1,2 Excess body fat, particularly central adiposity, is associated with a concomitant and persistent increase in lowgrade inflammation. Consumption of meals typical of the Western diet, that is, meals that are energy dense and provide a surplus of readily available carbohydrates and fat, induces an acute inflammatory stress in both healthy weight and overweight individuals.3−8 In contrast, population studies indicate that diets rich in fruits and vegetables are inversely associated with inflammatory stress.6,9−11 Likewise, higher intakes of fruits and vegetables are associated with lower prevalence of cancer, CVD, type 2 diabetes, and Alzheimer’s disease.12−21 Fruits and vegetables are rich sources of essential vitamins and minerals; however, they also contain an array of nonessential biologically active components, such as phenolic acids and polyphenols. In recent years, the health benefits of (poly)phenols have attracted the interest of the research community, food industry, and lay public as evidenced by an increasing number of publications and the rapid growth of the “functional” foods market, respectively.22−25 Polyphenols are a large group of phytochemicals found ubiquitously in the plant kingdom. Flavonoids are a major subclass of polyphenols and can be found in a variety of foods such as fruits,26 vegetables,27 nuts,28 wine,29 cocoa,30 soybeans,31 and olive oil.32 Berry fruits contain appreciable amounts of flavonoids, particularly anthocyanins, which are responsible for the distinctive red-blue-purple coloring of berries. A diet rich in plant flavonoids is associated with a lower risk of chronic disease development and specifically CVD mortality in men and women.22−24,33 Moreover, a higher intake of diet-derived anthocyanins is associated with lower risk of hypertension,34,35 myocardial infarction,36 type 2 diabetes,37 and cancer.38,39 © 2014 American Chemical Society
Special Issue: 2013 Berry Health Benefits Symposium Received: Revised: Accepted: Published: 3886
September 30, 2013 January 21, 2014 February 11, 2014 February 11, 2014 dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
Journal of Agricultural and Food Chemistry
Review
resulting in increased expression of genes, many of which are inflammation mediators.6 Hence, elevations in inflammatory molecules in the postprandial state are often accompanied, and perhaps preceded by, increases in oxidative stress markers.
effect in mixed fruit/mixed dietary polyphenol formulation were included in this review. Four studies were not included due to mixed fruit preparations45−47 or mixed polyphenol sources (i.e., berry plus tea).48
■
■
INFLAMMATION − GENERAL OVERVIEW Inflammation is the normal, protective, and (usually) temporary response of the innate immune system to pathogens and injury. However, with recurrent stimuli or inefficient regulation, chronic inflammation ensues. Quantifiable inflammatory responses are characterized by the production of proinflammatory molecules and anti-inflammatory cytokines acting as signals between immune cells to coordinate the inflammatory response. Cytokines are immune-modulatory molecules that control the inflammatory response. Nuclear factor kappa B (NF-κB), a redox-sensitive transcription factor, is a central orchestrator of the inflammatory response. Once NF-κB is activated, it stimulates the expression of a number of genes including those responsible for the production of cytokines (i.e., tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β), chemokines (i.e., monocyte chemoattractant protein (MCP)-1), adipokines (i.e., leptin, adiponectin), cell adhesion molecules (i.e., E-selectin, P-selectin, soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble intercellular adhesion molecule-1 (sICAM-1)), and acute phase proteins (i.e., (hs) C-reactive protein (CRP), fibrinogen).49 Other important mediators of inflammation include pattern recognition receptors (PRR) such as Toll-like receptors (TLRs) and kinases such as mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases (JNK). The inflammatory response can be triggered by stimuli such as endotoxin (lipopolysaccharide from bacteria), viruses, and changes in levels of reactive oxygen species (ROS), cellular redox status, fatty acids, cytokines, growth factors, and carcinogens among others.
MEASURING INFLAMMATORY STATUS Several biomarkers have been identified as appropriate indicators of inflammatory status in humans. Circulating concentrations of inflammatory molecules, such as acute phase proteins, cytokines, adipokines, and adhesion molecules, are the main measures used in research as well as in some clinical practices. Gene expression of transcription factors, receptors, and protein kinases in immune cells such as monocytes and macrophages help to identify mechanisms and pathways that are either stimulated or inhibited under varying conditions resulting in a particular inflammatory outcome. The various (pro-)inflammation markers that are measured in humans include the acute phase proteins (CRP, fibrinogen, serum amyloid A); cytokines and chemokines (TNF-α, IL-6, IL-1β, MCP-1); and adhesion molecules (E-selectin, P-selectin, sVCAM-1, sICAM-1). Anti-inflammatory markers that are measured and may be suppressed in inflammation include adiponectin and IL-10. Commonly measured molecular targets for assessing inflammation include suppressor of cytokine signaling (SOCS) and NF-κB.57 In general, modulation of these responses, negatively or positively, acutely or sustained under the various circumstances is measurable. Healthy individuals define fluctuations within a “normal” range and demonstrate a relative homeostatic flexibility. Assessing the effects of nutrition/ nutritional components on inflammation/inflammatory status has been suggested to include examining patterns of change in inflammatory markers against a reference range for “normal” or prevention of modulation induced by other factors (such as poor diet) and modulation from a less favorable range to a more favorable range.58 Other effects may include modulation from outside the reference range back into the reference range or modulation from within the range to outside the range. Even with this background, the study of nutrition and inflammation in chronic diseases (NCD) development/prevention/management is relatively new. Continued attention to the field and reviews such as this will help advance knowledge and constructive design of future research.
■
LIFESTYLE CONTRIBUTORS TO INFLAMMATION In addition to classic inflammatory stimuli (i.e., bacteria), inflammatory stress can also result from excess body fat and poor diet. Obesity is well recognized as a chronic, low-grade inflammatory state. Hotamisligil et al. first reported a positive relationship between adipose mass and expression of the proinflammatory gene TNF-α.50 Nowadays, it is well recognized that adipose tissue is much more than a static depot of fat but rather a dynamic secretory tissue that produces a large number of hormones and cytokines.51 In obesity, morphological changes in adipocytes result in altered secretory responses favoring an inflammatory state. Expanding adipose tissue mass recruits macrophages that produce pro-inflammatory proteins such as TNF-α, IL-6, and MCP-1 contributing further to the pro-inflammatory state. Elevated circulating inflammatory proteins are observed in obesity and explain the critical link between obesity and the development of insulin resistance, type 2 diabetes, and CVD.52 In addition to excess body adiposity, excess energy intake and poor dietary composition typical of Western eating patterns promote acute (postprandial) as well as cumulative sustained inflammatory responses in both obese and normal weight individuals.53−56 The postprandial period is a time of active oxidative metabolism and formation of ROS. Excess energy intake stimulates overproduction of ROS, resulting in metabolic oxidative stress and cellular redox imbalance. This change in a cell’s redox status activates redox-sensitive signaling molecules, such as NF-κB, JNK, and other stress-signaling molecules, ultimately
■
BERRIES, ANTHOCYANINS, AND BIOLOGICAL EFFECTS The botanical definition of a berry is different from the average consumer’s definition of “berries” in the marketplace. Botanically, berries are defined simply as fleshy fruit produced from a single ovary.59 In common English terms, “berries” are usually described as small edible fruits that are brightly colored in shades of red-blue-purple and do not have a pit, although they may contain seeds. Consumers typically identify strawberries, blueberries, blackberries, and raspberries as “berries”, but only blueberries are true berries according to the scientific definition of a berry. Despite the incongruent definitions, one unifying trait among “berries” (with few exceptions) is that they contain anthocyanins, which are pigment polyphenol compounds that give them their distinctive color. Anthocyanins constitute a significant percentage of berry polyphenols. Anthocyanins are a type of flavonoid; they are glycosylated polyhydroxy and polymethoxy derivatives of flavilium salts and possess a characteristic C6−C3−C6 carbon 3887
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
Journal of Agricultural and Food Chemistry
Review
Figure 1. Molecular structures of berry flavonoids. Reprinted with permission from ref 61. Copyright 2009 Hindawi Publishing Co.
Table 1. Major Anthocyanin (Polyphenols) in Selected Berries berry
botanical name
bilberry black currant black raspberry blueberry
Vaccinium myrtillus Ribes nigrum Rubus occidentalis Vaccinium corymbosum
chokeberry cranberry sea buckthorn berry strawberry wolfberry
Aronia melanocarpa Vaccinium macrocarpon Hippophae rhamnoides Fragaria × ananassa Lycium barbarum
major anthocyanins
ref
delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, malvidin-3-O-glucoside cyanidin-3-O-glucoside, -rutinoside; delphinidin-3-O-glucoside, -rutinoside cyanidin-3-O-glucoside, -2G-xylosylrutinoside, -rutinoside delphinidin 3-O-galactoside, -arabinoside; malvidin 3-O-galactoside, petunidin 3-O-galactoside cyanidin-3-glucoside, -galactoside, -arabinoside; cyanidin-3-xyloside peonidin 3-O-galactoside, -arabinoside; cyanidin 3-O-galactoside isorhamnetin-3-rutinoside, -galactoside, -glucoside; trace amounts of delphinidin, cyanidin, and peonidin pelargonidin; pelargonidin 3-O-glucoside; cyanidin 3-O-glucoside no documented anthocyanins
structure (Figure 1).60 The most commonly identified anthocyanins in berries are cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin. In addition to anthocyanins, berries contain several other (poly)phenolic compounds that may contribute to their anti-inflammatory potential, such as flavonols, phenolic acids, proanthocyanidins, and ellagitannins. Anthocyanins are uniquely characterized by the presence of an oxonium ion on the C ring and are highly colored (Figure 1).61 Selected details of the anthocyanin content of berries are presented in Table 1. Estimates of anthocyanin intake vary widely within and between populations as does the composition of anthocyanin intake, which is based on food and beverage selection. In France, anthocyanin intake is estimated to be ∼57 mg/day,62 and in Finland, an average intake of 47 mg/day of anthocyanins has been estimated.63 Wu et al. estimated that the daily consumption of anthocyanins in the United States in 2001− 2002 from fruits, vegetables, and beverages was 12.5 mg/day.64 Of the total anthocyanins consumed/day, almost 70% was accounted for by fruit intake (24 g of fruit contributing ∼8.7 g of anthocyanins). Apples (‘Red Delicious’) dominated the percent of fruit intake (∼50% of anthocyanin-containing fruit),
Mäaẗ tä-Riihinen (2004) Kapasakalidis (2006) Wang (2009) Cho (2004) Wu (2004) Zheng (2003) Mishra (2008) Wang (2002) Potterat (2010)
yet apples contributed only 8% of anthocyanin intake. In contrast, berries were only ∼13.5% of the fruit intake, but contributed ∼56% of the fruit-derived anthocyanins. Americans currently eat about half the dietary recommendation for fruit (1 cup equivalent/ day), and on the basis of these data, fruit variety, particularly of the red-blue-purple fruits, appears to be limited.65,66 Berry research has traditionally focused on their antioxidant properties. Berries (or their extracts) rank highly on in vitro antioxidant measures, such as oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant capacity (FRAP) analyses,67−69 and have been shown in various in vitro assay systems to mitigate oxidative stress.70−72 Polyphenols, including anthocyanins, have antioxidant capability; however, biologically, several studies suggest that their effects in vivo are not through antioxidant scavenging properties. Current dogma suggests polyphenols work via activation/inhibition of various cell signaling processes such as modulating kinase activity resulting in transcription factor activation (e.g., Nrf-2, NF-κB), altering receptor activation (PRR dimerization) as well as possible direct ligand activity (e.g., PPAR-γ).73,74 Berry extracts and anthocyanin preparations have been shown in various in vitro and in vivo animal models to affect specific steps in cell 3888
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
Journal of Agricultural and Food Chemistry
Review
Figure 2. Schematic representation of link between antioxidative and anti-inflammatory potential of dietary polyphenols. ROS, reactive oxygen species; OH•, hydroxyl radical; O2−•, superoxide radical; AP-1, activator protein-1; NF-κB, nuclear factor-kappa B; TNF-α, tumor necrosis factoralpha; IL, interleukin.
Black Currant. Black currants are native to parts of Europe and Asia and are an excellent source of vitamin C in addition to anthocyanin polyphenols. The major anthocyanins found in black currants include cyanidin-3-O-glucoside, cyanidin-3-Orutinoside, delphinidin-3-O-glucoside, and delphinidin-3-Orutinoside.91 The rutinosides are reported to be absorbed intact and, following ingestion, reach peak concentrations in circulation 1−2 h after intake.92 Black currant has been studied previously as part of mixed-fruit formulations; however, one study was identified that assessed markers of vascular inflammation after consumption of black currant fruit exclusively.93 Twenty healthy men and women drank a black currant (20% juice) or placebo juice devoid of polyphenols and vitamin C. No significant effects were observed on markers of (vascular) inflammation, sVCAM-1 or sICAM-1, 3 h after beverage intake.93 No additional inflammation associated data were available; hence, the anti-inflammatory effects of black currant in humans are inconclusive. Blueberry. Blueberries are well-known for their antioxidant effects in vitro and in vivo.94 In the past few years, data on blueberry consumption and inflammatory status have emerged, although results have been inconsistent. Four long-term (6−8 weeks) interventions showed no effect of blueberry supplementation on selected inflammatory markers,95−98 whereas one acute study reported significant improvements in inflammatory status attributable to blueberry intake in an exercise model.97 Basu and colleagues95 administered freeze-dried blueberry powder to 48 individuals with metabolic syndrome (MetS) for 8 weeks. The daily dose of 50 g of blueberry powder (equivalent to 350 g of the fresh fruit) was provided as a waterbased drink divided into two beverages per day. At the end of the intervention, blueberry intake did not elicit significant changes in fasting plasma hs-CRP, IL-6, adiponectin, or adhesion molecules (sICAM-1 and sVCAM-1) compared to the plain water control. Stull et al. conducted a similar chronic dosing of blueberries (45 g of freeze-dried blueberry powder per day for 6 weeks) in 32 obese men and women with insulin resistance.96 Blueberry powder (daily amount equivalent to 2 cups of fresh fruit) was blended into a yogurt plus milk beverage. At the end of the double-blinded, parallel arm intervention, there were no differences in changes in circulating hs-CRP, TNF-α, or MCP-1 in the blueberry group compared to the control. In a separate study of males presenting with at
signaling pathways that are known to be involved in chronic disease initiation and development75−82 including anti-inflammatoryspecific effects.83−88 Recent in vitro work with red wine and grapes has also shown anti-inflammatory effects that authors attribute to their specific anthocyanin content.89,90 During inflammation, cells involved in the inflammatory process, which are recruited to the damage site, take up oxygen and release ROS. In addition, inflammatory cells secrete cytokine and chemokine cell mediators, which help to further recruit inflammatory cells, generating yet more ROS. As a result, transcription factors such as NF-κB and activator protein-1 (AP-1) that encode pro-inflammatory genes are activated, leading to an increased secretion of cytokines. The presence of this vicious cycle supports a sustained environment of oxidative and inflammatory stress, which contributes to several chronic diseases. Dietary polyphenols are known to act as both antioxidants and anti-inflammatory molecules, thereby conferring considerable potential protective effects against development of inflammation-related chronic diseases (Figure 2). Overall, the preclinical literature suggests a role for berries in modifying inflammatory status, an effect that may be due to their polyphenol content in general or anthocyanins in particular. A major question that remains is whether these effects translate to humans.
■
EFFECTS OF “COMMON” BERRIES AND BERRY PRODUCTS ON INFLAMMATORY MARKERS IN HUMANS Berries that are readily available and widely consumed by the general population are those that are cultivated for wide commercial distribution. Strawberries, blueberries, cranberries, and black currant are among the most common. Eighteen human clinical trial investigations have been identified examining the effects of common berries on markers of inflammation, generally or specific to CVD risk. Two studies97,111 involved both an acute and a chronic intervention, which were counted as four separate investigations. Of the 18 studies/investigations, 5 were acute interventions (postprandial paradigm: 2.5−6 h study duration) and 13 were chronic feeding interventions (ranging between 3 and 12 weeks in duration). Blueberry, including wild blueberries, cranberries, and strawberries were investigated in 5/6, 4, and 7 of the clinical trials, respectively, and black currant was investigated in 1 of the 18 investigations (Table 2). 3889
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
3890
crossover, PC
parallel, 2-arm, PC; post- overweight, MF 6 week-interventionf
crossover, 2-arm, PC
crossover, 2-arm, PC
parallel, 2-arm, BC
postprandial
postprandial
3
6
24
esophageal cancer at risk, MF
overweight, MF
obese, MF
overweight, MF
MetS, MF
parallel, 2-arm, C
8
MetS, F
parallel, 1-arm, BC
abdominal obesity, M abdominal obesity, M MetS, MF CHD, MF
4
dose escalation, 3-phase, PC dose escalation, 3-phase, PC parallel, 2-arm, PC crossover, 2-phase, PC
CVD at risk, M
insulin resistant, MF trained athletes, MF trained athletes, MF exercisers, F
MetS, MF
healthy, MF
subjects
36
22−24
20
24
24
10/15
16
16/15 44
30
30
18
10
25
25
17/15
23/25
20
nc
freeze-dried powder; water-based beverage freeze-dried powder; water-based beverage freeze-dried powder, milk-based beverage, MF-HC meal freeze-dried powder in milk beverage with MF-HC meal freeze-dried powder; mixed into foods freeze-dried powder; milk-based beverage freeze-dried powder; water-based beverage
low-energy juice double-strength juice
juice cocktail
juice cocktail
freeze-dried powder; waterbased beverage
fruit in smoothie
freeze-dried powder; waterbased beverage freeze-dried powder; yogurtbased smoothie whole berries prior to exercise whole berries
20% juice
format
30 g/60 g
NA
≈320 g frozen berries 10 g
NA
94.7 mg
94.7 mg
94.7 mg
2006 mg
2006 mg
458 mg 835 mg
100 mg/125 mL
100 mg/125 mL
375 mg anthocyanins
168 mg GAE, 97 mg anthocyanins
NA
NA
1462 mg
1624 mg
NA
total phenolsd
10 g
10 g
50 g
50 g
480 mL 480 mL
125, 250, 500 mL
125, 250, 500 mL
25 g
200 g
250 g
375 g
45 g
50 g
250 mL
dose/day
Zunino (2011) Ellis (2011) Chen (2012)
↔ ↔ ↓ iNOS, COX-2, pNFκBp65, pS6
Ellis (2011)
↓ PAI-1, IL-1β
Ruel (2009)
↓ MMP-9
Edirisinghe (2011)
Ruel (2008)
↓ sICAM-1, sVCAM-1
↓ IL-6, hs-CRP
Riso (2013)
↔
Basu (2010)
McLeay (2012)
↔
↓ sVCAM-1
McAnulty (2011)
↑ NK cells
Basu (2009)
McAnulty (2011)
↑ IL-10
↔
Stull (2010)
↔
Basu (2011) Dohadwala (2011)
Basu (2010)
↔
↔ ↔
Jin (2011)
ref
↔
circulating biomarkerse
BC, baseline controlled; C, controlled; CAD, coronary artery disease; F, female; GM-CSF, granulocyte macrophage colony stimulating factor; HC, high carbohydrate; COX, cyclooxygenase; GAE, gallic acid equivalents; HF, high fat; hs-CRP, high-sensitivity C-reactive protein; IL, interleukin; iNOS, inducible nitric oxide synthase; M, male; MetS, metabolic syndrome; MF, moderate fat; MMP, matrix metalloproteinase; NA, not available; NF-κB, nuclear factor kappa B; PAI-1, plasminogen activator inhibitor-1; PC, placebo controlled; sICAM-1, soluble intercellular cell adhesion molecule-1; sVCAM-1, soluble vascular adhesion molecule-1; TG, triglyceride. bDuration represented in weeks unless otherwise specified. cNumber represents n/group in parallel design studies. dAs reported in referenced papers, using different methods (HPLC, LC-MS/MS, spectrophotometry, Folin−Ciocalteu method) and units of expression, eOnly biomarkers that showed statistically significant change have been specified. fPostprandial test meal did not include strawberry beverage.
a
strawberry
8 4
12
12
crossover, 2-phase, PC
exercise 60 h
cranberry
parallel, 2-arm, C
crossover, 2-phase, PC
parallel, 2-arm, C
postprandial, exercise 6
6
parallel, 2-arm, PC
6
wild blueberry
parallel, 2-arm, C
8
blueberry
crossover, 2-phase, PC
study design
postprandial
durationb (weeks)
black currant
berry
Table 2. Effects of Common (Wide Commercial Distribution) Berries on Inflammatory Markers in Humansa
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
Journal of Agricultural and Food Chemistry
Review
Ruel et al. used a dose escalation schema wherein subjects increased their daily intake of cranberry juice every 4 weeks starting with 125 mL, then moving to 250 mL, and finally ending their last 4 weeks of the 12 week study with 500 mL/day (total phenols, 100 mg/125 mL juice).105,106 No control group was used for comparison. At the end of 4 weeks, subjects had significantly lower plasma concentrations of sVCAM-1 and sICAM-1105 and MMP-9106 compared to baseline. Plasma soluble E-selectin remained unchanged. Two subsequent studies from different researchers recruited individuals with MetS107 and patients with coronary heart disease (CHD)108 and supplemented cranberry juice for 8 and 4 weeks, respectively. Amounts of cranberry juice did not differ remarkably between the two Ruel et al. studies and the Basu et al. and Dohadwala et al. studies (500 vs 480 mL); however, the dose of total phenols was nearly double (835 mg) in the Dohadwala et al. trial.108 Basu et al. reported no significant improvements in plasma hs-CRP or IL-6,107 and, similarly, Dohadwala and colleagues reported no significant change in circulating hs-CRP and sICAM-1 concentrations after 4 weeks of the double-strength juice.108 Whether a longer duration of consumption or an acclimation to higher doses over time is needed to observe effects is not clear. Likewise, much like the research with other berries, it is not clear what amounts of fruit may be necessary to impart anti-inflammatory benefits in the different populations being studied. Strawberry. Strawberries, widely consumed fruit in North America, contain a number of essential nutrients, such as vitamin C and potassium as well as polyphenolic components, including anthocyanins, ellagitannins, and various flavonols. Pelargonindin-3-O-glucoside is the main anthocyanin of strawberries, with lesser amounts of cyanidin glycosides.109,110 Published human studies assessing strawberry intake and inflammation outcomes have been from acute7,111 as well as long-term-feeding studies.111−114 The two acute postprandial studies tested a freeze-dried strawberry powder incorporated into a challenge meal paradigm to test the ability of strawberries to attenuate meal-induced inflammation. Edirisinghe et al. studied the acute postprandial effects of strawberries in a milkbased beverage (10 g of freeze-dried strawberry powder; 94.7 mg of total polyphenols) on selected inflammatory markers in 24 overweight individuals.7 A beverage matched for sensory attributes, total calories, and macronutrients was used as the placebo. Blood sampling was conducted at baseline (0 h) and up to 6 h post-challenge meal (with strawberry or placebo beverage). Inclusion of the strawberry drink with a high-calorie breakfast resulted in lower cumulative plasma concentrations of IL-6 and hs-CRP over the 6 h period following the meal. Concentrations of TNF-α, IL-1β, and plasminogen activator inhibitor-1 (PAI-1), a thrombolytic factor, did not differ between strawberry and placebo conditions. This group of men and women was then subjected to the same postprandial protocol (high-calorie challenge meal with placebo beverage only) after 6 weeks of daily supplementation with either the strawberry beverage or the placebo beverage.111 Contrary to the results of the acute study, there were no differences between treatments on post-challenge meal/postprandial IL-6 or hs-CRP after 6 weeks of strawberry intake or placebo intake. However, significant decreases in postprandial IL-1β and PAI-1 were observed in those individuals who consumed the strawberry beverage for 6 weeks. Whereas the effect of strawberry on endpoints of inflammation differed between study paradigms, an anti-inflammatory effect
least one CVD risk factor, no change in inflammatory markers (IL-6, TNF-α, hs-CRP) or adhesion molecule (sVCAM-1) after 6 weeks of supplementation with a wild blueberry drink (25 g of freeze-dried powder and 375 mg of anthocyanins) compared to a polyphenol-free control beverage was observed.98 These results are consistent with McAnulty et al., who also found no changes in fasting blood markers of inflammation (IL-1 receptor antagonist (ra), IL-6, IL-8, and IL-10) after study participants (trained athletes) ingested 250 g/day of whole blueberries for 6 weeks.97 However, blueberry consumption increased counts of natural killer (NK) cells in the study subjects. NK cells promote natural cellular toxicity, for example, in counteracting proliferation of cancerous cells,99 which suggests that blueberries may beneficially modulate the immunological profile separate from inhibiting pro-inflammatory molecules. The same researchers also investigated the acute effects of blueberries in an exercise-induced inflammation paradigm at the end of the 6 week experimental period. Subjects ingested either 375 g of blueberries (n = 13) or no blueberries (n = 12) 1 h prior to 2.5 h of treadmill running. Pro-inflammatory markers, including circulating total leukocyte count, IL-8, IL-6, and IL-1ra, and skeletal muscle NF-κB activity, were measured 1 h after completion of the exercise: no differences between the two groups on these measures were observed. However, blueberry supplementation increased the anti-inflammatory regulatory cytokine IL-10 following exercise, which coincided with reduced oxidative stress as measured by F2-isoprostanes. In a shorter duration exercise study (60 h), oxidative stress and inflammation were assessed after intense strenuous eccentric contractions of the quadriceps with blueberry (in a smoothie) or a placebo smoothie devoid of blueberry.100 Whereas a faster rate of recovery was observed in the blueberry group, improvements in oxidative stress were not observed until 36 h and inflammatory indicators of damaged muscle (creatine kinase and IL-6) were still elevated in both groups (P > 0.05, treatment effect). Collectively, the data supporting a beneficial association between blueberries and inflammatory status are unremarkable at present; however, the positive effects on selected beneficial immune/inflammation biomarkers97 warrant further investigations to understand dose/amount−response of blueberries, delivery, and metabolic status of subject population on outcomes, which may help to explain the differences between clinical and preclinical studies on anti-inflammatory effects elicited by blueberries. Cranberry. Cranberries, a popular fruit consumed as juice and jelly and in dried form are best known for their urinary tract infection-preventing benefits.101 Cranberries are rich in cyanidin- and peonidin-glycosides,102 pro-anthocyanidins (particularly the A-type), flavonols, and phenolic acids.103 Clinical investigation of the anti-inflammatory effect of cranberries is supported by in vitro studies; 104 however, the clinical results are mixed. Specifically, in two of four clinical trials with cranberry juice, Ruel and colleagues105,106 reported favorable anti-inflammatory effects of cranberry in a dose-escalation paradigm over 12 weeks in abdominally obese men (n = 30; waist circumference ≥ 90 cm), whereas in two other investigations no significant effect of cranberry juice supplementation was observed compared to placebo107,108 (Table 2). Distinct differences in study design, doses, supplement duration, and populations may account for at least part of the discrepancy in outcomes. 3891
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
Journal of Agricultural and Food Chemistry
■
Review
EFFECTS OF “LESSER-KNOWN” (LIMITED DISTRIBUTION) BERRIES AND BERRY PRODUCTS ON INFLAMMATORY MARKERS IN HUMANS Several berry fruits remain underutilized due mainly to harvest in the wild and local consumption.117 They are indigenous to different parts of the world, with limited cultivation for commercial distribution. However, many, if not all, of these berries are rich sources of anthocyanins118,119 and other polyphenolic compounds, which have been shown to mitigate the negative effects of oxidative and inflammatory stress in animal and in vitro experiments.37,120−122 This section of the review discusses human intervention studies of the less commonly known berries on selected clinically relevant inflammatory markers associated with chronic diseases. Details of the investigations are presented in Table 3. A total of 12 studies are presented in this section and include clinical trials on bilberry,123−125 black raspberry,126,127 chokeberry,128 sea buckthorn berry,124,129−131 wolfberry,132 and a berry mixture of lingonberry, bilberry, black currant, and sea buckthorn berry.133 All interventions were chronic feeding studies except for one postprandial experiment with sea buckthorn berries.131 Bilberry. Bilberries are rich sources of polyphenols. Anthocyanins comprise nearly half to three-fourths of the total polyphenol content of bilberries.134 Three clinical trials were identified that included markers of inflammation as study endpoints. All three investigations reported anti-inflammatory effects associated with bilberry consumption for 4−8 weeks. Karlsen et al. studied the effects of 330 mL of bilberry juice/day for 4 weeks in men and women (n = 62) who were at risk for CVD development based on the presence of at least one predisposing risk factor such as cigarette smoking, hypertension, or elevated LDL-cholesterol concentrations.123 At the end of the 4 weeks, bilberry supplementation compared to water control resulted in lower levels of circulating IL-6, hsCRP, and monokine induced by interferon-γ (MIG). However, the authors also reported an increase in plasma TNF-α, which was incongruent with the IL-6 and hs-CRP findings: these cytokines are known to be positively related. In contrast, Lehtonen et al. reported a decrease in plasma TNF-α concentration with frozen whole bilberries (equivalent to 100 g fresh fruit) at the end of a 33−35 day intervention in overweight and obese women (n = 110).124 In addition, while there was no impact on levels of hs-CRP, IL-6, or sICAM-1 concentrations, bilberries resulted in a decrease in plasma sVCAM-I and adiponectin. Epidemiological studies show that circulating adiponectin levels are inversely proportional to body fat and inflammation and positively associated with insulin sensitivity, and thereby adiponectin is inversely associated with CVD risk. According to the authors,124 the decreased adiponectin concentrations in response to bilberry consumption may be reflective of a lack of sufficient adaptation time required to observe changes in adiponectin concentrations in circulation135 and not necessarily a detrimental effect of bilberries. Kolehmainen et al. reported outcomes from an intervention in which men and women with MetS supplemented their habitual diet with bilberries.125 The parallel arm study included a 4 week run-in phase, an 8 week intervention, and a 4 week follow-up period. Individuals in the bilberry group consumed daily 200 g of berry puree (524 mg anthocyanins/100 g) plus 40 g of dried berries (832 mg anthocyanins/100 g; equivalent to 200 g of fresh berries) to provide a total dose equivalent to
was consistently found with strawberry consumption. Followup investigations to understand the different mechanisms involved with the observed responses under different feeding conditions are ongoing. Possible explanations for the different responses may be related to the milieu of strawberry metabolites circulating in an acute setting versus on the background of a dietary pattern including daily consumption of strawberries. Chronic feeding studies with strawberries were conducted in four different subject populations: overweight,111 individuals with MetS,112,113 obese,114 and patients at risk for cancer.115 Outcome measures were fasting concentrations of inflammatory markers following strawberry interventions ranging from 3 to 6 weeks. In overweight individuals (n = 24) who consumed 10 g of freeze-dried strawberry powder in a beverage daily for 6 weeks (compared to energy-matched control beverage), no significant treatment effects were reported on fasting concentrations of inflammatory markers (i.e., hs-CRP, IL-6, IL-1β).111 Consistent with these findings, no significant differences in fasting hs-CRP, complement C3, serum amyloid A, fibrinogen, IL-1β, IL-6, IL-8, TNF-α, leptin, sICAM-1, or sVCAM-1 were reported in obese men and women (n = 20) after 3 weeks of supplementation with freeze-dried strawberry powder (equivalent to ∼320 g of frozen fruit) incorporated into yogurt, cream cheese, and smoothie compared to the same foods without strawberry powder.114 In MetS subjects,112,113 50 g/day of freeze-dried strawberry powder (equivalent to ∼500 g of fresh fruit) prepared in water and drunk daily for 8 weeks significantly reduced sVCAM-1, but not sICAM-1.113 Individuals with MetS have higher blood levels of adhesion molecules,116 which are associated with endothelial inflammation and higher CVD risk. In a smaller (n = 16) and shorter duration (4 weeks) pilot study with MetS women ingesting 50 g of freeze-dried strawberry daily (no comparator group), no significant changes in plasma hs-CRP or adiponectin were observed.112 Chen et al. studied strawberry supplementation in patients with esophageal dysplastic lesions who are at risk for developing esophageal cancer.115 Using a parallel design study, subjects consumed either 60 or 30 g (n = 36 per group) of freeze-dried strawberry powder in the form of a water-based drink for a period of 6 months. At the end of the supplementation period, patients who had consumed the 60 g dose had significantly lower protein expression of iNOS (79.50% reduction), COX-2 (62.9% reduction), pNF-κB-p65 (62.6% reduction), and pS6 (73.2% reduction) in esophageal mucosa. Reductions in the same parameters were also observed in the group receiving 30 g of strawberry powder/day, but the changes were not statistically significant from baseline measurements. Overall, the available data on strawberry consumption support future research to better understand dose−response dynamics in different populations, acute versus chronic intake (eating) regimens, matrix effects, and the relationship between polyphenol metabolite profiles and (anti-)inflammation outcomes. Among the four types of berries reviewed in this section, cranberries and strawberries showed “inhibitory” effects on selected pro-inflammatory markers,7,105,106,111,113 whereas blueberry intake resulted in beneficial effects on immune function by increasing anti-inflammatory/regulatory molecules.97 In four studies in which a relative oxidative and inflammatory stress was imposed, after exercise,97,100 or after eating meals typical of Western meal composition,7,111 or in MetS113 or abdominal obesity,105,106 consuming cranberries, blueberries, and strawberries resulted in beneficial anti-inflammatory responses. 3892
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
90 days 33−35 days
3893
30 days
healthy, F
parallel, 2-arm, PC healthy, MF
parallel, 2-arm, C
60
10
90 110
10
31/30
58
10
15
15/12
31 110
nc
426 mg/ 100 mL
fruit juice
dried and ground; with yogurt-based meal
frozen puree air-dried, whole
juice
NA
120 mL
40 g
NA
NA
28 g NA ≈100 g fresh berries NA
300 mL
Eccleston (2002) Larmo (2008) Lehtonen (2011) Lehtonen (2010)
Amagase (2009)
↔ ↓ hs-CRP ↓ TNF-α ↔ ↑ IL-2, IgG, lymphocytes
Skoczyñska (2007)
↓ fibrinogen
250 mL
NA
Lehtonen (2010)
Sardo (2013)
↓ IL-6
45 g; with HFHC meal
↑ adiponectin
Mentor Marcel (2012)
1669 mg
↑ GM-CSF; ↓ IL-8
60 g
Kolehmainen (2012)
↓ hs-CRP, IL-12
Karlsen, (2010) Lehtonen (2011)
ref
≈400 g fresh berries NA
circulating biomarkerse ↓ IL-6, hs-CRP, IL-15, MIG; ↑ TNF-α ↓ TNF-α, sVCAM-1, adiponectin
total phenolsd
330 mL NA ≈100 g fresh berries NA
dose/day
traditional products plus oilh ≈163 g fresh berries; NA 0.5 g oil
fruit juice
freeze-dried powder; water slurry freeze-dried powder; with water
frozen puree plus dried
juice frozen, whole
format
BC, baseline controlled; C, controlled; Chol, cholesterol(emia); CVD, coronary artery disease; F, female; HFHC, high-fat high-carbohydrate; hs-CRP, high-sensitivity C-reactive protein; IL, interleukin; M, male; MetS, metabolic syndrome; MIG, monokine induced by IFN-γ; NA, not available; PC, placebo controlled; sVCAM-1, soluble vascular cell adhesion molecule-1; TNF-α, tumor necrosis factoralpha; IgG, immunoglobulin G. bDuration represented in weeks unless otherwise specified. cNumber represents n/group in parallel design studies dAs reported in referenced papers, using different methods (HPLC, LC-MS/MS, spectrophotometry, Folin−Ciocalteu method) and units of expression. eOnly biomarkers that showed statistically significant change have been specified. f4 week run-in period, and 4 week recovery period following intervention. gLingonberry, bilberry, black currant, sea buckthorn. hJuice, bread, berry powder, berry oil, dried berries.
a
wolfberry
parallel, 2-arm, PC healthy, MF crossover, 2-phase, overweight and BC obese, F crossover, 2-phase, normal weight, M PC
8
sea buckthorn berry
postprandial
parallel, 2-arm, PC healthy, M
20
mixed berriesg
parallel, 1-arm, BC hyper-chol, M
6
chokeberry
CVD at risk, MF overweight and obese, F MetS, MF
subjects
parallel, 1-arm, BC colorectal cancer, MF postprandial overweight, M
parallel, 2-arm, C crossover, 2-phase, BC parallel, 2-arm, C
design
1−9
8f
4 33−35 days
durationb (weeks)
black raspberry
bilberry
berry
Table 3. Effects of Lesser-Known (Limited Distribution) Berries on Inflammatory Markers in Humansa
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
Journal of Agricultural and Food Chemistry
Review
Although blood samples were collected for up to 360 min during the postprandial phase, TNF-α was measured only at baseline (0 min) and 60 min after meal consumption. The trajectory of the TNF-α response suggested that the meal was inducing an increase in TNF-α and that concomitant intake of sea buckthorn berries with the meal was attenuating the increase; however, this was not significant (p = 0.175). An insufficient sample size and high between-subject variability along with measuring a single inflammatory biomarker (TNF-α) at only two time points (0 and 60 min) are insufficient to understand the potential benefits of sea buckthorn berries on postprandial inflammation in humans, particularly when others have shown the response may not be evident until 3−6 h.7,54,142 In 2002, Eccleston et al. conducted a parallel arm intervention in men (n = 10/group) to study the effect of ingesting 300 mL of a sea buckthorn berry juice (containing 1182 mg/L of flavonoids) for 8 weeks.129 No effect of supplementation was found on plasma sICAM-1 concentrations, which was the only marker measured. Two recent human studies124,130 reported favorable effects of sea buckthorn berries on inflammatory markers. Two hundred and thirty-three subjects were supplemented for 90 days with either a frozen sea buckthorn puree or a placebo puree that had similar sensory attributes. The daily dose of 28 g of berry puree provided about 8.4 mg of flavonols. At the end of the intervention, consumption of sea buckthorn berries resulted in lower hs-CRP concentrations in serum compared to placebo.130 A similar anti-inflammatory effect was observed in the second published intervention in which overweight and obese women (n = 110) consumed air-dried whole sea buckthorn berries (representing approximately 100 g of fresh berries).124 Plasma TNF-α, hs-CRP, IL-6, sVCAM-I, adiponectin, and sICAM-1 concentrations were measured before and at the end of the 33−35 day intervention. Supplementation with the sea buckthorn berries resulted in lower circulating TNF-α concentrations, but no change was observed in hs-CRP. Lehtonen et al. studied the long-term effects of 20 weeks of supplementation of four different berries (bilberry, black currant, lingonberry, and sea buckthorn berry) in the form of traditional berry products, as well as berry oils.133 Sixty-one disease-free women were randomized to either the treatment group or a control group (lifestyle intervention but no additional berries). The active group consumed 3 servings of berry products daily plus 0.5 g of berry oil/day (equivalent to 163 and 245 g of fresh berries, respectively). The control group consumed lower amounts of berries per day (27 g). The 18 berry products, comprising breads, juices, berry powder, dried berries, and berry oils, were consumed according to a specified consumption cycle. At the end of the 20 week intervention with the mixture of berries, there was a significant increase in levels of adiponectin from baseline, but also an unfavorable increase in soluble adhesion molecules (sVCAM-1 and sICAM-1) in the active group; however, there were no significant treatment differences observed between active and control groups. No effect of berry supplementation was observed on hs-CRP and TNF-α levels in either group. Wolfberry. The results of a single intervention on wolfberry (also known as goji berry) in a sample population of 60 healthy men and women utilizing a parallel design have been reported by Amagase et al.132 Goji berries have been traditionally used in Chinese medicine and are rich sources of carotenoids, particularly zeaxanthin, but also flavonoids and betaine.143,144 To the best of our knowledge, goji berries are an example of
400 g/day of fresh bilberries. Control subjects consumed only their habitual diet but were allowed to occasionally include a maximum of 80 g of fresh berries/day. Compared to the control group, subjects in the bilberry group showed significant improvements in serum hs-CRP and IL-12 (p < 0.05), whereas IL-6 and LPS tended to be lower (p = 0.074 and p = 0.094, respectively). Bilberry supplementation did not affect leptin and adiponectin levels. Black Raspberry. Black raspberries are native to North America and are darkly pigmented, reflective of their high anthocyanin content (about 214−589 mg/100 g).136 Black raspberries exhibit potent antioxidative capacity in vitro, and a considerable amount of research on black raspberries pertains to their preventative effects on different cancers both in vitro and in animals.137,138 Two human interventions with black raspberries have been recently published. Patients with colorectal cancer were supplemented with 60 g/day freeze-dried powder for approximately 3 weeks (10−63 days).126 A daily dose of 60 g/day (20 g in a water slurry 3 times a day) provided 1669 mg of anthocyanins. Plasma levels of cytokines (IL-1β, IL-2, IL-6, IL8, IL-10, IL-12p70, granulocyte macrophage colony stimulating factor (GM-CSF), IFN-γ, and TNF-α) were measured before and after treatment with black raspberries. The berry intervention increased plasma GM-CSF concentrations. Patients receiving berries for more than 10 days also had decreased plasma concentrations of IL-8. An increase in IL-8 has been observed in colorectal carcinoma.139 Sardo et al. tested postprandial effects of 45 g of freeze-dried black raspberry powder consumed with an inflammatory high-fat high-carbohydrate (HFHC) meal in overweight men (n = 10). The postprandial investigation (up to 12 h) was preceded by 4 consecutive days of black raspberry intake (45 g/day). Under the control condition, no berry powder was consumed either preceding or during the postprandial testing day.127 The HFHC meal provided 1090 calories, 56 g of total fat, and 156 g of carbohydrates. The areas under the curve (AUC) for IL-6, TNF-α, and CRP during the postprandial period were compared between the black raspberry−HFHC meal regimen and the control condition. Black raspberry powder consumption with the HFHC meal resulted in a significant decrease in IL-6 AUC compared to no berry intake (HFHC meal alone). No effects were observed with black raspberry powder on TNF-α and CRP. Sea Buckthorn Berry. Sea buckthorn berries are native to India, China, and Eastern Europe and have traditionally been used in Eastern medicine.130 The polyphenols in sea buckthorn berries are composed mainly of flavonol glycosides (isorhamnetin-3-rutinoside, -galactoside, and -glucoside) but also contain smaller amounts of the anthocyanins delphinidin, cyanidin, and peonidin.118 In vitro studies indicate that sea buckthorn berries may have potent inhibitory effect on CVD risk markers including exerting anti-inflammatory activity.141 A single postprandial study131 and three chronic supplementation studies with sea buckthorn berries have been reported.124,129,130 In a relatively small group of men (n = 10), intake of dried and crushed whole sea buckthorn berries (equivalent to 200 g of fresh whole berries and providing 22.8 mg of flavonol glycosides/portion) did not significantly affect levels of TNF-α in circulation during a postprandial investigation.131 The study used a crossover design in which sea buckthorn berries were incorporated into a yogurt-based meal containing 50 g of glucose. The same meal without berries represented the control. 3894
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
Journal of Agricultural and Food Chemistry
Review
circulation when berries and berry products are consumed normally in the diet, that is, as “food”. Purifying active principles in berries and delivery in the form of berry extracts/ berry fractions is one way to increase “dose” and possibly circulating concentrations, assuming higher concentrations are needed to achieve benefit. A relatively large body of data from in vitro and in vivo animal experiments provides evidence for a protective role of purified polyphenolic compounds and berry fractions against increased inflammatory stress. A small number of clinical trials have been conducted to study the effects of berry extracts and purified anthocyanins from berries on inflammatory markers associated with chronic diseases (Table 4). Of the nine studies presented in this section, all involved chronic administration of either (1) purified anthocyanins146−148 or (2) extracts of black currant,149 chokeberry,150 cranberry,151,152 or sea buckthorn berry.124 Anthocyanins. Zhu et al. tested a commercially available purified anthocyanin supplement: 41% total anthocyanins/ capsule composed of 58% delphinidins and 33% cyanidins extracted from bilberries and black currants; the remaining 59% composed of nonpolyphenolic constituents.148 One hundred and fifty (n = 150) hypercholesterolemic men and women took the supplement (320 mg of anthocyanins/day) 30 min after breakfast and dinner meals. Supplementation with anthocyanins led to a significant decrease in serum sVCAM-1 concentrations compared to placebo (delta placebo vs delta anthocyanins, p = 0.023). In two other studies, anthocyanin extracts from bilberries and black currants (primary anthocyanins were cyanidin-3-O-β-glucosides and delphinidin-3-O-β-glucosides) were tested against a control maltodextrin supplement.146,147 The first intervention was conducted by Karlsen et al., in which 118 men and women were randomized to either the placebo or active treatment providing 300 mg anthocyanins/day (two capsules each in the morning and evening; 75 mg of anthocyanins/capsule) for a period of 3 weeks.146 Of the several inflammatory markers that were measured (hs-CRP, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, macrophage inflammatory protein (MIP)-1α, MIP-1β, immunoprotein (IP)-10, MCP-1, eotaxin, and regulated upon activation, normal T cell expressed and secreted (RANTES)), the anthocyanin-supplemented group showed a decrease in plasma levels of IL-8, RANTES, IFN-α, IL-4, and IL-13 compared to placebo. A subsequent study by the same group of researchers tested the anthocyanin supplement in 27 men with elevated blood pressure (137/85 ± 12/7 mmHg).147 Subjects ingested four capsules (80 mg of anthocyanin each) in the morning and four capsules in the evening separate from mealtimes to provide a total daily dose of 640 mg of anthocyanins (approximately equal to 100 g of a mixture of blueberries and black currants). The intervention lasted for 4 weeks. Endpoints measured were hs-CRP, TNF-α, IL-6, IL-4, MCP-1, CD40L, sICAM-1, and sVCAM-1, P-selectin, von Willebrand factor, and total nitric oxide. Anthocyanin supplementation had no effect on levels of cytokines, markers of endothelial function, or oxidative stress. Anthocyanin supplementation resulted in a significant increase in von Willebrand factor, a marker of endothelial function associated with CVD. The authors noted that although this may be an unfavorable effect, the clinical significance of increased von Willebrand factor relative to endothelial inflammation in this population is unknown.
berries that do not contain anthocyanins or, if they do, quantities are too low to detect. The intervention included supplementation of the berries as a fruit juice. A beverage with comparable taste, color, and flavor was used as the placebo control. Subjects consumed a total of 120 mL of juice/day in two equal doses after the morning and evening meals for a period of 30 days. At baseline and the end of the treatment period, the following immunological markers were measured: counts of CD4, CD8, lymphocytes, and NK cells, in addition to circulating concentrations of IL-2, IL-4, immunoglobulin (Ig) G, and IgA. At the end of the 30 days, significant increases were found in the levels of serum IL-2, IgG, and NK cells with consumption of the goji berry juice compared to placebo. Similar beneficial immunological changes were observed when baseline to phase end differences between the two groups were compared. Chokeberry. Chokeberries, which are native to parts of North America and Eastern Europe, are rich in the anthocyanins cyanidin-3-arabinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, and cyanidin-3-xyloside, with total anthocyanin concentrations varying from 357 to 1790 mg/100 g fresh weight.140 One investigation was identified reporting inflammation biomarkers after consumption of chokeberries.128 Fifty-eight nonmedicated hypercholesterolemic men drank 250 mL/day of 100% chokeberry juice (total polyphenol content = 40−70 mg/g dry weight) for 6 weeks followed by 6 weeks of discontinuation of juice intake (washout) and followed by another 6 weeks of juice intake. Fasting blood concentrations of hs-CRP and fibrinogen were measured at the beginning of the study and three more times at the end of each 6 week period (two juice periods and one washout period). Chokeberry juice intake did not significantly change hs-CRP concentrations in these subjects compared to baseline. However, circulating fibrinogen levels were lower at the completion of all phases compared to baseline, and significantly so at the end of the second chokeberry intervention phase (p < 0.01). Fibrinogen is a known coagulation factor, but also has a pro-inflammatory role in a number of chronic diseases: several studies suggest fibrinogen is a key regulator of inflammation.145 In summary, 10 of the 12 studies reviewed in this section showed a beneficial effect of lesser known berries on selected pro-inflammatory and adhesion molecules in circulation. The single study on wolfberry showed that intake increases beneficial immune cells and molecules in humans.132 The two studies that showed no effect of sea buckthorn intake may have been mainly due to the small sample of subjects (n = 10) or to study/sampling design. Compared to the commonly consumed berries such as strawberries and blueberries, the lesser known berries appear to exert an overall consistent anti-inflammatory effect in humans. These promising data support the value of including these berries in the diet and support research to advance cultivation and distribution for widespread sale and consumption.
■
EFFECTS OF BERRY EXTRACTS AND ANTHOCYANINS ON INFLAMMATORY MARKERS IN HUMANS The use of whole fruit and whole fruit products, comprising several beneficial components, has been demonstrated to mitigate inflammatory burden in some human intervention studies. Although little is known about dose−response, the lack of effect reported in some studies may be due to failure in achieving the required levels of relevant compounds in 3895
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
3896
crossover, 2-phase, BC crossover, 2-phase, BC
overweight and obese, F overweight and obese, F
at risk prostate disease, M
110
110
21
15
22
10
73
27
59
nc
Zhu (2011)
Lyall (2011)
↓ sVCAM-1 ↓ CK activity ↓ IL-6, CRP, sVCAM-1, sICAM-1, MCP-1; ↑adiponectin
240 mg anthocyanins
3 × 85 mg: 255 mg flavonoids (25% anthocyanins)
berry (ethanol) extract berry oil extract
↓ TNF-α, sICAM-1 ↑ hs-CRP; ↓ sVCAM-1, adiponectin
≈100 g fresh berries
Lehtonen (2011)
Lehtonen (2011)
Vidlar (2010)
↔
≈100 g fresh berries
Lee (2008)
↔
Naruszewicz (2007)
Hassellund (2013)
↔
8 × 80 mg: 640 mg anthocyanins 4 × 80 mg: 320 mg anthocyanins
Karlsen (2007)
ref
↓ IL-8, RANTES, IFN-α, IL-4, IL-13
circulating biomarkersd
4 × 75 mg: 300 mg anthocyanins
dose/day
extract powder, 3 × 500 mg: 1500 mg 500 mg capsules fruit powder, 3 × 500 mg: 1500 mg 500 mg capsules (∼1.65 mg anthocyanins)
extract, capsule
cFDP, capsule
capsules, 80 mg
capsules, 80 mg
capsules, 75 mg
format
BB, bilberry; BCt, black currant; BC, baseline controlled; C, controlled; CAD, coronary artery disease; Chol, cholesterol(emia); CK, creatine kinase; DM, diabetes mellitus; F, female; cFDP, concentrated freeze-dried powder; hs-CRP, high-sensitivity C-reactive protein; IFN-α, interferon alpha; IL, interleukin; M, male; PC, placebo controlled; RANTES, regulated upon activation, normal T cell expressed and secreted; sICAM-1, soluble intercellular cell adhesion molecule-1; sVCAM-1, soluble vascular cell adhesion molecule-1; TNF-α, tumor necrosis factor-alpha. bDuration represented in weeks unless otherwise specified. cNumber represents n/group in parallel design studies. dOnly biomarkers that showed statistically significant change have been specified.
a
33−35 days
33−35 days
parallel, 2-arm, C
6 months
sea buckthorn berry
parallel, 2- arm, PC type 2 DM, MF
12
cranberry
CAD on statins, MF
6
chokeberry
parallel, 2-arm, PC
exercise, pre/ post
hyper-chol, MF
hypertensive, M
healthy, MF
subjects
crossover, 2- phase, healthy, MF PC
crossover, 2-phase, PC parallel, 2-arm, PC
4
12
parallel, 2-arm, PC
design
3
duration (weeks)b
black currant
anthocyanins, purified (BB, BCt)
berry
Table 4. Effects of Berry Extracts/Fractions, Including Anthocyanins, on Inflammatory Markers in Humansa
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf4044056 | J. Agric. Food Chem. 2014, 62, 3886−3903
Journal of Agricultural and Food Chemistry
Review
Black Currant. An encapsulated concentrated black currant powder was supplemented to exercisers to study effects on exercise-induced oxidative and inflammatory stress.149 The dose was approximately equivalent to 48 g of fresh blackcurrants (providing 240 mg of anthocyanins and
