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There is now considerable scientific evidence that a diet rich in fruits and vegetables can improve human health and protect against chronic diseases. However, it is not clear whether different fruits and vegetables have distinct beneficial effects. Apples are among the most frequently consumed fruits and a rich source of polyphenols and fiber. A major proportion of the bioactive components in apples, including the high molecular weight polyphenols, escape absorption in the upper gastrointestinal tract and reach the large intestine relatively intact. There, they can be converted by the colonic microbiota to bioavailable and biologically active compounds with systemic effects, in addition to modulating microbial composition. Epidemiological studies have identified associations between frequent apple consumption and reduced risk of chronic diseases such as cardiovascular disease. Human and animal intervention studies demonstrate beneficial effects on lipid metabolism, vascular function and inflammation but only a few studies have attempted to link these mechanistically with the gut microbiota. This review will focus on the reciprocal interaction between apple components and the gut microbiota, the potential link to cardiovascular health and the possible mechanisms of action.
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Nutrients 2015, 7, 3959-3998; doi:10.3390/nu7063959
nutrients
ISSN 2072-6643
www.mdpi.com/journal/nutrients
Review
Apples and Cardiovascular Health—Is the Gut Microbiota a
Core Consideration?
Athanasios Koutsos 1,2, Kieran M. Tuohy 2 and Julie A. Lovegrove 1,*
1 Hugh Sinclair Unit of Human Nutrition and Institute for Cardiovascular and Metabolic Research
(ICMR), Department of Food and Nutritional Sciences, University of Reading, Reading RG6 6AP,
UK; E-Mail: a.koutsos@pgr.reading.ac.uk
2 Nutrition and Nutrigenomics Group, Department of Food Quality and Nutrition, Research and
Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Trento 38010, Italy;
E-Mail: kieran.tuohy@fmach.it
*Author to whom correspondence should be addressed; E-Mail: j.a.lovegrove@reading.ac.uk;
Tel.: +44 (0)-118-378-6418; Fax: +44 (0)-118-378-7708.
Received: 11 April 2015 / Accepted: 12 May 2015 / Published: 26 May 2015
Abstract: There is now considerable scientific evidence that a diet rich in fruits and
vegetables can improve human health and protect against chronic diseases. However, it is
not clear whether different fruits and vegetables have distinct beneficial effects. Apples are
among the most frequently consumed fruits and a rich source of polyphenols and fiber. A
major proportion of the bioactive components in apples, including the high molecular
weight polyphenols, escape absorption in the upper gastrointestinal tract and reach the
large intestine relatively intact. There, they can be converted by the colonic microbiota to
bioavailable and biologically active compounds with systemic effects, in addition to
modulating microbial composition. Epidemiological studies have identified associations
between frequent apple consumption and reduced risk of chronic diseases such as
cardiovascular disease. Human and animal intervention studies demonstrate beneficial
effects on lipid metabolism, vascular function and inflammation but only a few studies
have attempted to link these mechanistically with the gut microbiota. This review will
focus on the reciprocal interaction between apple components and the gut microbiota, the
potential link to cardiovascular health and the possible mechanisms of action.
Keywords: apples; juice; fiber; pectin; polyphenols; cardiovascular; gut microbiota; blood
lipid; cholesterol; vascular; inflammation
OPEN ACCESS
Nutrients 2015, 7 3960
1. Introduction
A high intake of plant derived foods such as fruits, vegetables and whole grains can have a
preventative effect against cardiovascular diseases (CVD) [1–3]. The mechanisms are not entirely clear
but plant polyphenols and fiber are considered the principal mediators. Apples are among the most
popular and frequently consumed fruits in the world, because of their availability throughout the year,
in a variety of forms including fresh fruit, juice, cider, concentrate and puree [4], and the general
perception that apples are good for health [5]. Epidemiological studies support the view that frequent
apple consumption is associated with a reduced risk of chronic pathologies such as cardiovascular
disease, specific cancers, and diabetes [6–10]. Data from intervention studies in humans and animals
suggest that apple intake may positively affect lipid metabolism [11–15], weight management [16],
vascular function [17,18] and inflammation [19–22]. Apples are a rich source of polyphenols and fiber.
The main polyphenol classes in increasing order are: Dihydrochalcones, flavonols, hydroxycinnamates
and flavanols (catechin and proanthocyanidins (PAs)) [23]. Readily absorbed polyphenols such as
flavanol monomers may be responsible for some of the health effects. However, larger polyphenol
molecules such as the PAs, a major class of apple polyphenols, together with pectin, the main soluble
fiber in apples and other cell wall components, reach the colon and undergo extensive bioconversion
by colonic microbiota producing metabolites that may have local intestinal effects whilst in the gut,
and systemic effects after absorption. Apple polyphenols and fiber may also beneficially modulate the
gut microbiota composition and activity [2427]. The gut microbiota may serve as a potential novel
target for the prevention of CVD [28].
In this review we will present the major apple components and their reciprocal interaction with the
gut microbiota; the evidence relating to the ability of apples to reduce CVD risk from in vitro and
in vivo studies in animals and humans focusing on lipid metabolism, vascular function, blood pressure
and inflammation; and finally the possible mechanisms which link the human gut microbiota and gut
function to reduced risk.
2. Apple Components
Apples are low in fat and high in carbohydrate, with fructose as the predominant sugar. Apples are
also a rich source of vitamins (mainly C and E), minerals (potassium and magnesium), triterpenoids,
such as ursolic acid, fiber (soluble and insoluble) and polyphenols (Table 1).
Table 1. Composition of apples (Malus domestica), raw with skin. USDA National
Nutrient Database for Standard Reference.
Components
Value per 100 g
Energy (kcal)
52
Energy (kJ)
218
Water (g)
85.56
Total carbohydrates (g)
13.81
§
Total dietary fiber (g)
2.21
§
Insoluble fiber (g)
1.54
§
Soluble fiber (g)
0.67
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Table 1. Cont.
Total sugars (g)
10.39
Fructose (g)
5.9
Glucose (dextrose) (g)
2.43
Sucrose (g)
2.07
Starch (g)
0.05
Protein (g)
0.26
Total lipid (fat) (g)
0.17
Fatty acids, total polyunsaturated (g)
0.051
Fatty acids, total monounsaturated (g)
0.007
Fatty acids, total saturated (g)
0.028
Vitamin C, total ascorbic acid (mg)
4.6
Thiamin (mg)
0.017
Riboflavin (mg)
0.026
Niacin (mg)
0.091
Vitamin B-6 (mg)
0.041
Folate, DFE (μg)
3
Vitamin A, RAE (μg)
3
Vitamin A, IU (IU)
54
Vitamin E (alpha-tocopherol) (mg)
0.18
Vitamin K (phylloquinone) (μg)
2.2
Calcium, Ca (mg)
6
Iron, Fe (mg)
0.12
Magnesium, Mg (mg)
5
Phosphorus, P (mg)
11
Potassium, K (mg)
107
Sodium, Na (mg)
1
Zinc, Zn (mg)
0.04
* Total polyphenols (mg)
110.20
* Flavanols (mg)
96.33
* Hydroxycinnamates (mg)
14.21
* Flavonols (mg)
5.66
* Dihydrochalcones (mg)
4.17
#
Anthocyanins (mg)
1.62
§ Data from Li et al., 2002 [29]; * data from Vrhovsek et al., 2004 [23]; # only in red apples.
2.1. Apple Fiber
Apples contain approximately 2%–3% fiber [29,30]. They are rich in insoluble fiber, including
cellulose and hemicullose, with pectin as the major soluble fiber containing homogalacturonans
(consisting of long chains of α-(1–4) linked galacturonic acids) and rhamnogalacturonans. The degree
of methylation, DM (or degree of esterification), of the galacturonic acid residues strongly influence
the functional and physicochemical properties [31–33]. It has been reported that apple pectin has
cholesterol lowering properties [34] and beneficial effects on glucose metabolism [35]. The structural
parameters of pectin, including molecular weight and DM, have a major influence on the degree of
these effects [32,34,36,37]. Moreover, pectin is a gelling agent and impacts on transit time, gastric
Nutrients 2015, 7 3962
emptying and nutrient absorption from the gut [34,38–41]. It is resistant to degradation by gastric acid
and intestinal enzymes and thus, reaches the colon where it is fermented by the gut microbiota into
short chain fatty acids (SCFA) [26,42–44].
2.2. Apple Polyphenols
The polyphenol content of apples has been extensively measured in various studies [4,23,45–47]
with only the most recent including the determination of oligomeric and polymeric PAs (representing
approximately 80% of apple polyphenols) [4,23]. PAs, also known as condensed tannins, are
oligomers and polymers of flavanols and are composed mainly of (−)-epicatechin units, although some
of the terminal units may be from (+)-catechin. The most common subclass of PAs is the procyanidins
(PCs) consisting of (epi)catechin units [48]. Vrhovsek et al. (2004) reported the polyphenol content in
apples representing 8 of the most widely cultivated varieties in south and west Europe [23]. The mean
concentration of total polyphenols was 110.2 mg/100 g of fresh fruit and ranged from 66.2 mg to
211.9 mg/100 g according to the following increasing order: Fuji, Braeburn, Royal Gala, Golden
Delicious, Morgenduft, Granny Smith, Red Delicious and Renetta [23]. Flavanols (catechin and PAs)
was the major class of apple polyphenols (71%–90%), followed by hydroxycinnamates (4%–18%),
flavonols (1%–11%), dihydrochalcones (2%–6%) and anthocyanins (1%–3%), only in red apples [23].
Similarly a later study by Wojdylo et al. (2008) on 67 old and new varieties grown in Western Europe
reported, flavanols (catechin and oligomeric PCs), 80%, hydroxycinnamic acids, 1%–31%, flavonols,
2%–10%, dihydrochalcones, 0.5%–5% and in red apples, anthocyanins, 1% [4]. The polyphenol
profiles among several varieties are similar, but the concentration range varies [45,49–52] due to
differences in cultivars, growing conditions (light availability), maturity, storage, extraction
procedures, analytical techniques and pre- or post-harvest factors [47].
Phenolic compounds in apples are not evenly distributed in the fruit tissue. Despite the small
contribution of apple peel (6%–8%) to whole fruit weight [53], peel contains a significantly higher
content of phenolics. In particular all the flavonols (quercetin conjugates) and anthocyanins, as well as
an important part of dihydrochalcones (phloridzin and phloretin glycosides) [23,46]. Moreover, the
peel contains large amounts of PCs, ()-epicatechin, (+)-catechin which also appear in the flesh but in
lower concentrations [54]. Flesh has higher chlorogenic and some dihydrochoalcones [54]. The high
content of polyphenols in the peel has been attributed to their defensive role against pathogens which
may protect the fruit [55]. Apple juice contains only small amounts of quercetin glycosides and
dihydrochalcones. Moreover, the technological processing is crucial. Clear apple juice has a small
polyphenol content due to the oxidative conditions during the pulping and pressing and the final
clarification process [56]. Cloudy apple juice on the other hand may maintain an important polyphenol
amount due to anaerobic conditions and the lack of the clarification step [56,57].
2.2.1. Absorption and Bioavailability of Apple Polyphenols
The health effects of polyphenols are likely to depend upon the initial dose, absorption and
bioavailability [58]. The absorption of polyphenols is influenced by various factors such as their
physicochemical properties (e.g., molecular size, degree of polymerization (DP), solubility, pKa),
biological factors (through their passage in the gastrointestinal tract), food matrix, interaction with
Nutrients 2015, 7 3963
other dietary components and gut microbiota composition [59,60]. Aglycones and a few glycosides,
mainly the low molecular weight polyphenols, can be directly absorbed in the small intestine [61].
High molecular weight polyphenols such as polymeric PAs reach the colon almost unchanged
where they are transformed by the intense metabolic activity of the gut microbiota, a requirement for
their absorption [61]. Glycosylated polyphenols require loss of their sugar moiety and esterified
phenolic acids are hydrolyzed. In the brush borders of the intestine and after absorption in the liver,
polyphenols undergo phase II metabolism leading to the formation of glucuronides, sulphates or
methylated derivatives [61]. An increasing evidence base indicates that the concentration of parent
polyphenols in human plasma is lower, sometimes by several orders of magnitude, than the
concentration of pure polyphenols or plant extracts observed to have potential health effects
in vitro. [59,61]. Moreover, bioconverted and conjugated forms of intact polyphenols could have
higher biological activity than their parent compounds [61]. Absorbed polyphenols can either circulate
in the blood reaching the target tissues or be resecreted into the intestine via the bile as a result of the
enterohepatic circulation. The latter fraction can be either deconjugated by gut microbes and absorbed,
or further metabolized by the gut microbiota [59,61,62]. A detailed review of the bioavailability and
metabolism of the apple derived polyphenols in humans is presented by Bergmann et al. [63].
PCs are the major polyphenols in apples and cloudy apple juice, with only the monomeric, and to a
lesser extent dimeric fractions, readily absorbed [64,65]. Oligomeric PCs may be cleaved into smaller
units during their passage through the gastrointestinal tract, however, the major fraction (90%) is not
absorbed and thus, reaches the colon [66]. Both in vitro and animal studies support the theory that a
high polymerization decreases intestinal absorption [67]. Fifty-eight percent of polyphenols present in
cloudy apple juice were absorbed or degraded, with the remaining detected in the ileostomy fluid [66].
Hydroxycinnamic acids are also an important group of polyphenols in apple products. In a study by
Hagl et al. (2011), 10 healthy ileostomy subjects consumed 0.7 L of apple smoothie containing 60% of
cloudy apple juice and 40% apple puree [60]. After 8 hours, 63% of the total polyphenols and
D-(–)-quinic acid were found in the ileostomy bags compared with 60.9% of the hydroxycinnamic
acids [60] with only 28.1% of hydroxycinnamic acids from pure cloudy apple juice [66]. Differences
in the amount consumed and the quantity of cell wall compounds in apple smoothie may be
responsible for the inconsistent results [60,66]. Hydroxycinnamic acids, including caffeoylquinic acids
and p-coumaroylquinic acids, may be metabolized or hydrolyzed in the small intestine leading to
D-()-quinic acid and caffeic or p-coumaric liberation which can be absorbed [60,66]. However, a
significant proportion of chlorogenic acid (5-caffeoylquinic acid), may reach the colon where it can be
further metabolized by the gut microbiota [59,60,67,68].
It has been reported that quercetin from apples is less bioavailable than that found in onions, due to
differences in the quercetin conjugates present [69], suggesting that the sugar moiety affects
absorption. [67]. Quercetin aglycones and glucosides, present in onions, are more bioavailable
compared to quercetin monoglycosides and quercetin rutinosides found in apples [69,70]. Other
studies reported that quercetin glucosides were more effectively absorbed compared to pure
quercetin [67]. The main quercetin glycosides detected in human ileostomy bags after the consumption
of apple smoothie or cloudy apple juice were 3-O-rhamnoside and 3-O-arabinoside, with higher
recovery rates after apple smoothie consumption (46.3%) [60] compared with the cloudy apple juice
Nutrients 2015, 7 3964
(2.9%) [66]. In the large intestine, microorganisms can cleave the glycosidic bonds and free quercetin
which can be absorbed or further metabolized by the gut microbiota [71].
The main dihydrochalcones include phloretin 2-O-glucoside and phloretin-2′-O-(2′′-O-xylosyl)
glucoside and are characteristic ingredients of apples and apple products. In a human study by Marks
et al. (2009) the absorption and metabolism of the major apple dihydrochalones were explored in
9 healthy and 5 ileostomy subjects after the consumption of 500 ml of apple cider [72]. Phloretin
glycosides were metabolized to phloretin 2´-O-glucuronide in both the healthy and ileostomy
volunteers. Moreover, phloretin-2′-O-(2′′-O-xylosyl) glucoside was present in the ileal fluid together
with the aglycone phloretin, two phloretin-O-glucuronides, and two phloretin-O-sulfates, but none of
the ingested phloretin 2-O-glucoside [72]. In total, 38.6% of the total dihydrochalcones were detected
in the ileal fluid. These findings are consistent with Kahle et al. (2007) and Hagl et al. (2011) [60,66].
3. The Human Gut Microbiota—Effects of Fiber and Polyphenols
The human gut is host to a diverse collection of microorganisms, comprising approximately
1012 microbial cells per gram of gut content and up to 1000 different species [73,74]. This collection of
microorganisms is called the gut microbiota. The human colon is dominated mainly by two phyla, the
Firmicutes and Bacteroidetes, representing more than 90% of all the phylotypes, followed by lower
relative abundances of Actinobacteria, Proteobacteria, Fusobacteria and Verrucomicrobia [75]. The gut
microbiota plays an important role in human health by increasing the efficiency of ‘energy harvest’
from the diet through the metabolism of nondigestible dietary components, maintaining immune
homeostasis, synthesizing vitamins, such as B12 and K, providing a barrier to invading pathogenic
microorganisms and reinforcing the intestinal epithelial cell tight junctions [76]. Bifidobacteria,
lactobacilli and butyrate producing bacteria, such as Faecalibacterium prausnitzii and Eubacterium
rectale, are commonly considered as health promoting bacteria involved in saccharolytic fermentation
producing SCFA [75,76]. In contrast the overgrowth of other bacteria, such as the Enterobacteriaceae
and certain clostridia groups, is associated with negative health implications. Aberrant gut microbiota
composition has been associated with metabolic diseases including obesity, type I and type II diabetes
and atherosclerosis, certain cancers and autoimmune diseases [77–79]. It has been shown that obese
people have an increase ratio of Firmicutes/Bacteroidetes compared to lean subjects [80], however,
this was not confirmed by later studies [81,82]. More recently it has been suggested that differences in
microbiota diversity may be more important, in these pathologies, than changes in specific bacteria
populations [83].
Gut microbiota profiles can be modified through diet [79,80,84]. The type and quantity of food
components that reach the colon have an important impact on microbiota composition and
activity [85]. Dietary fiber, a major substrate for colonic fermentation, plays an important role
against the development of chronic diseases such as type 2 diabetes, heart disease, obesity and
cancers [86–89]. This is particularly true for prebiotic fiber, a term first introduced in the mid-1990s
referring to a class of fiber that may beneficially alter the colonic microbiota [90]. According to the
recent definition by Gibson et al. (2010), a dietary prebiotic is ‘‘a selectively fermented ingredient that
results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus
conferring benefit(s) to host health’’ [91]. Well established prebiotics include inulin,
Nutrients 2015, 7 3965
fructooligosaccharides, galactooligosaccharides and lactulose, which have been consistently reported
to increase relative numbers of bifidobacteria and lactobacilli [91]. Moreover, fiber is the main energy
source for the gut microbiota leading to production of SCFAs butyrate, acetate and propionate [85].
These organic acids have multiple functions in the host, as key energy sources for intestinal mucosa,
liver, muscle or other peripheral tissues contributing about 7%–8% of daily energy requirements. But
more importantly they play a significant role in cell function, immune system, lipid metabolism, gut
motility and permeability, affecting the risk of gastrointestinal disorders, cancers and CVD [85,92,93].
Moreover, up to 90% of dietary plant polyphenols [94] including apples, reach the colon
intact [48,59,95,96]. The interaction with the gut microbiota is reciprocal, since commensal bacteria
transform polyphenols into simple aromatic metabolites while polyphenols have the ability to
modulate the gut microbiota composition, inhibiting some bacterial populations and stimulating
others [97,98]. Tzounis et al. (2011) showed that a high cocoa flavanol drink (494 mg/day)
significantly increased fecal bifidobacteria and lactobacilli, but decreased clostridia populations after
4 weeks, compared with a low cocoa flavanol drink (23 mg/day), in a randomized, double-blind,
controlled, crossover, human intervention trial of 22 subjects [99]. Similar prebiotic effects have been
shown in a study exploring the impact of red wine, dealcoholized red wine and gin consumption on gut
microbiota composition in 10 subjects for 20 days [100]. The intake of red wine increased the number
of Enterococcus, Bacteroides, and Prevotella genera and decreased the Clostridium genera and
Clostridium histolyticum group [100]. These effects were not observed after the gin consumption, used
as an alcohol control, which gave significant increased levels of fecal clostridia and Bacteroides, and
decreased Prevotellaceae [100]. Both red wine and dealcoholized red wine increased the levels of
Blautia coccoidesEubacterium rectale group, Bifidobacterium, Eggerthella lenta, and Bacteroides
uniformis, indicating the beneficial effects of wine polyphenols [100]. Finally, the beneficial observed
reductions in total cholesterol (TC) and C-reactive protein (CRP) were related to the changes in
bifidobacteria number [100]. However, these beneficial effects on gut health were not confirmed in a
recent placebo-controlled, crossover, human intervention study by Wallace et al. (2015), where a
boysenberry beverage (750 mg polyphenols, anthocyanins and ellagitannis/ellagic acid as the main
source) and an apple fiber beverage (7.5 g) were consumed separately and in combination [101].
There was no indication of significant differences in fecal bacteria or SCFA levels after 4 weeks
intervention [101]. Differences in polyphenol dose, class and the food matrix is a crucial factor to
consider when interpreting these results. The modulation of gut microbiota activity and composition by
apple components has been shown in in vitro and in vivo experiments, the majority in animals and only
a few in humans.
3.1. Impact of Apple Components on the Gut Microbiota Composition
3.1.1. In vitro Studies
Pectin is almost completely fermented in vitro and thus, can modify the human gut microbiota
composition [42–44]. Structural characteristics such as DM and molecular weight influence the
fermentability properties [31,33,102]. The speed of fermentation and selectivity by the human fecal
bacteria for low and high methylated pectins are inconsistent in different studies which may be due to
Nutrients 2015, 7 3966
variability in the fecal donor and in vitro experimental conditions [31,33,102]. Furthermore it was
reported that pectin fermentation was relatively common among Bacteroides spp. and only performed
by Eubacterium eligens among gram-positive anaerobes including species from Firmicutes and
actinobacteria [103,104]. Later studies show that Faecalibacterium prausnitzii, which belongs to the
Firmicutes, are able to utilize apple pectin and compete with known pectin utilizing species such as
Bacteroides thetaiotaomicron and Eubacterium eligens [105]. However, the fermentability of pectic
oligosaccharides (POS) differs from pectin. Comparing the two substrates Olano-Martin et al. (2002)
showed that POS increased bifidobacteria number whereas pectin increased Bacteroides and
Clostridium [102]. Similarly, it has been shown that apple POS increased bifidobacteria and
lactobacilli levels and decreased clostridia and Bacteroides, while pectin increased bifidobacteria,
clostridia, Bacteroides and eubacteria [106]. Thus, these studies indicate that selectivity towards
bifidobacteria may only be confirmed for POS, suggesting a prebiotic effect.
The effects of pure apple PAs on the gut microbiota composition are not well explored. In an
in vitro batch culture system, inoculated with human faeces, a decrease in SCFA concentration after
apple PAs fermentation was reported, suggesting reduction of beneficial saccharolytic fermentation,
although, the specific bacteria composition was not determined [107]. It has been shown that PAs
possess antimicrobial properties by inhibiting microbial enzymes [108], binding to bacterial cells
affecting membrane function [109] and complex formation with metal ions, including iron, affecting
bacterial growth [110]. Although there is evidence of a bacteriostatic effect of condensed tannins this
inhibition may be selective to particular bacterial species. Animal studies using flavanols from
blackcurrant extract, grape pomace or tannin rich diets modified the balance of gut microbiota
composition towards beneficial bacteria [111–113]. Moreover, it has been suggested that structural
differences of the bacterial cell walls and membranes may explain why gram-positive bacteria such as
clostridia are more sensitive to bactericidal effects of tannins compared to gram-negative Prevotella
and Bacteroides species [111].
3.1.2. Animal Studies
Short and long term consumption of whole apples and apple components modified the rat caecal
microbiota composition [26]. Pectin was considered as the main bioactive component after
administration of 7% of apple pectin for 4 weeks by increasing Clostridium coccoides populations and
the expression of genes encoding the butyryl-coenzyme A (CoA) transferase, which are present in
bacteria of the Clostridium cluster XIVa, Roseburia-Eubacterium rectale cluster and Faecalibacterium
prausnitzii, all known as important butyrate producers [26]. Furthermore, the pectin diet resulted in a
decrease of Bacteroides spp. and a higher level of butyrate [26]. Although pectin is an important
contributor to gut health, exploring the combined effects of apple polyphenols and fiber seems prudent.
Moreover, dietary fiber can form a complex with polyphenols referred as “antioxidant dietary
fiber” [114] protecting antioxidants which can reach the colon and have local effects in the gut [19].
Apple pomace, a by-product of juice production, and a high source of fiber and polyphenols, has been
shown to have beneficial effects by decreasing caecal pH and increasing SCFA in rats compared with a
control diet [114]. Similarly, apple pomace extraction juice colloids (5%) increased Bacteroidaceae,
caecal content weight and SCFA concentration, mainly acetate and propionate, indicating microbial
Nutrients 2015, 7 3967
fermentation of pectin [24]. An alcohol apple pomace extract rich in insoluble fiber further increased
butyrate concentration and Eubacterium rectale cluster compared to the juice colloids and the
control [24]. In addition, the same group reported an increase in Lactobacillus and Bifidobacterium
counts after the administration of juice extracted from apple pomace [25]. Moreover, the observed
elevated acetate was associated with pectin fermentation whereas the higher total SCFA were linked to
polyphenols, including quercetin-3-glucoside [25]. A synergistic interaction of apple pectin and
polyphenols have been reported on large intestinal fermentation and lipid metabolism [115], and rat
cecum fermentation [116]. In contrast, although an intake of 5% unprocessed apple pomace,
containing 61% of fiber and 0.23% of polyphenols increased caecal SCFA compared to a control diet,
reducing or removing the polyphenol fraction caused further beneficial effects by increasing the
glycolytic activity of caecal microbiota, beneficially modifying the ratio of caecal SCFA and
branched-chain fatty acids (BCFA), and decreasing caecal ammonia and colonic pH [117]. Finally, in a
recent ex vivo study the microbiota balance after in vitro fermentation of faeces from diet induced
obese mice with nondigestible apple compounds including dietary fiber, extractable and
non-extractable phenolics, resembled that in lean controls [118]. Nevertheless, it should be noted that
animals metabolize apple components differently from humans [26]. Moreover, the main site of
fermentation in rats is the cecum, whereas in humans this occurs in the colon. Thus, human studies are
necessary to explore these effects. A summary of the in vivo studies is presented in Table 2.
3.1.3. Human Studies
To our knowledge, only two human studies have explored the effects of apples on gut microbiota
(Table 2). In a small scale intervention study (n = 8), 2 apples per day for 2 weeks significantly
increased fecal bifidobacteria, while reducing Enterobacteriaceae and lecithinace-positive clostridia,
including C. perfringens [27]. A trend for increased levels of Lactobacillus, Streptococcus and
Enterococcus was also reported [27]. However, this study did not use culture independent
microbiology techniques and a control treatment, limiting the ability to accurately assess apple induced
changes. In a recent 4 week-study of 23 healthy subjects whole apple and pomace consumption
lowered fecal pH and resulted in differences in denaturing gradient gel electrophoresis (DGGE)
profile. However, a potential modulation of the gut microbiota population was not confirmed by
quantitative PCR [15]. There is a need to further explore the impact of apple consumption on both the
composition and metabolic output of the gut microbiota in suitable designed, well powered, controlled,
dietary intervention studies using the most up to date culture independent microbiological techniques.
Nutrients 2015, 7 3968
Table 2. Effects of apples and apple components on gut microbiota composition and activity.
Type of Study
Duration-Diets-Daily Dose
Results
Author
Animal
(Wistar rats)
6 weeks, 10 rats for each group:
Control diet or
5% apple pomace (AP) 1B-juice colloids extract
(54.3% soluble and 2.6% insoluble fiber) or
5% AP 4B-juice colloids extract, rich in soluble fiber
(78.3% soluble and 1.8% insoluble) or
5% alcohol AP extract, rich in insoluble fiber (22.9%
soluble and 73.3% insoluble)
FISH (caecal)
Plate count (feaces)
B-juice AP extracts:
Total SCFA, acetate and propionate: ↑, pH: ↓
Bacteroidaceae: ↑ (faeces)
Alcohol AP extract:
Total SCFA and butyrate: ↑, pH: ↓
Bacteroidaceae: ↑ (faeces)
Eubacterium rectale: ↑ (caecal)
Sembries et al.
(2003) [24]
Animal
(Wistar rats)
4 weeks, 12 rats for each group:
Control diet or
Extraction juice from apple pomace
Plate count Total SCFA and acetate: ↑, pH: ↓
Lactobacillus: , Bifidobacterium:
Primary bile acids and neutral sterols: ↑
Secondary bile acids: ↓
Sembries et al.,
2006 [25]
Animal
(Fischer rats)
4 weeks, 8 rats for each group:
Control diet or
10 g apple or
7% apple pectin
qPCR Apple:
Butyrate:↑, pH: , Bacteroides spp:
Apple pectin:
Butyrate:↑, pH: , Bacteroides spp: , Clostridium coccoides:
Licht et al.,
2010 [26]
Animal ex vivo
(mice)
Granny Smith apple fermented in vitro from faeces
from diet induced lean (control) and obese mice.
qPCR Firmicutes, Bacteroidetes, Enterococcus, Enterobacteriaceae,
Escherichia coli and Bifidobacterium abundances from obese mice
tended to be similar to lean mice after apple fermentation.
Condezo-Hoyos et al.,
2014 [118]
Human 2 weeks, 8 subjects:
2 apples
Plate count Bifidobacteria: ↑
Clostridia: , Enterobacteriaceae:
Shinohara et al.,
2010 [27]
Human
Randomized,
single blinded,
controlled,
crossover
4 weeks, 23 subjects:
Control: period of restricted diet or
550 g whole apples or
22 g apple pomace or
500 ml clear apple juice or
500 ml cloudy apple juice
qPCR No changes in bacteria composition.
pH:
Ravn-Haren et al., 2012
[15]
↑: significant increase; : significant decrease; SCFA: short chain fatty acids; FISH: fluorescence in situ hybridization; qPCR: quantitative polymerase chain reaction.
Nutrients 2015, 7 3969
3.2. Impact of the Gut Microbiota on Apple Components—Focus on Polyphenols
3.2.1. In vitro
A study by Deprez et al. (2000) was one of the first to show that PA polymers can be degraded by
the human colonic microbiota into low molecular weight aromatic acids with different hydroxylation
profile and aliphatic side chain length [119]. From the initial 14C labelled PAs, 9%–22% found in the
metabolite pool and the major microbial metabolites included 3-hydroxyphenylpropionic acid,
3-phenylpropionic acid, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid [119]. The
ability of the human gut microbiota to ferment apple PAs with different chain length was explored for
pure apple, enzymatically digested apple, isolated cell walls, isolated PAs, cell walls-PAs or ciders
from Marie Menard and Avrolles apple varieties [107]. The main microbial metabolites included
3-(3,4-dihydroxyphenyl)propionic acid, 3-(3-hydroxyphenyl)propionic acid, 3-phenylpropionic acid
and benzoic acid derived from apple flavanols and 2-(3,4-dihydroxyphenyl)acetic acid and
2-(3-hydroxyphenyl)acetic acid from flavonols [107]. The DP of PAs can affect microbial conversions.
Increasing the DP might suppress the extension of microbial metabolism and formation of phenolic
acids. Moreover, the isolated long chain PAs inhibited the formation of SCFA from cell walls and
carbohydrates naturally present in the fecal inoculum which was not noticeable when the PAs were
combined with the apple cell walls [107]. Differences in the extent of microbial degradation between
monomeric and oligomeric flavanols have been shown in other in vitro studies [120,121]. Recently,
Ou et al. (2014) explored the human microbial metabolism of ()-epicatechin, (+)-catechin, PC B2 and
partially purified apple PCs (Granny Smith variety) [122]. The major microbial metabolites of
()-epicatechin, (+)-catechin and PC B2 were benzoic acid, 2-phenylacetic acid, 3-phenylpropionic
acid, 5-(3′-hydroxyphenyl)-γ-valerolactone and 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and
accounted for over 70% of the total metabolites [122]. Fermentation of all the substrates including
apple PCs further produced 2-(3-hydroxyphenyl)acetic acid, 2-(4-hydroxyphenyl)acetic acid,
2-(3,4-dihydroxyphenyl)acetic acid, 3-(3-hydroxyphenyl)propionic acid and hydroxyphenylvaleric
acid. Apple PCs produced the lowest amount of metabolites but demonstrated the highest quantity of
3-(3-hydroxyphenyl)propionic acid. Moreover, 2-(3,4-dihydroxyphenyl)acetic acid was only
detectable from PC B2 and apple PCs. The lowest recovery of metabolites after 24 h fermentation were
observed for the purified apple PCs compared to the other substrates supporting the theory that PCs
might slow down the microbiota metabolism [122].
Hydroxycinnamic acids that reach the colon may undergo intensive microbial metabolism.
Chlorogenic acid has been shown to be converted to 3-(3-hydroxyphenyl)propionic acid and
benzoic acid [123] whereas Rechner et al. (2004), observed conversion to caffeic acid,
3-(3,4-dihydroxyphenyl)propionic acid, followed by the formation of 3-(3-hydroxyphenyl)propionic
acid and finally 3-phenylpropionic acid [124]. Colonic absorption of the last two metabolites can lead
to the production of 3-hydroxyhippuric acid and hippuric acid in the liver [124].
Quercetin glycosides may also reach the colon and could serve as a substrate for human gut
bacteria. It has been shown that once the quercetin molecule is released by the action of microbial
enzymes it can be degraded to 3,4-dihydroxyphenylacetic acid [125]. Phloroglucinol appears as a
transient intermediate which might be further degraded to acetate and butyrate [126]. Eubacterium
Nutrients 2015, 7 3970
ramulus and Flavonifractor plautii, among other bacteria are capable of metabolizing
quercetin [125,127]. Finally, it is worth mentioning that different fecal donors possess a different
intestinal microbiota composition causing a high individual variation and thus, caution is necessary
when comparing these in vitro results [107].
3.2.2. In VivoAnimal and Human Studies
The urinary metabolome of rats was investigated using an untargeted mass spectrometry based
approach after the consumption of 7% apple pectin or 10 g of raw apple compared to a standard
diet [128]. The metabolites and potential markers of apple intake that appear in urine included quinic
acid, m-coumaric, hippuric acid and 3-hydroxy hippuric acid, all related to gut microbial metabolism
predominantly of chlorogenic acid. Dihydroxyphenyl-γ-valerolactone an important microbial
metabolite of ()-epicatechin was also identified [128]. The metabolites related to pectin consumption
were pyrrole-2-carboxylic acid, 2-furoylglycine and 2-piperidinone. Gut microbial metabolism was
also suggested after alterations in plasma metabolite levels in rats fed with fresh apples [129].
Animal studies result in different bioconversions compared to humans. For example, although
human gut microbiota can metabolize PAs into low molecular weight phenolic acids, these may be
poorly metabolized in rats [130,131]. To our knowledge no human study has explored the formation of
polyphenol gut microbiota metabolites after apple consumption. However, in a recent study, changes
in the blood metabolome after different apple product consumption suggested altered gut
microbial metabolism [132]. Studies on cocoa derived products, which are a rich source of oligomeric
and polymeric PCs, with similar chemical structures to apples, identified in humans
colonic microbial-derived phenolic catabolites, mainly 3-hydroxyphenylpropionic acid,
3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid and 3-hydroxybenzoic acid [133].
4. Cardiovascular Disease Risk
Cardiovascular disease risk factors including, dyslipidemia, endothelial dysfunction and
inflammation are some of the biggest health challenges today. Epidemiological evidence suggests that
flavonoid consumption may reduce cardiovascular disease risk and apples, as a rich source, may be a
major contributor [7,9]. In a Finnish study of 10,054 men and women the link between flavonoid
intake and several chronic diseases was explored. Apple intake was strongly inversely correlated with
ischemic heart disease mortality, thrombotic stroke and total mortality [7]. Apples were also associated
with a reduced risk of coronary heart disease and total cardiovascular disease mortality, as shown in
the Iowa Women’s Health Study, where 34,489 subjects free of CVD were followed up for 16 years [9]
.
In contrast, a prospective study of 38,445 women [134] and a 5 year follow up of 805 elderly
men [135] found only non-significant inverse associations between CVD risk and apple consumption.
4.1. Lipid Metabolism
4.1.1. Animal Studies
The effects of whole apples or specific apple constituents on lipid metabolism have been
investigated in a number of animal studies (Table 3). Fiber, mainly pectin, was considered initially as
Nutrients 2015, 7 3971
the main apple component responsible for the cholesterol lowering properties [136–138] and as shown
by Sanchez et al. (2008) to a similar extent as oat β-glucan which is known for its hypocholesterolemic
effects [35]. Apple polyphenols can also play an important role. Unripe apples, rich in PCs,
significantly decreased serum and liver cholesterol, as well as atherogenic indices, after a
hypercholesterolemic diet [139]. Polyphenols and in particular catechin, epicatechin and PC B1 were
considered responsible for the lipid lowering effects of a portuguese apple variety (Bravo de Esmolfe)
in hypercholesterolemic rats [140]. Other studies indicated that apple polyphenols could compensate
the dyslipidemic effects of a high cholesterol diet [20,140–143]. However, the combined effect of
apple fiber and polyphenol proved more efficient in reducing plasma cholesterol levels [115], and
hepatic cholesterol [144] than the individual components. An apple diet consisting of 10% or 15%
lyophilized apple improved lipid metabolism and significantly decreased TC respectively [145,146].
Increasing the amount of lyophilized apple to 20%, lowered plasma cholesterol, low density
lipoprotein-cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and triacylglycerol
(TAG) concentrations in liver and heart in hypercholesterolemic genetically obese Zucker rats
compared to a control diet [147]. Apple pomace, reduced TC, TAG and serum atherogenic
index [117]. The importance of apple pomace on lipid metabolism was also demonstrated in a more
recent study [148] whereas Sembries et al., (2004) did not find effects on serum lipid levels [149].
Finally, apple peel had a higher positive effect on plasma lipids compared to the pulp which was
attributed to the higher content of bioactive components in the peel [150].
4.1.2. Human Studies
Apple pectin has been shown to decrease plasma TC in humans [151]. A meta-analysis concluded
that 1 g of pectin could decrease TC and LDL-C by 0.070 and 0.055 mmol/L respectively [37]. The
combined effect of apple fiber and gum arabic supplemented in an apple juice decreased TC (8%) and
LDL-C (14%) in mildly hypercholesterolemic men compared to the unsupplemented apple juice [152].
Pure apple polyphenols can also decrease TC and LDL-C in a dose-depended manner as was shown in
mild hypercholesterolemic subjects [11]. Although the main effects were observed after the highest
concentration (1500 mg), which is considerably higher with the amount found in an apple, a later study
by the same group found similar effects with 600 mg, together with a decrease in visceral fat [12]. In
contrast, a cloudy apple juice containing 800 mg of polyphenols had no effects on blood lipids but a
significant interaction between IL-6-174 G/C polymorphism and body fat reduction [13]. It was
identified in an early study that the consumption of 3 apples per day for 4 months significantly reduced
TC and increased HDL-C in 76 mildly hypercholesterolemic subjects compared to a control group
(less than 3 apples per week) [153]. The effects of dried apples were explored in a long intervention
parallel study (12 months) of 160 postmenopausal women compared to dried plum (comparative
control) [14]. TC concentration was significantly decreased at 6 months compared to the control, and
at 3 months within the intervention group. More recently, 23 healthy subjects consumed whole apples
(550 g), apple pomace (22 g), clear and cloudy apple juices (500 ml) daily, each for 4 weeks including
a control period [15]. Compared to the control period only, trends were observed for a reduction in
LDL-C after whole apple (6.7%), pomace (7.9%) and cloudy apple juice (2.2%) intake [15].
Nutrients 2015, 7 3972
Table 3. Effects of apples and apple components on lipid metabolism in animals.
Animal Type-
Number (n)-
Study Duration-Design
Diets-Daily Dose Results Author
Wistar rats
n = 12 each group
3 weeks/parallel
Control diet
or
15% lyophilized apple
TC: ↓ 9.3%
Faecal TS excretion: ↑
Faecal TC excretion: ↑
Aprikian et al., 2001 [145]
Wistar rats
n = 10 each group
40 days/parallel
Control cholesterol (3g/kg) diet
or
10% apple pomace fiber
TC: ↓ 18.4%
LDL-C: ↓ 31.2%
HDL phospholipids: ↑ 19%
TAG: ↓ 14.8%
Liver TC: ↓ 26.3%
Leontowicz et al., 2001[138]
Lean (Fa/-) and obese (fa/fa)
Zucker rats
n = 8 each group
3 weeks/parallel
Control diet
or
20% lyophilized apple
TC: ↓ 22% (Obese group)
LDL-C: ↓ 70% (Obese group)
HDL-C: ↓ 26% (Obese group)
Liver and heart TAG: ↓ (Obese group)
Faecal BA excretion: ↑ (Lean group)
Aprikian et al., 2002 [147]
Wistar rats
n = 8 each group
4 weeks/parallel
Control cholesterol (1%) diet
or
10% whole dry apples
TC: ↓ 20%
LDL-C: ↓ 32.6%
TAG: ↓ 17%
Liver TC: ↓ 29.6%
Leontowicz et al., 2002[146]
Nutrients 2015, 7 3973
Table 3. Cont.
Wistar mild
hypercholesteolemic rats
n = 10 each group
3 weeks/parallel
Control diet
or
Freeze dried pectin, 5% (PEC)
or
High polyphenol cider apple extract, 10%
(PL)
or
Mixed diet: PEC + PL
TC: ↓ 24% (PEC + PL)
TAG: ↓ 29% (PL)
↓ 35% (PEC + PL)
Liver TC and TAG: ↓ (PEC and PEC + PL)
Faecal BA excretion: ↓ (PEC and PEC + PL)
Faecal NS excretion: ↑ (PEC and PEC + PL)
Aprikian et al., 2003 [115]
Wistar rats
n = 8 each group
4 weeks/parallel
Control cholesterol (1%) diet
or
apple peel, 10% (Apeel)
or
apple pulp, 10% (Apulp)
TC: ↓ 21.6% (Apeel), ↓ 19.4% (Apulp)
LDL-C: ↓ 35.3% (Apeel), ↓ 33.3% (Apulp)
TAG: ↓ 18% (Apeel), ↓ 14.6% (Apulp)
Liver TC: ↓ 31.6% (Apeel), ↓ 27% (Apulp)
Leontowicz et al., 2003[150]
Sprague-Dawley rats
n = 8 or 9 each group
30 days/parallel
Control cholesterol (0.5%) diet
or
0.2% apple polyphenols rich in oligomeric
procyanidins (AP)
or
0.5% AP
or
1% AP
TC: ↓ (all treatments)
HDL-C: ↑ (1% AP), ↓ (0.2% AP)
HDL-C/TC: ↑ (0.5% and 1% AP)
Liver TC: ↓ (0.5% and 1% AP)
Atherogenic indices: ↓ (0.5% and 1% AP)
Faecal acidic and neutral steroid excretion: ↑ (0.5% and 1% AP)
Osada et al., 2006 [139]
Nutrients 2015, 7 3974
Table 3. Cont.
ApoE deficient mice (apoE-
KO)
n = 16 each group
4 months/parallel
Control diet
or
apple polyphenols (AP), equivalent to
1.6 g/day for humans
or
apple fiber (AF), equivalent to 50 g/day for
humans
or
Mixed diet: AP + AF
Liver TC: ↓ 22% (AP + AF)
Atherosclerotic lesions: ↓ (all treatments)
Auclair et al., 2008 [144]
Hypercholesterolemic
hamsters
n = 8 each group
12 weeks/parallel
Control atherogenic diet
or
apple (A)
or
apple juice (AJ)
Equivalent to daily consumption of 600 g
apples or 500 ml of juice for humans
TC: ↓ 11% (A)
↓ 24% (AJ)
Non HDL-C: ↓ 30% (A)
↓ 55% (AJ)
TC/HDL-C: ↓ 25% (A)
↓ 38% (AJ)
Aortic fatty streak area: ↓ 48% (A) and 60% (AJ)
Decorde et al., 2008 [142]
Golden Syrian hamsters
n = 13 each group
6 weeks/parallel
Control atherogenic diet, 0.1% cholesterol
or
0.3% apple polyphenols (AP)
or
0.6% AP
HDL-C: ↑14.7% (0.3% AP)
↑16.5% (0.6% AP)
Non HDL-C: ↓ 20% (0.3% AP)
↓ 36.7% (0.6% AP)
TAG: ↓ 31.9% (0.6% AP)
Faecal BA excretion: ↑ (0.3% and 0.6% AP)
Faecal NS excretion: ↓ (0.3% and 0.6% AP)
Lam et al., 2008 [143]
Zucker fatty rats
n = 10 each group
15 weeks/parallel
Control diet
or
High methoxylated apple pectin, 10%
(HMAP)
or β-glucan, 10%
TC: ↓ (HMAP and β-glucan)
HDL-C: ↑ (HMAP)
Non HDL-C: ↓ (HMAP and β-glucan)
TAG: ↓ (HMAP and β-glucan, higher effect for HMAP)
Sanchez et al., 2008 [35]
Nutrients 2015, 7 3975
Table 3. Cont.
Rabbits
n = 8 each group
2 months/parallel
Control cholesterol (1%) diet
or
5 ml apple juice (low dose, LD)
or
10 ml apple juice (high dose, HD)
TC: ↓ 75% (LD), ↓ 77% (HD)
LDL-C: ↓ 70% (HD)
HDL-C: ↑ 86% (HD)
TAG: ↓ 61% (LD), ↓ 59% (HD)
Atherosclerotic lesions: ↓ (LD and HD)
Setorki et al., 2009 [20]
Wistar rats
n = 8 each group
4 weeks/parallel
Control diet
or
5% AP: apple pomace (61% dietary fiber
(DF), 0.23% polyphenols (PP))
or
5% APE: ethanol extracted apple pomace
(66% DF, 0.1% PP)
or
5% APA: ethanol/acetone extracted apple
pomace (67% DF, 0.01% PP)
TC: ↓ 19% (AP and APA)
TAG: ↓ 26% (APA)
↓ 38% (AP)
Atherogenic index, log TAG/HDL-C: ↓ (AP)
Kosmala et al., 2011 [117]
Wistar hypercholesterolemic
rats
n = 8 each group
30 days/parallel
Control cholesterol (2%) diet
20% apples from 3 different varieties:
- Golden (G)
- Malapio da serra (MS)
- Bravo de esmolfe (BE) (highest
polyphenol and antioxidant
capacity)
TC: ↓ 21% (BE)
LDL-C: ↓ 20.4% (BE)
TAG: ↓ 27.2% (BE)
Serra et al., 2012 [140]
SpragueDawley rats
n = 8 each group
5 weeks/parallel
Control high fat diet
or
10% apple pomace (AP)
or
10% apple juice concentrate (AC)
TC: ↓ 23% (AP), ↓ 22% (AC)
LDL-C: ↓ 34% (AP), ↓ 32% (AC)
HDL-C: ↑ 12% (AP)
HDL-C/TC: ↑ (AP and AC)
TAG: ↓ 30% (AP), ↓ 27% (AC)
Liver TC: ↓19% (AP)
Liver TAG: 21% (AP), ↓ 10% (AC)
Atherogenic index: ↓ (AP and AC)
Cho et al., 2013 [148]
↑: significant increase compared to the control diet; : significant decrease compared to the control diet; TC: total cholesterol; LDL-C: low density lipoprotein-cholesterol; HDL: high density
lipoprotein; TAG: triacylglycerols; TS: total steroids; NS: neutral sterols; BA: bile acids.
Nutrients 2015, 7 3976
In contrast, some studies concluded that apples or apple components did not show any significant
beneficial effects with a limited number suggesting some adverse effects. The lipid lowering properties
of gum arabic-pectin supplementation was not observed in a study of 85 hypercholesterolemic
subjects [154]. Consumption of clear apple juice resulted in a significantly higher level of TC and
LDL-C compared to whole apple or apple pomace [15]. Blood lipid levels did not change after the
consumption of fresh whole apples [155,156] or apple juice [155]. Apple consumption increased TAG
levels in hypercholesterolemic overweight women in Brazil after a period of 12 weeks [16]. However,
a small but significant weight loss of 1.22 kg, was also reported after the fruit consumption, suggesting
beneficial modification of the energy intake and satiety [16]. Similarly, a control group (no apple
intake) of overweight men decreased serum TAG and very low density lipoprotein compared to the
intervention apple group [157]. The most recent human intervention studies are presented in Table 4.
In general, the majority of these human studies provide some evidence to support beneficial effects
on lipid metabolism, mainly TC reduction. In a mini-review it was reported that 3 apples per day could
reduce cholesterol by 5%–8% (approximately 0.5 mmol/L) [158]. The specific amount and type of
polyphenols and fiber that are responsible for these effects requires exploration. The delivery matrix of
these bioactive components is also crucial. Beneficial effects are shown with fresh or dry apples and
cloudy apple juice, however clear apple juice has been associated with adverse effects. Apple pomace
is also a valuable material since it contains an important fraction of the polyphenols and fiber of the
whole apple. More large scale randomized and well controlled human intervention studies are required
to confirm these effects and to explore the potential mechanisms using realistic doses that can be
successfully incorporated to a habitual human diet.
4.1.3. Potential Lipid-Lowering Mechanisms
Modulation of Bile Acid Enterohepatic Circulation
Bile acids (BAs) are known to play an important role in lipid metabolism. BA synthesis is
performed in the liver and regulated mainly by cholesterol -hydroxylase (CYP7A1), an enzyme that
converts cholesterol to cholic acid (CA) and chenodeoxycholic acid (CDCA) as the primary
forms [159]. Prior to their secretion via the gall bladder, BAs are conjugated with the amino acids
taurine or glycine to form bile salts. After a meal bile salts enter the duodenum, facilitating the
metabolism and the absorption of dietary lipids. Most bile salts are then reabsorbed in the distal ileum.
However, a small percentage escape reabsorption and are deconjugated by gut bacteria. These are then
converted into secondary bile acids, which are either excreted in the faeces or absorbed and return back
to the liver where together with the absorbed primary bile acids and salts are reconjugated and
resecreted completing one cycle of the enterohepatic circulation [159]. The enzyme responsible for the
deconjugation of bile salts is bile salt hydrolase (BSH), which has been isolated from several gut
bacteria including species of bifidobacteria and lactobacilli [160]. Once deconjugated, bile acids are
less soluble leading to reduced reabsorption and increased faeces excretion [161]. Consequently more
cholesterol is removed from the circulation for de novo synthesis [161].
Nutrients 2015, 7 3977
Table 4. Effects of apples and apple components on blood lipid levels in humans.
Subjects-Study Duration-Design
Diets-Daily Dose
Results
Author
25 healthy men/women
6 weeks
Randomized, crossover
340 g apple
or
375 ml apple juice
No changes: TC, LDL-C, HDL-C and TAG Hyson et al., 2000 [155]
49 hypercholesterolemic,
overweight women
12 weeks (35 women)
Randomized, parallel
300 g apple
or
300 g pear
or
60 g oat cookies
TC: ↓ (oat group)
TAG: ↑ (fruit group)
de Oliveira et al., 2003[16]
48 hypercholesterolemic
men/women
4 weeks
Randomized, double-blinded,
placebo-controlled, parallel
Control: supplement without polyphenols
or
Low dose: 300 mg apple polyphenols (AP)
or
Medium dose: 600 mg AP
or
High dose: 1500 mg AP
TC: ↓ 4.5% (from baseline for High dose)
LDL-C: ↓ 7.8% (from baseline for High dose)
No changes: HDL-C and TAG
Nagasako-Akazome et al.,
2005 [11]
15 elderly
4 weeks
Fresh apples (2 g/kg body weight,
approximately 1 apple)
No changes: TC, LDL-C, HDL-C and TAG Avci et al., 2007 [156]
48 moderately obese men/women
12 weeks
Randomized, double-blinded,
placebo-controlled, parallel
Control: capsules without polyphenols
or
600 mg apple polyphenols capsules
TC: (from baseline and control group)
LDL-C: (from baseline)
No changes: HDL-C and TAG
VFA: (from control group)
Adiponectin: ↑ (from control group)
Nagasako-Akazome et al.,
2007 [12]
Nutrients 2015, 7 3978
Table 4. Cont.
46 overweight, hyperlipidemic men
8 weeks
Randomized, controlled, parallel
Control: no apple intake
or
300 g apple
No changes: TC, LDL-C and HDL-C
TAG: (in control group compared with the apple group)
VLDL-C: (in control group compared with the apple group)
Vafa et al., 2011 [157]
68 overweight men
4 weeks
Randomized, blinded, placebo-
controlled, parallel
Control: beverage without polyphenols
or
750 ml cloudy apple juice
No changes: TC, LDL-C, HDL-C and TAG
% total body fat: ↓ (from control group)
Body fat mass: ↓ (only in IL-6-174 C/C variant compared with
G-allele carriers).
Barth et al., 2012 [13]
160 postmenopausal women
1 year
Randomized, single blinded,
controlled, parallel
Dried plum (comparative control)
or
75 g dried apples
TC: ↓ (from control group)
TC: ↓ 13% (from baseline)
LDL-C: ↓ 24% (from baseline)
TC:HDL-C: ↓ (from baseline)
LDL:HDL-C: ↓ (from baseline)
No changes: HDL-C and TAG
Chai et al., 2012 [14]
23 healthy men/women
4 weeks
Randomised, single blinded,
controlled, crossover
Control: period of restricted diet
or
550 g whole apples (WA)
or
22 g apple pomace (AP)
or
500 ml clear apple juice (AJ)
or
500 ml cloudy AJ
Treatment resulted in significant effects in TC and LDL-C.
Clear AJ: ↑ 5% TC, ↑ 6.9% LDL-C (compared with WA and
AP)
No changes: HDL-C and TAG
Ravn-Haren et al, 2012
[15]
Nutrients 2015, 7 3979
Table 4. Cont.
20 healthy young men/women
4 weeks
Randomized, crossover
500 ml of two cloudy apple juices:
510 mg/L catechin equivalent and 60 mg/L
vitamin C (VCR)
or
993 mg/L catechin equivalent and 22 mg/L
vitamin C (PR)
TC: 4% (VCR)
No changes: LDL-C, HDL-C and TAG
Soriano-Maldonado et al.,
2014 [162]
↑: significant increase; : significant decrease; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; VLDL-C: very low density lipoprotein cholesterol; HDL-C: high density
lipoprotein cholesterol; TAG: triacylglycerols; VFA: viscelar fat area.
Nutrients 2015, 7 3980
Animal studies showed that apple polyphenol supplementation increased the excretion of bile acids
in faeces [139,143]. This may be related to binding of polyphenols with bile acids enhancing their
excretion in the faeces (Table 2). Apple polyphenols and mainly PCs may directly enhance the activity
of CYP7A1, the first and rate-limiting enzyme in bile acid synthesis [139]. Moreover, it has been
suggested that catechins and PCs could inhibit intestinal cholesterol absorption by affecting micellar
solubility and may also increase direct excretion of cholesterol and lipids in faeces [163,164].
Apple pectin as a soluble, nondigestible and viscous fiber has the ability to increase bile acids
and/or cholesterol passage to the colon and faeces through formation of complexes and may also
interrupt the enterohepatic circulation and reduce plasma cholesterol levels [136,137,147,149]. In
contrast a decreased faecal BA and increased neutral sterol excretion after intake of apple pectin (alone
or combined with apple polyphenols) suggests cholesterol lowering properties in parallel with a high
rate of bile acid reabsorption [115]. Further studies are necessary to explore this discrepancy and
further assess the complete bile acid pool in circulation.
Additionally, intestinal microbiota may play a role in conversion of cholesterol to coprostanol
which is subsequently excreted in faeces [165]. Deconjugated BAs can also act as signalling molecules
affecting activation of the farnesoid X receptor (FXR), a nuclear receptor that inhibits hepatic de novo
lipogenesis and is responsible for other physiological processes which can impact on cholesterol
catabolism and lipid absorption from the intestine [166–169].
Modification of Lipid Metabolism
Apple polyphenols may affect lipid metabolism through several other mechanisms including,
activation of fatty acid β-oxidation and cholesterol catabolism in the liver [139,164,170], inhibition of
hepatic fatty acid synthesis [141,164], decreasing cholesterol esterification and secretion of
apoB-containing lipoproteins [171] and suppression of cholesteryl ester transport protein (CETP)
activity improving the distribution of cholesterol in lipoproteins [143]. Moreover, apple polyphenols
have been associated with a reduction in total body weight gain [24], adipose tissue
weight [141,148,172], visceral fat [12,164] and leptin levels [164]. PAs can inhibit the action of
digestive enzymes such as lipase and amylase with beneficial effects on lipid and glucose metabolism.
Oligomeric apple PCs have been shown to inhibit pancreatic lipase activity, with increased inhibition
associated with a high degree of polymerization, affecting postprandial TAG absorption [173].
Upregulation of lipoprotein lipase (LPL) activity has been suggested as an alternative mechanism of
TAG lowering [174]. PAs may also regulate lipid metabolism by activating FXR and by modulating
other nuclear receptors such as small heterodimer partner (SHP) and peroxisome proliferator-activated
receptors (PPARs) as well as transcription factors like steroid response element binding protein 1
(SREBP1) [175–177]. In addition, apple phenolic compounds have been associated with a reduction in
LDL oxidation, an important contributor to atherosclerosis, as has been shown in in vitro [178–180]
or in animal models [140,147,181].
Pectin has a major role in cholesterol lowering by inhibiting cholesterol absorption and uptake,
influencing micelle formation and affecting transit time [35,145]. The origin and physicochemical
properties of pectin, including molecular weight, DM and viscosity affect the efficacy of the
mechanisms [34,35,37,136,137,182]. For example, by increasing the DM a greater proportion of bile
acids are transported to the distal colon [183] increasing faecal excretion [149,183]. Production of
Nutrients 2015, 7 3981
SCFA can independently affect these activities. Butyrate plays a major role in colonic function, in
addition it has been shown to inhibit liver cholesterol synthesis, whereas acetate and propionate have
an impact on metabolic processes at a systemic level, and may possess opposing effects on lipid
metabolism [78]. While propionate may inhibit cholesterol synthesis, acetate could increase hepatic
lipogenesis, however, the results are inconsistent [78]. Finally, the potential lipid lowering effects of
apples are probably due to the combined/synergistic effects of polyphenols and fiber rather than the
individual components. Human intervention studies are necessary to support these mechanisms.
4.2. Vascular Function and Blood Pressure
Endothelial dysfunction is considered as an early marker in the pathogenesis of atherosclerosis and
its complications [184]. Thus, endothelial function can serve as an indication for cardiovascular health.
Nitric Oxide (NO) is an important endothelium-derived vasodilator produced from its precursor
L-arginine via the enzymatic action of endothelial NO synthase (eNOS) [185]. A defect in the NO
production is considered as the main mechanism of endothelial dysfunction and thus, impaired
endothelium dependent vasodilation. Flavonoids have been shown to increase NO status, yet few
human studies have explored the potential role of apple polyphenols on vascular function and blood
pressure. Higher flow mediated dilation (FMD) of the brachial artery, increased NO status and lowered
systolic blood pressure was observed in 30 healthy subjects after the acute intake of an apple blend
providing 184 mg of quercetin and 180 mg of ()-epicatechin [17]. Flavonoids can increase NO by
stimulating eNOS activity, protect NO from free radicals and inhibit the synthesis of vasoconstrictor
endothelin-1 [186,187]. Similarly, both a low and a high apple puree intake (230 g) containing 25 and
100 mg of epicatechin, respectively, increased plasma NO metabolite levels (at 6 h) and attenuated
platelet reactivity (at 2 and 6 h) acutely in a study of 25 healthy subjects, but showed no effects after
the daily consumption for 2 weeks [18]. No effect was reported in vascular function (assessed by
FMD) after a 4-week cross over intervention with 40 g of lyophilized polyphenol rich apples, in
30 hypercholesterolemic subjects [188]. Differences in the study design, food matrix, and polyphenol
composition may account for the different results. Thus, an improvement in vascular function with
dietary flavonoids has been shown mainly in acute studies with flavanol monomers, mainly
(−)-epicatechin, considered responsible for these effects [189]. In the chronic study these monomers
were not present in blood after a 10-hour fast which could explain the lack of effects. Although PAs
microbial metabolites were present in the blood these had no apparent effect. This has been supported
by a study claiming that gut microbial metabolites from PCs may not be responsible for the vascular
effects [190]. However, other chronic studies providing PC rich sources such as chocolate and tea have
shown beneficial effects and increased FMD (Hooper et al., 2008). Moreover, it has been shown
in vitro that oligomeric PCs from apples, cocoa, red wine and cranberries were responsible for the
inhibition of endothelin-1 mainly between the tetramer to heptamer range whereas monomers lacked
any activity [191,192]. Similarly, large oligomers from grape seed and cocoa were responsible for
endothelium-dependent vasodilation effects [192–194]. In addition, microbial polyphenol metabolites,
that also appear after apple intake, including benzoic acids, cinnamic acids, chlorogenic acid and
3-(3,4-dihydroxyphenyl)propionic acid have been shown to inhibit platelet activation and
aggregation [195–197], angiotensin converting enzyme (ACE) action [198] and increase eNOS
Nutrients 2015, 7 3982
expression [199]. Other studies have mainly focused on quercetin, a flavonol widely found in apples,
and the potential hypotensive effects, as reviewed by Larson et al. [200]. A daily administration of
730 mg of quercetin significantly decreased blood pressure in hypertensive, but not in prehypertensive
subjects after 28 days [201]. No change in blood pressure was reported with 1000 mg quercetin and
200 mg quercetin rhamnoglucoside in 27 normotensive subjects [202]. In contrast, a lower quercetin
dose (150 mg) reduced blood pressure in subjects homozygous for the apolipoprotein E3 genotype
only [203]. Apple peel has been shown to inhibit ACE activity in vitro, with quercetin-3-O-glucoside
and the metabolite quercetin-3-O-glucuronic acid the most effective, suggesting antihypertensive
properties [204]. Apples contain approximately 5 mg of quercetin per 100 g and thus any potential
hypotensive effect may be due to synergistic interaction of the other polyphenols. Further chronic
human studies are necessary to assess the impact of apple product consumption on vascular function
and blood pressure.
4.3. Inflammation
Inflammation plays a key role in the pathology of atherosclerosis and coronary heart disease. There
is a strong evidence base that polyphenols can exert anti-inflammatory/immunomodulatory
activities [205,206], however, few studies have focused on the effects of apple polyphenols. The
potential anti-inflammatory effect of apple PCs from 109 different cultivars was tested using cell-based
assays [207]. Cultivars with a high PC content were able to inhibit nuclear factor-kappa B (NF-κB), a
transcription factor involved in the induction of pro-inflammatory enzymes including
cyclooxygenase-2 (COX-2) and the inducible nitric oxide synthase (iNOS), as well as the expression
of inflammation related-genes such as those for tumor necrosis factor-alpha (TNF-α), interleukin-1β
(IL-1β), IL-6 and IL-8 [207]. Jung et al. (2009) reported that PC B1, PC B2 and phloretin from apple
juice extracts were related to an anti-inflammatory activity in vitro [208]. In an aninal study a high
cholesterol diet supplemented with apple juice significantly decreased CRP levels in rabbits [20]. The
administration of 7.6% lyophilized apple, rich in polyphenols, in particular PAs and hydroxycinnamic
acids (Marie Menard cider variety), for 3 months, reduced colonic inflammation compared to a low
polyphenol intake (Golden Delicious) in HLA-B27 transgenic rats, which develop spontaneous
intestinal inflammation [19]. Interestingly, the high polyphenol variety had a dramatic impact on
Bacteroides fragilis, a group of bacteria thought to be involved in the etiology or maintenance of
inflammatory bowel disease (IBD). Inhibition of the NF-kB transcription factor, suggested as a
potential mechanism [19] [207] has also been reported in other human cell studies with apple
extracts [209,210]. The beneficial effect of Marie Menard apples was further confirmed in rats
showing decreased colonic and systemic inflammation [22]. Furthermore apple pectin has been
associated with anti-inflammatory effects in animal studies by down regulating pro-inflammatory
cytokine expression and immunoglobulin production in the colon [211] and systematically by reducing
plasma TNF-α [21]. Citrus pectin has been reported to inhibit iNOS and COX-2 expressions in
lipopolysaccharide (LPS)-activated macrophages, with greater inhibition from the pectin with the
higher DM [212]. Studies in humans are scarce. In an epidemiological study of 8335 US subjects the
intake of apples was inversely associated with serum CRP levels [10]. Whereas an intervention study
of 77 subjects showed no association between apple consumption and anti-inflammatory activity [213].
Nutrients 2015, 7 3983
Gut microbiota composition may also modulate systemic inflammation. LPS, a constituent of
gram-negative bacteria triggers the secretion of pro-inflammatory molecules. Elevated levels of LPS in
blood circulation, mainly after a high fat diet contribute to metabolic endotoxemia which plays
a major role in the pathophysiology of the metabolic syndrome and the progression of
atherosclerosis [214–216]. Prebiotic dietary fiber, pectin and apple polyphenols through the potential
modification of the intestinal microbiota may reduce metabolic endotoxemia by improving gut barrier
function and reducing intestinal permeability and uptake of LPS [214,217–219]. In high fat fed mice,
prebiotic oligofructose reduced endotoxemia and inflammation by increasing bifidobacteria
levels [214]. Microbiota-derived polyphenol metabolites may also be responsible for these
anti-inflammatory effects. It has been reported that quercetin can exert anti-inflammatory effects in
rats, only when it is released from its glycosylated forms (e.g., quercetin 3-rhamnoside) by the action
of the intestinal microbiota. [220]. Other in vitro and in vivo experiments have indicated that
polyphenols found in apples may reduce intestinal inflammation in humans after microbial degradation
suggesting beneficial effects, locally, at the intestinal level. [221]. Finally systemic effects have been
reported in humans. Dihydroxylated phenolic acids, derived from the microbial degradation of PAs,
mainly 3,4-dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid, significantly inhibited
the secretion of pro-inflammatory cytokines including, TNF-α, IL-1β, and IL-6 in LPS stimulated
peripheral blood mononuclear cells from 6 healthy volunteers [222].
5. Conclusions
There is some supporting evidence to suggest that apples and apple components may beneficially
modulate CVD risk factors. The strongest effects are related to lipid metabolism where evidence shows
frequent apple consumption can reduce TC. Few studies have focused on vascular function and
evidence for an anti-inflammatory activity is also limited and derived mainly from in vitro and animal
studies. Microbiota-derived metabolites exert various biological activities and could contribute to the
beneficial effects observed. A potential prebiotic impact of apple ingestion may be an important
mechanism of CVD risk marker reduction. An increasing body of evidence highlights the important
regulatory role mediated by microbe:host co-metabolic processes, especially bile acid metabolism.
Both polyphenols, especially complex polyphenols such as PAs and dietary fiber, can influence the
enterohepatic circulation by binding bile acids in the intestine, in a manner similar to pharmaceutical
bile acid chelating agents, or by changing the profile of gut bacteria, modifying their potential to
deconjugate and hydrolyse bile acids into secondary bile acids. Both the quantity and relative chemical
profiles of bile acids returning to the liver will determine the regulatory potential of these important
cell signaling molecules. Moreover, dietary polyphenols may directly play a cell signaling role by
modulating transcription factors that regulate important physiological functions, such as intestinal
permeability, fat absorption, bile acid metabolism, hepatic lipid/cholesterol metabolism, glucose
homeostasis and systemic inflammation. However, many of the mechanisms that impact on
physiological processes related to CVD are in animal and in vitro models and remain to be
convincingly demonstrated in human studies. More suitable powered, randomized, controlled, long
term, human dietary intervention studies using metagenomic and metabolomic techniques are required
to progress this research area.
Nutrients 2015, 7 3984
Author Contributions
Athanasios Koutsos conducted the literature search and drafted the review. Julie A. Lovegrove and
Kieran M. Tuohy critically revised the manuscript. All authors read and approved the final version of
the paper.
Conflicts of Interest
The authors declare no conflict of interest
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©2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/4.0/).
... Apples are good source of polyphenols, and are the most commonly eaten fruits in the world [90]. Song et al. [91] showed that daily consume one or more apples could lower 28% risk of T2DM when without eating apples. ...
... Previous evidence has shown that diet is one of the most important contributors to the balance of both the gut microbiota and bile acid homeostasis [35]. In particular, population studies that demonstrate a higher consumption of fruits and vegetables with a high content in polyphenols are associated with the enhancement of the growth of the probiotic bacteria that actively interact with the bile acid metabolising activity [36]. In addition, to date, a number of polyphenols have been reported to exert bile acid sequestering activity [37,38]. ...
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... Apples, as one of the most popular fruits, are available in a wide range of products in our daily life, such as fresh apples, apple juice, apple cider, and apple puree [1]. The production of apples in 2019 was approximately 87.24 million tons in the world [2]. ...
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The main dietary sources of polyphenols are reviewed, and the daily intake is calculated for a given diet containing some common fruits, vegetables and beverages. Phenolic acids account for about one third of the total intake and flavonoids account for the remaining two thirds. The most abundant flavonoids in the diet are flavanols (catechins plus proanthocyanidins), anthocyanins and their oxidation products. The main polyphenol dietary sources are fruit and beverages (fruit juice, wine, tea, coffee, chocolate and beer) and, to a lesser extent vegetables, dry legumes and cereals. The total intake is ∼1 g/d. Large uncertainties remain due to the lack of comprehensive data on the content of some of the main polyphenol classes in food. Bioavailability studies in humans are discussed. The maximum concentration in plasma rarely exceeds 1 μM after the consumption of 10–100 mg of a single phenolic compound. However, the total plasma phenol concentration is probably higher due to the presence of metabolites formed in the body's tissues or by the colonic microflora. These metabolites are still largely unknown and not accounted for. Both chemical and biochemical factors that affect the absorption and metabolism of polyphenols are reviewed, with particular emphasis on flavonoid glycosides. A better understanding of these factors is essential to explain the large variations in bioavailability observed among polyphenols and among individuals.
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