Review

Role of Chlorogenic Acids in Controlling Oxidative

and Inflammatory Stress Conditions
Ningjian Liang and David D. Kitts *
Received: 8 October 2015; Accepted: 9 December 2015; Published: 25 December 2015

Departments of Food, Nutrition and Health, the University of British Columbia,

Vancouver, BC V6T-1Z4, Canada; ningjian.liang@alumni.ubc.ca
* Correspondence: david.kitts@ubc.ca; Tel.: +1-604-822-5560

Abstract: Chlorogenic acids (CGAs) are esters formed between caffeic and quinic acids, and represent
an abundant group of plant polyphenols present in the human diet. CGAs have different subgroups
that include caffeoylquinic, p-coumaroylquinic, and feruloyquinic acids. Results of epidemiological
studies suggest that the consumption of beverages such as coffee, tea, wine, different herbal infusions,
and also some fruit juices is linked to reduced risks of developing different chronic diseases. These
beverages contain CGAs present in different concentrations and isomeric mixtures. The underlying
mechanism(s) for specific health benefits attributed to CGAs involves mitigating oxidative stress, and
hence the related adverse effects associated with an unbalanced intracellular redox state. There is
also evidence to show that CGAs exhibit anti-inflammatory activities by modulating a number of
important metabolic pathways. This review will focus on three specific aspects of the relevance of
CGAs in coffee beverages; namely: (1) the relative composition of different CGA isomers present in
coffee beverages; (2) analysis of in vitro and in vivo evidence that CGAs and individual isomers can
mitigate oxidative and inflammatory stresses; and (3) description of the molecular mechanisms that
have a key role in the cell signaling activity that underlines important functions.

Keywords: chlorogenic acid isomers; coffee; antioxidant activity; oxidative stress; anti-inflammation;
inflammatory stress

1. Introduction
CGAs are phenolic acids with vicinal hydroxyl groups on aromatic residues that are derived
from esterification of cinnamic acids, including caffeic, ferulic and p-coumaric acids with quinic
acid. A number of conjugated structures, such as caffeoylquinic acids (CQA), dicaffeoylquinic
acids (di-CQA), feruloylquinic acids (FQA), and p-coumaroylquinic acids (p-CoQA), exist in several
isomeric forms in coffee beans. Coffee arguably is one of the most popular consumed beverages in
the world and is also a very rich source of CGAs. The major CGAs in coffee include 3-caffeoylquinic
acid (3-CQA), 4-caffeoylquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA), 3,4-dicaffeoylquinic
acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), and 4,5-dicaffeoylquinic acid (4,5-diCQA).
Additional, minor CGAs including 3-feruloylquinic acid (3-FQA), 4-feruloylquinic acid (4-FQA),
5-feruloylquinic acid (5-FQA), 3-p-coumaroylquinic acid (3-p-CoQA), 4-p-coumaroylquinic acid
(4-p-CoQA), and 5-p-coumaroylquinic acid (5-p-CoQA) are also present in traceable amounts in coffee
beverages [1]. CGA lactones are also present after primary thermal processing [2]. The chemical
structures of major CGAs are shown in Figure 1. Besides coffee, CGAs are also present widely
in beverages prepared from herbs, fruits (e.g., apples, pears, many berries), and vegetables, but
consumption from these sources is 5% to 10% of that from coffee beverage consumption [3]. The health
benefits of consuming coffee, tea, fruit juice, and vegetable juice referred to in many epidemiological
studies may be linked at least in part to the presence of CGAs in these food systems. There is

Nutrients 2016, 8, 16; doi:10.3390/nu8010016 www.mdpi.com/journal/nutrients

Nutrients 2016, 8, 16 2 of 20
Nutrients 2015, 7, page–page 

considerable  evidence  available  to  show  that  CGAs  exhibit  many  biological  properties,  including 
considerable evidence available to show that CGAs exhibit many biological properties, including
antibacterial,  antioxidant,  and  anti‐inflammatory  activities.  This  review  summarizes   
antibacterial, antioxidant, and anti-inflammatory activities. This review summarizes the known CGA
the  known  CGA  isomer  composition  present  in  different  beverages  and  discusses  recent 
isomer composition
developments  present
that  in potential 
point  to  differenthealth 
beverages and
benefits  discusses
that  recent
are  linked  developments
to  CGA  thatanti‐
antioxidant  and  point to
potential health benefits that
inflammatory attributes.  are linked to CGA antioxidant and anti-inflammatory attributes.

 
Figure 1. Chemical structures of 3‐CQA, 4‐CQA, 5‐CQA, 3,4‐diCQA, 3,5‐diCQA, 4,5‐diCQA, 3‐FQA, 
Figure 1. Chemical structures of 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA, 3-FQA,
4‐FQA, 5‐FQA, 3‐p‐CoQA, 4‐p‐CoQA, and 5‐p‐CoQA. 
4-FQA, 5-FQA, 3-p-CoQA, 4-p-CoQA, and 5-p-CoQA.
2. CGAs Content in Coffee Beans and Coffee Brew 
2. CGAs Content in Coffee Beans and Coffee Brew
CGA isomer composition in green coffee beans is complex and varies in part according to the 
CGA isomer
specific  coffee composition in green coffee beans
variety,  the  geographic location  where  isit complex
is grown, and
and varies in part according
the  processes used  in  post‐to the
specific coffeewashing/drying 
harvest,  variety, the geographic
procedures, location
all  of where
which  it is grown,
precedes  and the
roasting  of processes
the  coffee used
beans. inThe 
post-harvest,
most 
abundant CGA in green coffee beans is 5‐CQA, which accounts for 76%–84% of the total CGAs, or 
washing/drying procedures, all of which precedes roasting of the coffee beans. The most abundant
CGA approximately 10 g/100 g coffee beans [4,5]. Clifford and Ramirez‐Martinez [6] measured the CGA 
in green coffee beans is 5-CQA, which accounts for 76%–84% of the total CGAs, or approximately
composition in two major coffee plant species and reported that green Coffea Robusta beans grown in 
10 g/100 g coffee beans [4,5]. Clifford and Ramirez-Martinez [6] measured the CGA composition
Santos and Sao Paulo contained a higher content of CGAs compared to green Coffea arabica beans 
in two major coffee plant species and reported that green Coffea Robusta beans grown in Santos and
grown in Ghana and Uganda. Later research has confirmed this finding [7]. For example, Perrone et 
Sao Paulo contained a higher content of CGAs compared to green Coffea arabica beans grown in Ghana
al. [8] reported that total content of CGAs in green Coffea canephora beans grown at Conillon was 86 milligram
and Uganda.
per gram of Later research
dry weight, has confirmed
whereas the total CGA this finding
contents [7]. For
in green example,
Coffea arabica Perrone et al.
beans grown at [8]
Mundoreported
Novo that
total content
and Catuai ofVermelho
CGAs inranged Coffea
greenfrom 63 canephora beans
to 55 milligram pergrown
gram of atdry
Conillon
weight, was 86 milligram
respectively. per gram of
Farah et al. [2] 
dry weight, whereas the total CGA contents in green Coffea arabica beans grown at Mundo Novo and
reported that the total CGAs content in green Coffea Robusta beans was 28% higher than the average.
CatuaiIn addition to 5‐CQA, green coffee beans contain 3‐ and 4‐CGA, dicaffeolyquinic acids (3,4‐, 3,5‐, and 
Vermelho ranged from 63 to 55 milligram per gram of dry weight, respectively. Farah et al. [2]
reported that theferuloylquinic 
4,5‐diCQA),  total CGAs contentacids  (3‐, 
in 4‐, 
greenand Coffea
5‐FQA),  and  p‐coumaroylquinic 
Robusta beans was 28% higher (3‐p‐,  4‐p‐, 
thanand the 5‐p‐
average.
CoQA) acids [9].
In addition to 5-CQA, green coffee beans contain 3- and 4-CGA, dicaffeolyquinic acids (3,4-, 3,5-, and
Roasting  conditions  also  significantly  affect  the  total  CGAs  content  and  profile  in  processed 
4,5-diCQA), feruloylquinic acids (3-, 4-, and 5-FQA), and p-coumaroylquinic (3-p-, 4-p-, and 5-p-CoQA)
coffee beans. High‐temperature roasting will convert some CGAs into flavor and aroma compounds, 
acids [9].
or alternatively react them with other chemical components in the coffee bean or brew through at 
Roasting conditions also significantly affect the total CGAs content and profile in processed coffee
least five distinct reaction pathways: epimerization, decarboxylation, acyl migration, lactonization, 
beans. High-temperature roasting will convert some CGAs into flavor and aroma compounds, or
and dehydration [10]. The final product contributes to melanoidins in the coffee brew [11]. The idea 
alternatively react them with other chemical components in the coffee bean or brew through at least
2 
five distinct reaction pathways: epimerization, decarboxylation, acyl migration, lactonization, and
dehydration [10]. The final product contributes to melanoidins in the coffee brew [11]. The idea that
CGAs are a component of Maillard reaction melanoidin products was initially based on findings
Nutrients 2016, 8, 16 3 of 20

that used the Folin–Ciocalteu method for quantifying total phenolics [12]. This has since been
confirmed with quantitative analysis of phenolic derivatives recovered from high molecular weight
components isolated from coffee brews [13]. Many, if not all, of these reactions that lead to thermal
degradation of CGAs during coffee roasting are dependent on the intensity (e.g., time and temperature)
of roasting. The diverse and complex nature of products produced with roasting coffee beans vary
from relatively simple decarboxylation of quinic and cinnamic acids to simpler phenolic acids, to
more complex formation of chlorogenic lactones, derived from dehydration of the quinic acid moiety.
The latter evokes intermolecular ester bond formation when CGA is exposed to high heat treatment.
Nucleophilic amine- and thio- groups from peptides are also examples of conjugate additions involving
CGAs during heating coffee [10]. Hence, the more the coffee beans are roasted, the lower the content
of total CGAs. Moon et al. [14] reported that around 45%–54% of CGAs were lost in the light roasted
(230 ˝ C, 12 min) beans compared to the green beans, whereas more than 99% of CGAs were lost in
higher roasts (e.g., city roast, 250 ˝ C for 17 min; French roast, 250 ˝ C for 21 min). Although as much
as 99% of CGA could be lost with the greatest roasting, 5-CQA still remained the predominant CGA
isomer in roasted coffee. It is interesting that isomers 4-CQA and 3-CQA increased in some varieties of
light roasted coffee beans, which could be attributed to isomerization of CGAs [14]. The CGA isomer
content in roasted coffee beans has been characterized in decreasing order: CQA > diCQA > FQA >
p-CoQA [8]. CGAs composition in a coffee brew prepared from coffee beans with different roasting
degrees has also been comprehensively studied. Total CGAs ranged from 187.7 to 295.6 mg/100 mL
brew when prepared from light roasted coffee beans and from 24.2 to 41.3 mg/100 mL brew when
prepared from dark roasted coffee beans [15]. Another study reported that the total CGAs in espresso
coffee made from light, medium, and dark roasted coffee beans was 1060 mg/100 mL, 517 mg/100 mL,
and 340 mg/100 mL, respectively [16]. Regardless of the roasting degree, the content of the total CGAs
in espresso coffee from different sources ranged from 89 mg/100 mL to 811 mg/100 mL [17]. The CGA
isomer contents in a commercial coffee brew also decreased in the following order, CQA > FQA >
diCQA [18,19], which is different from the order reported in roasted coffee beans [8].
The specific procedures used to brew coffee beverages also affect the final content of CGAs,
since many factors influence the efficiency of elution of CGAs from ground roasted coffee beans.
Filtered coffee is the most widely consumed coffee brew, prepared by pouring boiled water over
ground coffee beans that are stationary on a paper filter. In contrast, espresso coffeemakers apply
high pressure to force a small amount of boiling water through ground coffee beans. The simplest
way of making a coffee brew is by pouring boiling water over the ground coffee beans and waiting
for the grounds to settle. All methods of brewing that vary in the ratio between the hot water and
ground coffee beans (v/w), the turbulence, pressure, and the contact surface and contact time will
produce a collective effect on the final CGAs profile. Tfouni et al. [15] reported that brews prepared by
boiling water without filtration had a higher content of CGAs than the corresponding filtered ones.
This result might be due to the greater contact of surfaces between the added water and the ground
coffee when simply boiled, compared to the filtered method. Ludwig et al. [20] compared the CGA
isomer composition in espresso coffee and filtered coffee and found that the espresso coffee brew
contained relatively more CGAs compared to filtered coffee. Strong pressure applied in espresso favors
the extraction efficiency of CGAs into this brew.

3. Chlorogenic Acid Content in Other Plant Sources

In addition to green and processed coffee beans being major sources of dietary CGAs, this group of
phenolic compounds is also present in fruits and vegetables; again, 5-CQA is the predominant isomer.
Fresh potatoes contain CGAs that range from 0.10 to 0.19 mg of 5-CGA per 100 g potato [21], which is
equivalent to 90% of the total phenolic compounds present in potato tubers [22,23]. 5-CQA, 5-FQA, and
3,5-diCQA 3,4-diCQA were also detected in different varieties of the vegetable Chicorium endivia [24].
Genetically modified tomatoes with increased CGA content have also been developed to enhance
their antioxidant properties [25]. Popular citrus fruits such as pears and apples are additional rich
Nutrients 2016, 8, 16 4 of 20

sources of CGAs. The content of 5-CQA in pears ranged from 0.02 to 3.72 mg per gram of fresh
fruit depending on the ripeness of the fruit [26] and type of cultivar [27]. Apples are a rich source
of CGAs with the core part having the highest level (2.10 mg per gram of dry fruit), followed by the
apple seed (1.10 mg per gram of dry fruit) and then apple flesh (0.48 mg per gram of dry fruit) [28].
CGAs are also present in some herbs. Wang et al. [29] studied the CGA profiles in beverages prepared
from chrysanthemum, purple sweet potato stem, kuding tea, and honeysuckle flower and reported
that 5-CQA and 3,5-diCQA were the dominant isomers with 3-CQA, 4-CQA, 3-FQA, 4-FQA, 5-FQA,
3,4-diCQA, and 4,5-diCQA relatively minor isomers in these beverages. CGAs are the main phenolic
compounds in the tea infusions prepared from the herb Artemisia annua [30]. In summary, CGAs are
widely present in the plant kingdom; many of these plants are important in the human diet. Beverages
prepared from coffee beans, fruits, vegetables, and herbs constitute important dietary sources of CGAs.
It is interesting to note that the “di-CGA” may contribute different taste qualities, such as producing
the bitter/metallic taste found in certain coffees. The significance of this in respect to the taste profile of
coffee could be particularly relevant to Robusta coffees, or blends of coffees that contain a proportion
of Robusta beans and hence higher amounts of di-CGA.

4. Bioavailability and Metabolism of CGAs

In a cultured gastric epithelial model, multiple CGA isomers showed transfer intact across the
gastric barrier at an acidic apical pH, with di-CQA having a relatively higher permeability coefficient
compared to CQA [31]. Experiments conducted in a rat model showed that CGAs are not hydrolyzed
in the stomach but absorbed in an intact form [32]. This could explain the early detection of CGA in
plasma within 30 min after coffee consumption. The intestinal absorption of 5-CQA has also been
studied in cell culture, using the human colon carcinoma cell line Caco-2, to model the intestinal
epithelium in vitro. The absorption rate for 5-CQA was 0.10% ˘ 0.08% at physiological concentrations
equivalent to gut lumen concentrations (0.1~1 mM) [33,34]. Transepithelial transport experiments
with CGA using Caco-2 intestine epithelia cultured monolayer [35] described bidirectional permeation
with no transport into the basolaterial side (e.g., 99% retained on apical side), regardless of pH
gradient. The permeation rate was concentration dependent and not saturable, thus indicating passive
diffusion. In addition, transport was inversely correlated with transepithelial electrical resistance,
indicating limited passive diffusion when intestinal junctions are tight. Similar results were observed
with caffeic acid, which is absorbed both from paracellular diffusion as well by a monocarboxylic
acid transporter (MCT). Strong evidence exists that the majority of CGA is not absorbed in the
proximal part of the gastrointestinal tract, unless transformed to caffeic and ferulic acids before being
absorbed [32]. Ferulic acid is more efficiently absorbed than CGA, having a monoanionic carboxyl
group and non-polar side chain or aromatic hydrophobic moiety that works well with MCT. This may
not be the same for CGA, with its noted ester group likely interfering with MCT. With subsequent
activity of esterases in both the small intestine mucosa and also microbial esterases in the large intestine,
respectively, transforming CGA to caffeic acid first, which is then transformed to m-coumaric acid
and phenylpropionic derivatives by gut microflora [36–38]. These main metabolites of CGA are also
transported across the intestine cell by MCT. Figure 2 is a diagram showing the absorption of CGA
when passing through human digestive tract. Digestion–balance studies conducted in rats reported
that around 9.2% ˘ 6.8% of CGAs was recovered in the urine 24 h after consumption of a 50 mg/kg
dose of CGAs [39]. Some of the original CGA dose was recovered as simpler phenolics, such as caffeic
and ferulic acids. Farah et al. [9] studied the pharmacokinetic profile and bioavailability of CGAs in
healthy human subjects and found that the apparent bioavailability of CGAs from a green coffee extract
was 33% ˘ 23%. Recovery was principally derived from CQA and diCQA, with poor absorption from
FQA. Urine was not the major route for excretion of CGAs in the human trial, but smaller metabolites
recovered suggested that the metabolism and excretion of CGA, which influence the overall elimination
kinetics in humans, could be quite variable and related to genetic polymorphisms. A similar result
(33% ˘ 17% CGAs) was reported in ileostomy subjects 24 h after consumption of a high dose (2.8 mmol)
of CQAs [40]. Further confirmation of this extent of digestion and absorption of CGA has come from
a study where coffee CGAs recovered in plasma were quantified from subjects consuming realistic
Nutrients 2016, 8, 16 5 of 20

Nutrients 2015, 7, page–page 

intakes.plasma 
A positivewere dose–response
quantified  from describing the absorption
subjects  consuming  realistic efficiency
intakes.  A was dependent
positive  on the intake
dose–response 
level [41].
describing the absorption efficiency was dependent on the intake level [41]. The bioavailability of the  factors
The bioavailability of the CGAs of coffee beverages can also be affected by various
that are CGAs of coffee beverages can also be affected by various factors that are external to the dietary source. 
external to the dietary source. For examples, milk fat added to a coffee beverage may increase
For  examples,  milk 
CGAs bioavailability fat  added 
[42] and, to  a  coffee 
moreover, beverage  may of
the concentration increase 
CGAs CGAs 
presentbioavailability 
in coffee will [42] 
alsoand, 
influence
moreover, the concentration of CGAs present in coffee will also influence bioavailability of CGAs 
bioavailability of CGAs [43]. At present, the influence of the type of food matrix, which influences
[43].  At  present,  the  influence  of  the  type  of  food  matrix,  which  influences  CGAs  digestion  and 
CGAs digestion and bioavailability, remains unclear and represents an interesting area for more
bioavailability, remains unclear and represents an interesting area for more research on factors that 
researchinfluence bioaccessibility of CGAs and other important dietary polyphenols. 
on factors that influence bioaccessibility of CGAs and other important dietary polyphenols.

 
Figure 2. Absorption of CGA when passing through human digestive tract. 
Figure 2. Absorption of CGA when passing through human digestive tract.
5. Antioxidant Activity of CGA Isomers 
5. Antioxidant Activity of CGA Isomers
5.1. Overview of Antioxidant and Prooxidant Mechanisms and Assays for Assessing Antioxidant Activity in 
5.1. Overview of Antioxidant and Prooxidant Mechanisms and Assays for Assessing Antioxidant Activity
Vitro and in Vivo 
in Vitro and in Vivo
Reactive oxygen species [44] and reactive nitrogen species (RNS) are generated endogenously 
by mitochondrial respiration and are contributed by exogenous exposure to oxidizing agents including 
Reactive oxygen species [44] and reactive nitrogen species (RNS) are generated endogenously by
ionizing radiation, heavy metals, and hypoxia [45]. The term ROS/RNS is used to include not only the 
mitochondrial respiration ‐and are contributed by exogenous exposure to oxidizing agents including
superoxide  anions  (O2 ),  hydroxyl  radicals  (∙OH),  nitric  oxide  radicals  (NO∙),  and  peroxyl  radicals 
ionizing(ROO∙), 
radiation,
but  heavy metals, and
also  non‐radical  hypoxia
oxidants,  [45].
such  The term ROS/RNS
as  hypochlorous  is used
acid  (HOCl),  to include
singlet  oxygen  (not only the
1O2), 

superoxide ´
anions (O2 ), hydroxyl radicals (¨ OH), 2nitric oxide radicals (NO¨ ), and peroxyl radicals
peroxylnitrite (ONOO −), and hydrogen peroxide (H O2), which are all capable of oxidizing important 
(ROO¨ ),biomolecules 
but also non-radical oxidants,
[33].  Antioxidant  suchrequired 
enzymes  as hypochlorous
to  maintain  acid (HOCl),
a  healthy  singlet
redox  oxygen (1 O2 ),
state  include: 
superoxide 
peroxylnitrite (ONOOdismutase 
´ ), and(SOD),  catalase, 
hydrogen glutathione, 
peroxide (H2 O glutathione  peroxidases,  and  reductase.  Dietary 
2 ), which are all capable of oxidizing important
components  such  as  vitamin  E,  vitamin  C,  and  phenolic  compounds  also  serve  as  non‐enzymatic 
biomolecules [33]. Antioxidant enzymes required to maintain a healthy redox state include: superoxide
antioxidants.  Together,  the  endogenous  and  dietary  derived  antioxidants  constitute  our  antioxidant 
dismutase (SOD), catalase, glutathione, glutathione peroxidases, and reductase. Dietary components
defense  system.  Antioxidant  phytochemicals  derived  from  beverages  constitute  a  major  amount  of 
such asdietary antioxidants with potential health benefits. In the case of CGAs, these health benefits are a 
vitamin E, vitamin C, and phenolic compounds also serve as non-enzymatic antioxidants.
Together, the endogenous and dietary derived antioxidants constitute our antioxidant defense system.
result of CGAs donating hydrogen atoms to reduce free radicals and to inhibit oxidation reactions. 
After donating hydrogen atoms, CGAs are oxidized to their respective phenoxyl radicals and these 
Antioxidant phytochemicals derived from beverages constitute a major amount of dietary antioxidants
phenoxyl radicals are quickly stabilized by resonance stabilization. However, pro‐oxidant activity of 
with potential health benefits. In the case of CGAs, these health benefits are a result of CGAs donating
hydrogen atoms to reduce free radicals and to inhibit 5  oxidation reactions. After donating hydrogen
atoms, CGAs are oxidized to their respective phenoxyl radicals and these phenoxyl radicals are quickly
stabilized by resonance stabilization. However, pro-oxidant activity of phytophenolics, such as caffeic
acid and CGA, respectively, occur when present in systems containing redox-active metals. The redox
cycling of CGA in the presence of oxygen is catalyzed by transition metals, such as Cu and Fe, to form
reactive oxygen species that are capable of damaging macromolecules, such as DNA and lipids [46].
Nutrients 2016, 8, 16 6 of 20

The extent of pro-oxidant activity of CGA depends on its metal reducing capacity, sequestering
behavior and the oxygen-reducing capacity, the latter of which greatly influences the stability/lifetime
of the CGA-derived phenoxyl radicals. Phenoxyl radicals generated from catechol ring-containing
phenolics, such as caffeic acid and CGA, in the presence of Cu ions are relatively short-lived radicals,
but nevertheless they can result in a pro-oxidant effect, such as DNA single strand breaks. This effect
has been shown to be small for this class of polyhydroxyl phenolics, and is relatively stronger with
caffeic acid than CGA. The primary reason for this difference is that caffeic acid is simpler p-coumaric
derivative, where CGA is esterified and contains a quinic-carbohydrate moiety. This difference evokes
a relatively weaker pro-oxidant effect for CGA compared to caffeic acid. Redox-inactive metals such as
aluminum and zinc can also enhance the pro-oxidative effect of CGA by producing a spin-stabilizing
effect on the CGA phenoxyl radical. The CGA-phenoxyl radical will then be available to induced lipid
peroxidation [47]. This potential pro-oxidant activity occurs under conditions that prolong the lifetime
of the phenoxyl radical.
Chemical-based assays, cell-based assays, and animal models have been established to gain
different levels of understanding about the antioxidant capacity of CGAs. Mechanisms of antioxidant or
pro-oxidant activity can be understood using chemical-based assays, where free radicals are artificially
generated to react with tested samples under fixed conditions of time and at defined conditions. At the
reaction endpoint, the amount of leftover free radicals is measured to reflect the free radical scavenging
capacity of the tested sample. Various chemical reactions have been used to generate free radicals.
Xanthine oxidase (XOD) utilizes hypoxanthine or xanthine as a substrate and O2 as a cofactor to
generate O2 ´ . A Fenton reaction between ferrous iron and H2 O2 produces ¨ OH. Sodium nitroprusside
breaks down to yield NO¨ and 2,21 -azobis(2-amidinopropane) dihydrochloride (AAPH) constantly
generates ROO¨ at a physiologically appropriate pH.
A cell-based model offers the potential to account for transcellular and paracellular transport
processes defining the absorption, and metabolism of the potential antioxidant molecule. There are
a variety of cell-based in vitro models available for studying antioxidant activity of different food
components, where chemical or physical stressors are used to induce oxidative stress prior to or
during exposure of cells to a potential antioxidant compound. The response is often measured
using defined biomarkers of redox status to reflect the antioxidant activity of the antioxidant test
compound. A commonly used redox biomarker for quantitating response of cells to oxidative stress
is the transcription factor, nuclear factor-E2-related factor 2 (Nrf2). Nrf2 is a member of the basic
leucine zipper NF-E2 family and plays an essential role in the antioxidant response element-mediated
expression of phase II detoxifying enzymes, including glutathione peroxidase (GPx), glutathione
reductase (GR), and superoxide dismutase (SOD) [48]. Genomic DNA integrity is another important
biomarker of redox status because reactive species continuously attack DNA structure and cause DNA
strand breaks, crosslinks, or sister chromatid exchanges, resulting in oxidative damage of DNA [49].
Furthermore, ROS affect the DNA methylation by oxidizing key enzymes involved in the methylation
process [45]. Moreover, ROS easily initiate lipid oxidation in vitro, leading to the accumulation of
lipid peroxidation products such as hydroperoxides and malondialdehyde (MDA), characteristic
components of the first and second stages of lipid oxidation reactions, respectively. In order to
gain more comprehensive understanding about the antioxidant activity of food components, such
as polyphenols in general and CGAs specifically, animal models have been employed to study the
affinity of plant phenolic compounds to mitigate oxidative stress. Table 1 is a summary of the results
obtained from chemical-based studies that have examined the antioxidant activity of CGAs. Table 2 is
a summary of the cell-based and animal studies describing the antioxidant activity of CGAs reviewed
in this section. In these studies, the chlorogenic acid referred to pertains mostly to 5-CQA.

5.2. Evidence from Chemical-Based Assays

All CGA isomers are potent antioxidants, as they possess one to two aromatic rings linked to
hydroxyl groups and the one-electron oxidation product of CGAs formed by the reaction with free
Nutrients 2016, 8, 16 7 of 20

radicals is rapidly broken down to non-free radical products [50]. Chemical-based assays have shown
that CGAs have the capacity to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, superoxide
anions (O2 ´ ), hydroxyl radicals (¨ OH) [51,52], 2,21 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
(ABTS) radicals [53], lipid oxidation [53,54], and peroxylnitrite (ONOO´ ) [54]. CGAs react with
difference sources of free radicals at different rate constants. The second-order rate constants for
5-CQA reacting with superoxide, hydroxyl radical, peroxyl radical, and peroxynitrite have been
determined to be 0.96 ˘ 0.01 ˆ 106 M´1 ¨ s´1 , 3.34 ˘ 0.19 ˆ 109 M´1 ¨ s´1 , 1.28 ˘ 0.11 ˆ 105 M´1 ¨ s´1 ,
and 1.6 ˘ 0.7 ˆ 105 M´1 ¨ s´1 , respectively [54]. This result shows that the relative efficiency of
5-CQA to react with free radicals is species-specific. Another important activity of CGAs towards
ROS-induced oxidative stress involves the radical damage caused to DNA, which can be quantified
to include DNA strand breakage. Six CGA isomers, namely 3-CQA, 4-CQA, 5-CQA, 3,5-diCQA,
3,4-diCQA, and 4,5-diCQA, were shown to exhibit a protective effect against H2 O2 -induced DNA
plasmid chromosome breaks [55]. Secondary byproducts of the oxidation reaction are also relevant in
initiating a mutagenic response as a result of DNA damage. One example concerns the oxidation of
chloride by H2 O2 , resulting in formation of HOCl, and subsequent reaction with amines to produce
NH2 Cl, an oxidant and potent mutagen. CGAs, specifically 5-CQA, have been shown to protect
against NH2 Cl-induced plasmid DNA breakage in cultured neutrophils [50]. Another example of
the anti-peroxidation activity of 5-CQA comes from the protection against LDL oxidation, which is
the initial step in the development of atherosclerosis. Studies based on incubating isolated LDL with
oxidizing agents in vitro showed that 5-CQA was effective at mitigating both copper-induced LDL
oxidation [56] and ferryl myoglobin-induced LDL oxidation [57]. These results correspond to other
studies that reported reduced MDA content in brain tissues when pre-treated with 5-CQA [53].

5.3. Evidence from Cell-Based Assays

Research directed at characterizing the antioxidant capacity of CGAs using cell-based models
has focused mostly on the 5-CQA isomer. 5-CQA protects against H2 O2 -induced oxidative stress
in human HaCaT cells [51]; it can activate Akt phosphorylation, increase the expression of FOXO
family genes in mesenchymal stem cells of bone marrow [58], and reduce apoptosis in primary
cortical neurons by upregulating antioxidant enzymes such as NADPH:quinine oxidoreductase 1 [59].
The protective effect of 5-CQA against oxidative stress stimulated by various oxidative stressors
(e.g., t-BHP, H2 O2 , FeSO4 ) has also been studied in PC12 cells, which used multiple biomarkers such
as the reduction of lipid peroxidation product, the extent of ROS formation, and GSH depletion to
evaluate the utility of CGAs to prevent oxidation [60]. 5-CQA showed effectiveness in protecting
against DNA damage with activity to inhibit methylation of the promoter region of the RAR beta
gene through increased formation of S-adenosyl-L-homocysteine in cultured human breast cancer cells
MCF-7 and MAD-MB-231, respectively [61]. 5-CQA also decreased the DNA damage by 4.49%~48.15%
in human blood lymphocytes caused by X-ray irradiation [62].
In addition to these examples of chemically induced oxidation, other studies have successfully
shown that CGAs are effective at reducing the damage caused by exposure to ultraviolet A light
(UVB) [63]. The photo-oxidation protection offered by 5-CQA in a mouse epidermal cell line [64]
and human HaCaT keratinocytes [51] was related to the effects of 5-CQA to trigger induction of Nrf2
transactivation and phase II enzyme activities.
There are only a few studies that have investigated the affinity of minor CGA isomers to modulate
redox status in biological systems. Three CGA isomers, namely 3-CQA, 4-CQA, and 5-CQA, have
affinity to protect against H2 O2 -induced apoptosis in PC12 cells by suppressing the mitochondrial
membrane depolarization caused by oxidative stress [65]. Recently, another research group reported
that 5-CQA and 3,5-diCQA had a protective effect against t-BOOH-induced ROS generation in HepG2
cells [66].
Nutrients 2016, 8, 16 8 of 20

Table 1. The antioxidant activity of CGA isomers evaluated by chemical assays.

Concentration/Exposure
Chemical Assays End-Point Measure CGA Isomer Results References
Time
DPPH assay DPPH 5-CQA 5–80 µM for 3 h 10%~90% inhibition on DPPH [51]
Xanthine/xanthine DMPO/¨ OOH
5-CQA 20 µM for 2.5 min Ó 30% ¨ OOH [51]
oxidase system adducts
FeSO4 + H2 O2 DMPO/¨ OH adducts 5-CQA 20 µM for 2.5 min Ó 51% ¨ OH [51]
FeSO4 + H2 O2 DMPO/¨ OH adducts 5-CQA 100–400 µM for 1 min Ó 50% to 80% ¨ OH [52]
Serials concentration for The ability of 100 g of CGA in scavenging
ABTS assay ABTS¨ + 5-CQA [53]
15 min ABTS¨ + is equivalent to 3.7 mmol Trolox
Rat brain homogenates +
MDA 5-CQA 1.56–6.25 µg/mL No significant inhibition of MDA [53]
sodium nitroprusside
Liposome system Second order rate of constant of the reactions of LOO¨
MDA 5-CQA 0.1–0.5 mM [54]
containing AAPH with CGA is 1.28 ˘ 0.11 ˆ 105 M´1 ¨ s´1
Pulse radiolysis to Second order rate of constant of the reactions of O2 -
O2 ´ 5-CQA 0.2–0.75 mM [54]
generate O2 ´ with CGA is 0.96 ˘ 0.01 ˆ 106 M´1 ¨ s´1
Fenton-type reaction to Second order rate of constant of the reactions of ¨ OH
¨ OH 5-CQA 0.1–0.75 mM [54]
generate ¨ OH with CGA is 3.34 ˘ 0.19 ˆ 109 M´1 ¨ s´1
Potassium phosphate to Second order rate of constant of the reactions of
ONOO´ 5-CQA 80 µM [54]
generate ONOO´ ONOO´ with CGA is 1.6 ˘ 0.7 ˆ 105 M´1 ¨ s´1
3-CQA, EC50 a 3-CQA:13.4 µg/mL
4-CQA, 4-CQA: 13.2 µg/mL
5-CQA, 5-CQA: 13.8 µg/mL
DPPH assay DPPH 5 µg/mL–60 µg/mL [55]
3,5-diCQA, 3,5-diCQA: 9.3 µg/mL
3,4-diCQA, 3,4-diCQA: 9.4 µg/mL
4,5-diCQA 4,5-diCQA: 7.5 µg/mL
3-CQA EC50 a 3-CQA: 91.4 µg/mL
4-CQA, 4-CQA: 87.5 µg/mL
5-CQA, 5-CQA: 91.5 µg/mL
ABTS assay ABTS¨ + 50 µg/mL–150 µg/mL [55]
3,5-diCQA, 3,5-diCQA: 77.6 µg/mL
3,4-diCQA, 3,4-diCQA: 77.4 µg/mL
4,5-diCQA 4,5-diCQA: 67.3 µg/mL
Nutrients 2016, 8, 16 9 of 20

Table 1. Cont.

Concentration/Exposure
Chemical Assays End-Point Measure CGA Isomer Results References
Time
3-CQA
4-CQA,
5-CQA, 4,5-diCQA > 3,5-diCQA > 3,4-diCQA >
FRAP assay Reducing power 25–125 µg/mL [55]
3,5-diCQA, 5-CQA = 4-CQA = 3-CQA
3,4-diCQA,
4,5-diCQA
3-CQA
4-CQA, Ó 43.1 to 62.4% DNA damage
DNA damage protective 5-CQA,
DNA damage 50 µg/mL [55]
effect assay 3,5-diCQA,
4,5-diCQA > 3,4-diCQA > 3,5-diCQA >
3,4-diCQA,
5-CQA > 4-CQA > 3-CQA
4,5-diCQA
Supercoiled DNA, Prevented a stepwise conversion of plasmid
Plasmid pUC18 + NH2 Cl nicked circular DNA 5-CQA 0.01 mM–1.23 mM DNA form supercoiled DNA, nicked circular [50]
and linear duple DNA and linear duplex DNA
LDL + copper Conjugated dienes 5-CQA 0.25–1.0 µM Ò lag time of LDL oxidation [56]
LDL + metmyoglobin + 1 molar ratio to
ROS 5-CQA Effectively blocked LDL oxidation [57]
H2 O2 metmyoglobin
a EC50 represents the concentration of the tested compound that results in half-maximal response.
Nutrients 2016, 8, 16 10 of 20

Table 2. Summary of studies that evaluated the capacity of CGAs to modulate oxidative stress in cell-based and animal-based models.

Concentration/Exposure Results Compared to the Control

Model End-Point Measure CGA Isomer References
Time without CGA Treatment
Cell-based assay
HaCaT cell + H2 O2 H2 O2 5-CQA 20 µM for 20 h Ó ROS [51]
Ó ROS
HaCaT cell + UVB ROS, DNA damage, cell viability 5-CQA 20 µM for 20 h Ó DNA damage [51]
Ò 13% cell viability
Ó Chromosomal condensation
Chromosomal condensation, cell
Mesenchymal stem cell + H2 O2 5-CQA 10 mM for 12 h Ó Cell apoptosis [58]
apoptosis, ROS
Ó ROS
Primary cortical NADPH: quinine oxido-reductase Ò NADPH: quinine oxido-reductase 1
5-CQA 12.5–100 µM for 1 h [59]
neurons + H2 O2 1, Cell viability Ò Cell viability
Differentiated neuronal PC12 Ò Cell viability
Cell viability, GSH 5-CQA 6.2–25 µM for 2 h [60]
cells + H2 O2 Attenuated GSH decrease
Did not change cell viability
Differentiated neuronal PC12
Cell viability, ROS, MDA 5-CQA 6.2–25 µM for 2 h Ó ROS level [60]
cells + FeSO4
Ó MDA
Differentiated neuronal PC12 Ò Cell viability
Cell viability, GSH 5-CQA 6.2–25 µM for 2 h [60]
cell + t-BHP Did not change GSH level
Human breast cancer
Global methylation status 5-CQA 1–20 µM for 8 days Ó Global methylation [61]
cell line MCF-7+
Human breast cancer cell line Did not change the global
Global methylation status 5-CQA 0.2–20 µM for 3 days [61]
MDA-MB-231+ methylation status
Human breast cancer
Global methylation status 5-CQA 20–50 µM for 2 days Ó Global methylation [61]
cell line T-47D+
Human lymphocyte + Ó Genetic damge index by
Genetic damage index 5-CQA 0.5–4 µg/mL [62]
X-ray radiation 4.49% to 48.15%
Ò GST
Mouse epidermal GST, NADPH: quinone
5-CQA 5–160 µM for 1 h Ò NADPH:quinone oxido-reductase [64]
cell line JB6 + UVB oxido-reductase, Nrf2
Ò Nrf2 nuclear translocation
Nutrients 2016, 8, 16 11 of 20

Table 2. Cont.

Concentration/Exposure Results Compared to the Control

Model End-Point Measure CGA Isomer References
Time without CGA Treatment
Cell-based assay

3-CQA, Protected mitochondrial membrane

Differentiated neuronal PC12 Mitochondrial membrane
4-CQA, 10 µM for 20 min depolarization through [65]
cell + H2 O2 depolarization
5-CQA Ó Caspase 9 activation
Human hepatoma HepG2 cell + 5-CQA,
ROS, GSH, GPx, GR, MDA 10–20 µM for 20 h Ó ROS, Ò GSH, Ò GR, Ó GPx, Ó MDA [66]
t-BOOH 3,5-diCQA
Animal Models
Oral administration at
Ó Plasma lipid hydroperoxides,
Type 2 diabetic rat model Lipid peroxidation, GSH, VC , VE 5-CQA 5 mg/kg body weight [67]
Ò GSH, Ò VC , Ò VE
daily for 45 days
Oral administration at
Lipid peroxidation, GST, SOD, Ó Lipid oxidation, Ò GST, Ò SOD,
Type 2 diabetic rat model 5-CQA 5 mg/kg body weight [68]
GPx, CAT Ò GPx, Ò CAT
daily for 45 days
Oral administration at
Methamphetamine induced
NO, MDA, SOD, GPx 5-CQA 60 mg/kg body weight, Ó NO, Ó MDA, Ò SOD, Ò GPx [69]
oxidative stress rat model
single dose
Intragastric
Cd induced brain impairment SOD, CAT, GPx, GSH, administration, 60 mg/kg Ò SOD, Ò CAT, Ò GPx, Ò GSH, ÒVC ,
5-CQA [70]
rat model VC , VE , MDA body weight daily for Ò VE , Ó MDA
30 days
Orally administered at
Scopolamine induced brain
MDA 5-CQA 3–9 mg/kg body weight, Ó MDA [71]
impairment rat model
single dose
Benzopyrene induced Eating 0.2% 5-CQA Ò GST,
GST, Cytochrome P-450 5-CQA containing diet for [72]
gastrointestinal pathogenesis Did not significantly change
rat model 10 weeks cytochrome P-450
Sodium pentobarbital induced Directly administrate
Vascular permeability in Attenuated the increased
intestinal ischemia-reperfusion 5-CQA 1 mM into jejunum, [73]
the small intestine vascular permeability
rat model single dose
Nutrients 2016, 8, 16 12 of 20

Table 2. Cont.

Concentration/Exposure Results Compared to the Control

Model End-Point Measure CGA Isomer References
Time without CGA Treatment
Animal Models
Orally administered at
Azoxymethane induced colon
GSH/GSSG ratio 5-CQA 0.1% 5-CQA containing Ò Hepatic GSH/GSSG ratio [44]
cancer mice model
diet for 20 weeks
UV irradiation induced Intradermal delivery of
Prevented erythema formation
erythema formation in Guinea Erythema 5-CQA 5-CQA at [74]
induced by UV irradiation
pig and Yucatan micropig 1.49 µmol/g skin
Gamma irradiation induced Orally administered at
Frequencies of micro-nucleated Ó Frequencies of micro-nucleated
chromosomal damage in 5-CQA 50–200 mg/kg body [75]
polychromatic erythrocytes polychromatic erythrocytes
mice model weight, single dose
Nutrients 2016, 8, 16 13 of 20

5.4. Evidence from Animal-Based Assays

Given the positive data from cell culture experiments that show antioxidant properties of specific
CGAs at both cellular and molecular levels, other studies conducted in rodent models have confirmed
these observations by examining the redox status in animals exposed to a variety of forms of oxidative
stress when fed CGAs. Furthermore, the efficacy of dietary intake of CGAs to prevent pathogenesis
of a wide range of chronic disease states has been examined. One example includes the side effect
of hyperglycemia that occurs with diabetes, and which leads to an increase in ROS production and
increased susceptibility to oxidative stress [76]. The antioxidant activity of 5-CQA in diabetic rat
models showed that feeding 5-CQA effectively reduced lipid hydroperoxide production and increased
the level of non-enzymatic antioxidants such as reduced glutathione and Vitamins C and E [67,68].
Other studies have shown that 5-CQA can alleviate the oxidative stress induced by methamphetamine
in rats by restoring liver SOD and GPx activities and preventing the accumulation of MDA [69].
The role of CGAs in prevention of environmental toxicity caused by heavy metal pollution,
specifically cadmium (Cd), has produced data showing protection against the induction of oxidative
stress in the central nervous system [77]. Pretreatment of rats with 5-CQA before Cd exposure
significantly restored the depleted levels of GSH, vitamin C, and vitamin E, and attenuated Cd-induced
MDA levels in brain tissue [70]. Other model systems used scopolamine, a muscarinic antagonist that
significantly increases MDA levels in the cortex and hippocampus [78]. The scopolamine-induced
amnesic mouse, an animal model to study Alzheimer’s disease, has produced data to show that 5-CQA
decreased the MDA level in both the frontal cortex and the hippocampus of scopolamine-induced
anemia in mice [71]. The anti-amnesic activity of 5-CQA was attributed to the affinity to reduce lipid
peroxidation in addition to reducing free radical scavenging activity [71]. Former studies have also
examined the role of 5-CQA to enhance detoxification of environmentally toxic residues derived from
polyaromatic hydrocarbon (PAHs) exposure in mice [72]. Dietary CGAs were effective at enhancing
gastrointestinal xenobiotic detoxification enzymes that are central to the detoxification of PAHs.
The more recent finding that 5-CQA protects against oxidative stress through its activation of Nrf2
nuclear translocation and upregulation of cellular antioxidant enzymes [64] confirms the observation
reported earlier in mice fed PAHs. ROS are also implicated in the development of ischemia/reperfusion
(I/R) injury in the intestine [72] and pathogenesis of colorectal cancer. An intestinal I/R model and
a colorectal cancer model were used to assess the ability of 5-CQA in alleviating oxidative stress in
these sections of the gastrointestinal tract. Sato et al. [73] reported in rats with I/R injury induced
by sodium pentobarbital, that dietary intake of CGA at concentrations ranging from 0.5 to 1.0 mM
was effective to improve the capillary permeability of the small intestine and the capacity to reduce
oxidative stress. In an azoxymethane-induced colon cancer mouse model, a 20% reduction in small
intestinal GSH levels occurred, pointing to an induced oxidative stress condition. Researchers found
that feeding diets containing 0.1% 5-CQA for 20 weeks attenuated azoxymethane-induced oxidative
stress by bringing GSH levels back to normal levels [44].
Ultraviolet radiation and gamma radiation can also trigger the generation of ROS and
consequently cause chromosomal damage in animals [63]. Intradermal delivery of 5-CQA in
guinea pigs during exposure to UVB reduced photooxidation-induced damage of skin attributed
to photooxidation stress [74]. Another study showed that oral administration of 5-CQA to mice at a
concentration of 100 mg/kg body weight before exposure to gamma radiation significantly reduced
chromosomal damage [75].
Since oxidative stress is implicated with a wide range of chronic diseases, it is challenging to
understand the role of specific antioxidants in different pathological and physiological conditions.
Numerous animal models have been used to identify a useful biomarker that will reflect the initiation
of oxidative stress so that the quality of the antioxidant can be evaluated. Despite using different
methodologies, there is strong evidence that CGAs are effective antioxidants that will protect against
oxidation reactions in vivo by up-regulating redox-related nuclear transcription factors involved in the
Nutrients 2016, 8, 16 14 of 20

expression of antioxidant enzymes. The majority of the studies are focused on the primary isomer of
CGA, 5-CQA, with a lesser amount of information available for other minor CGA isomers.

6. The Ability of CGA on Modulating Inflammatory Responses

6.1. Overview of Inflammation and Anti-Inflammatory Mechanisms

Inflammation is a physiological response to tissue injury caused by exogenous or endogenous
sources. Exogenous inducers include pathogen-associated molecular patterns, virulence factors,
allergens, foreign bodies, and toxic compounds [79]. Endogenous inducers of inflammation arise from
cell signaling in response to damaged or malfunctioning tissues [80,81]. It is believed that a controlled
inflammatory response is required to combat offending agents and result in the return of tissue
homeostasis. However, a dysregulated inflammatory response could lead to the failure of effective
resolution, thus leading to excessive tissue damage, resulting in acute or chronic disease states [82].
Anti-inflammatory drugs have been developed to resolve conditions of dysregulated inflammation
by targeting inflammatory mediators, or modulating the activity of cell signaling cascades involved
in responding to an inflammatory signal. The nuclear factor kappa B (NF-κB) pathway is a key
regulator of the release of pro-inflammatory cytokines, chemokines, and adhesion molecules [83].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs for the treatment
of inflammatory diseases [84]. The cyclooxygenase (COX) pathway is the major target for NSAIDs
because COX catalyzes fatty acid oxygenation to produce eicosanoids, which are the cardinal signs
of inflammation. Side effects of NSAIDs include a predisposition to ulcers and bleeding in the
stomach and intestines. Thus, there is increased interest in searching for novel agents that may have
anti-inflammatory activity, without inducing adverse side effects. Different cell lines triggered with
pathogen-associated molecular patterns (lipopolysaccharide (LPS)) and pro-inflammatory cytokines
(e.g., tumor necrosis factor-alpha (TNF-α), interleukin 1β (IL-1β), and interferon gamma (IFN-γ)) have
been used as cell-based inflammation models to study anti-inflammatory mechanisms. Also, animal
models of inflammatory bowel disease, rheumatoid arthritis, and injury-associated inflammation
have been successfully used to evaluate the anti-inflammatory effect of different components, such as
dietary bioactives.

6.2. CGA Suppression of Inflammation through Inhibition of Pro-Inflammatory Cytokines via Modulation of
Key Transcription Factors
Inflammatory bowel disease represents a chronic relapsing of disorders occurring in the
gastrointestinal tract that are characterized by intestinal inflammation and epithelial injury [85].
5-CQA has a protective effect against intestinal-related inflammation in both cell-based and
animal-based models. CGA has an anti-inflammatory effect in TNF-α and H2 O2 -induced human
intestine epithelia Caco-2 cells by down regulating IL-8 production [86]. Studies conducted with a
tea (Artemisia annua) containing CGAs showed a strong anti-inflammatory effect by decreasing the
secretion of pro-inflammatory cytokines IL-8 and IL-6 in Caco-2 cells stimulated with TNF-α, LPS,
IL-1β, and IFN-γ [30]. CGA also attenuated IL-1β, TNF-α, and IL-6 production in LPS-stimulated
murine RAW 264.7 macrophages and in BV2 microglial cells by effectively down regulating the
NF-κB pathway [87]. Previously, it has been reported that phenolic compounds lower the activity
of COX and subsequently prevent the synthesis of eicosanoids [88]. A cell study conducted
on murine RAW 264.7 macrophages confirmed that CGA showed anti-inflammatory activity by
suppressing LPS-induced COX-2 expression via attenuating the activation of NF-κB and JNK/AP-1
signaling pathways [89]. In animal studies, the oral administration of 5-CQA protected against
trinitrobenzenesulfonic acid-induced colitis in mice by reducing neutrophil infiltration and inhibition
of the NF-κB pathway [48]. A similar effect was also observed in the dextran sulfate sodium-induced
colitis model in mice [86] and in a carrageenan-induced paw edema model in rats [90]; in both cases a
suppression of pro-inflammatory cytokines was observed.
Nutrients 2016, 8, 16 15 of 20

Rheumatoid arthritis is another chronic inflammatory disorder characterized by the deterioration

of cartilage and bone. Chauhan et al. [91] observed that oral administration of 40 mg/kg of 5-CQA
effectively suppressed pro-inflammatory cytokines including TNF-α and IL-1β in an LPS-induced
knee joint inflammation rat model. This effect was similar to the standard treatment of administering
ibuprofen at 100 mg/kg.
The reduction of inflammation resulting in enhanced wound healing has also been reported
for CGAs. Oral administration of 5-CQA at a dose of 50 mg/kg/day accelerated wound healing
and decreased MDA and nitric oxide levels while elevating reduced-glutathione content in rats [92].
Oral administration of 5-CQA also alleviated hepatic ischemia and reperfusion-induced liver injury
by reducing inflammatory responses and increasing antioxidant defense systems [93]. There is
also evidence that 5-CQA can suppress IL-1β, TNF-α, and IL-6 production in CCl4-induced liver
inflammation and fibrosis in rats by inhibition of NF-κB activation [94]. Table 3 is a summary of cell
and animal studies on the anti-inflammatory activity of CGA.

Table 3. Summary of studies that evaluated the capacity of CGAs to modulate inflammatory stress in
cell-based and animal-based models.

Results Compared to the

End-Point CGA Concentration/
Models Control without CGA References
Measure Isomer Exposure Time
Treatment
Cell Models
Caco-2 + TNF-α
IL-8 5-CQA 0.5–2 mM Ó IL-8 [86]
and H2 O2
Caco-2 + cocktail of Mixture
Unknown composition
inflammatory IL-6, IL-8 of all Ó IL-6, Ó IL-8 [30]
of CGAs for 1 h
mediators CGAs
NO, IL-1β, TNF-α,
Ó NO, Ó IL-1β, Ó TNF-α, Ó IL-6,
RAW 264.7 + LPS cyclooxygenase-2, 5-CQA 2–20 µM for 24 h [87]
Ó cyclooxygenase-2, Ó NFκB
NF-κB, IL-6
Cyclooxygenase,
Ó Cyclooxygenase,
RAW 264.7 + LPS Prostaglandin E2, 5-CQA 12.5–37.4 µg/mL for 2 h [89]
Ó Prostaglandin E2, Ó NF-κB
NF-κB
Animal Models
Trinitrobenzenesulfonic Orally administration at
Myeloperoxidase, Ó Myeloperoxidase, Ó H2 O2 ,
acid induced colitis 5-CQA 20 mg/kg body weight, [48]
H2 O2 , NF-κB Ó NF-κB
mice model twice a day for 6 days
IL-1β, TNF-α, Ó IL-1β, did not significantly
Dextran sulfate
macrophage Orally administration at change the levels of TNF-α
sodium induced 5-CQA [86]
inflammatory 1 mM for 15 days and macrophage
colitis mice model
protein 2 inflammatory protein 2
Trinitrobenzenesulfonic Orally administration at
Myeloperoxidase, Ó Myeloperoxidase, Ó H2 O2 ,
acid induced colitis 5-CQA 20 mg/kg body weight [90]
H2 O2 , NF-κB Ó NF-κB
mice model twice a day
IL-1β, TNF-α, T
Ó IL-1β, Ó TNF-α, Ó T cells
Rheumatoid cells count, Th1 Orally administration at
5-CQA count, Ó Th1 cytokines, [91]
Arthritis rat model cytokines, 40 mg/kg body weight
Ò Th2 cytokines
Th2 cytokines
Wound healing Intraperitoneal injection
Wounds in Ò Wound healing speed,
speed, NO, 5-CQA at 50 mg/kg/day [92]
diabetic rat Ò GSH, Ó NO, Ó MDA
MDA, GSH for 15 days
MDA, GSH,
Orally administration at Ó MDA, Ò GSH, Ó TNF-α,
Liver injury TNF-α, NO,
5-CQA 2.5–10 mg/kg body Ó NO, Ó cyclooxygenase-2 [93]
rat model cyclooxygenase-2
weight, twice a day protein increase
protein increase

7. Summary and Conclusions

CGAs, which exist in green and roasted coffee beverages as well as fruit- and vegetable-based
juices, are a group of esters formed between quinic acid and some trans-cinnamic acids, such as caffeic,
ferulic, and p-coumaric acids. There has been considerable interest in understanding and quantitating
Nutrients 2016, 8, 16 16 of 20

the bioactivity of CGAs associated with antioxidant and anti-mutagenic activities related to oxidative
stress. The topic is complex with the knowledge that CGAs are not stable in high temperature processed
beverages, such as coffee [1,95]. This review highlights the cellular and molecular mechanisms that
explain the pharmacological benefits of consuming CGA-containing beverages. Clearly, in vitro and
in vivo data indicate that 5-CQA has antioxidant activity and can alleviate oxidative stress in various
disease models. This potential pro-oxidant activity occurs under conditions that prolong the lifetime
of the phenoxyl radical. The majority of the evidence to date indicates an anti-inflammatory activity
for 5-CQA that can be explained by its ability to down regulate pro-inflammatory cytokines, through
modulation of key transcription factors. A study reported that intravenous injection with 5-CQA at
high dosages caused a range of inflammatory reactions in rats [96]. However, in all reality, the CGA
dose infused does not relate to a typical dietary exposure. Future research is required to assess more of
the potential health benefits of CGA-containing beverages. This should involve understanding the
potential bioactivities of non-5-CQA isomers and the products of CGA transformation that occur in
the large intestine from microbial activity.

Acknowledgments: This work was funded by the Natural Sciences and Engineering Research Council of
Canada (NSERC) Discovery Grant (DDK). Ningjian Liang was a recipient of a University of British Columbia
PhD fellowship.
Author Contributions: Ningjian Liang wrote the manuscript with contributions made by David Kitts.
Conflicts of Interest: The authors declare no conflict of interest.

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