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An Outlook on Chlorogenic Acids—Occurrence,

Chemistry, Technology, and Biological Activities
a a
Rohit Upadhyay & L. Jagan Mohan Rao
a
Plantation Products, Spices and Flavour Technology Department , Central Food
Technological Research Institute (A constituent laboratory of Council of Scientific Industrial
Research, New Delhi, India) , Mysore , Karnataka , 570020 , India
Accepted author version posted online: 06 Jul 2012.

To cite this article: Rohit Upadhyay & L. Jagan Mohan Rao (2013): An Outlook on Chlorogenic Acids—Occurrence, Chemistry,
Technology, and Biological Activities, Critical Reviews in Food Science and Nutrition, 53:9, 968-984

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 Critical Reviews in Food Science and Nutrition, 53:968–984 (2013)
Copyright C Taylor and Francis Group, LLC
ISSN: 1040-8398 / 1549-7852 online
DOI: 10.1080/10408398.2011.576319

An Outlook on Chlorogenic
Acids—Occurrence, Chemistry,
Technology, and Biological Activities

ROHIT UPADHYAY and L. JAGAN MOHAN RAO

Plantation Products, Spices and Flavour Technology Department, Central Food Technological Research Institute (A constituent
laboratory of Council of Scientific Industrial Research, New Delhi, India), Mysore, Karnataka 570020, India
Downloaded by [University of Haifa Library] at 08:33 24 June 2013

Phenolics are widespread dietary antioxidants. Among these, chlorogenic acids (CGAs) received considerable attention
for their wide distribution and part of human diet with potential biological effects. CGAs (71 compounds), being esters of
derivatives of cinnamic acids with quinic acid are widely distributed in plant materials. Coffee is among the highest found
in plants, ranging from 4 to 14%. Besides, these are reported in plant foods such as apples, pears, carrot, tomato, sweet
potato, Phyllostachys edulis, oilseeds, Prunus domestica L, cherries, and eggplant. The traditional Chinese medicinal plants
such as flowers and buds of Lonicera japonica Thunb and the leaves of Eucommia ulmodies contained CGAs as bioactive
compound. These play an important role in the formation of roasted coffee flavor and have a marked influence on coffee cup
quality. CGAs are considered as main precursors of coffee flavor and pigments. Recent technological advancements in the
separation and purification of CGAs such as molecular-imprinted polymer technique; microwave-assisted extraction; pH
gradient counter current chromatography has also been described. The consumption of coffee correlated to several health
benefits such as reducing the risk of human chronic diseases such as inflammation, diabetes, and cardiovascular disease
owing to its antioxidant potential.

Keywords Chlorogenic acids, occurrence, chemistry, technology, biological activities, antioxidant, health benefits

INTRODUCTION The free acid, generated by the treatment of precipitate with

The trend to view many foods not only as sustenance but also
as medicine is increasing. Phenolics are the most widespread
dietary antioxidants and among these chlorogenic acid (CGA)
accumulates to high levels in some crop plants. A small quan-
tity of free quinic acid occurs in green coffee beans. A greater
quantity of quinic acid occurs as a series of esters (collectively)
generally known as CGA. CGAs (Table 1) are a family of es-
ters formed between certain trans-cinnamic acids (viz. caffeic,
coumaric, and ferulic acid) and (−)-quinic acid and are major
phenolic compounds in coffee, strawberries, pineapple, apple,
sunflower, and blueberries.
Possibly, the first report to describe these substances is at-
tributable to Robiquet and Boutron in the year 1837. In 1844,
Rochleder observed that caffeine in green coffee beans could
associate with an acid that could be precipitated with lead salts.
Address correspondence to Dr. L. Jagan Mohan Rao, Plantation Prod-
ucts, Spices and Flavor Technology Department, Central Food Technologi-
cal Research Institute, Mysore, Karnataka 570020, India. E-mail: ljnatpro@
yahoo.com
968
sulfuric acid, gave a yellow color on the addition of ammo-
nia. In 1846, Rochleder reported that the yellow ammonical
solution turned green on exposure to oxygen. He proposed an
empirical formula of C16 H9 O8 for the free acid. Later that year,
Payen reported the isolation of a crystalline potassium caffeine
chloroginate that formed some 3.5% of green coffee beans. An
empirical formula of C14 H8 O7 was proposed. Gorter and Griebel
reported a melting point of 202◦ C–203◦ C for green tinged, white
crystals of CGA. Gorter reported 206◦ C–207◦ C for pure white
crystals, and proposed an empirical formula of C32 H38 O19 . Al-
kaline hydrolysis at low temperatures yielded caffeic acid and
quinic acid in equimolar quantities. To reconcile this obser-
vation with the proposed empirical formula, Gorter suggested
that caffeic acid combined with quinic acid to produce hemi-
CGA, two molecules of which condensed to give CGA (Clarke
and Macrae, 1985). In 1920, Freudenberg reported that the en-
zyme tannase hydrolyze CGA to equimolar quantities of caffeic
acid and quinic acid (Table 1). In 1932, Fischer and Dangschat
deduced that CGA was 3-caffeoylquinic acid (3-CQA). Un-
der current International Union of Pure and Applied Chem-
istry (IUPAC) recommendations, 3-CQA is now designated
 AN OUTLOOK ON CHLOROGENIC ACIDS 969

Table 1 Structures of different types of chlorogenic acids (Sung et al., 2001; Rakesh et al., 2010)

No. Name Abbreviation R1 R2 R3

1 3-O-caffeoylquinic acid 3-CQA C H H

2 4-O-caffeoylquinic acid 4-CQA H C H
3 5-O-caffeoylquinic acid 5-CQA H H C
4 3-O-feruloylquinic acid 3-FQA F H H
5 4-O-feruloylquinic acid 4-FQA H F H
6 5-O-feruloylquinic acid 5-FQA H H F
7 3-O-p-coumaroylquinic acid 3-pCoQA pCo H H
8 4-O-p- coumaroylquinic acid 4- pCoQA H pCo H
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9 5-O-p- coumaroylquinic acid 5- pCoQA H H pCo

10 3-O-dimethoxycinnamoyl quinic acid 3-DQA D H H
11 4-O-dimethoxycinnamoyl quinic acid 4-DQA H D H
12 5-O-dimethoxycinnamoyl quinic acid 5-DQA H H D
13 3-O-sinapoylquinic acid 3-SiQA Si H H
14 4-O-sinapoylquinic acid 4-SiQA H Si H
15 5-O-sinapoylquinic acid 5-SiQA H H Si
16 3,4-di-O-caffeoylquinic acid 3,4-diCQA C C H
17 3,5-di-O-caffeoylquinic acid 3,5- diCQA C H C
18 4,5-di-O-caffeoylquinic acid 4,5-diCQA H C C
19 3,4-di-O-feruloylquinic acid 3,4-diFQA F F H
20 3,5-di-O-feruloylquinic acid 3,5-diFQA F H F
21 4,5-di-O-feruloylquinic acid 4,5-diFQA H F F
22 3,4-di-O-p-coumaroyl quinic acid 3,4-dipCoQA pCo pCo H
23 3,5-di-O-p-coumaroylquinic acid 3,5-dipCoQA pCo H pCo
24 4,5-di-O-p-coumaroylquinic acid 4,5- dipCoQA H pCo pCo
25 3-O-feruloyl-4-O-caffeoylquinic acid 3F-4CQA F C H
26 3-O-caffeoyl-4-O-feruloylquinic acid 3C-4FQA C F H
27 3-O-feruloyl-5-O-caffeoylquinic acid 3F-5CQA F H C
28 3-O-caffeoyl-5-O-feruloylquinic acid 3C-5FQA C H F
29 4-O-feruloyl-5-O-caffeoylquinic acid 4F-5CQA H F C
30 4-O-caffeoyl-5-O-feruloylquinic acid 4C-5FQA H C F
31 3-O-dimethoxycinnamoyl-4-O-caffeoylquinic acid 3D-4CQA D C H
32 3-O-dimethoxycinnamoyl-5-O-caffeoylquinic acid 3D-5CQA D H C
33 4-O-dimethoxycinnamoyl-5-O-caffeoylquinic acid 4D-5CQA H D C
34 3-O-dimethoxycinnamoyl-4-O-feruloylquinic acid 3D-4FQA D F H
35 3-O-dimethoxycinnamoyl-5-O-feruloylquinic acid 3D-5FQA D F H
36 4-O-dimethoxycinnamoyl-5-O-feruloylquinic acid 4D-5FQA H D F
37 3-O-p-coumaroyl-4-O-caffeoylquinic acid 3pCo-4CQA pCo C H
38 3-O-caffeoyl-4-O-p-coumaroylquinic acid 3C-4pCoQA C pCo H
39 3-O-p-coumaroyl-5-O-caffeoylquinic acid 3pCo-5CQA pCo H C
40 3–O-caffeoyl-5-O-p-coumaroylquinic acid 3C-5pCoQA C H pCo
41 4-O-caffeoyl-5-O-p-coumaroylquinic acid 4C-5pCoQA H C pCo
42 4-O-p-coumaroyl-5-O-caffeoylquinic acid 4pCo-5CQA H pCo C
43 3-O-p-coumaroyl-4-O-feruloylquinic acid 3pCo-4FQA pCo F H
44 3-O-p- coumaroyl-5-O-feruloylquinic acid 3pCo-5FQA pCo H F
45 4-O-p- coumaroyl-5-O-feruloylquinic acid 4pCo-5FQA H pCo F
46 4-O-dimethoxycinnamoyl-5-O-p-coumaroylquinic acid 4D-5pCoQA H D pCo
47 3-O-p-coumaroyl -4-O-dimethoxycinnamoylquinic acid 3pCo-4DQA pCo D H
48 3-O-p-coumaroyl-5-O-dimethoxycinnamoylquinic acid 3pCo-5DQA pCo H D
49 3-O-sinapoyl-5-O-caffeoylquinic acid 3Si-5CQA Si H C
50 3-O-sinapoyl-4-O-caffeoylquinic acid 3Si-4CQA Si C H
51 3-O- (3,5-dihydroxy-4-methoxy)cinnamoyl-4-O-feruloylquinicacid 3DM-4FQA DM F H
52 4-O-sinapoyl-3-O-caffeoylquinic acid 4Si-3CQA C Si H
53 3-O-sinapoyl-5-O-feruloylquinic acid 3Si-5FQA Si H F
54 3-O-feruloyl-4-O-sinapoylquinic acid 4Si-5FQA H Si F
 970 R. UPADHYAY AND L. J. M. RAO

Table 1 Structures of different types of chlorogenic acids (Sung et al., 2001; Rakesh et al., 2010) (Continued)

No. Name Abbreviation R1 R2 R3

55 4-O-sinapoyl-3-O-feruloylquinic acid 4Si-3FQA F Si H

56 4-O-trimethoxycinnamoyl-5-O-caffeoylquinic acid 4T-5CQA H T C
57 3-O-trimethoxycinnamoyl-5-O-caffeoylquinic acid 3T-5CQA T H C
58 3-O-trimethoxycinnamoyl-5-O-feruloylquinic acid 3T-5FQA T H F
59 3-O-trimethoxycinnamoyl-4-O-feruloylquinic acid 3T-4FQA T F H
60 4-O-trimethoxycinnamoyl-5-O-feruloylquinic acid 4T-5FQA H T F
61 3-O-dimethoxycinnamoyl-4-O-feruloyl-5-O-caffeoylquinic 3D-4F-5-CQA D F C
61 acid
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62 3,4,5-tri-O-caffeoylquinic acid 3,4,5-triCQA C C C

63 3,5-di-O-caffeoyl-4-O-feruloylquinic acid 3,5-diC-4FQA C F C
64 3-O-feruloyl-4,5-di-O-caffeoylquinic acid 3F-4,5-diCQA F F C
65 3,4-di-O-caffeoyl-5-O-feruloylquinic acid 3,4-diC-5FQA C C F
66 3-O-caffeoyl-4,5-di-O-feruloylquinic acid 3C-4,5-diFQA C F F
67 3,4-di-O-feruloyl-5-O-caffeoylquinic acid 3,4-diF-5CQA F F C
68 3,4-di-O-caffeoyl-5-O-sinapoylquinic acid 3,4-diC-5SiQA C C S
69 3-O-sinapoyl-4,5-di-O-caffeoylquinic acid 3Si-4,5-diCQA S C C
70 5-O-caffeoyl-4-methyl quinic acid 5C,4MQA H CH3 C
71 3-O-caffeoyl-1-methyl quinic acid 3C,1MQA C H H

C = caffeoyl, D = dimethoxycinnamoyl, F = feruloyl, pCo = p-coumaroyl, Si = sinapoyl, H = hydrogen, DM = 3,5-dihydroxy-4-methoxycinnamoyl, T =

trimethoxycinnamoyl, MQA = methyl quinic acid.

5-caffeoylquinic acid (5-CQA). 5-CQA is the only CGA
commercially available and has been extensively studied due
to its antioxidant activity. CGAs are free radical and metal scav-
engers; may interfere with glucose absorption; and has been
shown to modulate gene expression of antioxidant enzymes,
among other biological activities. CGA acts as an antioxidant
in plants and protects against degenerative, age-related diseases
in animals when supplied in their diet. The amount of CGA or
caffeic acid available to act as an antioxidant in vivo will de-
pend on absorption from the gut, which may be incomplete and
any subsequent metabolism, which may be extensive. A study
of human CGA metabolism showed that the unabsorbed CGA,
which reaches the colon, is hydrolyzed to caffeic acid and quinic
acid by the colonic microflora. Following dehydroxylation by
the colonic microflora, absorption and further metabolism in
the liver and kidney, benzoic acid is formed and conjugated to
glycine to form hippuric acid and half the ingested CGA appears
as urinary hippuric acid (Olthof et al, 2001). This metabolism
can be expected to considerably diminish the antioxidant activ-
ity of CGA in vivo as hippuric acid has no antioxidant activity.
The roasting of coffee beans dramatically increases their total
antioxidant activity. A roasting time of 10 minutes (medium-
dark roast) was found to produce coffee with optimal oxy-
gen scavenging and chain-breaking activities in vitro (Nicoli
et al., 1997). It has been reported that a 200 mL cup of Arabica
coffee contains between 70 and 200 mg CGA whereas a cup
of Robusta coffee contains between 70 and 350 mg (Clifford,
1999). It has been estimated that coffee drinkers may consume
about a gram of CGA each day. This amount of CGA, when
consumed regularly, appears to be sufficient to yield obvi-
ous therapeutic effects. Women are at greatest risk of form-
ing gallstones, about twice that for men. In a large study con-
ducted by Harvard University tracking of over 80,000 women,
it was found that regular coffee intake reduced gallstone
formation (Leitzmann, 2002). Further, coffee consumption
was associated with a lower risk of a variety of liver dis-
eases, including liver cirrhosis and liver cancer (Tverdal and
Skurtveit, 2003). This effect may come from a combination of
cholagogue action (keeping toxins and fats flowing with the
bile) and antioxidant effects. CGA is also found as a significant
component in certain commonly used medicinal herbs. Other
Chinese herbs known for their CGA content include chrysanthe-
mum flower, crataegus fruit, artemisia leaves, and epimedium
leaves. In Western herbal medicine, a herb especially known
for its CGA content is artichoke leaves; the extracts are usu-
ally standardized to 15% of this compound. Other medicinal
herbs known for content of CGA include burdock root, dande-
lion root, and echinacea root. The food industry today is facing
a problem where the traditional synthetic antioxidants such as
butylated hydroxyanisole (BHA) and butylated hydroxytoluene
(BHT) are becoming increasingly less acceptable to consumers;
they are looking for a new range of new antioxidants among
natural products to protect processed foodstuffs from rancidity
and off flavors, etc. Therefore, green coffee is a good source
for extraction of antioxidant conserve due to its high CGA
content.
 AN OUTLOOK ON CHLOROGENIC ACIDS 971

OCCURRENCE Table 2 Chlorogenic acid (CGA) content for different coffee species (% of
dry matter)

CGA is one of the most abundant polyphenols in the hu- Species or taxa CGA
man diet with coffee, fruits, and vegetables as its major sources.
C. sp. Bakossi 0.61-1.05
Around 71 different species of CGA (Table 1) have now been C. humblotiana 0.90-1.09
identified from different sources (Sung et al., 2001; Rakesh et al., C. salvatrix 1.74-3.00
2010). It is also an important bioactive compound and rich in C. eugenioides 4.55-6.08
some traditional Chinese medicine, such as flowers and buds C. sp. Moloundou 5.40-5.73
C. racemosa 4.78-5.57
of Lonicera japonica Thunb (L. japonica), and the leaves of
C. heterocalyx 6.11-6.52
Eucommia ulmodies. The CGAs are an important group of non- C. liberica Dewevrei 6.22-8.78
volatile compounds in green coffee beans. Although 30 different C. liberica Koto 8.81-8.89
species of CGA have now been identified in green beans (Clif- C. liberica liberica 8.75-10.7
ford et al., 2006a, 2006b), the vast majority of the compounds C. congensis 8.15-8.77
C.kapakata 8.84-10.7
found belong to three classes: CQAs (3-CQA, 4-CQA, and 5-
C. poscii 9.77-11.5
CQA), di-CQAs (3,4diCQA, 3,5diCQA, and 4,5diCQA), and C. stenophylla 6.76-9.39
feruloylquinic acids (FQA). The levels of CGA in green beans
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have been reported to vary from approximately 7.88 to 14.4%
dry matter basis (dmb) for Coffea canephora (robusta) and ap-
proximately 3.4–4.8% dmb for Coffea arabica (Ky et al., 1997).
In addition to different total CGA levels, the relative amounts of
CQA, diCQA, and FQA have also been shown to vary in mature
C. canephora and C. arabica grain. Ky et al. (2001) have found
that CQA, diCQA, and FQA represent roughly 67%, 20%, and
13%, respectively, of the total CGA content of C. canephora va-
riety versus 80%, 15%, and 5%, respectively, of the total CGA
content of C. arabica variety. In addition to being found in
coffee, these compounds are also found at significant levels in
plant foods such as apples, pears, tomato, potato, and eggplant
(Castelluccio et al., 1995; Niggeweg et al., 2004).
Wild Coffee Species
Two coffee species, C. canephora and C. arabica, are culti-
vated worldwide. However, these species only represent a small
proportion of the worlds coffee genetic resources as, in the
wild, the Coffea subgenus (Coffea genus, Rubiaceae family)
includes more than 80 species or taxa endemic to intertropical
forest zones of Africa, Madagascar, Mauritius, Comoros, and
Réunion (Charrier, 1978; Anthony, 1992). Campa et al. (2005)
described the CGA content (Table 2) in a series of wild Cof-
fea genetic resources, including 15 species and 6 new taxa that
have not yet been botanically described. Similar variations are
noted in caffeine content. Their study highlighted a discontin-
uous distribution of CGA content and enabled them to define
five qualitative classes in the caffeine–CGA relationship. The
CGA content evaluated for the 21 species or taxa, ranged from
0.6% dmb in C. sp. Bakossi to 12.7% dmb in C. sp. Nkoumbala.
The CGA bean content range differed between eastern, central,
and western Africa. The lowest range with higher content was
recorded in western Africa, where CGA varied from 8.8% in
C. liberica subsp. liberica to 14.4% in C. canephora. In eastern
Africa, CGA content varied from 1.5% in C. pseudozangue-
bariae to 11.5% dmb in C. pocsii. The range was similar in
central Africa (Central African Republic, Congo, Cameroon),
C. humilis 7.87-10.3
C. sp. Congo 7.65-10.3
C. sp. N’ gongo2 10.1-11.3
C. brevipes 10.4-12.3
C. canephora 9.76-14.4
C. sp. N’ koumbala 11.1-12.7
Note: Campa et al. (2005).
where CGA content ranged from 0.6% in C. sp. Bakossi to
12.7% in C. sp. Nkoumbala.
Fruits as Source
Phenolic compound are ubiquitous in plant kingdom, being
found in all fruits and vegetables. CGA is the most important
cinnamic acid derivative in fruits, which is sometimes a predom-
inant phenolic compound. In 1983, Möller and Herrmann de-
tected 88–731 mg/kg of 3-CQA, 15–129 mg/kg of 5-CQA, and
56 mg/kg of 4-CQA in fresh plum. In case of other stone fruits,
cherries contained 3-CQA and 3-O-p-coumaroylquinic acid as
major hydroxycinnamates, followed by 5-CQA and other sub-
ordinate compounds such as 4-CQA. In apricots and peaches,
3-CQA and 5-CQA were predominant and other components
were present in slightly lower amounts. In the pome fruits, ap-
ples and pears contained principally 5-CQA and other minor
compounds (Möller and Herrmann, 1983). Concerning these
stone and pome fruits, 4-CQA was contained only in cherries
and only as a minor component. Therefore, the content of 4-
CQA in prune is fairly high. It might be supposed that a high
amount of 4-CQA was obtained by isomerization among CGA
isomers, because isomerization of some plant components oc-
curred during the extraction with protic solvent (Nakatani et al.,
1991). Generally, plum refers to the fruits of the genus Prunus,
such as Prunus domestica, P. salicina, P. subcordiata, and
P. insititia (Pijipers et al., 1986). Among P. domestica, the
so-called prune is the dried fruit of some cultivars of P. do-
mestica, which are available for making dried fruits. It is re-
ported that neo-CGA (3-O-caffeoylquinic acid, 3-CQA) was
a major hydroxycinnamate (541 mg/kg) in the fruit of plum.
CGA (5-O-caffeoylquinic acid, 5-CQA) was also contained at a
 972 R. UPADHYAY AND L. J. M. RAO
concentration of 73 mg/kg, and crypto-CGA (4-O-caffeoyl-
quinic acid, 4-CQA) was found at 9 mg/kg as a minor component
(Herrmann, 1989). According to Hermann (1993) and Amiot
et al. (1995), the primary phenolic in pear juice is CGA and
the secondary phenolic is epicatechin. Besides that, polyphe-
nols represent the third most abundant constituent in grapes and
wines after carbohydrates and fruit acids. The most common
polyphenolic acids in grapes include cinnamic acids (caffeic,
ferulic, chlorogenic, and neo-CGAs). Tanrioven and Eksi (2005)
studied the phenolic content of pear juice from seven different
varieties. They have reported that total amount of polyphenol in
pear juice varied from 196 to 456 mg/L. The main phenolics in
pear juice were CGA, epicatechin, caffeic acid, and coumaric
acid.
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Other Sources
Three CGA derivatives from the leaves of bamboo Phyl-
lostachys edulis have been isolated and identified as 3-O-(3’-
methylcaffeoyl) quinic acid, 5-O-caffeoyl- 4-methoxylquinic
acid, and 3-O-caffeoyl-1-methoxylquinic acid (Table 1, No 4,
70, 71, Sung et al., 2001). They all showed strong free radical
and superoxide scavenging activities and inhibition of lipid per-
oxidation. CGA have also been reported in oilseeds. Soya bean
has been reported to have coumestrol, prunetin, formononetin,
CGA, caffeic acid, ferulic acid, p-coumaric acid, salicyclic acid
as minor phenolic compounds whereas sunflower has CGA,
caffeic acid, and quinic acid as major phenolic compound.
Flower buds of Lonicera japonica Thunb are traditionally used
as herbal medicine in the treatment of a wide range of ailments
including syphilitic skin diseases, tumors, bacterial dysentery,
colds, enteritis, pain, and swellings. These are reported to con-
tain CGA (Pharmacopoeia Commission of People’s Republic
of China, 2005). CGA, a major bioactive component in the
flower buds, has received more and more attention because of
its antivirus, anticancer, and anti-inflammatory activities (Naka-
mura et al., 1997; Jiang et al., 2001; Jin et al., 2005). Sun
et al. (2009) have reported the occurrence of CGAs in carrot
while carrying out studies on antioxidant phytochemicals and
antioxidant capacity of biofortified carrots (Daucus Carota L.)
of various colors. Their research indicated CGA was a ma-
jor antioxidant in all carrots. Marques and Farah (2009) re-
ported the contents of CGAs and related compounds in medic-
inal plants and infusions. In their study, they determined the
CGA composition of 14 dried medicinal plants by high perfor-
mance liquid chromatography-ultraviolet detector (HPLC-UV)
and liquid chromatography-diode array detector-electrospray
ionization-mass spectrometry (LC-DAD–ESI-MS). The plants
with the highest CGA contents were Ilex paraguariensis,
Bacharis genistelloides, Pimpinella anisum, Achyrochine sat-
ureioides, Camellia sinensis, Melissa officinalis, and Cymbo-
pogon citratus, with 84.7 mg/100 g–9.7 g/100 g, dry weight.
I. paraguariensis and B. genistelloides were good sources of
both CQA and diCQA compounds, while C. sinensis contained
more CQA and A. satureioides more diCQA compounds.
CHEMISTRY
CGA [(1,3,4,5-tetrahydroxycyclohexane carboxylic acid 3-
(3,4-dihydroxycinnamate); Table 1] is a kind of polyphenol
derivative which widely exists in higher plants like fruits, veg-
etables, berries, and in selected traditional Chinese medicines
(Delage et al., 1991; Zhang et al., 1996a). Besides possessing
the antioxidant activity, inhibiting hypertension, and stimulat-
ing the flowering of plants, it also affects the activity of trypsin,
amylase, and other enzymes (Zhu and Xiao, 1991). Moreover,
CGA is the main component of producing the bitter taste in
crude coffee and thus deliberative elimination of CGA from the
instant coffee was adopted extensively to improve the taste of
various kinds of coffees.
A number of the CGA series have yet to be obtained in
the crystalline form because of difficulty in purifying these sub-
stances, whether obtained by synthesis or isolation. Those mem-
bers of CGA series that have been crystallized have generally
been found to have very small crystals and very few crystal-
lographic data have been published. Two forms of crystalline
5-CiQA have been reported. Short needles obtained from the
aqueous ethanol melted at 146◦ C, whereas the prisms occasion-
ally obtained in ethyl acetate-petroleum ether melted at 166◦ C
(Clarke and Macrae, 1985).
Species Classes According to CGA Content
According to the Newman and Keuls test, two discontinu-
ities split the CGA content range into three classes. The first
class consisted of four species with low CGA content (mean
1.4% dmb). Its range (0.8–2.2% dmb) showed significant inter-
specific differences. The second class included four species. Its
CGA content ranged from 5.2 to 6.3% dmb, with a mean of 5.6%
dmb. The third class consisted of 13 species with high CGA con-
tent (mean 9.8% dmb). As in first class, its range (7.6–11.9%
dmb) showed significant between-species variations, but with a
large overlap. Some investigators (Farah et al., 2008), using liq-
uid chromatography-mass spectrometry (LC-MS) and synthetic
standards, reported major and minor CGAs and CGAs lactones
isomers (CGL) in green and roasted samples of economically
relevant Brazilian C. arabica and C. canephora coffee culti-
vars. 1-FQA, 1-feruloylquinic lactone, and 3,4-di-FQA were
quantified in C. arabica and C. canephora. Contents of 3- and
4-p-coumaroylquinic lactones were reported in C. canephora
and 3,4-di-p-coumaroylquinic acid was identified in C. arabica.
A total of 14 CGA compounds were identified in green C. ara-
bica and C. canephora samples, according to their molecular
weight and retention time: 3-CQA, 4-CQA, 5-CQA, 3-FQA, 4-
FQA, 5-FQA, 3-p-CoQA, 4-p-CoQA, 5-p-CoQA, 3,4-diCQA,
3,5-diCQA, 4,5-diCQA, 3,4-diFQA, and 3,4-di-p-CoQA.
Solubility
The CGA, in general, and the less polar diCQA, in partic-
ular, are more soluble in the lower alcohols or alcohol–water
 AN OUTLOOK ON CHLOROGENIC ACIDS
mixtures. These are insoluble in benzene, chloroform, and
petroleum ether. The greater polarity and water solubility is
associated with the more solvatable-free equatorial hydroxyl
groups rather than the sterically hindered free axial hydroxyl
groups. Water solubility declines 1-acyl > 3-acyl > 4-acyl >
5-acyl. Certainly, the first two are much more polar than the
last two. An aromatic hydroxy group with the acylating residue
raises the polarity whereas a methoxy group reduces it. The wa-
ter solubility declines 5-GQA > 5-CQA > 5-CoQA > 5-FQA.
The diCQA are less water soluble than structurally related CQA,
but more soluble in ethyl acetate, butyl acetate, and acetone. The
1, 5-lactones are much less polar than free acids, but appear to
be soluble in butyl acetate, ethyl acetate, acetone, and methanol
(Clarke and Macrae, 1985).
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Dissociation Constant
Dissociation constant for the carboxylic group of quinic acid,
4-CQA and 5-CQA, fall in the range of 3.40–3.60. Correspond-
ing values for the coffee bean diCQA and 5-CQA in 50% alcohol
are in the range 4.20–4.50. These data are consistent with the
carboxy equatorial conformation of the quinic acid. In water,
the more acidic aromatic hydroxyl group in the CQA has a pKa
value of 8.45 (Clarke and Macrae, 1985).
Organoleptic Properties of Chlorogenic Acids
The organoleptic properties of CGAs have received scant at-
tention. The 5-CQA is much less acidic than free quinic acid
and slightly bitter. In contrast, a mixture of the three diCQA in
essentially equal proportions has been described by the volun-
teers as having a bitter, lingering after taste, metallic bitter taste
down both sides of tongues. These are the sensation produced
by diCQA. Both 5-CQA and this mixture have a threshold val-
ues in water and coffee brew in the range 0.05–0.1 mg/mL.
Studies on cylitols have shown that bitter notes are produced
as deoxy sites are introduced and/or hydroxyl groups are acy-
lated. The increase in bitterness in the series quinic acid (no
bitterness), 5-CQA (some bitterness), and diCQA (more bitter-
ness) is consistent. The astringency of these diCQA was reduced
by the presence of 5-CQA and a competitive mechanism had
been proposed (Ohiokpehai et al. 1982; Clarke and Macrae,
1985).
Studies in Water
Certain salts of caffeic acids and CGAs have been reported
to be sweet tasting and sweetness enhancing. King and Solms
(1981) have shown that 5-CQA and the potassium salt of the 5-
CQA–caffiene complex increase the water solubility of certain
odor volatiles, thereby raising their threshold values. In contrast,
973
Maga (1978) reported the synergistic lowering of odor threshold
values when certain phenols are mixed (Clarke and Macrae,
1985).
Studies in Coffee Beverage
The various CGAs will be extensively ionized at beverage
and palate pH values, but earlier reports considered that only
phosphoric acid (pK = 1.96) makes a significant contribution
to beverage acidity, even in mild roasts. However, one can ex-
pect an organoleptic contribution from some of the chlorogenate
anions and even in weak brews (0.5% w/v solids) the level of
5-CQA will often exceed the taste threshold reported (Ohiokpe-
hai et al., 1982). The diCQA concentration could exceed the
lower limit proposed for its taste threshold in some brews, es-
pecially strong brews of certain soluble powders, but with nor-
mal commercial products the CQA:diCQA molar ratio would
almost certainly mask the peculiar note of the diCQA. How-
ever, it would seem wise to avoid beans from immature fruit
and discolored green beans since the higher than usual content
might give rise to undesirable sensory properties in the bev-
erages (Clarke and Macrae 1985). Multiple linear regression
analysis on four grades of Brazilian green arabicas was able to
explain almost 70% of the variations in organoleptic quality by
variations in green coffee bean composition. There is general
and superficially tempting belief that the lower CGAs contents
of the arabicas are causally associated their superior beverage
quality. While a modest correlation is plausible, much has yet
to be learnt about the fate of CGAs before this belief can be
properly evaluated (Clarke and Macrae 1985). CGA are known
to be important determinants of coffee flavor. They contribute
to the final acidity (Trugo and Macrae, 1984) and confer astrin-
gency (Carelli et al., 1974; Clifford and Wight, 1976; Variyar
et al., 2003) and bitterness (Trugo, 1984) to the beverage. As a
result of Maillard and Strecker’s reactions, bitterness increases
during roasting due to release of caffeic acid and formation of
lactones and other phenol derivatives responsible for flavor and
aroma (Maria et al., 1994; Ginz and Enhelhardt, 1995; Variyar
et al., 2003).
Effect of Roasting
In addition to their relevance for plant physiology and for a
potential use in the pharmacology field, CGA takes part in the
generation of color, flavor, and aroma of coffee during roasting
(Trugo and Macrae, 1984; Moreira et al., 2000; Montavón et al.,
2003). Due to their thermal instability, CGA may be almost
completely degraded into phenol derivatives when submitted to
intense roasting conditions. It was also reported that the total
CGA in green coffee beans was reduced significantly by roast-
ing at 205◦ C for four different time periods as presented in
Fig. 1: light is 7 minutes for Arabica and 5 minutes for Robusta;
 974
100
95
Percentage reduction in Chlorogenic acid
90
85
80
75
70
65 60.9%
59.7%
60
55
50
45
40
35
30
25
20
15
10
5
0 main lactone, with maximum levels of 230 and 254 mg% (dmb),
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Light
Figure 1 Effect of roasting on chlorogenic acid content of green coffee beans
(Arabica and Robusta).
medium is 10 minutes for Arabica and 7 minutes for Robusta;
dark is 13 minutes for Arabica and 14 minutes for Robusta; or
very dark is 19 minutes for Arabica and 16 minutes for Robusta
(Trugo and Macrae, 1984). The rate of reduction was 60.9% for
light, 67.7% for medium, 88.8% for dark, and 96.5% for very
dark in Arabica and 59.7% for light, 76.4% for medium, 93.0%
for dark, and 98.0% for very dark in Robusta. The total level of
CGA was 68.8 mg/g of dry green bean in Arabica green bean
and 88.0 mg/g in Robusta green bean. These levels reduced
to 26.9 mg/g for light, 22.2 mg/g for medium, 7.71 mg/g for
dark, and 2.42 mg/g for very dark in roasted Arabica coffee and
35.4 mg/g for light, 20.7 mg/g for medium, 6.15 mg/g for dark,
and 1.76 mg/g in roasted Robusta coffee.
Changes in Chlorogenic Acid
During roasting, part of CGA is isomerized, part is trans-
formed into quinolactones due to dehydration and formation
of an intramolecular bond, and part is hydrolyzed and degraded
into low-molecular weight compounds (Trugo, 1984; Trugo and
Macrae, 1984; Leloup et al., 1995; Clifford, 2000; Farah et al.,
2005). CGA also participate in the formation of polymeric ma-
terial like melanoidins (Menezes, 1994; Steinhart and Luger,
1997). Drastic roasting conditions may produce losses of up
to 95% of CGA (Trugo, 1984), with 8–10% being lost for ev-
ery 1% loss of dry matter (Clifford, 1997, 1999, 2000). Total
CGA content in commercial roasted coffee ranges from about
0.5 to 7%, depending on the type of processing, roasting de-
gree, blend, and analytical conditions. CGA contents in light to
medium roasted coffees still stand out when compared to most
food sources of CGA (Farah et al., 2001). In relation to changes
in CGA individual subgroups and isomers during roasting, at
the beginning of the roasting process, isomerization of CGA
R. UPADHYAY AND L. J. M. RAO
96.5% 98% occurs. The levels of the substitutes of the 5-position of the
93%
quinic acid decrease substantially while those of the substitutes
88.8%
in the 3- and 4-positions increase in some cases to almost dou-
76.4% ble their original levels (Trugo and Macrae, 1984; Leloup et al.,
67.7% 1995; Farah et al., 2005). According to Leloup et al. (1995),
at this roasting stage, diCQA may be partially hydrolyzed into
monoesters and caffeic acid, which may be again hydrolyzed,
decarboxylated, and degraded to a range of simple phenols.
Levels of volatile phenols increase along the process (Mor-
eira et al., 2000). CGL formation occurs after 6–7% of weight
loss (Farah et al., 2005). About 7% of CGA in regular Ara-
bica coffee and 5.5% in Robusta coffee seem to be transformed
into 1, 5-γ -quinolactones during the roasting process. The 3-
caffeoylquinide or 3-caffeoylquinic-1, 5-lactone (3-CQL) is the
in Arabica and Robusta coffees, respectively. The second major
Medium Dark Very dark
lactone is 4-caffeoylquinic-1, 5-lactone (4-CQL), with average
contents of 116 and 139 mg%, for Arabica and Robusta coffees,
respectively (Farah et al., 2005). 3-CQL and 4-CQL are ex-
pected to be the major 1,5-γ -quinolactones, since CQAs are the
main CGA and only those CGA isomers that lack a substitute
in the 5-position are able to form a 1,5-γ -quinolactone. Lactone
formation of 3-CQA is favored relative to 4-CQA because of
steric hindrance of the ester group in axial position of the equato-
rial conformer (Farah et al., 2005). Lactones from FQA, diCQA,
and p-CoQA, in order of relevant and other minor quinides, have
also been identified in roasted coffee (Flores-Parra et al., 1989;
Scholz and Maier, 1990; Scholz-Bottcher et al., 1991; Farah
et al., 2005). The content of total CGL increases until about
14% weight loss, that is light-medium roast, reaching average
levels of 398 and 424 mg% (dmb) for Arabica and Robusta cof-
fees, respectively, and decreasing gradually thereafter (Bennat
et al., 1994; Farah et al., 2005).
Factors Affecting Extraction of Chlorogenic Acids
into the Beverage
The extraction of CGAs into the beverage depends on the
degree of grinding, the proportion of coffee to water, the brew-
ing method, the water temperature, and length of time coffee
is in contact with water, but domestic brewing substantially ex-
tracts CGA (80–100%) and CGL from roasted coffee (Clifford,
1997, 2000). Higher temperatures under 100◦ C result in greater
extraction of CGA (Trugo and Macrae, 1984; Clifford, 1987).
Extraction rate increases over the first 10 minutes commonly
employed in domestic brewing. The highest extraction rate of
CGA usually occurs in the first 2 minutes at 93◦ C, increas-
ing less rapidly thereafter (Merrit and Proctor, 1959; Clifford,
1987). Domestic extraction will result in 70–200 mg of CGA
per 200 mL cup, in the case of Arabica coffee and 70–350 mg of
CGA per 200 mL cup in Robusta coffee (Clifford, 1997). Keep-
ing coffee brews at an elevated temperature reduces the contents
of both CQA and CQL in the brew (Bennat et al., 1994; Schrader
et al., 1996).
 AN OUTLOOK ON CHLOROGENIC ACIDS 975

Chlorogeic Acids in Commercial Brewed Coffees
The major CGAs identified in commercial brewed coffees are
three caffeolylquinic acids (3-CQA, 4-CQA, and 5-CQA), three
FQAs (3-FQA, 4-FQA, and 5-FQA), and three diCQAs (3, 4-
diCQA, 3, 5-diCQA, and 4, 5-diCQA). Shibamoto and Fujioka
(2008) analyzed commercial brewed coffees (seven regular and
five decaffeinated) for CGAs and caffeine by HPLC. The total
CGAs ranged from 5.26–17.1 mg/g in regular coffees and from
2.10–16.1 mg/g in decaffeinated coffees. Among CGAs, 5-CQA
was present at the highest level, ranging from 2.13–7.06 mg/g
coffee, and comprising 36–42% and 37–39% of the total CGA in
the regular and decaffeinated coffees, respectively. CGA isomer
contents were, in decreasing order, 5-CQA > 4-CQA > 3-CQA
> 5-FQA > 4-FQA > 3-FQA > 3, 4-diCQA > 4, 5-diCQA, 3,
5-diCQA.
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Biomimetic Sensor Based on a New Tetra Nuclear Copper
(II) Complex
A new tetra nuclear copper (II) complex, which mimics the
active site of catechol oxidase, was synthesized and charac-
terized by infrared spectrophotometry (IR), carbon, hydrogen,
and nitrogen elemental analysis (CHN), and electronic spectro-
scopic and 1H NMR analyses. The title complex [Cu2 (μ-OH)
(bpbpmp-NO2)] 2 [ClO4]2 (Fig. 2) was employed in the con-
struction of a novel biomimetic sensor and used in the determi-
nation of CGA by square wave voltammetry. The performance
and optimization of the resulting biomimetic sensor were stud-
ied in detail. The best response of this sensor was obtained for
75:15:10% (w/w/w) ratio of the graphite powder: nujol: Cu (II)
complex, 0.1 mol/L phosphate buffer solution (pH 7.0), with fre-
quency, pulse amplitude, and scan increment at 30 Hz, 100 mV,

and 3.0 mV, respectively. The CGA concentration was linear in

the range of 5.0 × 10 − 6 to 1.45 × 10 − 4 mol/L (r = 0.9985)
with a detection limit of 8.0 × 10 − 7 mol/L. This biomimetic
sensor demonstrated long-term stability (250 days; 640 determi-
TECHNOLOGICAL ADVANCEMENTS IN SEPARATION
nations) and reproducibility, with a relative standard deviation
AND PURIFICATION
of 10.0%. The recovery study of CGA in coffee samples gave
values from 93.2 to 106.1% and the concentrations determined
The reported analytical methods for CGA include infrared
showed good agreement when compared with those obtained us-
spectrometry (Fabian et al., 1994), difference spectra spec-
ing capillary electrophoresis at the 95% confidence level. This
trophotometry (Tono and Fujita, 1995), 1H nuclear magnetic
sensor showed good long-term stability and good performance
resonance (NMR) spectroscopy (Berregi et al., 2003), liquid
in terms of sensitivity, detection limit, response, and linear cali-
chromatography (Rehwald et al., 1994; Bicchi et al., 1995), gas
bration range. In addition, it was shown to be accurate, reliable,
chromatography, and chemiluminescence (Sakaki, 1982). This
simple, and quick to prepare, and of low cost, offering an al-
paper reviews the recent technological advancements in the sep-
ternative analytical procedure for the determination of CGA in
aration and purification of CGAs from different sources.
coffee (Vieira et al, 2008).

ANALYTICAL METHODS

Chemiluminescent (CL) Method

Wang et al., 2007b reported to detect CGA by flow injec-

tion CL (FI-CL) method, based on the CL reaction of the acidic
potassium permanganate with CGA in the presence of formalde-
hyde as an enhancer. The CL intensity difference of the acidic
potassium permanganate and formaldehyde in the presence of
CGA from the CL intensity without CGA was linear with the
concentration of CGA in the range from 5.0 × 10−8 to 5.0 × 10−5
g/mL with a detection limit of 5.7 × 10−9 g/mL when the sam-
pling rate was 150 injections per hour. The method was applied
successfully to the determination of CGA in fruits with recov-
eries in the range of 100 ± 6%. Compared with the reported CL
methods, the big advantage of the proposed method is to utilize
a cheap, stable, unharmful oxidant reagent and to avoid using
expensive instruments. The developed FI-CL method could be Figure 2 Structure of the [Cu2 (μ-OH) (bpbpmp∗ -NO2 )] 2 [ClO4]2 com-
adopted officially for quantitative detection of CGA in medicine, plex ∗ {bpbpm:-(2-[N-bis-(2-pyridylmethyl) amino methyl]-4-methyl-6-[N-(2
fruits, and foods in future. pyridylmethyl) (2-hydroxy-5-nitrobenzyl)amino methyl]}.
 976 R. UPADHYAY AND L. J. M. RAO
EXTRACTION/ SEPARATION METHODS
Microwave-Assisted Extraction (MAE)
MAE is a process that uses microwave energy along with
solvent to extract target compounds from various matrices. The
highly localized temperature and pressure can cause selective
migration of target compounds from the material to the sur-
roundings at more rapid rate and with similar or better recov-
eries compared with conventional extractions. MAE was used
for extraction of interested components from a wide variety of
sample matrices and has been used as a promising alternative
sample preparation technique for a number of applications (Gao
et al., 2006; Zhou and Liu, 2006; Rostagno et al., 2007; Zhang
and Xu, 2007). Compared to conventional methods, MAE can
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considerably reduce both extraction time and solvent consump-
tion. An efficient MAE technique has been developed to recover
CGA from flower buds of Lonicera japonica Thunb. The yield of
CGA rapidly reached 6.14% within 5 minutes under the optimal
MAE conditions, that is, 50% ethanol as extraction solvent, 1:10
(w/v) of the solid/liquid ratio and 60◦ C of extraction tempera-
ture. The MAE showed obvious advantages in terms of short
duration and high efficiency to recover CGA from raw plant
materials in comparison with conventional heat-reflux extrac-
tion. The mechanism of the enhanced extraction by microwave
assistance was studied by observing cell destruction of plant
material after MAE treatment by scanning electron microscopy.
The enhanced extraction was related partly to a greater extent
of cell rupture of the plant materials, and this was observed by
scanning electron microscopy. The plant materials were signifi-
cantly destroyed due to the cell rupture during MAE treatment.
In comparison to the heat-flux extraction, the MAE showed ob-
vious advantages in terms of short duration and high efficiency
to extract CGA from the plant materials. This is mainly due to
the fact that microwave energy is delivered efficiently to materi-
als through molecular interaction with the electromagnetic field
and offers a rapid transfer of energy to the extraction solvent
and raw plant materials.
pH-Gradient Counter Current Chromatography (CCC)
The pH-gradient CCC method for separation of CGA has
been successfully established. As well known, pH-related CCC
techniques such as pH-peak-focusing and pH-zone-refining
CCC, offer various advantages over conventional CCC meth-
ods, such as large sample capacity, high concentration of eluted
fractions, and enrichment and detection of minor components
present in a large quantity of the crude sample (Ito and Ma,
1996). Recently, some new developments of the pH-related
CCC, such as pH-modulated stepwise elution CCC (Yang et al.,
1999, 2001), and pH-gradient CCC (Liu et al, 2004) were estab-
lished to separate hydroxylanthraxquinones and cinnamic acid,
which display the higher partition efficiency resulting in that
complex target compounds separated clearly. A pH-gradient
CCC was used for the isolation of natural antioxidant CGA
from Lonicera japonica Thunb using an upright coil planet cen-
trifuge with three multilayer coils connected in series. Lu et al.
(2004) developed a high-speed CCC method for separation of
CGA from Flos lonicera. The pH-gradient CCC method effec-
tively used to isolate and purify CGA from flowers and buds of
L. japonica (Wang et al., 2008).
Centrifugal Partition Chromatography
The salting-out gradient method was successfully tested with
the separation of the major CGAs(hydroxycinnamoylquinic
acids) present in green coffee beans (5-CQA, 5-FQA, and 3,5-
diCQA) using ethyl acetate-hexane as the stationary phase and
an ionic gradient of LiCl (5.0, 2.5, and 0.1 M) as the mobile phase
in one case and (NH4 )2 SO4 /KNO3 (3.0 and 1.5 M/1.5 M) in an-
other. Regio-isomers of each CGA obtained by base-catalyzed
isomerization were also separated by centrifugal partition chro-
matography (CPC) using isocratic elution. The best resolution
for both FQAs and diCQAs was achieved with a chloroform–n-
butanol–0.01 M pH 2.5 phosphate buffer (84:16:100; v/v)
system, while CQAs were best isolated using chloroform–n-
butanol–0.01 M pH 2.5 phosphate buffer/5.0 M LiCl (82:18:100;
v/v) system (Gonzalez and Verpoorte, 2009).
Direct Lipase-Catalyzed Lipophilization of Chlorogenic Acid
from Coffee Pulp in Supercritical Carbon Dioxide
Coffee pulp constitutes 40–50% of the fresh weight of the
coffee cherries. Developing alternative technologies for effi-
ciently using this abundant agro industrial by-product represents
an important challenge in tropical regions. Alike coffee beans,
coffee pulp has been recognized as a potential source of phenolic
acids with promising applications, from which CGA (5-cafeoyl
quinic acid, 5-CGA) has been found to be a major constituent
(31–42%; Pandey et al., 2000; Ramirez-Coronel et al., 2004; Le-
pelley et al., 2007). However, the biological availability of phe-
nolic acids and particularly their applications in oil-based prod-
ucts may be limited due to their polar nature. There has been a
growing interest in the biotransformation of natural antioxidants
for the design and improvement of nutraceuticals and foods
beneficial to health. Esterification of phenolic acids has been
proposed for increasing their lipophilicity and, consequently,
to obtain multifunctional antioxidants with enhanced bioactiv-
ity and bioavailability. Structuring lipophilic phenolics in this
way results in amphiphilic molecules with numerous combined
beneficial properties and was widely recognized to improve
their oxidative stability and miscibility (Figueroa-Espinoza and
Villeneuve, 2005; Jayaprakasam et al., 2006; Sabally et al.,
2006; Weitkamp et al., 2006). Recent researches reported the
functionalization of 5-CGA through the enzymatic lipophiliza-
tion by esterifying the carboxylic acid moiety of the quinic acid
with a fatty alcohol (Figueroa-Espinoza and Villeneuve, 2005).
 AN OUTLOOK ON CHLOROGENIC ACIDS
The esterification of 5-CGA with fatty alcohols, catalyzed by li-
pase (Novozym 435) in free and added solvent systems, reached
40–75% after 30 days (Guyot et al., 2000). Even though rela-
tively high yields were obtained, the characteristic heterogeneity
of this reaction resulted in extremely slow conversion rates. Most
recently, an alternative chemoenzymatic strategy was proposed
where 5-CGA was first chemically esterified with methanol and
then transesterified with fatty alcohols (Giraldo et al., 2007). Al-
though the overall yield of this two-step reaction was 61–93%
after 96 hours, a 9-hour methanol-mediated esterification and
the subsequent removal of this toxic reagent represent a ma-
jor drawback limited due to their polar nature. There has been
a growing interest in the biotransformation of natural antioxi-
dants. The use of supercritical carbon dioxide (SC CO2 ) as an
alternative solvent for lipase-catalyzed esterifications has been
a subject of much research due to its adjustable solvation and fa-
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vorable transport properties (Laudani et al., 2007; Knez, 2009).
Hence, opportunities arise to integrate 5-CGA biotransforma-
tion and the extraction/fractionation of less polar derivatives by
a simultaneous SC CO2 process. Adjusting SC CO2 density by
manipulating either temperature or pressure, above their critical
limit (31.1◦ C and 73.8 bar, respectively), allows the control of
mass-transfer limitations. It is also important to consider that
given the poor solubility of 5-CGA in SC CO2 , the addition of a
polar co-solvent would be necessary. Therefore, altogether with
the biocatalyzer load, temperature, pressure, and co-solvent are
considered major reaction parameters.
SEPARATION AND PURIFICATION METHODS
Separation and Purification by Molecularly Imprinted
Polymer (MIP) Monolithic Stationary Phase
Molecularly imprinting is a technique used for preparing
polymer by arranging functional monomers around a template
compound and by fixing it with cross-linker in solution. Re-
moval of the template from the obtained polymer matrix pro-
duces vacant recognition sites that exhibit recognition ability
and have a predesigned selectivity to the template molecule
and structurally related compounds (Remcho and Tan, 1999;
Sellergren, 2003). Lately, MIPs attracted growing attention es-
pecially due to its capability of molecular recognition, easiness
to prepare, and tolerance to harsh environmental conditions,
like high temperature, high pressure, acid, base, and even or-
ganic solvent, makes MIP a very promising separation material
in solid phase extraction and sample preconcentration. Extrac-
tion of CGA from the leaves of E. ulmodies was an efficacious
approach to obtain this compound (Zhang et al., 1996b). Based
on the functional groups and the molecular geometric shape of
CGA (target product) and co-existed compounds mentioned as
above (regarded as the impurities) in the E. ulmodies leaves ex-
tract (Swatsitang et al., 2000), it is suitable to design specific
recognition cavities with no-covalent approach. Thus, investi-
977
gators directed their attention to applying molecular-imprinting
technique. Furthermore, a crucial question needs to be taken into
account is the choice of the template compound for the prepara-
tion of the MIP. After study on the chemical constitution of the
E. ulmodies leaves extract and the molecular structure character
of CGA and the impurity compounds, it was found that there
exist a great “shape” difference between CGA molecule and
the impurity molecules and relatively small “shape” difference
among the impurity molecules (Barnes et al., 1950; Takeshi
et al., 1987; Ma et al., 1997). This offered a new idea to utilize
the impurity molecule as the template for the preparation of the
MIP, and such MIP was then applied for the purification of CGA
by the weak retention of the product molecule on the MIP. Thus,
caffeic acid molecule, a typical compound in the impurities, was
chosen as the template for the preparation of the MIP monolithic
stationary phase in chromatographic column by an in situ syn-
thesis method. The retention behavior of CGA, the template,
and the other impurity molecules coexisted in the E. ulmodies
leaves extract on the MIP monolith was studied. The adsorption
capacity of the monolithic polymer to these compounds was de-
termined by frontal analysis technique. The retention behavior
of CGA, the template, and the impurity compounds coexisted
in the leaves extract on the polymer monolith was studied. The
results show a weak adsorption of CGA on the polymer mono-
lith, which may result from a great “shape” difference of the
CGA molecule with the template molecule. The monolith was
successfully applied to the separation and purification of CGA
from the leaves extract of E. ulmodies, resulting in high purity
CGA. This approach offered researchers a new way to sepa-
rate and purify bioactive constituents from traditional Chinese
medicine.
Preparative High-Speed CCC (HSCCC)
CGA, an ester formed between caffeic acid and quinic acid,
is a major phenolic compound in the traditional Chinese medic-
inal herb Flos lonicerae. The separation and purification of
CGA from the crude extract of Flos lonicerae was achieved
by HSCCC. HSCCC is a useful method for rapid chromato-
graphic purification employing highly efficient fractionation by
a hybrid technique of liquid–liquid counter-current distribution
and liquid chromatography, in conjunction with the use of cen-
trifugal force. The centrifugal force field generated from both
rotational and synchronous planetary motion of coiled columns
containing two immiscible liquid phases provides vigorous mix-
ing between stationary and mobile phases, as well as retention
of a very large fraction of the stationary phase. HSCCC elimi-
nates the irreversible adsorptive loss of samples onto the solid
support matrices used in conventional column chromatography
and HPLC. Furthermore, it permits excellent sample recovery
and can be employed for preparative-scale separation in a com-
pletely straightforward manner. Chen et al. (2004) performed
high acid, highly polar two-phase solvent system containing
 978 R. UPADHYAY AND L. J. M. RAO
n-butanol–acetic acid–water (4:1:5) run on a preparative scale.
The upper phase was used as the mobile phase in the head-to-tail
elution mode. A 300-mg quantity of the crude extract containing
5.97% CGA was loaded on a 342-mL HSCCC column. Dou-
ble separations were performed with the same solvent system
yielding 16.9 mg CGA at 94.8% purity with approximately 90%
recovery. The overall results indicate that HSCCC is a power-
ful technique in separating and purifying bioactive components
from natural products.
BIOLOGICAL ACTIVITIES
CGAs and caffeic acid are phytochemicals found abundantly
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in plants including coffee, fruits, and vegetables (Herrmann,
1989; Clifford et al., 2006a). Particularly, CGAs have been con-
sidered as main precursors of coffee flavor and pigments during
roasting and often used as compounds assessing the quality of
coffee (Fung et al., 1988; Borrelli et al., 2002; Almeida et al.,
2006). Coffee is probably one of the most popular and widely
consumed drinks worldwide, and the consumption of coffee
has been reported to be positively correlated to reducing risk
of human chronic diseases such as inflammation, diabetes, and
cardiovascular disease (Boyer and Liu, 2004; Farah et al, 2006).
Because coffee contains high levels of CGAs, daily intake of
CGAs are estimated about 100–500 mg/80 kg coffee drinkers
(Olthof et al., 2001; Nardini et al., 2002; Gonthier et al., 2003;
Lafay et al., 2006). The biological activities of CGAs are briefly
illustrated in Fig. 3.
Figure 3 Illustration of biological activities of chlorogenic acids.
Absorption and Metabolism of Chlorogenic Acid
For CGAs to exert the purported biological effects in hu-
mans, it should be absorbed and enter into the blood circula-
tion. Therefore, the information about absorption and bioavail-
ability of CGAs are vital in evaluating their purported health
effects in vivo. However, there is considerable disparity regard-
ing the absorption of CGAs, particularly the hydrolysis of the
ester bond of CGAs. Some reports suggest that the bioavail-
ability of CGAs is largely dependent on its metabolism by
microorganisms in the gut, and most of the ingested CGAs
might be absorbed/metabolized and found as its metabolites
in plasma (Olthof et al., 2001; Nardini et al., 2002; Gonthier
et al., 2003). Meanwhile, there is also a report indicating that
CGAs ingested in rats can be absorbed and found as intact
forms (100–170 μg/L) in plasma, even though CGAs may be
metabolized in the gut (Lafay et al, 2006).
Potential as Antioxidants
The antioxidant activities of CGAs are preserved by inhibit-
ing the formation of reactive oxygen species (ROS) or by scav-
enging them. As a result, CGAs may play beneficial role in
the prevention of certain oxidative diseases. The observed phar-
macological activities of medicinal plants relate to the CGAs
and their constituents. Despite the abundance of biological data
demonstrating the antioxidant activities of CGAs, it remains
controversial whether these compounds are potent antioxidants
or pro-oxidants. Chlorogenic and caffeic acids switch from
 AN OUTLOOK ON CHLOROGENIC ACIDS
anti- to pro-oxidant activity, depending on their concentration,
on the presence of free transition metal ions, or on their redox
status. Epidemiological, biological, and biochemical data also
concur to support the beneficial role of CGAs and other dietary
polyphenolic compounds in human health. In particular, CGAs
have been reported to exert inhibitory effects on carcinogenesis
in the large intestine, liver, tongue, and a protective action on
oxidative stress in vivo. Other research has examined the protec-
tive effects of phenolic CGAs against oxidative damage (Kasai
et al., 2000). It is also an antioxidant and an inhibitor of the
tumor-promoting activity of phorbol esters. CGA and caffeic
acid are antioxidants in vitro and might therefore contribute to
the prevention of cardiovascular disease (Lincoln et al., 2000)
and type 2 diabetes mellitus (Paynter et al., 2006). It is claimed
to have antifungal (Bowels and Miller, 1994), antibacterial (de
Sotillo et al., 1998), and antiviral (Jassim and Naji, 2003) effects
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with relatively low toxicity and side effects, alongside properties
that do not lead to antimicrobial resistance. Potential uses are
suggested in pharmaceuticals, foodstuffs, feed additives, and
cosmetics. CGA is marketed under the trade name Svetol in
Norway and the United Kingdom as a food active ingredient
used in coffee, chewing gum, and mints to promote weight re-
duction. Antioxidant phytochemicals from edible and medicinal
plants have been proposed as the major dietary antioxidants pro-
viding health benefits (Kasai et al., 2000; Garcia-Alonso et al.,
2006; Rangkadilok et al., 2007). CGA is polyphenolic antiox-
idant as protective functional factor and beneficial in oxidative
stress-related diseases (Delcy et al., 2002, 2006; Jin et al., 2005;
Suzuki et al., 2006; Wang et al., 2007a).
Neuroprotective Effects
CGA may protect PC12 cells against apoptosis by oxidative
stress from methylmercury (MeHg) exposure, and may exert
neuroprotective effects through its antioxidant actions. In order
to investigate the antioxidant effect of CGA against MeHg in
PC12 cells, Li et al. (2008) used MeHg as neuronal oxidative
agent and PC12 cells were preincubated with or without CGA
were exposed to different MeHg dose for different time. The
activity of glutathione peroxidase (GPx), caspase-3, ROS, and
reduced glutathione (GSH) level were also determined. CGA
protected PC12 cells from MeHg-induced apoptosis by a mech-
anism that was related to GSH, ROS, GPx, and caspase-3. The
data implied that the extraneous antioxidants may contribute to
the sensitivity of neuronal cells to oxidative injury. CGA could
be of considerable preventive measure to some free radical-
associated health problems of metal-induced neurotoxicity, as
well as several neurodegenerative diseases and ageing. In order
to elucidate the action of CGA, the protective effects of CGA
were compared to vitamin E. CGA was effective at protecting
PC12 cells against MeHg-induced damage in dose-dependent
manner. CGA not only suppressed the generation of ROS, the
decrease of activity in GPx, and the decrease of GSH, but also
attenuated caspase-3 activation in PC12 cells by MeHg. CGA
979
eventually protected PC12 cells against MeHg-induced apopto-
sis. The results highlighted that CGA may exert neuroprotective
effects through its antioxidant actions.
Protective Effect Against Cardiovascular Diseases
CGA (5-CQA) and caffeic acid have beneficial effects on
cardiovascular diseases via suppressing P-selectin expression
on platelets. Potential effects of these acids on P-selectin ex-
pression were due to its significant involvement in platelet ac-
tivation (Park, 2009). First, the effects of 5-CQA and caffeic
acid on cyclooxygenase (COX) enzymes were determined due
to their profound involvement in regulating P-selectin expres-
sion on platelets. At the concentration of 0.05 μM, 5-CQA and
caffeic acid were both able to inhibit COX-I enzyme activity by
60% and 57%, respectively. At the same concentration, 5-CQA
and caffeic acid were also able to inhibit COX-II enzyme ac-
tivity by 59% and 56%, respectively. As expected, 5-CQA and
caffeic acid were correspondingly able to inhibit P-selectin ex-
pression on the platelets by 33% and 35%, at the concentration
of 0.05 μM. In animal studies, 5-CQA and caffeic acid orally ad-
ministered to mice were detected as intact forms in the plasma.
Also, P-selectin expression was respectively reduced by 21%
and 44% in the plasma samples from mice orally administered
5-CQA (400 μg per 30 g body weight) and caffeic acid (50 μg
per 30 g body weight). These data suggest that both 5-CQA and
caffeic acid orally administered can be absorbed and suppress
P-selectin expression on mouse platelets and therefore shown
to have significant protective effect against cardiovascular
diseases.
Potent α-Glucosidase Inhibitors
Ma et al. (2008) described the synthetic methods and the
α-glucosidase inhibitory activity of CGA derivatives with alkyl
chains of different lengths and orientations. α-Glucosidases play
important roles in the digestion of carbohydrates and biosynthe-
sis of viral envelope glycoproteins. Inhibitors of α-glucosidase
are promising candidates for the development of antitype II di-
abetics and anti-AIDS drugs. The synthesis of mono- and dike-
tal/acetal derivatives of CGA and their α-glucosidase inhibitory
activity are reported.
Inhibition of Lipopolysaccharide (LPS)-Induced
COX-2 Expression
CGA also inhibits LPS-induced inflammatory response
[AU1] in RAW 264.7 cells (Shan et al., 2009). CGA signifi-
cantly decreased LPS-induced upregulation of COX-2 at protein
and mRNA levels in RAW 264.7 cells and as a result it inhib-
ited prostaglandin E2 (PGE2 ) release from LPS-treated RAW
264.7 cells. In the further experiments, LPS-induced activation
 980 R. UPADHYAY AND L. J. M. RAO
of nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase
(JNK)-c-Jun-activator protein (AP-1) pathway was suppressed
significantly by CGA. In addition, CGA did not affect phospho-
rylation of extracellular signal-regulated kinase 1/2 (ERK1/2)
and p38. In conclusion, CGA suppresses LPS-induced COX-2
expression via attenuating the activation of NF-κB and JNK/AP-
1 signaling pathways suggesting that CGA, the polyphenol com-
pound in the food, could exert anti-inflammatory effects through
inhibiting PGE2 production.
Effects on the Sleep–Wakefulness Cycle
The effect of CGA on the sleep–wakefulness cycle in rats
was investigated (Shinomiya et al., 2004) and compared with
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those of caffeic acid (the metabolite of CGA) and dihydrocaffeic
acid (the metabolite of caffeic acid). A significant prolongation
of sleep latency was observed with CGA and caffeic acid at a
dose of 500 and 200 mg/kg, respectively. On the other hand, no
remarkable effects were observed with dihydrocaffeic acid even
at a dose of 500 mg/kg. Caffeine caused a significant increase
in sleep latency and waking time and decrease in nonrapid eye
movement sleep time at a dose of 10 mg/kg. In contrast, CGA
and its metabolites had no significant effects on each sleep state.
From these results, it may be concluded that CGA caused a
mild arousal effect compared with that of caffeine, and the ef-
fect of CGA may have occurred through its metabolite caffeic
acid.
Modification of Plasma and Liver Concentrations
of Cholesterol, Triacylglycerol, and Minerals
Selected derivatives of CGAs are hypoglycemic agents and
may affect lipid metabolism. Concentrations of cholesterol and
triacylglycerols are of interest due to their association with dis-
eases such as noninsulin-dependent-diabetes-mellitus and obese
insulin resistance. As little is known about the effects of CGA in
vivo, studies using obese, hyperlipidemic, and insulin resistant
(fa/fa) Zucker rats were conducted (Sotillo and Hadley, 2002)
to test the effect of CGA on fasting plasma glucose, plasma and
liver triacylglycerols, and cholesterol concentrations. Addition-
ally, the effects of CGA on selected mineral concentrations in
plasma, spleen, and liver were determined. CGA did not promote
sustained hypoglycemia and significantly lowered the postpran-
dial peak response to a glucose challenge when compared to the
same group of rats before CGA treatment. In CGA-treated rats,
fasting plasma cholesterol and triacylglycerols concentrations
significantly decreased by 44% and 58%, respectively, as did
in liver triacylglycerols concentrations (24%). Researchers did
not find differences in adipose triacylglycerols concentration.
Significant differences in the concentration of selected min-
erals in plasma, liver, and spleen were found in CGA-treated
rats. In vivo, CGA was found to improve glucose tolerance, de-
creased some plasma and liver lipids, and improves mineral pool
distribution.
Prevention of Septic Arthritis Caused by Candida albicans
Lee et al. (2008) determined the effect of CGA on septic
arthritis caused by Candida albicans, a major etiological agent
that causes fungal arthritis. Results showed that the CGA treat-
ment reduced approximately 40% of the edema at the peak of
septic arthritis. This antiarthritic activity appeared to be medi-
ated by a complete inhibition of nitric oxide (NO) production
from macrophages and suppression of T-cells proliferation. Fur-
thermore, CGA also inhibited growth of C. albicans yeast cells
and caused no hemolysis. These data indicate that CGA, which
has antifungal and antiarthritic effects, can safely be adminis-
tered into the blood circulation for treatment of septic arthritis
due to C. albicans. In addition, in respect to antiseptic arthritis,
it can be suggested that the anticandidal effect of CGA may be
helpful as an all-in-one treatment of the candidal arthritis.
CONCLUSIONS
The objective of this review is to emphasis the importance
about the occurrence, chemistry, technology, and biological ac-
tivities of CGAs. CGAs received considerable attention for their
wide distribution and potential biological effects. It is an impor-
tant group of nonvolatile compounds in green coffee beans and
widely distributed in plant materials, their content in green cof-
fee is among the highest found in plants. It is also reported at
significant levels in plant foods such as apples, pears, carrot,
tomato, sweet potato, P. edulis, oilseeds, P. domestica L, cher-
ries, eggplant, and traditional Chinese medicinal herbs, such as
flowers and buds of Lonicera japonica Thunb and the leaves
of E. ulmodies. It plays an important role in the formation of
roasted coffee flavor and has a marked influence in determin-
ing coffee cup quality. The advancement in the development
of newer techniques has significantly improved the extraction
yield for large-scale separation and purification operations of
CGA. This area provides an immense opportunity to exploit
the newer technological advancements in analytical chemistry
to improve the efficiency of extraction of CGAs from different
sources. Besides that, this review has also emphasized on sev-
eral beneficial health effects, which have been attributed to and
largely explained by its potent antioxidant activities. Some in
vitro and in vivo potential pharmacological properties reported
are hypoglycemic, antihypertensive, antiviral, antifungal, hep-
atoprotective, neuroprotective, and immunoprotective activities.
Whether the antioxidants characteristic of coffee are protective
against chronic diseases such as cardiovascular disease and can-
cer remains to be determined. Considerable research needs to
done to explore further the health attributes of CGAs and their
underlying mechanism.
 AN OUTLOOK ON CHLOROGENIC ACIDS
ACKNOWLEDGMENTS
The authors are thankful to the Director, Central Food Tech-
nological Research Institute, Mysore, Head, Plantation Prod-
ucts, Spices and Flavor Technology, and Head, Human Re-
source Development Department, Central Food Technological
Research Institute, Mysore for their keen interest and encour-
agement.
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