Substituent Eects on in Vitro Antioxidizing Properties, Stability, and

Solubility in Flavonoids
Merichel Plaza,*
,
Tania Pozzo,

Jiayin Liu,

Kazi Zubaida Gulshan Ara,

Charlotta Turner,

and Eva Nordberg Karlsson

Department of Chemistry, Centre for Analysis and Synthesis, and

Department of Chemistry, Biotechnology, Lund University, P.O.
Box 124, SE-221 00 Lund, Sweden
ABSTRACT: Antioxidants are widely used by humans, both as dietary supplements and as additives to dierent types of
products. The desired properties of an antioxidant often include a balance between the antioxidizing capacity, stability, and
solubility. This review focuses on avonoids, which are naturally occurring antioxidants, and dierent common substituent
groups on avonoids and how these aect the properties of the molecules in vitro. Hydroxyl groups on avonoids are both
important for the antioxidizing capacity and key points for further modication resulting in O-methylation, -glycosylation,
-sulfation, or -acylation. The eects of O-glycosylation and acylation are discussed as these types of substitutions have been most
explored in vitro concerning antioxidizing properties as well as stability and solubility. Possibilities to control the properties by
enzymatic acylation and glycosylation are also reviewed, showing that depending on the choice of enzyme and substrate,
regioselective results can be obtained, introducing possibilities for more targeted production of antioxidants with predesigned
properties.
KEYWORDS: antioxidant, solubility, stability, enzymes, avonoid, acylation, glycosylation, methylation, sulfation

INTRODUCTION
Antioxidants are molecules that diminish oxidative stress and
prevent or delay oxidation by scavenging free radicals. On the
market, antioxidants are divided into two major groups:
functional food ingredients and antioxidants for preservation.
1
Use of antioxidants derived from natural resources is for both
purposes gaining more and more attention.
Polyphenols are secondary metabolites, widespread among
plant species, and are the most common antioxidants in the
human diet.
26
In addition, due to their presence in various
types of biomass, polyphenols have potential as additives to
industrially produced products.
7
Polyphenolic antioxidants are
composed of at least one aromatic ring with one or more
hydroxyl groups as well as other substituents.
8
The major type
of polyphenols is the avonoids, composed of a benzene ring
(A), condensed with a six-membered pyran ring (C) carrying a
phenyl ring (B) in the 2- or 3-position (Figure 1). Flavonoids
have high antioxidizing capacity, which has in vitro been shown
to be higher than those of vitamins E and C.
9,10
Their capacity
to act like antioxidants was recognized already in the 1930s,
11
when they were called vitamin P (a term that, however,
nowadays has been abandoned). Although avonoids have high
antioxidizing capacity in vitro, they may be less ecient in vivo,
and more knowledge on the rate and extent of their absorption,
metabolism, and tissue or cell distribution, is needed to
elucidate their role in disease prevention.
12
Reactive oxygen species (ROS), namely, superoxide anion
(O
2

), hydroxyl radical (OH

), hydrogen peroxide (H
2
O
2
),
and hypochlorous acid (HOCl), attack biological macro-
molecules (e.g., DNA and proteins) under conditions of
oxidative stress. ROS are generated as unwanted byproducts of
regular oxygen metabolism by all aerobic organisms and can in
vivo give rise to a number of chronic degenerative diseases (e.g.,
arthritis, cancer, cardiovascular diseases, diabetes, inammatory
diseases, ischemia-reperfusion injury, and neurodegenerative
diseases).
1317
Flavonoids act as inhibitors of enzymes involved
in the generation of ROS (e.g., xanthine oxidase, protein
kinases, enzymes activated by calmodulin, cyclooxygenase,
lipoxygenase, and NADPH oxidase) and also chelate pro-
oxidant metal ions (e.g., copper and iron), thus preventing ROS
formation while their free radical scavenging capability is
kept.
18,19
A proposed way to combat health risks imposed by ROS is to
adopt diets involving consumption of foods rich in antioxidants
(precluding progression of chronic diseases, diminishing
mortality rates caused by the same),
6
which, for example, has
been identied as benecial under conditions of hyper-
tension.
20
Metabolism of dietary avonoids results in the
Received: December 11, 2013
Revised: March 17, 2014
Accepted: March 21, 2014
Published: March 21, 2014
Figure 1. Classication of avonoids according to IUPAC.
26
Three
dierent structural backbones with ring labeling and atom numbering
of the C-ring are shown.
Review
pubs.acs.org/JAFC
2014 American Chemical Society 3321 dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333
formation avonoid metabolites that may or may not have
similar eects in vivo as their precursors. Hence, it makes sense
to investigate related compounds (with dierent substituent
patterns) in vitro to shed light on the inuence of structural
changes introduced during metabolism.
21
Antioxidants are also used as additives to dierent
compounds to prolong their life span.
22,23
Sectors of industry
with interest in antioxidants include the food industry, but also
plastics and rubber, gas and fuel, lubricants, adhesives, and
cosmetics. Increased interest in obtaining products from
renewable resources in these sectors (e.g., oils that are more
oxidation prone when derived from renewables) has led to a
growing market.
24
For industrial use it is important that the
antioxidant can be dissolved together with the target compound
and also that its action will proceed over a signicant time span,
keeping sucient stability.
The antioxidizing power of avonoids is along with other
physicochemical characteristics (i.e., stability and solubility)
important for function and depends on the substituents that
decorate the avonoid backbone. The focus of this paper is to
review the inuence of some major substituents occurring
naturally, or as a consequence of in vitro modications, in
dierent subclasses of avonoids (especially focusing on
hydroxyl groups and their modication by glycosylation and
acylation). This is followed by a discussion of current
knowledge of the inuence of these substituent groups in
relation to their application, including methodologies to
measure antioxidizing properties, solubility, and stability.
Finally, current attempts to modify substituent patterns in
vitro by biotechnological methods are reviewed.

OVERVIEW OF NATURAL FLAVONOIDS AND

THEIR SUBSTITUENTS
Flavonoids are classied according to chemical structure, and
their functionality depends on this structure including the
relative orientation of dierent substituent groups on the
molecule.
25
As avonoids have attracted the attention of
researchers in various areas (ranging from chemistry to biology,
pharmaceutical sciences, and medicine), they have been named
by diverse criteria. The numbers of subclasses vary, depending
on the classication system used. On the basis of the backbone
structure the IUPAC nomenclature recommends classication
of the avonoids into avones, isoavan, and neoavone
(Figure 1).
26
However, in the literature avonoids have been
divided into many groups, which are not all correct; thus, to
avoid the confusion about nomenclature IUPAC is running a
project called Recommendations on Nomenclature of
Flavonoids (www.iupac.org). The classication that we nd
most sensible is the one shown in Table 1, which is built on the
major dietary components, named as avones, avonols,
avanones, isoavones, avan-3-ols, and anthocyanidins.
Other minor groups of avonoids include chalcones,
Table 1. Structures of the Major Dietary Groups of Flavonoids and a Few Examples
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3322
dihydrochalcones, dihydroavonols, avan-3,4-diols, coumarins,
and aurones.
27
The exact structure of avonoids varies broadly within the
dierent classes as a result of substitutions, and in plants the
various substituent groups include, for example, OH groups, O-
and C-methyl groups; methylenedioxy groups; O- and C-
prenylation; furano, pyrano, and aromatic substitution;
esterication, sulfation, and chlorination.
2830
There are also
many avonoids available in nature in oligomeric forms, and
one example is the tannins widely found in tea.
31
Glycosyl groups (furanose and pyranose groups) are
common in avonoids and are connected via either O- or C-
glycosylation. In the C-glycosides (found, e.g., in rice and
cereals) the glycosyl group is linked to an aglycone carbon,
usually at the C6- or C8-position.
32
More commonly,
substitutions in avonoids occur via reactions with the hydroxyl
groups of the aglycone backbone, and O-glycosides are
examples of this. The wide structural variation among avonoid
glycosides is thus inuenced by several factors including
linkage, number and nature of the sugars, and the hydroxylation
pattern of the aglycone, which in turn determines the position
of O-glycosylation. In nature, C-linked glycosylation is mostly
found in the avone group. In general, dicots or higher families
have a tendency to accumulate avone O-glycosides, whereas
phylogenetically more primitive families such as ferns or
gymnosperms produce more avonol O-glycosides.
30,33
The O-
glycosylated avonoids are due to bioavailability frequently
selected for analysis of potential eects on antioxidizing
capacity, involving, for example, enzymatic modication trials
in vitro (discussed below). A list stating the type and position
of the O-linked glycosides and acyl groups of various types of
avonoids is given in Table 2. The substituents are aecting not
only the antioxidizing properties but also the stability and
solubility of the compounds. In the coming section we start by
focusing attention on the antioxidizing capacity, including
methods to obtain this on naturally occurring and dierently
substituted avonoids.

MEASURING THE ANTIOXIDIZING PROPERTIES

The capacity of an antioxidant has, according to Biedrzycka and
Amarowics,
34
been dened by Rice-Evans et al.
9
and is
determined by (i) its reactivity as a hydrogen- or electron-
donating agent (depending on its reduction potential), (ii) the
fate of the resulting radical (controlled by the capacity to
stabilize and delocalize the unpaired electron), (iii) the
reactivity with other antioxidants, and (iv) the transition
metal-chelating potential.
Antioxidizing (or reducing) properties can be displayed as
either antioxidant activity or capacity. Antioxidant activity is
molecule specic and can be described as a rate constant of a
reaction between a specic antioxidant and a specic oxidant,
whereas antioxidant capacity measures the amount (in moles)
of a given free radical scavenged by a dened sample.
35
Measurements of antioxidant capacity can hence, depending on
sample composition, yield scavenging ability either of an
individual antioxidant or of the combined action of a mixture of
antioxidants.
Flavonoids are known to be ecient scavengers of free
radicals, such as OH

, O
2

, and LOO

(lipid peroxide radicals).

9
Their potential as antioxidants is predicted by their reducing
properties as hydrogen- or electron-donating agents, and
evaluation is made using a variety of antioxidant assays, with
dierent strengths and limitations. It is also dicult to compare
data from investigations that use dierent methods, and thus it
is recommendable to use a combination of assays. For this
purpose, it is advisable to pick methods that are validated,
standardized, and widely reported. A number of such methods
are summarized in Table 3, along with a brief description of the
analysis principle for the respective method. These methods,
for example, include oxygen radical absorbance capacity
(ORAC), Trolox equivalent antioxidant capacity (TEAC),
2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antiox-
idant power (FRAP), cyclic voltammetry total reducing
capacity (CV), FolinCiocalteu reducing capacity (FC), cupric
reducing antioxidant capacity (CUPRAC), and total radical-
trapping antioxidant parameter (TRAP).
Antioxidant assays can mainly be divided into one of the two
groups: hydrogen atom transfer (HAT)-based assays
52
or
electron transfer (ET)-based assays (see Table 3). HAT-based
assays measure the capacity of an antioxidant to quench free
radicals by hydrogen atom donation, and the majority involve a
competitive reaction scheme. In these assays, thermal
decomposition of azo-compounds generates peroxyl radicals,
resulting in competition between the antioxidant and the
substrate.
40
ET-based assays measure the ability of a potential
antioxidant to transfer one electron to reduce any compound
(which could be metals, carbonyls, and radicals). In principle,
ET-based assays measure the capacity of an antioxidant to
reduce an oxidant probe that changes color upon reduction
40
according to the following scheme:
+
+
probe (oxidant) antioxidant
reduced probe oxidized antioxidant
Table 2. Overview of Generally Occurring Flavonoid Glycosides and Acyls in Nature
subclass of
avonoids position monosaccharide disaccharide acyl group
avones 5, 6, 7, 2, 4 glucose, galactose, xylose,
rhamnose, arabinose, mannose
gentiobiose, rutinose, cellobiose,
diglucose, dirhamnose
2-methylbutyric acid, 3-hydroxy-3-
methylglutaric acid, acylated sugar
avonols 3, 7, 5, 4 glucose, rhamnose, xylose rutinose, isovaleric, vinylpropionic acid, acylated
sugar
avanones 5, 6, 7, 4 glucose, rhamnose, galactose,
fucose, arabinose
rhamnose-glucose acylated sugar, p-coumaroyl
avanonols 3, 7 glucose, rhamnose, arabinose rhamnose-galactose acylated sugar, isobutyric acid
isoavons 7 glucose not reported malonic acid
avan-3-ols
(catechin)
7 glucose not reported
anthocyanidins 2, 3, 4, 5, 6, 7, 3, 5 glucose, galactose, rhamnose, xylose sophorose, rutinose, sambubiose,
robinobiose, gentiobiose
p-coumaric acid, caeic acid, malonic acid,
ferulic acid, sinapic acid
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3323
T
a
b
l
e
3
.
I
n
V
i
t
r
o
A
n
t
i
o
x
i
d
a
n
t
C
a
p
a
c
i
t
y
A
s
s
a
y
s
a
s
s
a
y
s
b
y
h
y
d
r
o
g
e
n
a
t
o
m
t
r
a
n
s
f
e
r
r
e
a
c
t
i
o
n
s
a
s
s
a
y
s
b
y
e
l
e
c
t
r
o
n
-
t
r
a
n
s
f
e
r
r
e
a
c
t
i
o
n
m
e
t
h
o
d
O
R
A
C
a
T
R
A
P
b
T
E
A
C
c
D
P
P
H
d
C
U
P
R
A
C
e
F
R
A
P
f
C
V
g
F
C
h
p
r
o
b
e
A
A
P
H
i
(
p
e
r
o
x
y
l
r
a
d
i
-
c
a
l
g
e
n
e
r
a
t
o
r
)
A
A
P
H
i
(
p
e
r
-
o
x
y
l
r
a
d
i
c
a
l
g
e
n
e
r
a
t
o
r
)
A
B
T
S

+
r
a
d
i
c
a
l
j
D
P
P
H

r
a
d
i
c
a
l
d
b
i
s
(
n
e
o
c
u
p
r
o
i
n
e
)
c
o
p
p
e
r
(
-
I
I
)
c
a
t
i
o
n
f
e
r
r
i
c
2
,
4
,
6
-
t
r
i
p
y
r
i
d
y
l
-
s
-
t
r
i
a
-
z
i
n
e
c
o
m
p
l
e
x
t
u
n
g
s
t
a
t
e

m
o
l
y
b
d
a
t
e
c
o
m
p
l
e
x
e
s
p
r
i
n
c
i
p
l
e
o
f
m
e
a
s
u
r
e
m
e
n
t

u
o
r
e
s
c
e
n
c
e
d
e
c
a
y
a
l
o
n
g
t
i
m
e
d
u
e
t
o
o
x
i
d
a
t
i
o
n
o
f
p
r
o
b
e
i
s
i
n
h
i
b
i
t
e
d
b
y
a
n
t
i
-
o
x
i
d
a
n
t
s
o
x
y
g
e
n
c
o
n
-
s
u
m
p
t
i
o
n
A
B
T
S

+
r
a
d
i
c
a
l
c
a
t
i
o
n
j
i
s
r
e
d
u
c
e
d
b
y
a
n
t
i
o
x
i
-
d
a
n
t
s
,
c
a
u
s
i
n
g
a
b
s
o
r
b
-
a
n
c
e
d
e
c
r
e
a
s
e
a
t
4
1
4
o
r
7
3
4
n
m
D
P
P
H

r
a
d
i
c
a
l
d
r
e
-
d
u
c
e
c
a
u
s
i
n
g
a
b
-
s
o
r
b
a
n
c
e
d
e
c
r
e
a
s
e
a
t
5
2
0
n
m
b
i
s
(
n
e
o
c
u
p
r
o
i
n
e
)
c
o
p
p
e
r
(
-
I
I
)
c
a
t
i
o
n
i
s
r
e
d
u
c
e
d
b
y
a
n
t
i
o
x
i
d
a
n
t
s
,
c
a
u
s
i
n
g
a
b
-
s
o
r
b
a
n
c
e
d
e
c
r
e
a
s
e
a
t
4
5
0
n
m
f
e
r
r
i
c
2
,
4
,
6
-
t
r
i
p
y
r
i
d
y
l
-
s
-
t
r
i
a
-
z
i
n
e
c
o
m
p
l
e
x
i
s
r
e
d
u
c
e
d
b
y
a
n
t
i
o
x
i
d
a
n
t
s
c
a
u
s
i
n
g
a
b
s
o
r
b
a
n
c
e
i
n
c
r
e
a
s
e
d
a
t
5
9
5
n
m
i
n
t
e
n
s
i
t
y
o
f
a
n
o
d
i
c
c
u
r
r
e
n
t
i
s
i
n
-
c
r
e
a
s
e
d
d
u
e
t
o
o
x
i
d
a
t
i
o
n
o
f
a
n
t
i
-
o
x
i
d
a
n
t
c
o
m
p
o
u
n
d
s
a
t
t
h
e
s
u
r
f
a
c
e
o
f
t
h
e
e
l
e
c
t
r
o
d
e
t
u
n
g
s
t
a
t
e

m
o
l
y
b
d
a
t
e
c
o
m
p
l
e
x
e
s
a
r
e
r
e
d
u
c
e
d
b
y
a
n
t
i
o
x
i
d
a
n
t
s
,
c
a
u
s
i
n
g
a
b
s
o
r
b
a
n
c
e
i
n
c
r
e
a
s
e
a
t
7
5
0
n
m
t
e
c
h
n
i
q
u
e

u
o
r
o
m
e
t
r
i
c
o
x
y
g
e
n
e
l
e
c
-
t
r
o
d
e
s
p
e
c
t
r
o
p
h
o
t
o
m
e
t
r
i
c
s
p
e
c
t
r
o
p
h
o
t
o
m
e
t
r
i
c
s
p
e
c
t
r
o
p
h
o
t
o
-
m
e
t
r
i
c
s
p
e
c
t
r
o
p
h
o
t
o
m
e
t
r
i
c
e
l
e
c
t
r
o
c
h
e
m
i
c
a
l
c
e
l
l
s
p
e
c
t
r
o
p
h
o
t
o
m
e
t
r
i
c
q
u
a
n
t
i

c
a
t
i
o
n
n
e
t
A
U
C
k
e
x
p
r
e
s
s
e
d
a
s
T
r
o
l
o
x
e
q
u
i
v
a
-
l
e
n
t
s
l
a
g
t
i
m
e
e
x
-
p
r
e
s
s
e
d
a
s
T
r
o
l
o
x
e
q
u
i
v
a
l
e
n
t
s
T
r
o
l
o
x
e
q
u
i
v
a
l
e
n
t
s
E
C
5
0
l
,
T
r
o
l
o
x
e
q
u
i
v
-
a
l
e
n
t
s
T
r
o
l
o
x
e
q
u
i
v
a
l
e
n
t
s
f
e
r
r
o
u
s
i
o
n
s
e
q
u
i
v
a
l
e
n
t
s
o
x
i
d
a
t
i
o
n
p
o
t
e
n
t
i
a
l
(
E
1
/
2
)
,
i
n
t
e
n
s
i
t
y
o
f
t
h
e
a
n
o
d
i
c
c
u
r
r
e
n
t
(
I
a
)
,
a
r
e
a
u
n
d
e
r
t
h
e
a
n
o
d
i
c
w
a
v
e
(
S
)
g
a
l
l
i
c
a
c
i
d
e
q
u
i
v
a
l
e
n
t
s
r
e
f
e
r
e
n
c
e
s
3
6

3
8
3
6
,
3
9
3
6
,
4
0
,
4
1
4
2
3
6
,
4
3

4
8
3
6
,
4
9
3
6
,
5
0
3
6
,
5
1
a
O
R
A
C
,
o
x
y
g
e
n
r
a
d
i
c
a
l
a
b
s
o
r
b
a
n
c
e
c
a
p
a
c
i
t
y
.
b
T
R
A
P
,
t
o
t
a
l
r
a
d
i
c
a
l
-
t
r
a
p
p
p
i
n
g
a
n
t
i
o
x
i
d
a
n
t
p
a
r
a
m
e
t
e
r
.
c
T
E
A
C
,
T
r
o
l
o
x
e
q
u
i
v
a
l
e
n
t
a
n
t
i
o
x
i
d
a
n
t
c
a
p
a
c
i
t
y
.
d
D
P
P
H
,
2
,
2
-
d
i
p
h
e
n
y
l
-
1
-
p
i
c
r
y
l
h
y
d
r
a
z
y
l
.
e
C
U
P
R
A
C
,
c
u
p
r
i
c
r
e
d
u
c
i
n
g
a
n
t
i
o
x
i
d
a
n
t
c
a
p
a
c
i
t
y
.
f
F
R
A
P
,
f
e
r
r
i
c
r
e
d
u
c
i
n
g
a
n
t
i
o
x
i
d
a
n
t
p
o
w
e
r
.
g
C
V
,
c
y
c
l
i
c
v
o
l
t
a
m
m
e
t
r
y
t
o
t
a
l
r
e
d
u
c
i
n
g
c
a
p
a
c
i
t
y
.
h
F
C
,
F
o
l
i
n

C
i
o
c
a
l
t
e
u
r
e
d
u
c
i
n
g
c
a
p
a
c
i
t
y
.
i
A
A
P
H
,
2
,
2

-
a
z
o
b
i
s
(
2
-
a
m
i
d
i
n
o
p
r
o
p
a
n
e
)
d
i
h
y
d
r
o
c
h
l
o
r
i
d
e
.
j
A
B
T
S

+
,
2
,
2

-
a
z
i
n
o
b
i
s
(
3
-
e
t
h
y
l
b
e
n
z
o
t
h
i
a
z
o
l
i
n
e
-
6
-
s
u
l
f
o
n
i
c
a
c
i
d
)
r
a
d
i
c
a
l
c
a
t
i
o
n
.
k
A
U
C
,
a
r
e
a
u
n
d
e
r
t
h
e
c
u
r
v
e
t
h
a
t
r
e
p
r
e
s
e
n
t
s
t
h
e
a
n
a
l
y
t
i
c
a
l
p
r
o
p
e
r
t
y
m
o
n
i
t
o
r
e
d
a
l
o
n
g
t
i
m
e
.
l
E
C
5
0
,
e

c
i
e
n
t
c
o
n
c
e
n
t
r
a
t
i
o
n
.
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3324
After completion (no more change in color), the degree of
color change is proportional to the concentration of
antioxidant.
40

ANTIOXIDANT CAPACITIES OF DIFFERENTLY

SUBSTITUTED FLAVONOIDS
Structures of A-, B-, and C-Rings and Their Hydrox-
ylation. The phenolic groups of avonoids (B-ring, Figures 1
and 2) supply readily available H-atoms ensuring that the
radicals produced can be delocalized over the avonoid
structure.
53
During the radical scavenging reaction, the
avonoid molecule donates a hydrogen atom to the reacting
radical,
54
as shown in Figure 2. The avonoid free radical
formed in the reaction is more stable than the ROS that
donates the electron due to the delocalization of the electron in
the benzene ring (A-ring, Figures 1 and 2).
In vitro experiments have shown that substituents on the
molecule aect the scavenging capability. Important for high
antioxidizing capacity are the number and arrangement of
hydroxyl groups on the aromatic backbone (A-, B-, and C-
rings)
5557
(Figure 3). Hydroxyls on the B-ring are important
for the hydrogen-donating capacity. An o-dihydroxy group
(catechol group) gives higher stability to the radical structure
and takes part in electron delocalization
58
(Figure 3A). A
pyrogallol group (hydroxyl groups at the 3-, 4-, and 5-
positions on the B-ring) enhances antioxidant capacity
compared to avonoids with single hydroxyl groups in ring
B
59
(Figure 3B). Electron delocalization from ring B is
promoted by a C-2, C-3 double bond combined with the 4-
oxo function in the pyran ring (C-ring),
58
which also
contributes to the antioxidant capacity (Figure 3C). In line
with this, measurements using the TEAC assay (Table 3) show
that quercetin presents enhanced antioxidant capacity com-
pared to catechin
60
(see Table 1 for structures). The 3- and 5-
OH groups (in the A- and C-rings, respectively) neighboring
the 4-oxo function of the C-ring (Figure 3D) also promote high
antioxidant capacity.
61
The presence of a free 3-OH group
(Figure 3E) seems important as further modication (e.g., 3-
glycosylation) leads to reduction of antioxidizing capacity.
55,62
In the A-ring, pairwise hydroxyl substitutions involving position
5 or 8 promote high antioxidant capacity. This is true for both
5,8- and 7,8-hydroxylation (Figure 3F), whereas corresponding
groups at the 5,7-positions had little inuence. A single 7-
hydroxyl also had little eect, whereas a single 5-hydroxyl group
(next to the 4-oxo function) increased antioxidant capacity.
63,64
Following these criteria, a structureantioxidant capacity
relationship of avonoids (quercetin > quercetin-3-O-rutinoside
> kaempferol > luteolin) was proposed by Brown et al.
65
Quercetin (Table 1) has all requirements to exercise eective
antioxidant function, mainly due to the presence of both a
catechol group in the B-ring and a 3-hydroxyl group in the C-
ring. Several studies
66,67
have also reported that avanones with
hydrogenated C-2 and C-3 (Table 1) are in principle inactive,
indicating the importance of the double bond between C-2 and
C-3 (Figure 3C), whereas luteolin (with the C-2, C-3 double
bond and a 3,4-dihydroxyl group) has relatively high
capacity.
66
The antioxidizing capacity of the anthocyanin
molecule (with a structurally dierent C-ring, Table 1) is,
however, high and in the same range as that of quercetin. The
high capacity of the anthocyanin molecule has been attributed
to its electron delocalization ability combined with its ability to
form resonance structures upon changes in pH. The completely
conjugated structure of anthocyanin allows electron delocaliza-
tion, resulting in very stable radicals, which from this
perspective is favorable.
59,68
Moreover, the positively charged
oxygen atom in the C-ring makes it a better hydrogen-donating
antioxidant compared to oligomeric proanthocyanidins and
other avonoids.
69
Clearly, the antioxidant capacity of avonoids is related to a
combination of these chemical and structural elements.
Generally, the higher degree of hydroxyl groups present in
the avonoid ring, the stronger the free radical scavenging will
be. It is also apparent that hydroxyl groups of avonoids are
frequently sites of further modication, modulating antioxidant
capacity by glycosylation, methylation, or, more rarely,
acylation or sulfation.
Glycosylation Decreases Antioxidant Capacity. Most
avonoids are glycosylated in nature, and the position and
nature of the sugar substituents are species specic.
70
Glycosylated avonoids generally show decreased antioxidant
capacity compared to the corresponding aglycones,
71
but
glycosylation also modulates parameters, for example, solubility
and stability, as discussed below. Zielinska et al.
72
determined
antioxidant properties of quercetin and its glucosides from
onion by the CV assay (Table 3) and showed that highest
antioxidant capacity was obtained using the aglycone. The
order of the antioxidant capacity was quercetin > quercetin-3-
Figure 2. Structures of avonols and their oxidized forms. R = OH,
quercetin; R = H, kaempferol.
Figure 3. Basic structural features that characterize antioxidant
functionality in polyphenols. Areas circled in red show features that
are critical for ecient antioxidant function. Letters A, B, and C denote
the ring structures of the avonoid moiety.
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3325
O-glucoside > quercetin-4-O-glucoside > quercetin-3,4-O-
diglucoside. These results are in agreement with a report by
Rice-Evans et al.
55
indicating that the antioxidant properties of
quercetin glucosides were most aected by glycosylation of the
hydroxyl group in the catechol group of the B-ring.
Plumb et al.
73
have also reported that antioxidant capacities
of avonol glycosides from tea decreased as the number of
glycosidic moieties increased. Besides the total number of
glycosidic moieties, the type of glycosylation (O- or C-) and the
position and structure of the sugar play important roles. C-
Glycosylation in the A-ring was in this case shown to decrease
antioxidant capacity,
74
and this eect may be caused by the
properties of the sugar molecule. Flavonoid glycosidic moieties
occur most frequently as O-glycosides at the 3- or 7-position.
The 7-glycoside (in ring A) resulted in a more pronounced
decrease of capacity than 3-glycosylation in the C-ring.
75
Obviously, the presence of substituents also aects the
bioavailability (uptake) of avonoids by changing the solubility
in hydrophilic solvents.
Acylation Is Mostly Investigated after in Vitro
Modication. A way to improve the hydrophobic nature of
avonoids is, for example, to connect fatty acids to the
hydroxyls by esterication. Acylated avonoids may occur in
natural sources (Table 2), but these modications are not as
common as glycosylation and do normally not encompass long
hydrophobic molecules. In this eld, in vitro acylation of
avonoids by fatty acids has been made and the eect of these
substitutions on antioxidant capacity monitored (see also
enzymatic modication, below). Hydroxyl groups on the sugar
of a avonoid glycoside (and not the backbone) are common
targets for acylation and generally lead to reduced antioxidant
capacity, although the results appear to be dependent on the
model assay chosen to measure the resulting antioxidizing
capacity. Acylation of quercetin-3-rutinoside via glucose with
lauric acid (n-dodecanoic acid), more so than palmitic acid
(hexadecanoic acid), negatively inuenced overall metal
chelation when compared to unesteried quercetin-3-O-rutino-
side.
76
Acylation of the glucoside in quercetin-3-O-glucoside
with fatty acids esters by Salem et al.,
77
on the other hand,
resulted in the enhanced scavenging capacity of the quercetin-
3-O-glucoside-ester against ABTS radicals but decreased it
against DPPH and the superoxide radical. Furthermore,
quercetin-3-O-glucoside esters exhibited an antioxidant capacity
that depended on the acyl chain length, where the antioxidant
capacity decreased with increasing acyl chain length.
Other Substituents. Methylation and sulfation of the free
hydroxyl groups are examples of other substitutions, and these
follow the trends seen for glycosylation and acylation in
generally reducing antioxidant capacity. Rimbach et al.
78
studied the eect of sulfation and found that genistein-4-
sulfate was a less ecient antioxidant than genistein and
genistein-4,7-disulfate even less ecient. This indicates that
sulfation masks important hydroxyl groups of the isoavone
molecule and decreases the antioxidant capacity of the resulting
compound.
O-Methylated quercetin is also an example of a less potent
peroxyl radical scavenger than unsubstituted quercetin, and
several studies have reported that steric eects perturb the
planarity of O-methylation, which may induce the suppression
of antioxidant capacity.
75,79
Investigations, mainly using assays
based on DPPH or ABTS (Table 3), have shown that the B-
ring is especially sensitive to the position of methoxyl groups.
Reduction of the antioxidant capacity was seen for 2-O-
methyl/4,6-hydroxy substitution in the B ring, whereas 2,6-
hydroxy/4-O-methyl substitution resulted in a lower eect.
80
Zima et al.
81
also showed that O-dihydroxy substitution of the
avanone B ring resulted in higher capacity than methoxylated
compounds or compounds with para-located hydroxyl groups.
Accordingly, reduction of antioxidant capacity by methylation
depends on a combination of free hydroxyl groups and the
position of methoxyl groups in the avonoid.
Antioxidizing Eects Also Depend on the Chosen
Model System. It is apparent that the antioxidant capacity of
avonoids has been studied in many dierent model systems to
gain insights on structureantioxidant capacity relationships
and that the choice of assay system (depending on its
mechanism) may aect the result obtained. In a recent study
performed by Ishimoto et al.,
52
antioxidative properties of ve
typical avonoids were assessed (kaempferol, quercetin,
myricetin, isorhamnetin, and quercetin-3-glucuronide, Table
1) by the ORAC assay (Table 3). Despite previous ndings that
an increase in the number of hydroxyl groups in the B ring of a
avonol is generally correlated with an increase in antioxidative
capacity, it was found that the ORAC level for kaempferol (with
one hydroxyl group in the B-ring) was 1.5-fold higher than that
of quercetin and myricetin (with two and three hydroxyl groups
in their respective B-rings). It was also shown that the ORAC
score of quercetin was comparable to the scores of both its
methylated metabolite (isorhamnetin) and its 3-O-glucuro-
nide.
52
In the same study, the ORAC score of ()-epigallo-
catechin gallate was 1.5 times lower than that of its methylated
metabolite, ()-epigallocatechin 3-O-(3-O-methyl)-gallate.
On the other hand, (+)-catechin and ()-epicatechin both
showed remarkable antioxidative capacity (ORAC levels of 9.0
and 10.0 mol Trolox equiv/mol) compared to all 3- and 4-O-
methyl-(+)-catechins and 3- and 4-O-methyl-()-epicatechins
(ORAC values in the range of 5.76.5 mol Trolox equiv/mol).
This shows that the scavenging ability depends not only on the
structure of the antioxidant (discussed above) but also on the
target molecule chosen to monitor oxidation.
In conclusion, antioxidant capacities are dependent on many
specic structural features, of which one is the number and/or
position of free hydroxyl groups of avonoids. It is, however,
also important to note that the choice of model system for
measuring antioxidant capacity inuences the results. Anti-
oxidant assays dier from each other in terms of reaction
mechanisms, oxidant and target/probed species, reaction
conditions, and the form in which results are expressed.
36
Therefore, it is prudent to use more than one type of
antioxidant assay and also consider the relevance of the assay in
relation to putative use of the assayed molecule.

STABILITY AND SOLUBILITY ARE OTHER

IMPORTANT PROPERTIES MODIFIED BY
FLAVONOID SUBSTITUENTS
Most studies involving avonoids have focused on the
antioxidizing capacity. There are, however, other properties of
importance, especially for use and selection of antioxidants for
applied purposes. Such properties include the stability and
solubility of the molecules. With increased industrial interest for
use of antioxidants, the choice of antioxidant for a certain
application will be dependent on both the possibility to dissolve
the antioxidant and the possibility to prolong the shelf life of
the antioxidant or of product compounds to which the
antioxidant is added.
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3326
Substituents (such as the O-glycosyl groups connected to the
hydroxyl groups in the avonoids) not only regulate the
antioxidizing capacity but also inuence both the stability and
solubility of the molecules. Although less attention has been
given to these factors, they are important for successful use.
Glycosylation frequently occurs in natural avonoids and, in
addition, both glycosylation and acylation have been done in
vitro, to tune naturally occurring antioxidants to more desired
properties for applied use. The eect of glycosylation and
acylation on both stability and solubility is thus reviewed
according to our current knowledge in the eld.
Eect of Glycosylation on Stability. Relatively little
information is available on the eect of glycosylation on the
stability of avonoid molecules. A few examples are however
found where glycosylation has been shown to contribute to
stability. For example, Buchner et al. measured the changes in
concentration of rutin (also called quercetin-3-O-rutinoside or
sophorin) and quercetin, in aqueous solution at 100 C with air
perfusion, and the results showed that rutin had higher stability
compared to quercetin.
82
In another study of metal-catalyzed
oxidative degradation, rutin and quercetin were dissolved in
phosphate buer containing Fe
2+
and Cu
2+
. It was also found
that rutin had a slower degradation rate by monitoring the
concentration changes during the degradation process.
83
Other
oxidation studies (e.g., oxidation by peroxidase) of rutin and
quercetin also reached the same conclusion.
84
In the study by
Buchner et al.,
82
both quercetin and rutin were degraded in
aqueous solutions at pH 5 and 8. Again, and at both conditions,
the degradation of quercetin seemed to be much faster
compared to that of rutin.
Rutin is glycosylated at a single point involving a disaccharide
linked to the 3-OH on the quercetin C-ring. The stability of
avonoids is, however, also inuenced by the number of
glycosylated hydroxyl groups. As an example, studies have
shown that boiling in water at 100 C caused a greater decrease
in quercetin-4-glucoside content than in the content of
quercetin-3,4-diglucoside,
85,86
making the diglucoside more
stable than the monoglucoside. The degradation was successive,
and, for example, quercetin diglucosides almost completely
broke down to aglycone via the monoglucose derivative by
hydrolytic enzymes.
8789
Acylation Can Further Promote Stability. Although the
mechanism is not known, the introduction of acyl groups has
also been shown to enhance the thermostability of avonoids.
In Ishihara and Nakajimas study, in vitro monoacylation of
quercetin-3-glucoside via a glucoside hydroxyl with nine
dierent aromatic carboxylic acids all improved the thermo-
stability and light-resistivity of the resulting avonoids.
90
It was
suggested that the reason for the improved stability by acylation
might be that the intra- and intermolecular interactions,
between the avonoid skeleton and the aromatic ring in the
acyl moiety, would prevent the degradation of the molecule.
90
A similar observation was reported by Fossen et al.
91
that
complex anthocyanins such as petanin, which contains one
aromatic acyl group, showed higher color intensity and stability
than cyanidin-3-glucoside at pH 4.08.1.
In conclusion, the addition of the above substituents
increased the stability of avonoids. The additional sugar or
acyl moiety could protect the aglycone from degradation to
some extent, although so far there are no studies available to
conrm the mechanism behind this.
Eect of Glycosylation on Solubility. Solubility is
another important parameter that will determine whether it is
possible to utilize an antioxidant as an additive. Solubility in oils
can be desired to prevent oxidation,
92
and in addition solubility
in water has previously been shown to improve the uptake of
avonoids supplied in the diet.
93
There is, however, very
limited data on direct measurement of solubility of dierently
substituted avonoids, like, for example, comparisons of
glycosylated and nonglycosylated compounds. Moreover,
some solubility data were obtained by comparing extraction
94
or chromatography data,
95
which makes it dicult to compare
the solubility between dierent studies. Octanolwater
distribution constant (log K
ow
) values are related to water
solubility, and such values can be used as index values for
comparing the hydrophilicity of the avonoids.
96,97
Frequently,
available comparisons, however, report water solubility at a
given temperature for dierent avonoid compounds.
The presence of a sugar moiety usually increases the
solubility of the avonoids in water solutions. For example,
quercetin has a low solubility in water at 25 C (about 0.92 g/
L).
98
Glycosylated quercetin
99
as well as other glycosylated
avonols
94
was reported to have considerably enhanced water
solubility compared to the corresponding aglycone, although it
is dicult to nd exact numbers on the improvement from the
literature. In the study of Chen et al.
94
it is reported that the
higher solubility in water of glycosylated avonols led to
improved extraction yields. There was, however, no quantitative
measurement of the solubility of the avonols in the above
studies. The position of the sugar moiety and the number of the
bound sugars also play important roles. For example, the
solubility of quercetin-3,4-diglucoside, quercetin-3-glucoside,
and quercetin-4-glucoside in water solution decreases in the
same order.
96,97
On the other hand, glycosylation will decrease
the lipophilicity of the avonoid aglycones. The review by
Chebil et al., for example, shows that the solubility of rutin
(quercetin-3-rutinoside) and isoquercitrin (quercetin-3-gluco-
side) was decreased in acetone and acetonitrile as compared to
the solubility of the aglycone quercetin.
100
Eect of Acylation on Solubility. If an increased
lipophilicity is desired, acylation, particularly with long-chain
fatty acids or vinyl esters from C4 to C18 in length, increases
the solubility of avonoids in organic solvent, that is, reecting
the lipophilicity of the avonoids.
76,101
Such reactions have
been done in vitro predominantly as enzymatic biotranforma-
tions. Another example is the use of aromatic acids such as
hydroxycinnamic acids in acylation reactions, which have been
shown to decrease the solubility of anthocyanins in cell
cultures, which are hydrophilic environments.
95
However,
acylation with small inorganic acids such as sulfuric acid
instead increases the solubility of quercetin in water solution.
95
Therefore, it is reasonable to assume that acylation with
hydrophilic acids will increase the hydrophilicity of the targeted
avonoids and vice versa.
All in all, although there are not so many studies comparing
the absolute solubility data, this is very important in future
avonoid research. Existing literature data support the
conclusion that the solubility of avonoids is aected by the
hydrophilicity of the additional moiety either through
glycosylation or acylation. Adding hydrophilic groups will
increase the hydrophilicity of the avonoid; for example,
glycosylation will increase the solubility of avonoids in
aqueous solution, whereas acylation will increase or decrease
the solubility of avonoids in aqueous solution by adding
hydrophilic or hydrophobic acyl groups. Therefore, by choosing
an appropriate method, it is possible to modify the solubility of
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3327
avonoid in vitro according to the need of the study, which of
course, as described above, will also aect the antioxidant
properties of the compounds. Enzymes are typically used to
make selective modications on avonoids, and below some
eorts in this eld will be shown.

ENZYMATIC METHODS FOR IN VITRO

MODIFICATION OF FLAVONOIDS
The use of enzymes and microorganisms has by the European
Commission been identied as a key enabling technology in
replacing nonrenewable chemicals and materials with renewable
alternatives,
102104
and for the purpose of avonoid mod-
ication, enzymes classied as hydrolases (EC 3.x.x.x) are
biocatalysts of special interest. Hydrolases can, depending on
the reaction conditions, catalyze both hydrolysis and synthesis
reactions and thus modify the content of glycosyl groups (using
glycoside hydrolases (EC 3.2.1.x)
105
) or acyl groups (using
lipases or carboxyl esterases (EC 3.1.1.x)
100
) in avonoids in
biotransformation reactions. These biotransformations may
aect both the antioxidative power and the physicochemical
properties of the target molecule, and the benets of in vitro
modication of avonoid structures using enzymes include
production possibilities in amounts that make bioavailability
trials as well as proper investigations of physicochemical
properties of designed molecules possible. In addition, novel
compounds can be created, thus increasing the natural
avonoid diversity in the laboratory.
92,106108
When it comes
to practical use of avonoids in pharmaceuticals, cosmetics,
food, or other industrial applications, the relatively low
solubility and stability in lipids or water (dependent on the
molecule chosen), can also be improved by designed
modications,
109,110
avoiding current limitations.
Enzymatic Acylation Frequently Requires Glycosy-
lated Acceptor Flavonoids. The enzymes most commonly
used for in vitro modication to date are lipases, which have
successfully been used to acylate avonoids, using dierent
esters (transesterication) and organic acids (direct esterica-
tion) as acyl donors, that, depending on the selected donor, can
favor solubility of the product in lipids. Most lipases, however,
require glycosylated avonoid acceptor molecules for function,
and they selectively acylate hydroxyl groups on glycosides and
do not modify free hydroxyls on the avonoid aglycone. Eorts
in this eld using lipases and carboxyl esterases (also including
the protease subtilisin) have been carefully reviewed by Chebil
and co-workers,
97
and it was shown that among the enzymes
thus far used, it was only lipase PS from Pseudomonas cepacia
(now renamed Burkholderia cepacia)
90,100
and carboxyl
esterases from Streptomyces rochei and Aspergillus niger
100,111
that successfully acylated hydroxyls on a nonglycosylated
phenolic antioxidant acceptor, which in both reported cases
was catechin. A limited number of donor molecules were tried
for these enzymes and were for lipase PS restricted to vinyl
acetate (an ester of fossil origin), whereas for the carboxyl
esterases a short series of esters were used, including ethyl
acetate, ethyl propionate, phenyl propionate, and phenyl
butyrate.
90,100,111
Most commonly, free or immobilized forms of lipase B from
Candida antarctica (CalB) have been used in various synthetic
approaches for acylation of glycosylated avonoids, using a
range of donor molecules via both direct esterication and
transesterication. The reaction conditions are largely inuenc-
ing the yield, and immobilized CalB clearly showed the best
conversion at water activities close to zero. The observed
conversion yields vary largely between dierent studies, using
dierent acyl donors. Dierent vinyl esters are relatively
commonly used for this purpose
100
and, for example, the
synthetic reaction in the production of 6-O-((+) catechin 7-O-
-D-glucopyranoside) cinnamate from vinylcinnamate reached a
yield of 70%.
112
Among acyl donors tested in reactions
involving CAL-B to obtain products with increased solubility
in lipids, high conversion yields have in some cases been
reported using fatty acid donors with carbon chains up to C18.
For example, 71% yield has been noted in rutin palmitate
synthesis using palmitic acid (C16:0) as donor in reactions
using the immobilized form of CAL-B (Novozym435).
113
Table 4. Enzymatic Glycosylation of Flavonoids Using Glycoside Hydrolases
source enzyme donor acceptor product reference
Aspergillus niger cellulase 4NP-Fuc catechin catechin 4--D-fucopyranoside 112
Bacillus stearothermophilus -D-glucosidase maltose catechin catechin 7--D-glucopyranoside 112
catechin 5--D-glucopyranoside
Bacillus sp. -amylase dextrin catechin catechin 7--D-maltoside 112
catechin 5--D-maltoside
Leuconostoc mesenteroides glucansucrase sucrose luteolin luteolin-3--D-glucopyranoside 123
luteolin-4--D-glucopyranoside
Leuconostoc mesenteroides glucansucrase sucrose quercetin quercetin-3--D-glucopyranosides 123
quercetin-4-D-glucopyranosides
Leuconostoc mesenteroides glucansucrase sucrose myricetin myricetin-3--D-glucopyranoside 123
myricetin-4-- D-glucopyranoside
Pencillium decumbens cellulase maltose quercetin quercetin-3-rutinoside 94
Humicola insolens synthase -glycosyl uoride luteolin luteolin-5--D-glycosyl 119
Cel7B_E197S
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3328
From the available literature it can also be seen that the
enzymes are to a certain extent regioselective. For example,
CAL-B has by dierent authors been reported to prefer either
the primary alcohol (C6-OH)
114
or the secondary alcohol at
C4-OH
115
of the sugar for the acylation reaction. Most papers
indicate monoacylation, but for isoquercitrin (quercetin-3-
glucoside) diacylation on C6-OH and C3-OH has also been
shown.
100
Glycosides Can Be both Removed and Added by
Glycoside Hydrolases. The hydrophilicity of avonoids can
also be varied by changes in the glycosidic group content.
Glycoside hydrolases (GH) either catalyze hydrolysis or
transglycosylation of glycosidic groups, and are in nature taking
part in processes such as (i) degradation and assimilation of
exogenous glycosides, (ii) recycling/remodeling of cellular
components, or (iii) modication of biological activity of free
glycosides.
104,116
These activities can of course also be utilized
in vitro to deglycosylate avonoids, for example, using
thermostable -glucosidase,
117
as well as to selectively
glycosylate avonoid molecules. The relationship between
hydrolytic or transglycosylation activity varies between dierent
enzymes and dierent GH families and can be modulated if the
right conditions are found in the reaction, such as high sugar
concentration and controlled water activity. Compared to the
use of lipases, rather limited attempts have thus far been made
in this eld, but interest is increasing, and in addition to the use
of natural enzymes, some nucleophile-mutated variants for
improved performance have also been constructed and
analyzed.
118,119
A problem encountered is that glycoside
hydrolases require higher water activity (generally a
w
> 0.6)
than lipases,
120,121
whereas many avonoid aglycones have low
solubility in water.
Successful trials are summarized in Table 4 and include the
use of a cellulase from Aspergillus niger and an -amylase from a
Bacillus sp., in reactions with 4-nitrophenyl--L-fucoside (4NP-
Fuc) as sugar donor molecules and catechin aglycone as
acceptor molecule. Although successful, the yields achieved
under the conditions used were not so high, up to 26%.
112
Attempts have also been made to optimize the conditions for
glycosylation of the avonoids. Quercetin glycosylation trials at
various temperatures, pH values, solvents, and substrates using
a Penicillium decumbens cellulase (4060 C, pH 67, 3060%
ethanol/water v/v) were made with glucose or maltose as the
glycosyl donor. In this case, conversions up to 30% were
reached.
122
Later, the same cellulase was used to assist in
extractions of avonoid compounds in mild solvents such as
ethanol/water followed by transglycosylation of avonoids,
converting them into more polar molecules, but the highest
transglycosylation yield was only 27% and was achieved using
maltose and isorhamnetin.
94
To overcome low glycosylation yields, an enzyme with higher
natural transglycosylation activity, a glucansucrase from
Leuconostoc mesenteroides, was used for glucosylation carried
out in aqueous/organic solvents to improve avonoid solubility.
Conversion yields did, however, not improve so much, but
varied depending on the avonoid used, resulting in a
conversion to glycosylated forms (Table 4) of luteolin (8%),
quercetin (4%), and signicantly higher for myricetin (49%).
123
In addition, using this enzyme, an -linked glucoside
(quercetin--D-glucopyranoside) not occurring in natural
isolates was obtained.
124
A new approach to glycosylate avonoids is to use GHs,
mutated in the catalytic nucleophile, so-called glycosynthases,
to switch the enzyme mechanism to synthesis only. This
methodology is established for oligosaccharide synthesis but
has seldom been used for the modication of avonoids. As the
nucleophile of the enzyme is replaced, this approach requires
that either uorinated sugars or external nucleophiles are
available in the reaction mixture.
125,126
In Yang et al.,
119
a high-
throughput mass spectrometry-based method was used to assay
biocatalysts and substrates for glycosynthesis, demonstrating
that nding the right donor and acceptor is crucial for the
glycosynthesis reactions. The glycosynthase Cel7B_E197S
from Humicola insolens was found to catalyze the reaction,
transferring an -glycosyl uoride (LacF) into the avonoid,
forming a glycosidic bond with a specicity for position 4 and
6-linked OH groups on the avonoid, with a rather high
glycosylation yield (7595%) compared to previous attempts
using glycosidases and glycosyltransferases.
127
In addition, fair
yields (up to 40%) were obtained using a glycosynthase of
Thermotoga neapolitana -glucosidase 3B, using a monogluco-
sylated quercetin as substrate in reactions obtaining the
diglucoside, but required a second mutation in the active site
for function.
128
In summary, avonoids are natural antioxidants, and they are
getting more attention being molecules from renewable
resources that can be utilized as additives in many types of
products, ranging from food and pharmaceuticals to cosmetics
and eventually larger scale industrial products. To eciently use
and select the antioxidants, a detailed understanding of the
interplay between their activity, stability, and solubility is greatly
aiding selection of suitable candidates. Substituents connected
to their hydroxyl groups denitely have a role in the
determination of these properties, and enzymatic routes to
modify such substituents and properties are likely to play a role
in future modication of natural avonoids for more selective
applications.

AUTHOR INFORMATION
Corresponding Author
*(M.P.) E-mail: Merichel.Plaza@chem.lu.se, merichelpla@
gmail.com. Phone: +46 765855167. Fax: +46 46 222 82 09.
Funding
We acknowledge nancial support from the Swedish Research
Council Formas for funding the SuReTech research collabo-
ration (229-2009-1527). T.P., K.Z.G.A., and E.N.K. also
acknowledge support from the FP7 framework program
AMYLOMICS. C.T. acknowledges the Swedish Research
Council for funding (2010-333).
Notes
The authors declare no competing nancial interest.

REFERENCES
(1) Transparency market research, Antioxidants Market Global
Industry Analysis, Market Size, Market Share, Growth and Forecast,
20122018, http://www. transparencymarketresearch. com/
antioxidants-market.html#sthash.VtcXD4kh.dpuf (accessed Nov 8,
2013),
(2) Ghosh, D.; Scheepens, A. Vascular action of polyphenols. Mol.
Nutr. Food Res. 2009, 53, 322331.
(3) Cohen, S. D.; Kennedy, J. A. Plant metabolism and the
environment: implications for managing phenolics. Crit. Rev. Food Sci.
2010, 50, 620643.
(4) Van Dorsten, F. A.; Grun, C. H.; Van Velzen, E. J. J.; Jacobs, D.
M.; Draijer, R.; Van Duynhoven, J. P. M. The metabolic fate of red
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3329
wine and grape juice polyphenols in humans assessed by
metabolomics. Mol. Nutr. Food Res. 2010, 54, 897908.
(5) Bennick, A. Interaction of plant polyphenols with salivary
proteins. Crit. Rev. Oral Biol. Med. 2002, 13, 184196.
(6) Scalbert, A.; Manach, C.; Morand, C.; Re me sy, C.; Jime nez, L.
Dietary polyphenols and the prevention of diseases. Crit. Rev. Food Sci.
Nutr. 2005, 45, 287306.
(7) Ekman, A.; Campos, M.; Lindahl, S.; Co, M.; Bo rjesson, P.;
Nordberg Karlsson, E.; Turner, C. Bioresource utilisation by
sustainable technologies in new value-added biorefinery concepts
two case studies from food and forest industry. J. Cleaner Prod. 2013,
57, 4658.
(8) Harborne, J. B.; Simmonds, N. W. In Biochemistry of Phenolic
Compounds; Harborne, J., Ed.; Academic Press: New York, 1964; pp
77127.
(9) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Antioxidant
properties of phenolic compounds. Trends Plant Sci. 1997, 2, 152159.
(10) Blokhina, O.; Virolainen, E.; Fagerstedt, K. V. Antioxidants,
oxidative damage and oxygen deprivation stress: a review. Ann. Bot.
2003, 91, 179194.
(11) Benthsath, A.; Rusznnyak, S.; Szent-Gyo rgy, A. In Flavonoids in
Health and Disease; Dekker: New York, 1936; pp 137161.
(12) Hollman, P. C. H.; Cassidy, A.; Comte, B.; Heinonen, M.;
Richelle, M.; Richling, E.; Serafini, M.; Scalbert, A.; Sies, H.; Vidry, S.
The biological relevance of direct antioxidant effects of polyphenols for
cardiovascular health in humans is not established. J. Nutr. 2011, 141,
989S1009S.
(13) Dhalla, N. S.; Temsah, R. M.; Netticadan, T. Role of oxidative
stress in cardiovascular diseases. J. Hypertens. 2000, 18, 655673.
(14) Mezzetti, A.; Cipollone, F.; Cuccurullo, F. Oxidative stress and
cardiovascular complications in diabetes: isoprostanes as new markers
on an old paradigm. Cardiovasc. Res. 2000, 47, 475488.
(15) Sayre, L. M.; Smith, M. A.; Perry, G. Chemistry and
biochemistry of oxidative stress in neurodegenerative disease. Curr.
Med. Chem. 2001, 8, 721738.
(16) Miulescu, R. D.; Da noiu, S.; Purca rea, V.; Ionescu-Trgovis e, C.
Factors that may influence the risk of cancer in diabetes. Farmacia
2012, 60, 602614.
(17) Chiurchiu , V.; MacCarrone, M. Chronic inflammatory disorders
and their redox control: from molecular mechanisms to therapeutic
opportunities. Antioxid. Redox Signal. 2011, 15, 26052641.
(18) Ursini, F.; Maiorino, M.; Morazzoni, P.; Roveri, A.; Pifferi, G. A
novel antioxidant flavonoid (IdB 1031) affecting molecular mecha-
nisms of cellular activation. Free Radical Biol. Med. 1994, 16, 547553.
(19) Middleton, E., Jr.; Kandaswami, C.; Theoharides, T. C. The
effects of plant flavonoids on mammalian cells: Implications for
inflammation, heart disease, and cancer. Pharmacol. Rev. 2000, 52,
673751.
(20) Zhan, C. D.; Sindhu, R. K.; Pang, J.; Ehdaie, A.; Vaziri, N. D.
Superoxide dismutase,catalase and glutathione peroxidase in the
spontaneously hypertensive rat kidney: effect of antioxidant-rich diet.
J. Hypertens. 2004, 22, 20252033.
(21) Hollman, P. C. H.; Katan, M. B. Absorption metabolism and
health effects of dietary flavonoids in man. Biomed. Pharmacother.
1997, 51, 305310.
(22) Garca-Jime nez, J. F.; Valencia, M. C.; Capita n-Vallvey, L. F.
Simultaneous determination of antioxidants, preservatives and sweet-
ener additives in food and cosmetics by flow injection analysis coupled
to a monolithic column. Anal. Chim. Acta 2007, 594, 226233.
(23) Chaithongdee, D.; Chutmanop, J.; Srinophakun, P. Effect of
antioxidants and additives on the oxidation stability of Jatropha
biodiesel. Nat. Sci. 2010, 44, 243250.
(24) Ceresana, Market Study: Antioxidants, http://www.ceresana.
com/en/market-studies/additives/antioxidants/ (accessed Nov 9,
2013).
(25) Cody, V. Crystal and molecular structures of flavonoids. Prog.
Clin. Biol. Res. 1988, 280, 2944.
(26) IUPAC. Compendium of Chemical Terminology (the Gold
Book), 2nd ed.; compiled by McNaught, A. D., Wilkinson, A.;
Blackwell Scientic Publications: Oxford, UK, 1997; XML on-line
corrected version http://goldbook.iupac.org (2006) created by Nic,
M., Jirat, J., Kosata, B.; updates compiled by Jenkins, A. ; ISBN 0-
9678550-9-8; DOI:10.1351/goldbook.
(27) Del Rio, D.; Rodriguez-Mateos, A.; Spencer, J. P. E.; Tognolini,
M.; Borges, G.; Crozier, A. Dietary (poly)phenolics in human health:
structures, bioavailability, and evidence of protective effects against
chronic diseases. Antioxid. Redox Signal. 2013, 18, 18181892.
(28) Harborne, J. B. Nature, distribution and function of plant
flavonoids. Prog. Clin. Biol. Res. 1986, 213, 1524.
(29) Harborne, J. B. Flavonoids in the environment: structure-activity
relationships. Prog. Clin. Biol. Res. 1988, 280, 1727.
(30) Quideau, S. Flavonoids. Chemistry, biochemistry and
applications. Angew. Chem., Int. Ed. 2006, 45, 67866787.
(31) Brune, M.; Rossander, L.; Hallberg, L. Iron absorption and
phenolic compounds: importance of different phenolic structures. Eur.
J. Clin. Nutr. 1989, 43, 547557.
(32) de Rijke, E.; Out, P.; Niessen, W. M.; Ariese, F.; Gooijer, C.;
Brinkman, U. A. Analytical separation and detection methods for
flavonoids. J. Chromatogr., A 2006, 1112, 3163.
(33) Harborne, J. B.; Williams, C. A. Advances in flavonoid research
since 1992. Phytochemistry 2000, 55, 481504.
(34) Biedrzycka, E.; Amarovics, R. Diet and health: apple
polyphenols as antioxidants. Food Rev. Int. 2008, 24, 235251.
(35) MacDonald-Wicks, L. K.; Wood, L. G.; Garg, M. L.
Methodology for the determination of biological antioxidant capacity
in vitro: a review. J. Sci. Food Agric. 2006, 86, 20462056.
(36) Magalhaes, L. M.; Segundo, M. A.; Reis, S.; Lima, J. L.
Methodological aspects about in vitro evaluation of antioxidant
properties. Anal. Chim. Acta 2008, 613, 119.
(37) Cao, G.; Verdon, C. P.; Wu, A. H. B.; Wang, H.; Prior, R. L.
Automated-assay of oxygen radical absorbency capacity with the cobas
fara II. Clin. Chem. 1995, 41, 17381744.
(38) Ou, B. X.; Huang, D. J.; Hampsch-Woodill, M.; Flanagan, J. A.;
Deemer, E. K. Analysis of antioxidant activities of common vegetables
employing oxygen radical absorbance capacity (ORAC) and ferric
reducing antioxidant power (FRAP) assays: a comparative study. J.
Agric. Food Chem. 2002, 50, 31223128.
(39) Somogyi, A.; Rosta, K.; Pusztai, P.; Tulassay, Z.; Nagy, G.
Antioxidant measurements. Physiological. Physiol. Meas. 2007, 28,
R41R55.
(40) Huang, D. J.; Ou, B. X.; Prior, R. L. The chemistry behind
antioxidant capacity assays. J. Agric. Food Chem. 2005, 53, 18411856.
(41) Dai, J.; Mumper, R. J. Plant phenolics: Extraction, analysis and
their anatioxidant and anticancer properties. Molecules 2010, 15,
73137352.
(42) Brandwilliams, W.; Cuvelier, M. E.; Berset, C. Use of a free-
radical method to evaluate antioxidant activity. Food Sci. Technol.
LWT 1995, 28, 2530.
(43) Beer, C.; Myers, R. A.; Sorenson, J. H.; Bucci, L. R.
Comprehensive comparison of the antioxidant activity of fruits and
vegetables based on typical serving sizes from common methods. Curr.
Top. Nutraceut. Res. 2004, 2, 227250.
(44) Boga, M.; Hacibekiroglu, I.; Kolak, U. Antioxidant and
anticholinesterase activities of eleven edible plants. Pharm. Biol.
2011, 49, 290295.
(45) Cronin, J. R. Comparing antioxidant values with the ORAC
method. Alternat. Complement. Ther. 2004, 10, 167170.
(46) Gulc in, I. Antioxidant activity of food constituents: an overview.
Arch. Toxicol. 2012, 86, 345391.
(47) Triantis, T.; Stelakis, A.; Dimotikali, D.; Papadopoulos, K.
Investigations on the antioxidant activity of fruit and vegetable
aqueous extracts on superoxide radical anion using chemiluminescence
technique.s. Anal. Chim. Acta 2005, 536, 101105.
(48) Zujko, M. E.; Witkowska, A. M. Antioxidant potential and
polyphenol content of selected food. Int. J. Food Prop. 2011, 14, 300
308.
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3330
(49) Benzie, I. F. F.; Strain, J. J. The ferric reducing ability of plasma
(FRAP) as a measure of antioxidant power: the FRAP assay. Anal.
Biochem. 1996, 239, 7076.
(50) Magalhaes, L. M.; Segundo, M. A.; Reis, S.; Lima, J. L. F. C.
Methodological aspects about in vitro evaluation of antioxidant
properties. Anal. Chim. Acta 2008, 613, 119.
(51) Singleton, V. L.; Rossi, J. A. Colorimetry of total phenolics with
phosphomolybdic and phosphotungstic acid reagents. Am. J. Enol.
Vitic. 1965, 16, 144147.
(52) Ishimoto, H.; Tai, A.; Yoshimura, M.; Amakura, Y.; Yoshida, T.;
Hatano, T.; Ito, H. Antioxidative properties of functional polyphenols
and their metabolites assessed by an ORAC assay. Biosci., Biotechnol.,
Biochem. 2012, 76, 395399.
(53) Burda, S.; Oleszek, W. Antioxidant and antiradical activities of
flavonoids. J. Agric. Food Chem. 2001, 49, 27742779.
(54) Jacob, J. K.; Tiwari, K.; Correa-Betanzo, J.; Misran, A.;
Chandrasekaran, R.; Paliyath, G. Biochemical basis for functioanl
ingredient design from fruits. Annu. Rev. Food Rechnol. 2012, 3, 79
104.
(55) Rice Evans, C. A.; Miller, N. J.; Paganga, G. Structure-
antioxidant activity relationships of flavonoids and phenolic acids. Free
Radical Biol. Med. 1996, 20, 933956.
(56) Chen, Z. Y.; Chan, P. T.; Ho, K. Y.; Fung, K. P.; Wang, J.
Antioxidant activity of natural flavonoids is governed by number and
location of their aromatic hydroxyl groups. Chem. Phys. Lipids 1996,
79, 157163.
(57) Cao, G. H.; Sofic, E.; Prior, R. L. Antioxidant and prooxidant
behavior of flavonoids: structure-activity relationships. Free Radical
Biol. Med. 1997, 22, 749760.
(58) Bors, W.; Heller, W.; Michel, C.; Saran, M. Flavonoids as
antioxidants: determination of radical-scavenging efficiencies. Methods
Enzymol. 1990, 186, 343355.
(59) Van Acker, S. A. B. E.; Van Den Berg, D. J.; Tromp, M. N. J. L.;
Griffioen, D. H.; Van Bennekom, W. P.; Van Der Vijgh, W. J. F.; Bast,
A. Structural aspects of antioxidant activity of flavonoids. Free Radical
Biol. Med. 1996, 20, 331342.
(60) Salah, N.; Miller, N. J.; Paganga, G.; Tijburg, L.; Bolwell, G. P.;
Rice-Evans, C. Polyphenolic flavanols as scavengers of aqueous phase
radicals and as chain-breaking antioxidants. Arch. Biochem. Biophys.
1995, 322, 339346.
(61) Bors, W.; Michel, C. Chemistry of the antioxidant effect of
polyphenols. Ann. N.Y. Acad. Sci. 2002, 957, 5769.
(62) Villano, D.; Fernandez-Pachon, M. S.; Troncoso, A. M.; Garcia-
Parrilla, M. C. Comparison of antioxidant activity of wine phenolic
compounds and metabolites in vitro. Anal. Chim. Acta 2005, 538,
391398.
(63) Hu, J. P.; Calomme, M.; Lasure, A.; Debruyne, T.; Pieters, L.;
Vlietinck, A.; Vandenberghe, D. A. Structureactivity relationship of
flavonoids with superoxide scavenging activity. Biol. Trace Elem. Res.
1995, 47, 327331.
(64) Wei, H. C.; Bowen, R.; Cai, Q. Y.; Barnes, S.; Wang, Y.
Antioxidant and antipromotional effects of the soybean isoflavone
genistein. Proc. Soc. Exp. Biol. Med. 1995, 208, 124130.
(65) Brown, J. E.; Khodr, H.; Hider, R. C.; Rice-Evans, C. A.
Structural dependence of flavonoid interactions with Cu
2+
ions:
implications for their antioxidant properties. Biochem. J. 1998, 330,
11731178.
(66) Furusawa, M.; Tanaka, T.; Ito, T.; Nishikawa, A.; Yamazaki, N.;
Nakaya, K.; Matsuura, N.; Tsuchita, H.; Nagayama, M.; Iinuma, M.
Antioxidant activity of hydroxyflavonoids. J. Health Sci. 2005, 5, 376
378.
(67) de Martino, L.; Mencherini, T.; Mancini, E.; Aquino, R. P.; de
Almeida, L. F. R.; de Feo, V. In vitro phytotoxicity and antioxidant
activity of selected flavonoids. Int. J. Mol. Sci. 2012, 13, 54065419.
(68) Ka hko nen, M. P.; Heinonen, M. Antioxidant activity of
anthocyanins and their aglycons. J. Agric. Food Chem. 2003, 51,
628633.
(69) Kong, J. M.; Chia, L. S.; Goh, N. K.; Chia, T. F.; Brouillard, R.
Analysis and biological activities of anthocyanins. Phytochemistry 2003,
64, 923933.
(70) Williamson, G.; Plumb, G.; Garcia-Conesa, M. Glycosylation,
esterication, and polymerization of avonoids and hydroxycinna-
mates: eects on antioxidant properties. In Plant Polyphenols 2; Gross,
G., Hemingway, R., Yoshida, T., Branham, S., Eds.; Springer: New
York, 1999; Vol. 66, pp 483494.
(71) Ben Sghaier, M.; Skandrani, I.; Nasr, N.; Franca, M. G. D.;
Chekir-Ghedira, L.; Ghedira, K. Flavonoids and sesquiterpenes from
Tecurium ramosissimum promote antiproliferation of human cancer
cells and enhance antioxidant activity: a structure-activity relationship
study. Environ. Toxicol. Pharm. 2011, 32, 336348.
(72) Zielinska, D.; Wiczkowski, W.; Piskula, M. K. Determination of
the relative contribution of quercetin and its glucosides to the
antioxidant capacity of onion by cyclic voltammetry and spectrophoto-
metric methods. J. Agric. Food Chem. 2008, 56, 35243531.
(73) Plumb, G. W.; Price, K. R.; Williamson, G. Antioxidant
properties of flavonol glycosides from tea. Redox Rep. 1999, 4, 1316.
(74) Mora, A.; Paya, M.; Rios, J. L.; Alcaraz, M. J. Structure-activity
relationships of polymethoxyflavones and other flavonoids as
inhibitors of non-enzymic lipid peroxidation. Biochem. Pharm. 1990,
40, 793797.
(75) Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. Flavonoid
antioxidants: chemistry, metabolism and structure-activity relation-
ships. J. Nutr. Biochem. 2002, 13, 572584.
(76) Lue, B. M.; Nielsen, N. S.; Jacobsen, C.; Hellgren, L.; Guo, Z.;
Xu, X. Antioxidant properties of modified rutin esters by DPPH,
reducing power, iron chelation and human low density lipoprotein
assays. Food Chem. 2010, 123, 221230.
(77) Salem, J. H.; Humeau, C.; Chevalot, I.; Harscoat-Schiavo, C.;
Vanderesse, R.; Blanchard, F.; Fick, M. Effect of acyl donor chain
length on isoquercitrin acylation and biological activities of
corresponding esters. Process Biochem. 2010, 45, 382389.
(78) Rimbach, G.; Weinberg, P. D.; de Pascual-Teresa, S.; Alonso, M.
G.; Ewins, B. A.; Turner, R.; Minihane, A. M.; Botting, N.; Fairley, B.;
Matsugo, S.; Uchida, Y.; Cassidy, A. Sulfation of genistein alters its
antioxidant properties and its effect on platelet aggregation and
monocyte and endothelial function. Biochim. Biophys. ActaGen. Subj.
2004, 1670, 229237.
(79) Dugas, A. J., Jr.; Castan eda-Acosta, J.; Bonin, G. C.; Price, K. L.;
Fischer, N. H.; Winston, G. W. Evaluation of the total peroxyl radical-
scavenging capacity of flavonoids: structure-activity relationships. J.
Nat. Prod. 2000, 63, 327331.
(80) Mathiesen, L.; Malterud, K. E.; Sund, R. B. Hydrogen bond
formation as basis for radical scavenging activity: a structure-activity
study of C-methylated dihydrochalcones from Myrica gale and
structurally related acetophenones. Free Radical Biol. Med. 1996, 22,
307311.
(81) Zima, A.; Hos ek, J.; Treml, J.; Muselk, J.; Suchy , P.; Praz anova ,
G.; Lopes, A.; Z

emlic ka, M. Antiradical and cytoprotective activities of

several C-geranyl-substituted flavanones from Paulownia tomentosa
fruit. Molecules 2010, 15, 60356049.
(82) Buchner, N.; Krumbein, A.; Rohn, S.; Kroh, L. W. Effect of
thermal processing on the flavonols rutin and quercetin. Rapid
Commun. Mass Spectrom. 2006, 20, 32293235.
(83) Makris, D. P.; Rossiter, J. T. Heat-induced, metal-catalyzed
oxidative degradation of quercetin and rutin (quercetin 3-O-
rhamnosylglucoside) in aqueous model systems. J. Agric. Food Chem.
2000, 48, 38303838.
(84) Takahama, U. Spectrophotometric study on the oxidation of
rutin by horseradish peroxidase and characteristics of the oxidized
products. Biochim. Biophys. ActaGen. Subj. 1986, 882, 445451.
(85) Makris, D. P.; Rossiter, J. T. Domestic processing of onion bulbs
(Allium cepa) and asparagus spears (Asparagus of f icinalis): effect on
flavonol content and antioxidant status. J. Agric. Food Chem. 2001, 49,
32163222.
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3331
(86) Lombard, K.; Peffley, E.; Geoffriau, E.; Thompson, L.; Herring,
A. Quercetin in onion (Allium cepa L.) after heat-treatment simulating
home preparation. J. Food Compos. Anal. 2005, 18, 571581.
(87) Lindahl, S.; Ekman, A.; Khan, S.; Wennerberg, C.; Bo rjesson, P.;
Sjo berg, P. J. R.; Nordberg Karlsson, E.; Turner, C. Exploring the
possibilities to use a thermostable mutant of -glucosidase for rapid
hydrolysis of quercetin glucosides in hot water. Green Chem. 2010, 12,
159168.
(88) Rhodes, M. J. C.; Price, K. R. Analytical problems in the study of
flavonoid compounds in onions. Food Chem. 1996, 57, 113117.
(89) Price, K. R.; Rhodes, M. J. C. Analysis of the major flavonol
glycosides present in four varieties of onion (Allium cepa) and changes
in composition resulting from autolysis. J. Sci. Food Agric. 1997, 74,
331339.
(90) Ishihara, K.; Nakajima, N. Structural aspects of acylated plant
pigments: stabilization of flavonoid glucosides and interpretation of
their functions. J. Mol. Catal. B: Enzym. 2003, 23, 411417.
(91) Fossen, T.; Cabrita, L.; Andersen, O. M. Colour and stability of
pure anthocyanins influenced by pH including the alkaline region.
Food Chem. 1998, 63, 435440.
(92) Viskupicova, J.; Danihelova, M.; Ondrejovic, M.; Liptaj, T.;
Sturdik, E. Lipophilic rutin derivatives for antioxidant protection of oil-
based foods. Food Chem. 2010, 123, 4550.
(93) Hollman, P. C.; de Vries, J. H.; van Leeuwen, S. D.; Mengelers,
M. J.; Katan, M. B. Absorption of dietary quercetin glycosides and
quercetin in healthy ileostomy volunteers. Am. J. Clin. Nutr. 1995, 62,
12761282.
(94) Chen, S.; Xing, X.-H.; Huang, J.-J.; Xu, M. S. Enzyme-assisted
extraction of flavonoids from Ginkgo biloba leaves: improvement effect
of flavonol transglycosylation catalyzed by Penicillium decumbens
cellulase. Enzyme Microb. Technol. 2011, 48, 100105.
(95) Gla gen, W.; Seitz, H. Acylation of anthocyanins with
hydroxycinnamic acids via 1-O-acylglucosides by protein preparations
from cell cultures of Daucus carota L. Planta 1992, 186, 582585.
(96) Murota, K.; Shimizu, S.; Chujo, H.; Moon, J. H.; Terao, J.
Efficiency of absorption and metabolic conversion of quercetin and its
glucosides in human intestinal cell line Caco-2. Arch. Biochem. Biophys.
2000, 384, 391397.
(97) Rothwell, J. A.; Day, A. J.; Morgan, M. R. A. Experimental
determination of octanolwater partition coefficients of quercetin and
related flavonoids. J. Agric. Food Chem. 2005, 53, 43554360.
(98) Razmara, R. S.; Daneshfar, A.; Sahraei, R. Solubility of quercetin
in water + methanol and water + ethanol from (292.8 to 333.8) K. J.
Chem. Eng. Data 2010, 55, 39343936.
(99) Calias, P.; Galanopoulos, T.; Maxwell, M.; Khayat, A.; Graves,
D.; Antoniades, H. N.; dAlarcao, M. Synthesis of inositol 2-phosphate-
quercetin conjugates. Carbohydr. Res. 1996, 292, 8390.
(100) Chebil, L.; Humeau, C.; Falcimaigne, A.; Engasser, J. M.;
Ghoul, M. Enzymatic acylation of flavonoids. Process Biochem. 2006,
41, 22372251.
(101) Lue, B. M.; Guo, Z.; Xu, X. Effect of room temperature ionic
liquid structure on the enzymatic acylation of flavonoids. Process
Biochem. 2010, 45, 13751382.
(102) Blumer-Schuette, S. E.; Kataeva, I.; Westpheling, J.; Adams, M.
W.; Kelly, R. M. Extremely thermophilic microorganisms for biomass
conversion: status and prospects. Curr. Opin. Biotechnol. 2008, 19,
210217.
(103) Commission of the European Communities. Communication
from the Commission to the Eurpean Parliament, the Council, the
European Economic AND Social Committee and the Committee of
the Regions. Preparing for our future: Developing a common strategy
for key enabling technologies in the EU, 2009; pp 111.
(104) Turner, P.; Mamo, G.; Nordberg Karlsson, E. Microb. Cell Fact.
2007, 6, 932.
(105) Ara, K. Z. G.; Khan, S.; Kulkarni, T.; Pozzo, T.; Nordberg
Karlsson, E. In Advances in Enzyme Biotechnology; Shukla, P., Pletschke,
B., Eds.; Springer: Dordrecht, The Netherlands, 2014; in press, ISBN
978-81-322-1093-1.
(106) Riva, S. Enzymatic modification of the sugar moieties of natural
glycosides. J. Mol. Catal. B: Enzym. 2002, 1920, 4354.
(107) Wang, A.; Zhang, F.; Huang, L.; Yin, X.; Li, H.; Wang, Q.;
Zeng, Z.; Xie, T. New progress in biocatalysis and biotransformation of
flavonoids. J. Med. Plant Res. 2010, 4, 847856.
(108) Bernhardt, P.; OConnor, S. E. Opportunities for enzyme
engineering in natural product biosynthesis. Curr. Opin. Chem. Biol.
2009, 13, 3542.
(109) Smith, A. J.; Kavuru, P.; Wojtas, L.; Zaworotko, M. J.; Shytle,
R. D. Cocrystals of quercetin with improved solubility and oral
bioavailability. Mol. Pharm. 2011, 8, 18671876.
(110) Xiao, M.; Shao, Y.; Yan, W.; Zhang, Z. Measurement and
correlation of solubilities of apigenin and apigenin 7-O-rhamnosylglu-
coside in seven solvents at different temperatures. J. Chem. Thermodyn.
2011, 43, 240243.
(111) Sakai, M.; Suzuki, M.; Nanjo, F.; Hara, Y. 3-O-acylated
catechins and methods of producing same. EP 0618203, 1994.
(112) Gao, C.; Mayon, P.; MacManus, D. A.; Vulfson, E. N. Novel
enzymatic approach to the synthesis of flavonoid glycosides and their
esters. Biotechnol. Bioeng. 2000, 71, 235243.
(113) Ardhaoui, M.; Falcimaigne, A.; Engasser, J. M.; Moussou, P.;
Pauly, G.; Ghoul, M. Acylation of natural flavonoids using lipase of
Candida antarctica as biocatalyst. J. Mol. Catal. B: Enzym. 2004, 29,
6367.
(114) Danieli, B.; Luisetti, M.; Sampognaro, G.; Carrea, G.; Riva, S.
Regioselective catalysed by acylation of polyhydroxylated natural
compounds Candida antarctica lipase B (Novozym 435) in organic
solvents. J. Mol. Catal. B: Enzym. 1997, 3, 193201.
(115) Ardhaoui, M.; Falcimaigne, A.; Ognier, S.; Engasser, J. M.;
Moussou, P.; Pauly, G.; Ghoul, M. Effect of acyl donor chain length
and substitutions pattern on the enzymatic acylation of flavonoids. J.
Biotechnol. 2004, 110, 265271.
(116) Faure, D. The family-3 glycoside hydrolases: from house-
keeping functions to host-microbe interactions. Appl. Environ.
Microbiol. 2002, 68, 14851490.
(117) Khan, S.; Pozzo, T.; Megyeri, M.; Lindahl, S.; Sundin, A.;
Turner, C.; Nordberg Karlsson, E. Aglycone specificity of Thermotoga
neapolitana -glucosidase 1A modified by mutagenesis, leading to
increased catalytic efficiency in quercetin-3-glucoside hydrolysis. BMC
Biochem. 2011, 1211.
(118) Pozzo, T.; Plaza, M.; Romero-Garca, J.; Faijes, M.; Nordberg
Karlsson, E.; Planas, A. Glycosynthases from Thermotoga neapolitana
-glucosidase 1A: a comparison of -glucosyl fluoride reactions with
reactions using exogenous nucleophile. J. Mol. Catal. B: Enzym. 2014,
accepted.
(119) Yang, M.; Davies, G. J.; Davis, B. G. A glycosynthase catalyst
for the synthesis of flavonoid glycosides. Ange. Chem. Int. Ed. 2007, 46,
38853888.
(120) Hansson, T.; Andersson, M.; Adlercreutz, P. Influence of water
activity on the competition between -glucosidase-catalysed trans-
glycosylation and hydrolysis in aqueous hexanol. Enzym. Microb.
Technol. 2001, 29, 527534.
(121) Ljunger, G.; Adlercreutz, P.; Mattiasson, B. Enzymic synthesis
of octyl--glucoside in octanol at controlled water activity. Enzym.
Microb. Technol. 1994, 16, 751755.
(122) Xu, M. S.; Luo, M. F.; Xing, X. H.; Chen, H. Z. Characteristics
of quercetin transglycosidation catalysed by Penicillium decumbens
glycosidase. Food Bioprod. Process. 2006, 84, 237241.
(123) Bertrand, A.; Morel, S.; Lefoulon, F.; Monsan, P.; Remaud-
Simeon, M. Leuconostoc mesenteroides glucansucrase synthesis of
flavonoid glucosides by acceptor reactions in aqueous-organic solvents.
Carbohydr. Res. 2006, 341, 855863.
(124) Moon, Y. H.; Lee, J. H.; Jhon, D. Y.; Jun, W. J.; Kang, S. S.;
Sim, J.; Choi, H.; Moon, J. H.; Kim, D. Synthesis and characterization
of novel quercetin--D-glucopyranosides using glucansucrase from
Leuconostoc mesenteroides. Enzym. Microb. Technol. 2007, 40, 1124
1129.
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3332
(125) Cobucci-Ponzano, B.; Strazzulli, A.; Rossi, M.; Moracci, M.
Glycosynthases in biocatalysis. Adv. Synth. Catal. 2011, 353, 2284
2300.
(126) Perez, X.; Faijes, M.; Planas, A. Artificial mixed-linked -
glucans produced by glycosynthase-catalyzed polymerization: tuning
morphology and degree of polymerization. Biomacromolecules 2011,
12, 494501.
(127) Williams, S. J.; Withers, S. G. Glycosyl fluorides in enzymatic
reactions. Carbohydr. Res. 2000, 327, 2746.
(128) Pozzo, T. Thermostable Glycosidases and Glycosynthases as
Biocatalysts in Green Chemistry Applications. Doctoral thesis, Lund
University, 2012; ISBN 978-91-89627-91-8.
Journal of Agricultural and Food Chemistry Review
dx.doi.org/10.1021/jf405570u | J. Agric. Food Chem. 2014, 62, 33213333 3333