plants

Review
Flavones: From Biosynthesis to Health Benefits
Nan Jiang 1,2 , Andrea I. Doseff 2,3 and Erich Grotewold 1,2, *
1 Center for Applied Plant Sciences, The Ohio State University, Columbus, OH 43210, USA;
[email protected]
2 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA; [email protected]
3 Department of Physiology and Cell Biology, 305B Heart and Lung Research Institute,
The Ohio State University, Columbus, OH 43210, USA
* Correspondence: [email protected]; Tel.: +1-614-292-2483

Academic Editor: Ulrike Mathesius

Received: 26 April 2016; Accepted: 16 June 2016; Published: 21 June 2016

Abstract: Flavones correspond to a flavonoid subgroup that is widely distributed in the plants,
and which can be synthesized by different pathways, depending on whether they contain C- or
O-glycosylation and hydroxylated B-ring. Flavones are emerging as very important specialized
metabolites involved in plant signaling and defense, as well as key ingredients of the human diet,
with significant health benefits. Here, we appraise flavone formation in plants, emphasizing the
emerging theme that biosynthesis pathway determines flavone chemistry. Additionally, we briefly
review the biological activities of flavones, both from the perspective of the functions that they play in
biotic and abiotic plant interactions, as well as their roles as nutraceutical components of the human
and animal diet.

Keywords: flavone; flavone synthase; biological activities; health benefits; cytochrome P-450;
Fe2+ /2-oxoglutarate-dependent dioxygenases; C-glycosyl transferases

1. Introduction
Flavonoids represent a large subgroup of the phenolic class of plant specialized metabolites.
They are widely distributed throughout the plant kingdom [1]. The basic flavan skeleton that
forms all flavonoids is a 15-carbon phenylpropanoid core (C6 -C3 -C6 system), which is arranged
into two aromatic rings (A and B) linked by a heterocyclic pyran ring (C) (Figure 1A). According to the
oxidation status and saturation of the heterocyclic ring, flavonoids are divided into several groups,
which include flavones, flavanones, isoflavones, flavonols, 3-deoxy flavonoids, and anthocyanins [1].
Flavones comprise one of the largest groups, which are characterized by the presence of a double
bond between C-2 and C-3, and the attachment of the B ring to C-2 (Figure 1A; [1]). As is the
case with other flavonoids, flavones have a diversity of functions that have contributed in making
plants adapt to a terrestrial environment including: (i) protection against UV radiation [2] and
oxidative stress [3]; (ii) interspecies interactions (pathogen resistance [4], symbiosis [5], protection from
herbivory [6], and allelopathy [7]); and (iii) plant development (copigmentation with anthocyanins [8]
and lignification [9]). In addition to their physiological, biochemical, and ecological functions to
plants, flavones also exert biological activities on animals, providing important nutritional value [10].
Key to fulfilling these multiple roles, flavones are characterized by a high degree of chemical diversity
provided by modifications of the chemical backbone, which include hydroxylation, O-/C-glycosylation,
O-methylation, and acylation [1]. This review focuses on the multiple and specialized metabolic
pathways responsible for flavone biosynthesis, discusses their roles in plant biology and briefly
summarizes the recent progress towards establishing the mechanisms by which flavones provide
health benefits.

Plants 2016, 5, 27; doi:10.3390/plants5020027 www.mdpi.com/journal/plants

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Figure 1. Structures of some important flavonoids discussed in this review: (A) flavones, including
Figure 1. Structures of some important flavonoids discussed in this review: (A) flavones, including
aglycones, C-/O-glycosyl flavones, and O-methylated flavones; and (B) selected flavonoids
aglycones, C-/O-glycosyl flavones, and O-methylated flavones; and (B) selected flavonoids mentioned
mentioned in the text belonging to the flavanone, isoflavone, and flavonol groups.
in the text belonging to the flavanone, isoflavone, and flavonol groups.
Plants 2016, 5, 27 3 of 25
Plants 2016, 5, 27 3 of 24

2.2. Flavone
Flavone Biosynthesis:
Biosynthesis: Multiple
Multiple and
and Specialized
Specialized Biosynthesis
Biosynthesis Pathways
Pathways Result
Result in
in Flavone
Flavone
ChemicalDiversity
Chemical Diversity

2.1.
2.1. Multiple
Multiple Enzymes
EnzymesCan
CanForm
Formthe
theFlavone
FlavoneBackbone
Backbone

2.1.1.
2.1.1. Biosynthesis
Biosynthesisof
ofFlavone
FlavonePrecursors
Precursors
In
In most
most species,
species, the
the biosynthesis
biosynthesis of of the
the flavone
flavone backbone
backbone originates
originates from
from thethe general
general
phenylpropanoid pathway followed by the flavonoid biosynthetic branch
phenylpropanoid pathway followed by the flavonoid biosynthetic branch [11]. As part of the [11]. As part of
the phenylpropanoid pathway, phenylalanine is deaminated to cinnamic
phenylpropanoid pathway, phenylalanine is deaminated to cinnamic acid by phenylalanine acid by phenylalanine
ammonia-lyase
ammonia-lyase (PAL).
(PAL). p-coumaric
p-coumaric acid
acid isis then
then formed
formed from
from cinnamic
cinnamic acid
acid by
by cinnamic
cinnamic acid
acid
4-hydroxylase (C4H), which catalyzes the introduction of an hydroxyl group on the
4-hydroxylase (C4H), which catalyzes the introduction of an hydroxyl group on the phenyl ring. The phenyl ring.
The carboxyl
carboxyl group
group of p-coumaric
of p-coumaric acid
acid is isthen
thenactivated
activatedtoto form
form p-coumaroyl-coenzyme
p-coumaroyl-coenzyme A A (CoA),
(CoA),
through
through the formation of a thioester bond with CoA, a process catalyzed by p-coumaroyl-CoAligase
the formation of a thioester bond with CoA, a process catalyzed by p-coumaroyl-CoA ligase
(4CL)
(4CL)(Figure
(Figure2,2,[11]).
[11]).

Figure 2. Multiple and specialized flavone biosynthetic pathways. Purple path: Newly identified
Figure 2. Multiple and specialized flavone biosynthetic pathways. Purple path: Newly identified
biosynthetic pathway for root-specific flavones in Scutellaria baicalensis. PAL: phenylalanine
biosynthetic pathway for root-specific flavones in Scutellaria baicalensis. PAL: phenylalanine ammonia
ammonia lyase; C4H: cinnamate-4-hydroxylase; 4CL: p-coumaroyl-CoA ligase; 4CL-like: cinnamic
lyase; C4H: cinnamate-4-hydroxylase; 4CL: p-coumaroyl-CoA ligase; 4CL-like: cinnamic acid
acid specific CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; FNS: flavone synthase;
specific CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; FNS: flavone synthase;
F2H: flavanone-2-hydroxylase; F3′H: flavanone-3′-hydroxylase; F6H: flavanone-6-hydroxylase; OGT:
F2H: flavanone-2-hydroxylase; F3’H: flavanone-3’-hydroxylase; F6H: flavanone-6-hydroxylase;
O-glycosyltransferase; FOMT: flavonoid O-methyltransferase; CGT: C-glycosyltransferase; RHM:
OGT: O-glycosyltransferase; FOMT: flavonoid O-methyltransferase; CGT: C-glycosyltransferase;
UDP-rhamnose synthase.
RHM: UDP-rhamnose synthase.
Plants 2016, 5, 27 4 of 25

After this, p-coumaroyl-CoA is condensed with three malonyl-CoA to form chalcone by

the chalcone synthase (CHS) enzyme (Figure 2, [12]). Chalcone synthases belong to the type
III polyketide synthase family, present in most plant species [13]. The mechanism of CHS
action demonstrated that as a starter molecule, p-coumaroyl-CoA binds to a cysteine residue
in the active site of CHS and a tetraketide intermediate is formed subsequently by a series of
decarboxylative condensation reactions that involves adding three malonyl-CoA extender molecules.
Chalcone (4,2’,4’,6’-tetrahydroxychalcone) is then produced by the intramolecular cyclization of the
tetraketide intermediate [13].
Chalcone is subsequently isomerized into a flavanone (e.g., naringenin or eriodictyol) in a
stereo-specific fashion by the action of chalcone isomerase (CHI, Figure 2; [14]). CHI is a very efficient
enzyme and highly specific to the chalcone substrate. However, recent studies have shown that the CHI
fold is derived from fatty-acid-binding proteins, which have no enzymatic activity and associate with
saturated fatty acids [15]. How this enzyme was co-opted to the flavonoid pathway remains unclear,
although CHI enzymes (or their products) might participate in ways that are not well understood in
intracellular transport processes [16].
Finally, flavones are synthesized from flavanones by the introduction of a double bond between
the C-2 and C-3 positions by a group of enzymes known as flavone synthase (FNS; Figure 2; [17]).
Flavone synthases have the unique characteristic that two distinct enzyme types can catalyze the
conversion of equivalent substrates (flavanones) to identical products (flavones), by rather different
mechanism [17].

2.1.2. Flavone Synthase I (FNSI) Class

The FNSI class belongs to the superfamily of soluble Fe2+ /2-oxoglutarate-dependent dioxygenases
(2-ODDs) and catalyzes the conversion of flavanones to flavones (Figure 2; [17]). The desaturation of
the flavanones’ C-ring involves two steps consisting of the initial elimination of the C-3 β-configured
hydrogen, followed by the elimination of the C-2 hydrogen [18]. The first FNSI enzyme was identified
from leaflets of Petroselinum crispum cv. Activity assays confirmed that FNSI converted 14 C-radiolabeled
flavanones to the corresponding flavones without forming a detectable reaction intermediate [19].
Following these initial studies, FNSI enzymes were identified from a number of species of the
Apiaceae family, and it was believed for many years that FNSI-type enzymes were restricted to the
Apiaceae [20,21]. However, recent studies showed that the rice FNSI enzyme OsFNSI-1 converts the
flavanone (2S)-naringenin into apigenin in vitro [22], indicating that 2-ODDs with FNS activity are
more widely distributed than initially believed. Indeed, a FNSI enzyme (PaFNSI) was cloned from the
liverwort Plagiochasma appendiculatum [23]. Recombinant PaFNSI not only had FNSI activity, but was
also able to catalyze the conversion of flavanones to 2-hydroxyflavanones, and therefore displayed
flavanone-2-hydroxylase (F2H) activity in vitro [23]. Further analyses indicated that Tyr240 was the
critical residue for F2H activity, since the Tyr240Pro mutant converted naringenin to apigenin, but could
not produce 2-hydroxynaringenin [23]. As described later in this review, these findings are significant
because separate cytochrome P450 (CYP) enzymes can also have F2H or FNS (FNSII) activity.
Recently, FNSI enzymes have been characterized from maize (Zea mays) and Arabidopsis
(Arabidopsis thaliana) [24]. ZmFNSI-1 and its Arabidopsis counterpart, AtDMR6, harbor in vitro FNSI
activity. While dmr6 Arabidopsis mutants show increased resistance to various pathogens including
Pseudomonas syringae, dmr6 plants transgenic for ZmFNSI-1 are equally susceptible to the pathogen
as wild-type plants. The dmr6 mutants accumulate higher salicylic acid levels than wild-type plants,
and the increased salicylic acid levels present in dmr6 are likely responsible for the increased tolerance
to Pseudomonas syringae [25]. Thus, a feedback relationship between flavones (e.g., apigenin) and
the salicylic acid metabolic pathways was proposed [24]. Possibly, to increase success in the plant,
P. syringae (and other pathogens [26]) induce flavone accumulation, which results in the decrease of
salicylic acid, and therefore increased pathogen susceptibility [27].
Plants 2016, 5, 27 5 of 25

FNSI might have evolved from flavanone 3β-hydroxylases (FHTs), 2-ODDs that catalyze the
conversion of (2S)-flavanones to (2R,3R)-dihydroflavonols [18], by gene duplication and functional
diversification [20]. Gebhardt et al. (2007) demonstrated that site-directed mutagenesis of critical amino
acids converted the activity of FHTs to that of FNSI. After homology modeling analyses between FHT
and FNSI, seven critical amino acids were identified around the active site. Results of site-directed
mutagenesis from one to seven substitutions of FHT indicated that a minimal of three amino acids
(Ile131Phe, Met106Thr, and Asp195Glu or Ile131Phe, Leu215Val, and Lys216Arg) were sufficient to
result in partial FNSI activity. All seven amino acid substitutions were sufficient to change the FHT
activity completely to FNSI [20].

2.1.3. Flavone Synthase II (FNSII) Class

FNSII enzymes correspond to oxygen- and NADPH-dependent cytochrome P450 (CYPs)
membrane-bound monooxygenases, which are widespread among the higher plants [17]. So far,
all characterized FNSII enzymes belong to the CYP93B subfamily for dicots and to the CYP93G
subfamily for monocots. Most FNSII enzymes convert flavanones into flavones directly by
introducing a double bond between the C-2 and C-3 residues in flavanones, as we described earlier
for FNSI [17]. FNSII enzymes were first identified from snapdragon and Torenia petal cDNA
libraries. AFNS2 (CYP93B3) and TFNS5 (CYP93B4) catalyzed the direct conversion of flavanones to
flavones [28]. Later, an oxylipin-induced FNSII (CYP93B16) from soybean (Glycine max) cell cultures
was characterized by enzymatic assays [29]. For the CYP93G subfamily, rice CYP93G1 was the first
characterized monocot FNSII [30]. Recently, three FNSII (LjFNSII-1.1, LjFNSII-2.1, and LmFNSII-1.1)
from Lonicera japonica and Lonicera macranthoides were confirmed to convert eriodictyol and naringenin
directly to luteolin and apigenin, respectively. Site-directed mutagenesis analyses showed that the
basic amino acid Lys242 is important for FNSII catalytic activity [31].

2.1.4. 2-Hydroxyflavanones as Flavone Precursors

Most flavones are synthesized by the direct conversion of flavanones, as described in the
previous section. However, some flavones, particularly those harboring C-glycosylation, use instead
2-hydroxyflavanones as precursors, which are produced from flavanones by F2H CYP enzymes.
For example, members from the Fabaceae were confirmed to have F2H activity (CYP93B1 from
Glycyrrhiza echinata [32] and CYP93B10/11 from Medicago truncatula [33]). The two M. truncatula FNSII
genes have distinct tissue-specific expression patterns, with MtFNSII-1 (CYPB10) highly expressed in
roots and seeds, while MtFNSII-2 (CYPB11) is highly expressed in flowers and siliques [33]. In the
Poaceae, a sorghum (Sorghum bicolor) pathogen-induced FNSII (CYP93G3) [34], CYP92G2 from rice [35],
and CYP93G5 from maize [36], all have been shown to have F2H activity. Once flavanones are
2-hydroxylated by F2H enzymes, they appear to serve as substrates for C-glycosyl transferase enzymes,
followed by a dehydration reaction (see Section 2.2). Table 1 lists all of the so far characterized FNSI,
FNSII, and F2H plant enzymes.

Table 1. Overview of functionally characterized plant FNSI, FNSII, and F2H enzymes.

Protein Accession No. Species Function Ref.

CYP93B1 AB001380 Glycyrrhiza echinata (Licorice) F2H [32]
CYP93B2 AF156976 Gerbera hybrida FNSII [37]
CYP93B3 AB028151 Antirrhinum majus (Snapdragon) FNSII [28]
CYP93B4 AB028152 Torenia hybrida FNSII [38]
CYP93B6 AB045592 Perilla frutescens var. Crispa FNSII [39]
CYP93B10 DQ354373 Medicago truncatula (Barrelclover) F2H [33]
CYP93B11 DQ335809 Medicago truncatula (Barrelclover) F2H [33]
Plants 2016, 5, 27 6 of 25

Table 1. Cont.

Protein Accession No. Species Function Ref.

CYP93B16 GU658027 Glycine max (Soybean) FNSII [29]
CYP93B24 KT963453 Scutellaria baicalensis FNSII [40]
CYP93B25 KT963453 Scutellaria baicalensis FNSII [40]
CYP93G1 AK100972 Oryza sativa (Rice) FNSII [30]
CYP93G2 AK099468 Oryza sativa (Rice) F2H [35]
CYP93G3 XP_002461286 Sorghum bicolor (Sorghum) F2H [34]
CYP93G5 GRMZM2G167336 Zea mays (Maize) F2H [36]
LjFNSII-1.1 KU127576 Lonicera japonica FNSII [31]
LjFNSII-2.1 KU127578 Lonicera japonica FNSII [31]
LmFNSII-1.1 KU127580 Lonicera macranthoides FNSII [31]
PcFNSI AY230247 Petroselinum crispum (Parsley) FNSI [19]
DcFNSI AY817675 Daucus carota (Wild carrot) FNSI [21]
AgFNSI AY817676 Apium graveolens (Celery) FNSI [21]
CmFNSI AY817677 Conium maculatum FNSI [21]
AcFNSI DQ683350 Aethusa cynapium FNSI [20]
AaFNSI DQ683352 Angelica archangelica (Wild celery) FNSI [16]
CcFNSI DQ683349 Cuminum cyminum FNSI [16]
OsFNSI-1 NP_922524 Oryza sativa (Rice) FNSI [22]
PaFNSI KJ439220 Plagiochasma appendiculatum (Liverwort) FNSI/F2H [23]
ZmFNSI-1 GRMZM2G099467 Zea mays (Maize) FNSI [24]
AtDMR6 AT5G24530 Arabidopsis thaliana (Arabidopsis) FNSI [24]

2.1.5. Evolutionary Relationships between Flavone Synthase II (FNSII), Flavanone-2-Hydroxylase

(F2H), and Isoflavone Synthase (IFS) in the CYP93 Subfamily
Isoflavonoids are characteristic of the leguminous plants (Fabaceae) [41], but are also found in
multiple other plant species [42]. Formation of the isoflavone skeleton requires two steps: The first
one is a CYP-dependent oxidative aryl migration of flavanones to yield a 2-hydroxyisoflavanone.
IFS facilitates the removal of the 3β-hydrogen from the flavanone substrate, followed by
migration of the side phenyl (B ring) from C-2 to C-3 and final hydroxylation at C-2 to form
2-hydroxyisoflavanone [43]. The next one is the introduction of a double bond between C-2 and
C-3, and is catalyzed by a dehydratase [44]. The first IFS (CYP93C2) was identified from Licorice [45].
Mutagenesis studies revealed that Ser310 and Leu371 regulate the aryl migration and the formation of
the by-product 3-hydroxyflavanone [43,46]. In addition, Lys375 in CYP93C2 is essential for the aryl
migration, since mutagenesis of Lys375 to Thr disrupted the aryl migration and generated only the
by-product 3-hydroxyflavanone [41,46].
The ancestral CYP93 protein may have had FNS activity, converting flavanones to flavones directly
(Figure 3) [46]. F2H might have evolved by a deletion of eight amino acid residues from the ancestral
CYP93 protein, resulting in the lack of dehydratase activity, which introduces the double bond between
C-2 and C-3 of the 2-hydroxylated flavanones [32]. During the radiation of the Leguminosae, it
was proposed that gene duplication was followed by mutations of Ser310 and Leu371 in one of the
two paralogs. These mutations lead to flavanone 3β-hydroxylase activity, which removes a proton
at C-3 of the substrate, instead of at C-2 [43]. Finally, the unique aryl migration function of IFS
was generated by further mutations, specifically the replacement of Val with Lys at position 375
(Figure 3) [46].
Plants 2016, 5, 27 7 of 25
Plants 2016, 5, 27 7 of 24

Figure 3. Proposed molecular evolutionary process of FNSII, F2H, IFS, and F3H from an ancestral
Figure 3. Proposed molecular evolutionary process of FNSII, F2H, IFS, and F3H from an ancestral
CYP93 [43,46]. Amino acid numbers refer to positions in CYP93C2. FNSII: flavone synthase II; F2H:
CYP93 [43,46]. Amino acid numbers refer to positions in CYP93C2. FNSII: flavone synthase II;
flavanone 2-hydroxylase; IFS: 2-hydroxyisoflavanone synthase; F3H: flavanone 3β-hydroxylase.
F2H: flavanone 2-hydroxylase; IFS: 2-hydroxyisoflavanone synthase; F3H: flavanone 3β-hydroxylase.
2.2. Biosynthesis of O- and C-glycosyl Flavones
2.2. Biosynthesis of O- and C-glycosyl Flavones
2.2.1. Backbone or Decoration First?
2.2.1. Backbone or Decoration First?
In plants, most flavones exist decorated by methylation, glycosylation or other modifications.
In plants, most
Glycosylation flavones
is one of theexist
mostdecorated
common by methylation,
modifications glycosylation
and is crucial foror other
multiplemodifications.
chemical
Glycosylation is one of the most common modifications and is crucial
properties characteristic of flavonoids in the cell, such as increased solubility and stabilityfor multiple chemical
[1].
properties
O-linked glycosides are the most common types of of glycosylations, in which the flavone backbone[1].
characteristic of flavonoids in the cell, such as increased solubility and stability
O-linked glycosides
(aglycone) is linkedareto the most
sugar common
moieties typesone
through of ofofglycosylations,
the multiple -OH in groups
which the flavone
present backbone
in flavones
(aglycone)
(Figure 2) is linked to sugar glycosylation
[47]. However, moieties through one ofby
can occur thedirect
multiple -OHofgroups
linkage carbonpresent
residuesin flavones
in the
(Figure 2) [47].
flavonoid andHowever, glycosylation
sugar backbones, can occur
resulting by direct
in C-glycosyl linkage[48].
flavones of carbon residues
Actually, in theflavones
C-glycosyl flavonoid
andandsugar
theirbackbones,
derivatives resulting C-glycosyl
have beeninfound flavones
in almost [48].
all plant Actually,
phyla, C-glycosyl
ranging flavones
from liverworts and their
(Frullania
derivatives have been found in almost all plant phyla, ranging from liverworts (Frullania polysticta [49]
Plants 2016, 5, 27 8 of 25

and Plagiochila jamesonii [50]), ferns (Asplenium viviparum [51] and Trichomanes [52]), to8 offlowing
Plants 2016, 5, 27 24
plants including monocots (hair grass Deschampsia antarctica [53], barley Hordeum vulgare [54,55],
polysticta [49] and Plagiochila jamesonii [50]), ferns (Asplenium viviparum [51] and Trichomanes [52]), to
and maize [56–58]) and dicots (black calla Arum palaestinum [59], weed silene Silene conoidea [60],
flowing plants including monocots (hair grass Deschampsia antarctica [53], barley Hordeum vulgare
yarrow Achillea setacea [61], wild hop Bryonia alba [62], colocynth Citrullus colocynthis [63],
[54,55], and maize [56–58]) and dicots (black calla Arum palaestinum [59], weed silene Silene conoidea
cucumber Cucumis
[60], yarrow sativus
Achillea [64], [61],
setacea bottle gentian
wild Gentiana
hop Bryonia albaandrewsii [65], geraniums
[62], colocynth Pelargonium
Citrullus colocynthis [63], [66],
Scutellaria
cucumber baicalensis
Cucumis [67], [64], bottleAbrus
sativusjequirity gentianprecatorius [68], Glycyrrhiza
Gentiana andrewsii [65], geraniums eurycarpa [69],[66],
Pelargonium lupine
Lupinus hartwegii [70], shy plant Mimosa pudica [71], orchid Ornithocephalus [72],
Scutellaria baicalensis [67], jequirity Abrus precatorius [68], Glycyrrhiza eurycarpa [69], lupine Lupinus purple passionflower
Passiflora incarnate
hartwegii [70], [73], milkworts
shy plant Mimosa Polygala
pudica telephioides
[71], orchid[74], globeflower[72],
Ornithocephalus Trollius ledebourii
purple [75], quince
passionflower
Passiflora
Cydonia oblongaincarnate [73], Citrus
[76], lemon milkworts
[77], Polygala
and fieldtelephioides
pansy Viola [74], globeflower
arvensis [78]). Trollius ledebourii [75],
quince Cydonia
Compared withoblonga [76], lemon
O-linked Citrus [77],
glycosides, and field pansy
C-glycosides Viola arvensis
are much [78]). since C-C bonds are
more stable,
Compared with O-linked glycosides, C-glycosides
largely resistant to glycosidase action or acid hydrolysis [79]. Intramolecular are much more stable, since
C-CC-C bondscoupling
phenol are
largely resistant to glycosidase action or acid hydrolysis [79]. Intramolecular C-C phenol coupling
reactions were found in the biosynthesis of isoquinoline alkaloid (CYP80G2, [80]) and morphine
reactions were found in the biosynthesis of isoquinoline alkaloid (CYP80G2, [80]) and morphine
(CYP719B1, [81]). Some members from the lyase family (e.g., norcoclaurine synthase [82], strictosidine
(CYP719B1, [81]). Some members from the lyase family (e.g., norcoclaurine synthase [82],
synthase [83], and
strictosidine tyrosine
synthase [83],phenol lyase [84])
and tyrosine phenol and oxidoreductases
lyase [85–87] also
[84]) and oxidoreductases can also
[85–87] catalyze
can the
formation of C-C bonds.
catalyze the formation of C-C bonds.
Two Two independent
independent metabolic
metabolic pathways
pathwaysare are responsible
responsible for forthetheformation
formation of O-
of O- andand C-glycosyl
C-glycosyl
flavones [35,36].
flavones O-glycosylation
[35,36]. O-glycosylationoccursoccursafter
after the flavone
flavonebackbone
backbone is is generated
generated by FNSI
by FNSI or FNSII,
or FNSII,
depending
depending of the of plant
the plant species.
species. Thisisiscalled
This calledthe
the“backbone
“backbone first”
first”pathway.
pathway. In In
contrast, C-glycosyl
contrast, C-glycosyl
flavone
flavone biosynthesis
biosynthesis requires
requires twotwo stpes.First,
stpes. First, the
the flavanone
flavanone substrates
substrates arearehydroxylated
hydroxylated by F2H to to
by F2H
form 2-hydroxylflavanones. The open-circular (dibenzoylmethane) form
form 2-hydroxylflavanones. The open-circular (dibenzoylmethane) form of the 2-hydroxylflavanone is of the 2-hydroxylflavanone
is then glycosylated by a glycosyltransferase. After conjugation of the sugar moiety, the
then glycosylated by a glycosyltransferase. After conjugation of the sugar moiety, the closed-circular
closed-circular form of the products (2-hydroxylflavanone C-glycosides) is dehydrated to produce
form of the products (2-hydroxylflavanone C-glycosides) is dehydrated to produce the corresponding
the corresponding flavone C-glycosides, in a process referred as “decoration first” pathway (Figure
flavone C-glycosides, in a process referred as “decoration first” pathway (Figure 4) [79].
4) [79].

Figure 4. A proposed mechanism for the conversion of 2-hydroxynaringenin to vitexin and

Figure 4. A proposed mechanism for the conversion of 2-hydroxynaringenin to vitexin and isovitexin
isovitexin by CGT and dehydration [79]. CGT: C-glycosyltransferase.
by CGT and dehydration [79]. CGT: C-glycosyltransferase.
Plants 2016, 5, 27 9 of 25

2.2.2. O- and C-glycosyl Transferases

O- and C-glycosylation are catalyzed by UDP-glycosyltransferases (UGTs), members of the
glycosyltransferase family 1 (GT1), and use nucleotide sugars as donors [88]. Genes encoding
for enzymes that catalyze flavone C-glycosylation at either C-6 or C-8 have been identified from
rice (OsCGT) [79], maize (UGT708A6) [89], Fagopyrum esculentum M. (buckwheat UGT708C1 and
UGT708C2) [90], soybean (UGT708D1) [91], Gentiana triflora (Japanese gentian GtUF6CGT1) [92], and
Mangifera indica (Mango MiUGT13) [93]. Residues His20, Asp85, and Arg292) within the N-terminal
acceptor-binding pocket seem necessary for the C-glycosyl transferase (CGT) activity. Substitution of
Asp85 and Arg292 with Ala in UGT708D1 resulted in loss of CGT activity, while the His20Ala mutant
protein also lost CGT activity but aquired a new O-glycosyl transferase (OGT) activity. Thus, Asp85
and Arg292 are essential for enzymatic activity, while Ala substitution of His20 also resulted in loss
of CGT activity but aquired a new O-glycosyl transferase (OGT) activity [91]. Maize UGT708A6
is a bifunctional O-/C-glycosyltransferase that catalyzes the formation of both C- and O-glycoside
links with 2-hydroxyflavanones and flavanones, respectively [89]. Protein sequence alignment among
maize UGT708A6 with known UGTs showed that UGT708A6 not only contains the conserved residues
(Asp91 and Arg287), which are restricted to the 2-hydroxyflavanone CGTs, but also has the conserved
His-Asp residues corresponding to the active site of OGTs [89,91]. Interestingly, Japanese gentian
GtUF6CGT1 can catalyze flavone C-glucosylation by directly adding a glucose group to the C6
position of the flavone skeleton, without the 2-hydroxyflavanone intermediate [92]. Recently, the novel
benzophenone C-glycosyltransferase (MiCGT) from mango, which is involved in the biosynthesis of
mangiferin, showed unusual substrates (both sugar acceptor and donors) and catalytic promiscuity [93].
MiCGT formed only C-glycosides with 2,4,6-tri-hydroxy acceptors, both C- and O-glycosides with
2,4-di-hydroxyl acceptors, and only O-glycosides with 2- or 4-mono-hydroxy acceptors. Thus, results
from this study indicate that the number and position of the electron-donating hydroxy groups in the
A-ring provide critical determinants for the C- or O-glycosylation capacity of MiCGT [93].
The biosynthesis of the maize C-glycosylflavone maysin provides a good example of the
sophistication of the pathways involved in flavone formation (Figure 2). Maysin is a maize
host-plant resistance factor against the corn earworm (Helicoverpa zea) and fall armyworm
(Spodoptera frugiperda) [94–97]. Maysin derives from the conversion of eriodictyol to 2-hydroxy
eriodictyol by a flavanone 2-hydroxylase (F2H1, CYP93G5) [36]. Subsequently, 2-hydroxy eriodictyol
is C-glycosylated by UGT708A6 [89], and dehydrated to form isoorientin (luteolin 6-C-glucoside).
It is unclear yet if this dehydration is spontaneous or enzymatic. Two additional enzymatic steps
are required for the conversion of isoorientin to maysin. A rhamnose residue is first incorporated
onto the glucose moiety of isoorientin to form rhamnosylisoorientin (isoorientin 2”-O-rhamnoside)
by a rhamnosyl transferase. Maysin is then formed by dehydration of the glucose moiety in
rhamnosylisoorientin to 4-keto-6-deoxy glucose by a rhamnose synthase [98]. The identification
of the last two steps of the pathway was possible due to the availability of mutants which accumulate
3-deoxyanthocyanins, providing a salmon color to the silks, and mutants in two salmon silk loci had
been previously identified [99,100].
In some species, mono-glycosylated flavones can be further decorated to form di-C-glycosyl
flavones. A number of di-C-glycosyl flavones possessing C-glucosyl and C-arabinosyl moieties
were identified as allelopathic compounds in Desmodium incanum against the parasitic weed
Striga hermonthica [101]. Sequential C-glycosylation steps are involved in the biosynthesis of
these di-C-glycosyl flavones. First, the mono-glucosylated 2-hydroxyflavanone intermediates
were formed from C-glucosylation of 2-hydroxynaringenin with further C-arabinosylation.
Subsequently, the 2-hydroxyflavanone products were dehydrated to yield di-C-glycosyl flavones,
including two 6,8-di-C-glycosylated isomers, isoschaftoside and schaftoside [102].
Plants 2016, 5, 27 10 of 25

2.2.3. Evidence for Dehydratase Activity

The last step in the formation of C-glycosyl flavones is the dehydration of the 2-hydroxyflavanone,
so far poorly understood. Both enzyme-catalyzed reactions by dehydratases and spontaneous
dehydration were observed, and may co-exist. Dehydration of the maize glucosylated product
is likely to be spontaneous, since no 2-hydroxyflavanone C-glycosides were identified following the
in vitro reaction [89]. On the other hand, specific dehydratases seem to operate in buckwheat and
rice [79,90]. If the dehydration process were spontaneous, all glycosylated 2-hydroxylflavanones (equal
molar equivalents of the 6C- and 8C-glucosyl 2-hydroxylflavanones) should be dehydrated to the
corresponding flavones. In contrast, flavone 6C-glucosides preferentially accumulated in planta [90].
These results suggest that a dehydratase activity is required to regulate the regioisomer ratios, which
resulted in flavone 6C-glucosides to accumulate in buckwheat and rice [79,90]. A similar situation is
found in the dehydration of 2-hydroxyisoflavanones to form isoflavones by 2-hydroxyisoflavanone
dehydratase. Two 2-hydroxyisoflavanone dehydratases were characterized from Glycyrrhiza echinata
and soybean [103]. Since the spontaneous dehydration of 2-hydroxyisoflavanone was slow and
negligible compared to the enzyme-catalyzed reaction, the dehydration of 2-hydroxyisoflavanones to
form isoflavones was proposed to be primarily enzyme dependent in plant cells [103].

2.3. Dedicated Biosynthetic Pathway for 4’ Deoxyflavones (B-Ring Deoxyflavonoids)

B-ring deoxyflavonoids that lack the -OH group at the 4’ position of the B-ring, were
first discovered by chemical analysis of chitin-elicited cactus Cephalocereus senilis cultures [104].
Pinocembrin, a non-hydroxylated flavanone, is converted from cinnamic acid through cinnamoyl-CoA
and 2’,4’,6’-trihydroxychalcone intermediates as a consequence of the characterization of a single
form of CoA ligase being active on both cinnamate and 4-coumarate. Unusually, CHS and CHI from
C. senilis cultures were active with cinnamoyl-CoA and 2’,4’,6’-trihydroxychalcone, respectively [104].
To synthesize these unusual deoxyflavonoids, metabolic compartmentalization between PAL and
CoA ligase has been proposed, bypassing the C4H reaction and leading to the formation of
cinnamoyl-CoA, which was incorporated into B-ring deoxyflavonoids via non-discriminating CHS
and CHI activities [104].
Recently, this specialized pathway for 4’ deoxyflavonoids biosynthesis was characterized
from roots of Scutellaria baicalensis [40], a traditional Chinese medicinal plant, whose active
compounds include baicalin, baicalein, wogonoside, wogonin, neobaicalein, visidulin I and oroxylin
A. Pinocembrin, rather than naringenin (mono-hydroxylated flavanone), was identified as the
intermediate in the formation of these root-specific 4’-deoxyflavones. Several key enzymes have been
characterized in this specialized pathway, including a cinnamic acid specific CoA ligase (4CL-like),
which uses cinnamic acid to form cinnamoyl-CoA (bypassing 4-coumaric acid for addition of CoA), a
specific isoform of CHS in roots, and a FNSII which is specific for pinocembrin and which generates
chrysin as a product. This root-specific flavones pathway was successfully reconstituted in tobacco by
co-expression of the characterized enzymes [40]. These findings revealed a great level of plasticity in
the pathways involved in generation of flavonoids with rare modifications.

2.4. Other Flavone Modifications

Plants also produce other specialized flavones derivatives by hydroxylation, O-methylation/
O-demethylation, and acylation [1]. Some hydroxylated flavones are methylated by O-methyltransferases
(OMTs), which transfer methyl groups from S-adenosyl-L-methionine (donor) to the -OH groups
of flavones (acceptors). In the past few years, the biosynthesis of poly-methoxylated flavones
was defined in sweet basil (Ocimum basilicum) [105], an aromatic herb belonging to the mint
family. The glandular trichomes accumulating lipophilic flavones with hydroxyl groups at positions
5, 6, 7, 8, and 4’ and up to four O-methyl residues at positions 6, 7, 8 and 4’ in this plant
provided an ideal system to investigate the biosynthesis of poly-methoxylated flavones. First, a
Plants 2016, 5, 27 11 of 25

set of cation-independent methyltransferases (ObFOMT1-6) which catalyze regioselective 6-, 7-,

and 4’-O-methylations was identified [106]. Two flavone 8-O-methyltransferases (ObPFOMT-1 and
ObF8OMT-1) catalyze O-methylation at C8 [107]. Compared to O-methylation, O-demethylation is very
rare in plant specialized metabolism. A flavone 7-O-demethylase (ObF7ODM1), which regiospecifically
catalyzes the 7-O-demethylation of methoxylated flavones (gardenin B and 8-hydroxysalvigenin) was
identified [108]. Recently, certain sequential catalytic reactions were found in the biosynthesis of
some polymethoxylated flavones (nevadensin and pilosin) in sweet basil. To synthesize nevadensin
and pilosin, 7-O-methylated flavone was required to be formed first as a precursor for the following
6-hydroxylation by flavone-6-hydroxylase (CYP82D33). Subsequently, the 7-unmethylated flavones
(nevadensin and pilosin) are generated by ObF7ODM1 through the removal of the 7-methyl group [109].
Some rice OMTs (e.g., ROMT9, ROMT15, and ROMT17) were also identified to be functional in tricin
(3’,5’-dimethoxyflavone) biosynthesis [110,111].

3. Biological Activities of Flavones

3.1. Biological Activities of Flavones in Plants

3.1.1. Abiotic Protection

Flavonoids, including flavones, provide a shielding mechanism against the harmful effects of
UV-B radiation (280–315 nm) by accumulating in both the epidermises and the outermost cell layers
of the mesophyll tissues. UV-B radiation causes cellular damage by generating photoproducts in
DNA and direct damage to proteins and lipids [2]. Flavones have two major absorption peaks: in
the 290–400 nm range provided by the cinnamoyl part of the molecule; and in the range 240–285 nm
provided by the benzyol part [112]. Thus, plants take full advantage of these broad absorption spectra
from the structure properties of flavones. For example, in developing leaves of barley, saponarin
(isovitexin-7-O-glucoside) and lutonarin increase under UV-B irradiation, decreasing DNA and protein
damage [2]. Two UV-absorbing flavones, maysin and rhamnosylisoorientin, accumulate in maize
leaves of high-altitude lines in response to increased UV-B radiation levels. The biosynthesis of these
flavones is controlled by a UV-B regulated P1-homologous transcription factor expressed in maize
leaves [113]. In response to oxidative stress baicalein and its derivatives act as H2 O2 scavengers in
Scutellaria baicalensis [3]. Hydrogen peroxide is a substrate of peroxidases and a major active compound
in the elimination of reactive oxygen species (ROS). In response to an elicitor, baicalein 7-O-glucuronide
is hydrolyzed to the aglycone baicalein by a β-glucuronidase. 6,7-dehydrobaicalein was then formed
through oxidation of the released baicalein by peroxidases. Thus, H2 O2 is effectively consumed and
detoxified during the peroxidase reaction [3].

3.1.2. Biotic Protection

Phytoalexins are a heterogeneous group of low molecular mass specialized metabolites with
anti-microbial activity [4]. Some phytoalexins flavones include simple aglycones such as luteolin in
sorghum resists Colletotrichum sublineolum growth [34]), C-glycosyl flavones (isoorientin/orientin, and
isovitexin/vitexin in cucumber [114] and flax [115] against fungal infection), and O-methylated flavone
(tricin inhibits the spore germination of fungal pathogens in rice [116,117])
Other than pathogenic microbes, arbuscular mycorrhiza (AM) fungus and nitrogen fixing bacteria
Rhizobium and Frankia provide the most prevalent examples of symbiosis in the plant kingdom [118,119].
Multiple metabolic changes, including the alteration of flavonoid expression in the roots of the host
plants, occur before and after the penetration of the host plant’s root by microorganisms [120]. In the
Legume family, flavones have been identified as signaling molecules and regulators in the development
of root nodulation. In 1986, luteolin and 7,4’-dihydroxyflavone (DHF) released by roots of alfalfa
(Medicago sativa) and white clover (Trifolium repens) were identified as interacting with nodulation
(Nod) factor (NodD) to activate transcription of other nodulation genes (nodABC and nodFE) in
Plants 2016, 5, 27 12 of 25

Rhizobium meliloti and R. trifolii, respectively [5,121]. Researchers further demonstrated that DHF in
Medicago truncatula nodulation are not only important for the induction of Sinorhizobium meliloti nod
genes in the rhizosphere by released DHF [33], but also plays an essential role for sustaining Nod factor
induction during nodulation by root internal DHF [122]. Luteolin-7-O-glucoside serves as an attractant
of R. meliloti resulting in enhanced nodulation, thus indirectly regulating the growth of alfalfa [123].
Flavones also act as regulators in a plant-AM fungus interaction during the pre-colonization and
cell-to-cell stages. Chrysin and luteolin showed a stimulatory effect on the pre-symbiotic hyphal
grow of several Glomus and Gigaspora species, and also affected root colonization by increasing the
number of entry points of tomato (Lycopersicum esculentum) [124]. In melon (Cucumis melo) roots,
isovitexin 2”-O-glucoside accumulated under low phosphate conditions to stimulate mycorrhizal
colonization [120]. Apigenin showed a mycorrhiza formation-stimulating activity during root
colonization of soybean by the AM fungus Glomus mosseae [125].
Plant allelopathy is a biological phenomenon by which plants produce special compounds,
including flavones, to interfere with the growth and/or reproduction of other plant species [126].
It is not just plant-plant interference, but can also involve soil-mediated chemical intervention.
Tricin was found in rice root exudates to act as an allelochemical inhibiting the growth of the weeds
Echinochloa crus-galli, Cyperus difformis and C. iris [127–129]. The C-glycosyl flavone isoschaftoside from
cattle forage legume (Desmodium uncinatum) root exudate was identified as an allelochemical capable
of inhibiting growth of the parasitic weed Striga hermonthica [7,101].
Flavones are also produced by plants as inhibitory compounds against herbivores, including
insects and nematodes [1]. We mentioned previously the effects of maize silk maysin on corn earworm and
fall armyworm [94–97]. In insect herbivore (Spodoptera littoralis larvae)-damaged alfalfa (Medicago sativa)
plants, increased levels of apigenin make alfalfa unpalatable to the larvae [130]. Three flavone
C-glycosides (schaftoside, isoschaftoside, and neoschaftoside) are the main compounds responsible
for an ingestion inhibiting activity to the sucking deterrent brown planthopper, Nilaparvata lugens,
in rice phloem sap [131]. Tricin was identified as anti-feedant to the brown planthopper [132] and
boll weevil [133] in rice, and an aphid-feeding deterrent in wheat [134]. Recently, the C-glycosyl
flavone adonivernith (orientin 2”-O-xylopyranoside) was overproduced in the carpel walls of the
European globeflower Trollius europaeus after infection by Chiastocheta fly larvae, resulting in larval
growth inhibition [135,136]. Finally, O-methyl-apigenin-C-deoxyhexoside-O-hexoside was found to be
induced in oats tissues attacked by the nematodes Heterodera and Pratylenchus [137].

3.1.3. Plant Development

Copigmentation is a phenomenon that increases color intensity and stability. This process is
achieved by the formation of complex associations between pigments (anthocyanins) and other
colorless flavonoids (e.g., flavones), or metallic ions [1]. For instance, isovitexin is involved in the
bluing effect with anthocyanins on the flower color of Japanese garden iris (Iris ensata) [138]; vitexin
or orientin enhance the color intensity and the stability of anthocyanins in blueberry juice [139]; and
a supramolecular metal complex pigment, metalloanthocyanin, consists of stoichiometric amounts
of anthocyanins, flavones, and metal ions with a fix ratio at 6:6:2, respectively [8,140]. Recently, an
unusual covalently linked anthocyanin-flavone C-glycoside dimer was isolated from the leaves of
Oxalis triangularis [141]. Unexpectedly, tricin was identified as a monomer in monocot lignins, which
are complex phenylpropanoid polymer in plant cell walls [142–145]. Indeed, during lignification, tricin
acts as a nucleation site that initiates lignin polymer chains in monocots [9]. It is yet unclear whether
the tricin that participates in lignin formation derives from the general flavonoid pathway, or whether
there is a specialized route or flavone pool that feeds into lignin.

3.2. Molecular Interactions of Flavones with Other Molecules

In addition to the chemical diversity of flavone structures, different interactions between flavones
and other molecules are also critical for fulfilling multiple biological functions in plants. Here, we
include some representative interactions, such as those involving lipids, nucleic acids, and proteins [1].
Plants 2016, 5, 27 13 of 25

3.2.1. Interactions with Lipids: Flavone-Membrane Interaction

How flavones interact with membrane lipids is mainly determined by the lipophilicity and planar
structure of the compounds [146]. This has significant consequences for the ability of flavonoids in
general to cross cellular membranes. The relative hydrophobicity of flavones is related to the number
and position of -OH groups [147]. By using dipalmitoyl phosphatidyl choline (DPPC) lipid bilayers to
represent a biological membrane, the orientation and localization of flavones was demonstrated [148].
The results showed that 5- and 7-hydroxy flavones interact with the lipid surface, while 6-hydroxy
flavone and chrysin localize adjacent to the glycerol backbone. It was proposed that flavone -OH
groups interact with polar groups on the membrane through H-bonding [147]. By analyzing the
interaction of structurally different flavones with membranes, these studies showed that flavone
A- or C-rings orient towards the lipid-water interface while the B-ring penetrates into the hydrophobic
core [147]. The hydrophilic nature of flavones increases with the number of -OH groups. As flavones
become more hydrophilic, their membrane localization shifts towards the aqueous environment [149].
This may explain why apolar flavones, such as chrysin, locate closer to the membrane center, whereas
luteolin shows a higher propensity towards the polar region [150]. The studies also showed that the
presence of the C-5 -OH enhances flavone lipophilicity [147].

3.2.2. Interactions with Nucleic Acids

The interaction between flavones and DNA is proposed as a mixed mode of intercalation
(between the base pairs) and groove binding [151]. Typically, flavones prefer to bind higher order
DNA structures such as tri- and tetraplexes [152]. Similar as in the interaction with lipids, -OH
substituents were found to be also important for DNA binding. Flavones hydroxylated at the
7 position, such as 7-hydroxyflavone and 5,7-dihydroxyflavone, were identified as the most relevant
for DNA binding [153]. Apigenin could intercalate into the base pairs of calf thymus DNA, forming an
apigenin-DNA complex largely driven by hydrophobic interactions [154]. The interaction between
apigenin and yeast RNA was also investigated. The carbonyl groups of the RNA bases are involved
in H-bond formation with apigenin, which also binds RNA through G-C and A-U bases through
intercalation [155].

3.2.3. Interactions with Proteins

Flavone-protein interactions have various biological effects. Flavones could bind human serum
albumin (HSA) for transportation through plasma [156]. Two flavones, baicalin and baicalein,
interacted with HSA in the vicinity of the Trp214 residue which is located in the hydrophobic cavity of
subdomain IIA [156]. The binding capacity between flavones and serum albumin is affected by A-ring
hydroxylation [157]. As more hydroxyl groups are introduced, the intra-molecular H-bonds within
flavones are easily formed, and the affinities for serum albumins decrease [157]. Moreover, chrysin
interacts with bovine serum albumin (BSA) through hydrophobic interactions [158]. Flavones also act
as modulators of several biological activities. These include inhibitory effects of 8-prenyl-chrysin on the
P-glycoprotein activity [159] and of several flavones on CYP-dependent monooxygenases (e.g., chrysin
and apigenin on CYP1A2 [160], acacetin and hesperetin on CYP1B1 [161]; and chrysin, apigenin, and
acacetin on CYP19 [162]); increasing sensitivity of GABAA receptor by interaction with apigenin [163],
chrysin [164,165], or wogonin [164,165]; and competitive inhibition of xanthine oxidase by apigenin
and isovitexin [166]. In human breast cancer cells, apigenin can bind to 160 cellular targets, which
include the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing
regulation, mRNA stability, and mRNA transport [167] (see below).

3.3. Major Flavone Health Benefits

Flavones are non-essential nutrients that provide additive nutraceutical value to our diet.
Their health beneficial activities have been historically recognized across different cultures.
Plants 2016, 5, 27 14 of 25

Flavonoids, including flavones, have received increasing attention due to their anti-inflammatory,
anti-microbial and anti-cancer activities. However, the molecular mechanisms responsible for these
activities are just starting to be deciphered.
One of the first beneficial effects ascribed to flavones were anti-oxidant activities, based on
the ability of these compounds to scavenge reactive oxygen species (ROS). Structural-functional
relationship analyses identified luteolin as one of the most potent inhibitors of xanthine oxidase [168],
a key enzyme in ROS production. Reduction of ROS by apigenin prevents endothelial damage during
acute inflammation and restores mitochondrial function [169].
Most of the anti-inflammatory and anti-microbial activities attributed to flavones seem to be
centered on their ability to regulate the Toll receptor (TLR)/NFκB axis. This is a central pathway
in the host-pathogen interplay in mammals [170], responsible for the expression of inflammatory
mediators, including tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) and cyclooxygenase-2
(COX-2), an enzyme mediating the conversion of arachidonic acid to prostaglandins. Notably, great
similarities are found between the mammalian TLR/NFκB and plant pathogen defense pathways,
suggesting that flavones may regulate evolutionary conserved targets [171]. Studies from our group
showed that in macrophages and in animal models, apigenin reduces the phosphorylation of the
NFκB p65 subunit, required for its transcriptional activity [172,173]. Inhibition of p65 phosphorylation
reduces the expression of inflammatory cytokines, limiting the cell damage characteristic of acute
inflammation. Other flavones, such as the acacetin and wogonin, abundant in saffron seeds and
scutellaria, inhibit COX-2 by halting NFκB nuclear localization [174]. Overall, glycosides show
less anti-inflammatory activity than aglycones, probably a consequence of their reduced cellular
absorption [175]. Combination of the C-glycosyl flavones orientin and isoorientin reduced the
production of the inflammatory mediator molecule High Mobility Group Box-1 (HMGB-1), but this
effect was not observed when either of the flavones was used alone or in combinations of vitexin and
isovitexin, suggesting high specificity in their mechanisms of action [176].
Recent studies identified additional mechanisms responsible of the anti-inflammatory activity
of flavones, including the regulation of non-coding RNAs. Large microRNA screenings showed that
apigenin, or consumption of celery foods which have a high content of apigenin, reduce microRNA155
(miR155) expression, a main inflammatory regulator [177]. miR155 binds to 3’-UTR regions of
several inflammatory cytokines, suggesting an additional mechanism by which flavones can restore
homeostasis during acute inflammation, independent of their anti-oxidant activity. So far, studies in
Arabidopsis indicate that the accumulation of anthocyanin follows patterns regulated by miR156 [178],
but whether flavones or other flavonoids themselves induce miRs in plants is yet to be determined.
Consistent with the ability of flavones to regulate inflammation, interventions with the
Mediterranean diet, which is rich in flavonoids, showed improved cardiac function, reduced
hypertension and obesity [179,180]. Epidemiological studies highlighted the beneficial effects of
this diet in metabolic function [181].
Flavones also affect leukocyte migration. This has profound effects in both inflammation and
cancer. Luteolin reduces Rho GTPases activity, decreasing leukocyte migration thereby resulting in the
prevention of inflammation and neuronal damage [182]. Apigenin inhibits leukocyte migration by
affecting the Janus kinase 3 (JAK3), a non-receptor tyrosine kinase [183]. Flavones’ ability to reduce
cell migration has great impact in cancer, suggesting alternative therapeutic approaches to reduce
metastasis. Apigenin reduced breast cancer cell migration, by inhibiting mitogen activated protein
kinases (MAPK), including ERK and JNK [184].
Flavones’ anti-carcinogenic activity promotes apoptosis of cancer cells at doses that are cell-type
specific. Leukemias in general seem more susceptible to flavones, undergoing caspase-dependent
apoptosis at low micromolar ranges [185]. In contrast, higher flavone concentrations are needed to
induce apoptosis of cancer cell lines from solid tumors including prostate, lung and skin cancer [185–187].
The anti-carcinogenic effect of flavones is given in part by their ability to induce DNA damage, and is
accompanied by cell cycle arrest at G1 or G2, depending on the particular cell type. Interestingly, the
Plants 2016, 5, 27 15 of 25

ability of apigenin to induce cell death in cancer cells is independent of ROS production [188],
supporting a beneficial role of flavones independent of their anti-oxidant activity. Flavones, such
as apigenin, induce the phosphorylation of heat shock protein 27, an inhibitor of apoptosis highly
expressed in cancers, by promoting the activity of the p38 kinase [189]. Yet, this effect is not direct and
the direct target remains to be identified. Maysin induces apoptosis of PC-3 prostate cancer cells, by
the mitochondrial-intrinsic pathway, but had no effect in lung, colon or stomach cancer cell lines [190].
Anti-cancer activity may also be due to inhibition of the NFκB pathway. In silico predictions suggest
that apigenin associates with IKKα, a kinase upstream of NFκB [191]. However, in models of acute
inflammation, apigenin reduces IKKβ without affecting IKKα [172]. Additional experiments will
be needed to further understand the specific contributions of the molecular networks responsible
for the anti-carcinogenic effects of apigenin and other flavones. Higher levels of NFκB activity and
COX-2 are common in both cancers and acute inflammation, suggesting shared mechanisms of action.
Identification of the direct targets will highly contribute to understand the molecular mechanism
related to flavones and health. The use of PD-Seq (phage display high-throughput sequencing),
a novel approach for small target identification, identified several targets, suggesting that dietary
compounds, unlike pharmaceuticals, may target several molecules. Many of the identified targets were
validated using independent strategies, suggesting that most are biologically relevant targets [167].
Using PD-Seq, we found that apigenin associates with several RNA binding proteins, including
hnRNPA2. hnRNPA2 regulates alternative splicing and is highly expressed in tumors. The treatment
of breast cancer cells with apigenin changed aberrant splicing isoforms found in cancer cells to
isoforms commonly found in non-malignant cells. These results highlight the existence of additional
mechanisms involved in the health beneficial effects of flavones and prompt the need of future studies
in the area.

4. Conclusions and Future Prospects

From all of the above, it is clear that flavones are not only important compounds for the biology
of plants and for human health, but they also provide convenient specialized metabolites to better
understand the chemical complexity of plants, and how different enzymes have evolved to use the
same substrates to produce identical products, sometimes in the same organism. The characterization
of the flavone pathway in a number of different plant species provides a valuable resource of clones
and enzymes for metabolic engineering to produce organisms with enhanced tolerance to biotic and
abiotic stress conditions, or for improved animal and human nutrition. Finally, the identification of
proteins that specifically interact with flavones provides the next frontier in establishing how related
specialized metabolites can have such a diversity of biological activities.

Acknowledgments: This work was supported by Agriculture and Food Research Initiative competitive USDA
National Institute of Food and Agriculture grants #2015-67013-22810 to Erich Grotewold and #2015-67017-23187
to Erich Grotewold and Andrea I. Doseff.
Author Contributions: Nan Jiang, Andrea I. Doseff, and Erich Grotewold wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this review:
PAL phenylalanine ammonia-lyase
C4H cinnamic acid 4-hydroxylase
CoA coenzyme A
4CL p-coumaroyl: CoA ligase
4CL-like cinnamic acid specific CoA ligase
CHS chalcone synthase
CHI chalcone isomerase
FNS flavone synthase
2-ODD Fe2+ /2-oxoglutarate-dependent dioxygenase
Plants 2016, 5, 27 16 of 25

F2H flavanone-2-hydroxylase
F3’H flavanone-3’-hydroxylase
F6H flavanone-6-hydroxylase
CYP cytochrome P450
IFS isoflavone synthase
UGTs UDP-glycosyltransferases
CGT C-glycosyl transferase
OGT O-glycosyl transferase
HID 2-hydroxyisoflavanone dehydratase
OMT O-methyltransferase
FOMT flavonoid O-methyltransferase
RHM UDP-rhamnose synthase

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