Plant Physiol.
(1997) 115: 1405-1 41 2
Flavonoid-Peroxidase Reaction as a Detoxification
Mechanism of Plant Cells against H 2 0 2
H i d e o Yamasaki*, Yasuko Sakihama, and N o r i k a t s u l k e h a r a
Laboratory of Cell and Functional Biology, College of Science, University of the Ryukyus,
Nishihara, Okinawa 903-01, Japan
Recent studies have revealed that dietary flavonoids are potent
radical scavengers, acting in a manner similar to ascorbate and
a-tocopherol. However, it is still not clear whether flavonoids have
a similar antioxidative function in plants. We examined the possi-
bility that flavonoids could function as stress protectants in plant
cells by scavenging H,O,. Two major flavonoids, quercetin and
kaempferol glycosides, were isolatedfrom leaves of the tropical tree
Schefflera arboricola Hayata. Both glycosides and aglycones of
isolated flavonols were oxidized by H,O, in the presence of horse-
radish peroxidase and/or in a soluble fraction of S. arboricola leaf
extract. The rates of oxidation were in the order quercetin >
kaempferol > quercetin glycoside >> kaempferol glycoside. Judging
from the effects of inhibitorssuch as KCN, pchloromercuribenzoate,
and 3-amino-1H-l,2,4-triazole, we conclude that guaiacol peroxi-
dase in the soluble fraction catalyzes H,O,-dependent oxidation of
flavonols. In the flavonol-guaiacol peroxidase reaction, ascorbate
had the potential to regenerate flavonols by reducing the oxidized
product. These results provide further evidence that the flavonoid-
peroxidase reaction can function as a mechanism for H,O, scaveng-
ing in plants.
~
Flavonoids are the most common secondary metabolites
in vascular plants, with the exception of betalain in a few
families (Stafford, 1994).As components in the human diet,
flavonoids have recently received considerable attention as
efficient antioxidants, in addition to ascorbate, a-tocopherol,
and carotenoids. Numerous in vitro studies have shown that
flavonoids can directly scavenge molecular species of active
oxygen: superoxide (O,-), hydrogen peroxide (H202),hy-
droxyl radical (.OH),singlet oxygen ('O,), or peroxyl radical
(Bors et al., 1990, 1994; Yamasaki et al., 1996).
In plants the photosynthetic electron transport system is
the major source of active oxygen species. To avoid
oxygen-mediated toxicity, chloroplasts have evolved a
highly developed detoxification system, which has been
termed the ascorbate-glutathione cycle (Foyer, 1993).It had
been proposed that flavonoids scavenge active oxygen spe-
cies, such as O,- or 'O,, photoproduced in the chloroplast
in a manner similar to a-tocopherol and carotenoids. How-
ever, this is unlikely in vivo because flavonoids are largely
localized in vacuoles (Charriere-Ladreix and Tissut, 1981)
and radicals cannot readily diffuse into vacuoles from chlo-
roplasts. Therefore, in plant cells the participation of fla-
* Corresponding author; e-mail yamasaki@sci.u-ryukyu.ac.jp;
fax 81-98-895-5376.
1405
vonoids in the scavenging of 'O, and/or oxygen radicals
(0,- and .OH) may be limited to nearby generating sites,
i.e. only in the vacuoles and partly in the cell wall. How-
ever, unlike other active oxygen species, H,O, is stable and
able to diffuse across membranes.
The toxicity of H,O, itself is relatively weak compared
with that of other active oxygen species, but in the presence
of O,-, H,O, can generate highly reactive hydroxyl radi-
cais via the metal-catalyzed Haber-Weiss reaction. Thus,
the scavenging of H,O, in cells is critica1to avoid oxidative
damage. Plant cells are prone to produce H,O, not only
under stressed conditions, but also from regular metabo-
lism. Accumulation of flavonoids seems to be pronounced
in tissues under such conditions. Food chemical studies
have revealed that flavonols can act as electron donors for
peroxidase (Miller and Schreier, 1985; Schreier and Miller,
1985). Takahama (198813, 1989) has proposed that this
flavonol-peroxidase reaction may function as an H,O,-
scavenging system in vivo as well as in vitro. In contrast to
our understanding of pharmacological properties of fla-
vonoid pigments, however, there is little information on
whether flavonols can contribute to the scavenging of H,O,
in vivo.
In this study the participation of a flavonol-peroxidase
system in H,O, detoxification is examined in contrast to
the well-established ascorbate-APX system in plants. Re-
sults obtained from the field-grown tropical plants further
support the H,O,-scavenging function of flavonoids.
MATERIALS A N D M E T H O D S
Leaves of the tropical tree Sckefflera arboricola Hayata
were harvested from a field on Okinawa island, a subtrop-
ical region of Japan. The age of the plants was between 10
and 15 years. The sampling period was the summer season,
from July to October. A11 materials analyzed were har-
vested from the canopy of the trees, where the light envi-
ronment was nearly constant (maximum 2200 pmol quanta
m-'s-') .
lsolation of Flavonols
Leaves with a removed midrib were homogenized for
30 s with a 3-fold volume of 80% MeOH at room temper-
~
Abbreviations: APX, ascorbate peroxidase; GuPX, guaiacol per-
oxidase; MeOH, methanol; SF, Schefflera arboricola flavonoid.
1406 Yamasaki et al.
ature. The homogenate was filtered through eight layers of
gauze and further through a paper filter (No. 2, Advantec,
Tokyo, Japan) in vacuo to remove cellular debris. To ex-
clude contaminants of photosynthetic pigments, such as
chlorophylls or carotenoids, the MeOH extract was passed
through a Sep-Pak C,, filter cartridge (Waters). Subse-
quently, the same volume of 100% acetone was added to
the filtrate (final ratio, acetone:MeOH:H,O, 5:4:1, v/v). The
precipitates caused by acetone were removed by centrifu-
gation (5000g for 3 min). This step efficiently separated
flavonols from contaminants in the MeOH extract. After-
ward, a 3-fold volume of diethyl ether was added to the
supernatant. Phase separation was carried out by centrifu-
gation at 20009 for 3 min. A brownish precipitate was
obtained and washed again with diethyl ether. The concen-
trate was diluted with 50% MeOH and subjected to
reverse-phase column chromatography (Lobar RP-18,
Merck, Darmstadt, Germany). The concentrate was eluted
with acetic acid:MeOH:H,O (5:25:75, v/v) at a detection
wavelength of 346 nm. The peak fractions were collected,
and flavonoid glycosides were adsorbed to a Sep-Pak C,
filter cartridge (Waters). After rinsing with distilled water,
the adsorbed flavonol glycosides were eluted with a small
volume of MeOH (100%) and used for subsequent bio-
chemical assays.
HPLC and TLC Analysis
HPLC analysis was carried out with a reverse-phase C,,
column (SC,,AR, 4.6 X 50 mm; Nacalai tesque, Kyoto,
Japan). The solvent for elution contained acetic acid:
MeOH:H,O in a ratio of 5:25:75 (v/v) for 10 min, 5:51:49 for
a subsequent 8 min, and 5:90:10 for 7 min at a flow rate of
1.0 mL min-l. Peak areas were monitored at 370 nm with
a Chromatopac integrator (C-R3A, Shimadzu, Kyoto, Ja-
pan). The identification of flavonoids was confirmed by
cellulose TLC (Funacel SF, Funakoshi, Tokyo, Japan) as
previously described (Yamasaki et al., 1995a).
Spectroscopic Analysis
Determination of chlorophyll, total flavonoids, and
ascorbate were carried out with a UV-160A spectropho-
tometer (Shimadzu). Leaves with midribs removed were
divided into halves. Halves of leaves were cut into small
pieces and were homogenized for 30 s with a known vol-
ume of solvent (80% acetone for chlorophylls and 100%
MeOH for flavonoids) at room temperature. The superna-
tant obtained by centrifugation was used for spectropho-
tometric determination. To entirely exclude chlorophylls
and carotenoids, the supernatant was passed through a
Sep-Pak C,, cartridge before the measurement of fla-
vonoids. This preparation was also used for the HPLC
analysis.
The sample preparation for the determination of ascor-
bate content was similar to that described above. The ho-
mogenization was carried out with a 0.1 M phosphate
buffer (pH 6.8) at 0°C. The homogenate was filtered
through four layers of gauze. The supernatant obtained by
centrifugation (20,0009 for 2 min at OOC) was passed
Plant Physiol. Vol. 115, 1997
through a cellulose nitrate filter (DISMIC 25, Advantec) to
remove proteins, and further through a Sep-Pak C, filter
cartridge to exclude UV-absorbing compounds such as
polyphenols. The ascorbate content was determined with
the enzymatic method (Takahama, 1992). DTT at 100 p~
was added before the measurement to reduce dehy-
droascorbate.
Measurement of the Peroxidase Activity
For sample preparation leaves were homogenized with a
10-fold volume of buffer containing 5% (w/v) polyvinyl
polypyrrolidone, 1 mM EDTA, 10 mM sodium ascorbate,
and 50 mM potassium phosphate (pH 7.0). The supernatant
obtained by centrifugation (20,00Og, 2 min at OOC) was
passed through a cellulose acetate filter (DISMIC 25) to
exclude membrane fractions. After addition of sorbitol
(20% at final [w/v]) for protection of the APX, the soluble
fraction obtained was applied to a gel-filtration column to
remove low-molecular-interference compounds, using an
Econopak 10 DG column (Bio-Rad)equilibrated with buffer
containing 1 mM EDTA, 20% sorbitol (w/v), and 10 mM
potassium phosphate (pH 7.0). Enzyme activity was mea-
sured as described previously (Amako et al., 1994). The
kinetics of flavonol oxidation were monitored at 380 and
360 nm for aglycones and glycosides, respectively. Ascor-
bate oxidation in the presence of flavonol was monitored at
265 nm, at which leve1 there was no significant overlap of
the absorption changes to cause difficulties in interpreta-
tion (Takahama and Oniki, 1992).Kinetics parameters were
determined from the absorbance decrease using reported
extinction coefficients (Takahama and Egashira, 1991).
RESULTS
Quercetin and Kaempferol Clycosides as the Major
Flavonoids in S. arboricola Leaves
In S. arboricola leaves two major flavonoids were identi-
fied as yellow fluorescent spots by two-dimensional TLC
under UV light (not shown). We have isolated these fla-
vonoids with C,, reverse-phase column chromatography
(Fig. 1).Judging from the ratio of A346to A,, as a measure
of purity, fractions showing a value below 0.9 were col-
lected and concentrated to use as the flavonoid preparation
for the subsequent assays. Each preparation was resolved
as a single peak upon retesting by HPLC.
The spectral profile of the isolated flavonoids showed
two characteristic absorption maxima in the UV range,
corresponding to bands I and I1 of flavone/flavonols
(Markham, 1993). For convenience, the flavonoids isolated
here are tentatively designated as SF1 and SF2 in elution
order. The absorption maxima of SF1 in 100% MeOH were
258 and 359 nm, and those of SF2 were 266 and 346 nm (Fig.
1, insets).
The R, value of the aglycones obtained after acid-
hydrolysis treatment of SFs corresponded to that of authen-
tic quercetin (0.62) and kaempferol (0.47) on cellulose TLC
(Fig. 2), indicating that SF1 is a quercetin glycoside and that
SF2 is a kaempferol glycoside. This was confirmed by
 H2O2-Scavenging Function of Flavonoids
o
VI
<
20 30 40 50
Fraction No.
Figure 1. Separation of SFs with C IH reverse-phase column chroma-
tography. The concentrate (2 ml) of flavonoid was applied to the
column and eluted with a solvent (acetic acid:MeOH:H2O, 5:25:75,
v/v). The dashed line shows the ratio of Ai4fl to /V,f)5 of each fraction
(5 ml). SF1 and SF2 represent SFs 1 and 2 purified from S. arboricola
in this study. Absorption spectra in 100% MeOH are shown in the
insets.
another solvent system, in which the R F values of quercetin
and SF1 aglycone in «-BuOH:acetic acid:H2O (4:1:2, v/v)
were 0.79 and those for kaempferol and SF2 aglycone in the
same solvent were 0.90. Absorption spectra of those hydro-
lysates in MeOH were also consistent with those of authen-
tic quercetin and kaempferol (not shown). The structure of
flavonoids can be deduced from the responses of their UV
spectra to various shift reagents (Markham, 1993). Results
Rf
apigenin (4'-OH)
kaempferol (3, 4'-OH)
quercelin (3. 3', 4'-OH)
myricetin (3. 3', 4' 5'-OH)
Figure 2. TLC of the aglycone moiety of the flavonoids isolated from
S. arboricola. The aglycones were obtained after acid hydrolysis of
isolated SFs 1 and 2. Forestal (acetic acid:HCI:H2O, 30:3:10, v/v) was
used as the developer. Lane 1, Authentic aglycones (apigenin,
kaempferol, quercetin, and myricetin); lane 2, SF1 aglycone; and
lane 3, SF2 aglycone. The chemical structure of flavone/flavonol is
represented on the right. Among these, only apigenin, lacking 3-OH,
is a flavone and the others are flavonols. The number of OH groups
of the B ring represents the only structural difference between
kaempferol, quercetin, and myricetin.
1407
shown in Table I suggest that the three positions of both
quercetin and kaempferol are glycosylated in SF1 and SF2.
These results are consistent with the identification of
the major leaf flavonoids contained in S. arboricola as fla-
vonol glycosides, namely quercetin-3-glycoside (SF1) and
kaempferol-3-glycoside (SF2).
H2O2-lnduced Oxidation of Flavonols in the Presence of
Horseradish Peroxidase
In vitro experiments have shown that flavonols are po-
tent antioxidants capable of directly scavenging various
active oxygen species (Bors et al., 1990). However, the
production and scavenging of active oxygen is generally a
localized event. Therefore, a direct scavenging function of
flavonoids appears to be less significant for the overall
detoxification mechanism in vivo. Along with these non-
enzymatic reactions, there has also been evidence to sug-
gest that quercetin and kaempferol may scavenge H2O2 in
conjunction with peroxidase (Miller and Schreier, 1985;
Schreier and Miller, 1985; Takahama, 1986). In broad bean
(Vicia faba L.) leaves it has been demonstrated that fla-
vonols in epidermal (Takahama, 1988b) and mesophyll
(Takahama, 1989) cells are oxidized in vivo by externally
added H2O2. Takahama and Egashira (1991) have sug-
gested that peroxidase catalyzes this H2O2-induced flavo-
nol oxidation. Apart from these observations, there is little
information on whether leaf flavonols act as electron do-
nors for the peroxidase reaction in a manner similar to
ascorbate. If this reaction could function as a common
detoxification system of leaves against H2O2, flavonoids
isolated from other species may also be oxidized by H2O2.
Isolated SFs were oxidized in vitro with peroxidase in
the presence of H2O2 (Fig. 3). The nonenzymatic oxidation
of the flavonoids by H2O2 was negligible under these con-
ditions (Fig. 3, A-D, top traces). However, quercetin and
kaempferol both were rapidly oxidized by the addition of
H2O2 in the presence of horseradish peroxidase (Fig. 3, A
and B). Similarly, H2O2 oxidized both SF1 and SF2 as their
glycosides in conjunction with horseradish peroxidase (Fig.
3, C and D). The rates of the oxidation were in the order
quercetin > kaempferol > SF1 » SF2. These results clearly
show that SF1 and SF2 can also act as electron donors for
the peroxidase reaction in addition to the corresponding
aglycones. As previously noted in the case of quercetin and
its glycosides (Takahama, 1986), the reactivity of SF glyco-
sides was considerably lower than that of their aglycones.
Table I. UV spectral data of the flavonoid glycosides isolated from
S. arboricola
Absorption Maxima
Flavonoid
MeOH NaOMe AICI3 AlCU/HCI
nm
SF1 359, 303sh'' 402, 273 417, 303 400, 358
258 271 303, 269
SF2 346, 302sh 396, 325 398, 346 397, 346
266 274 306, 276 306, 276
" sh, Shoulder.
1408 Yamasaki et al. Plant Physiol. Vol. 115, 1997

cells contain two molecular families of peroxidases: GuPXs,

-E 0.2 A -4 -HRP
such as horseradish peroxidase, and APXs. Both activities
are inhibited by KCN, but p-chloromercuribenzoic acid
0.2
inhibits only APXs (Amako et al., 1994). Table I1 shows the
O
W effect of inhibitors on the peroxidase-mediated oxidation of
c
a,
flavonols by H,O, in the presence of the leaf extract. The
o specificity of inhibitors was clearly observed with a soluble
c
P 0.1 o. 1 fraction of leaf extract when pyrogallol and ascorbate were
O + HRP used as electron donors for the peroxidase reactions. KCN
v)

9 virtually completely suppressed the oxidation of SF1, sug-

gesting the participation of peroxidase in the soluble frac-
tion. The oxidation of SF1 was not prevented by p-
n O
“o 1 2 0 1 2
chloromercuribenzoic acid at 200 p ~ whereas
, ascorbate
oxidation was completely inhibited. Another possibility is
that catalase, which is known to exhibit peroxidase activity
IC 4 -HRP under certain conditions, was responsible for the oxidation
of flavonol glycosides. However, we eliminated this possi-
E
o 0.08 - bility, because the oxidation of SF1 was not affected by 10
a 0.08
(3
Y
mM aminotriazole, an inhibitor of catalase (data not
a,
o shown). These overall data suggest that SFs can be oxidized
C
([I by GuPXs rather than APXs in leaves.
Q
Ô 0.04- + HRP 0.04
8
a
Cooperative Function of Ascorbate to the
01 I B ‘o Flavonol-Peroxidase Reaction
o 1 2 O 5 10 15 Figure 4 shows the effect of ascorbate on the oxidation of
Time (min) flavonols by peroxidase. Under the same conditions to
those in Figure 3, ascorbate in the reaction medium com-
Figure 3. Oxidation of flavonols by H,O, in conjunction with horse-
radish peroxidase. A, Quercetin; 6, kaempferol; C, SF1 (quercetin-
pletely suppressed the apparent H,O,-induced oxidation
3-glycoside);and D, SF2 (kaempferol-3-glycoside). The reaction me- of SF1 even in the presence of horseradish peroxidase (Fig.
dium (1 mL) contained 50 mM potassium phosphate (pH 6.5) and 30 4A, top trace). One explanation for thís result would be the
p~ flavonol. H,O, was added to reaction medium at the times preferential oxidation of ascorbate by peroxidase instead of
indicated by the arrows (final concentration, 30 p ~ ) -HRP,
. In the SF1, as proposed by Mehlhorn et al. (1996). However, as
absence of horseradish peroxidase; +HRP, horseradish peroxidase shown in Figure 4B (top trace), the activity of ascorbate in
(final, 30 milliunits mL-’) was present before adding H,O,. the GuPX reaction is very low. Nevertheless, H,O, caused
rapid oxidation of ascorbate when SF1 was also present in
the reaction medium (Fig. 4B, bottom trace). A similar
This decrease in reactivity was most pronounced in SF2; phenomenon was also observed in the case of SF2 (not
the oxidation rate of SF1 was decreased to 20% of quercetin shown). Obviously, ascorbate does not act as a primary
by glycosylation, whereas it was decreased to 2% of
kaempferol in the case of SF2. The relative K , values for
quercetin, kaempferol, SF1, and SF2 were 28 2 5, 14 2 4,
180 ? 36, and 1080 2 271 p ~respectively,
, in the presence Table II. Effects of peroxidase inhibitors on the rate of oxidation of
of 1 mM H20,. The relative V,,, for the oxidation of flavonols by leaf extract
quercetin, kaempferol, SF1, and SF2 were 16, 7, 2, and 0.7 A soluble extract was prepared from leaves as described in “Ma-
mmol mg-’ protein min-l, respectively, in the presence of terials and Methods.” A reaction mixture contained 50 mM potassium
phosphate (pH 7.0), 0.1 mM H,O,, and 50 FL of soluble extract in a
1mM H,O,. Because a similar decrease of the reactivity was
total volume of 1 mL. As the electron donor, 40 p~ SF1, 20 mM
also observed in the case of commercially available pyrogallol, or 1 mM ascorbate was present in a reaction mixture.
kaempferol-3-rutinoside (not shown), the decrease may be Values are the means ? so of three measurements. The control rates
ascribed to the significance of the 3-OH substitution pat- were 0.021, 1.37, and 2.1 7 pmol min-’ mg-’ protein in oxidation of
tern (Takahama, 1986). SF1, pyrogallol, and ascorbate, respectively.
Relative Activity
Inh ibitor
Oxidation of Flavonols by H,O, in Conjunction with the SF1 Pvroeallol Ascorbate
Soluble Fraction of Leaf Extract %

Similar to the results obtained with horseradish peroxi- Control 1 O0 1 O0 1 O0

dase, H,O, oxidized quercetin, kaempferol, SF1, and SF2 in 200 /.LM p-chloromercuribenzoate 64 f 1 O 122 f 11 823
1 mM KCN 5?4 1+-1 1152
the presence of the soluble fraction of leaf extract. Plant
 H,O,-Scavenging Function of Flavonoids 1409

Figure 4. Cooperative effect of ascorbate on the

peroxidase-dependent oxidation of flavonol by

s
0.10
- + ASA 2 0.20 - -1 -SFI H,O,. A, Suppression of the flavonol oxidation

y
(\1 by ascorbate. The oxidation of SF1 was moni-

-\
(o
(*, tored at 360 nm in the presence (+ASA) or
0.08 absence (-ASA) of 50 /.LM ascorbate. B, Stimu-

E
C
O
0.06 f
.$
.-5
a 0.16
a,
O
c 0.12
0.08
lation of ascorbate oxidation by flavonol. The
oxidation of ascorbate (initial concentration, 50
X p ~ was ) monitored at 265 nm in the presence
o 0.04 ( + S F l ) or absence (-SFl) of 30 /.LM SF1. The
G
v) go experimental conditions of the top trace in A
0.02 2 0.04 - and the bottom trace in B were identical except
for the monitoring wavelength. Other experi-
- mental conditions were similar to those in Fig-
O 0- ure 3 (H,O,, 30 ~LM;horseradish peroxidase, 30
O 60 120 180
milliunits mL-’).
Time (s)

electron donor for the peroxidase reaction, but may be
consumed to reduce the oxidized product(s) of flavonols.
Quantitative Relationship between Flavonoids and
Ascorbate in Leaves during Leaf Development
It is generally accepted that the ascorbate-APX reaction is
the most significant H,O,-scavenging mechanism in plant
cells (Asada, 1992). Thus, plant cells contain high concen-
trations of ascorbate, which serves as the electron donor for
this reaction (Foyer, 1993). This is particularly pronounced
in leaves because of a high requirement for detoxification
of active oxygens produced during photosynthesis (Grace
and Logan, 1996). Figure 5 shows the changes in chloro-
phyll and ascorbate content during leaf development. The
leaf area of S. arboricola increased significantly with devel-
opmental age. A rate of increase in leaf area was approxi-
mately 1 cm2 d-l under field conditions. Concomitantly,
the apparent leaf color changed from yellow-green to deep-
green during development. Chlorophyll content was well
correlated with leaf area ( r = 0.72), and ascorbate pool size,
calculated from the amount of ascorbate plus dehy-
droascorbate, also increased with leaf area (Y = 0.67). This
clearly shows the close relationship between photosyn-
thetic and detoxification capacity in leaves. Because the
number of chloroplasts in the mesophyll cell did not
change during leaf development (data not shown), the
age-dependent changes in ascorbate content can be attrib-
uted to an increase in antioxidative capacity concomitant
with the development of thylakoid lamella.
In contrast to these metabolites, flavonoid levels esti-
mated from the A,,, of MeOH extracts decreased with leaf
area, showing a negative correlation to chlorophyll ( r =
-0.61) and ascorbate (r = -0.61). Similar results were
obtained by HPLC analysis when flavonoid contents were
compared between young and mature leaves (Table 111). In
particular, the content of SF1 (quercetin glycoside) was
strongly dependent on the leaf age, but SF2 (kaempferol
glycoside) was less dependent. The content ratio of SF1 to
SF2 decreased during leaf development (Table 111). These
age-dependent changes in flavonoid content were less ap-
parent in shade leaves, which experienced maximum irra-
diances of approximately 200 pmol m-’ s-’ at noon and in
which the flavonoid levels were low, even in young leaves
(data not shown).

100 I o 0.12 I o
t Nf
-E,O
0.1 1 &- 0 . 4 1
6
g 0.0%- 2 0.3

i’
v v
a, VI
m
w 2
e 0.06 - e 0.2
8 9m
0 0 . 9 -
:‘= 0.04
ii
0.1
’

o
o
I I I I I
- 0 0.02 - 0
O 10 20 30 40 50 10 20 30 40 O 10 20 30 40 50
Leaf area (cm2) Leaf area (cm2) Leaf area (cm2)
Figure 5. Correlation between ascorbate and flavonoid contents during leaf development. Chlorophyll content is repre-
sented by chlorophyll a + b, and ascorbate i s represented by ascorbate + dehydroascorbate. Flavonoid levels were
estimated from A,,, of the MeOH extract.
1410 Yamasaki et al.
Table 111. Changes in flavonoid contenfs during leaf development
in S. arboricola
Values are the means ? SD of four to six measurements. Leaf area
was used as a measure of leaf age.
Leaf Area SF1 S F2 SFl/SF2
cmz pmol g- ’ fresh wt
Young (2.5 -+ 0.5) 4.24 2 2.58 2.02 2 0.07 2.0 ? 1.1
Middle (10.9 2 1.1) 2.96 2 0.90 3.37 ? 0.66 0.9 ? 0.3
Mature (26.3 ? 1.7) 1.29 2 0.27 2.35 ? 0.45 0.6 ? 0.1
DISCUSSION
Flavonoids as Electron Donors to Peroxidase
The present study has demonstrated that flavonol glyco-
sides in leaves of S. arboricola have the potential to act as
reducing agents in a manner similar to ascorbate (Figs. 3
and 4). The concept of antioxidative function of flavonoids
is not novel, as seen in the “vitamin P” concept proposed 60
years ago (Bors et al., 1990), but has been largely bypassed
in the physiological research of plants.
The chemical basis of the antioxidative potential of fla-
vonoids has been ascribed to the hydroxy groups present
in their structures. Bors et al. (1990) have suggested three
structural features that are important determinants for the
radical-scavenging potential of flavonoids: (a) the o-
dihydroxy (catechol) structure in the B ring; (b) the 2,3-
double bond in conjunction with 4-0x0 function; and (c) the
presence of 3- and 5-OH groups (see Fig. 2). Similar to
nonenzymatic radical-scavenging efficiency, the electron-
donating activity of flavonoids to peroxidases also requires
these structures (Takahama, 1986; Takahama and Egashira,
1991). Among them, the 3-OH group is the most’significant
determinant of electron-donating activity (Takahama and
Egashira, 1991); aglycones are oxidized much faster than
3-glycosides (Fig. 3). However, it is unlikely that aglycones
act as substrates for peroxidase in vivo because they are
localized in the nonaqueous phase. Thus, the catechol
structure in the B ring, rather than 3-OH, may be actually
more important in determining the efficiency in vivo. In
this context, quercetin glycosides, which dominated the
flavonoid profile of young leaves (Table III), are postulated
to be superior electron donors than kaempferol glycosides
in vivo.
Participation of Vacuolar Peroxidase in the H,O,-lnduced
Oxidation of Flavonoids
The inhibitor experiments (Table 11) suggest that fla-
vonoids are oxidized to a greater extent by GuPX than APX
in leaf extracts. Although GuPXs are known to be localized
in vacuoles and apoplasts, the former dominate the total
activity of leaf extracts (Takahama and Egashira, 1991).
Results in Table 11, therefore, can be largely explained by
the enzymatic activity of vacuolar GuPXs in S. arboricola.
Takahama and Egashira (1991) have isolated a basic per-
oxidase from vacuoles of broad bean and demonstrated
that flavonols are good electron donors to vacuolar perox-
idase. Because the concentrations of flavonols in vacuoles
Plant Physiol. Vol. 11 5, 1997
are 100 times higher than the K, values for the vacuolar
peroxidase, they have suggested that the flavonols in vacu-
oles can function as electron donors to vacuolar peroxidase
in vivo (Takahama and Egashira, 1991). Results obtained
~
from S. arboricola are consistent with these observations.
A Flavonoid Redox Cycle as the
H,O,-Scavenging Mechanism
When quercetin is oxidized in vitro by the peroxidase-
H,O, system, dimerized and trimerized quercetin are pro-
duced as the major oxidized products (Schreier and Miller,
1985). Ascorbate can reduce the primary oxidized product
of flavonols (probably a flavonoid radical) and conse-
quently inhibits the subsequent formation of degraded
products (Takahama, 1986; Jan et al., 1991). A scheme that
can account for these reactions is:
2 FlavOH + H202+ 2 FlavO .f 2 H20 (1)
2 FlavO + 2 ASA -+ 2 FlavOH + 2 MDA (2)
MDA + MDA + ASA + DHA (3)
H202+ ASA + 2 H 2 0+ DHA (4)
where FlavOH is a flavonoid containing a free hydroxyl
group, FlavO. is a flavonoid phenoxyl radical, MDA is the
monodehydroascorbic acid radical, ASA is ascorbic acid,
and DHA is dehydroascorbic acid. Reaction 1 is catalyzed
by peroxidase, whereas reactions 2 and 3 proceed nonen-
zymically. If ascorbate is absent, polymerization products
of flavonoids, similar to the case of tannin formation, may
be irreversibly generated. The results shown in Figure 4
can be accounted for by the sum of reactions 1, 2, and 3,
namely, no apparent oxidation of flavonoids, as shown in
reaction 4. If ascorbate regeneration by the cytosolic DHA
reductase and glutathione reductase system is coupled
with reaction 4, it is possible that the vacuolar flavonoid-
peroxidase system could function as an H,O,-scavenging
mechanism, as previously proposed in broad bean (Taka-
hama, 1992).
A Role of Flavonoids in U V Tolerance
With respect to their proposed functions in leaves, fla-
vonoids are thought to be primarily involved in the pro-
tection against UV light. It has long been proposed that
flavonols act as interna1 UV-screening molecules for pro-
tecting photosynthetic tissues (Koes et al., 1994; Shirley,
1996). Analyses of mutants defective in flavonoid biosyn-
thesis have indicated the importance of flavonoids for UV
tolerance (Lois and Buchanan, 1994; Shirley, 1996). Re-
cently, however, Landry et al. (1995) have demonstrated
that a mutant of Arabidopsis thaliana defective in ferulic acid
hydroxylase Vah 1 ) is more susceptible to UV damage than
a chalcone isomerase-deficient mutant (tt 5). They suggest
that hydroxycinnamate derivatives are more important
than flavonoids for UV tolerance (Landry et al., 1995). Like
flavonols, hydroxycinnamate derivatives also have antioxi-
dative properties (Castelluccio et al., 1995) and act as elec-
tron donors to GuPX (Takahama, 1988a). UV light induces
 H,O,-Scavenging Function of Flavonoids 1411

A ---- ASA m

H20

hv

B c
V

Figure 6. A proposed diagram for the protective function of flavonoids during stress and growth. A, Scheme of the
H,O,-scavenging mechanism by flavonoids. vPX, Vacuolar peroxidase; F, flavonoid; F., flavonoid radical; ASA, ascorbic
acid; DHA, dehydroascorbic acid; hv, light energy; and cDHAR, cytosolic dehydroascorbic acid reductase. The diffusive
nature of H,O, enables vPX to scavenge it in vacuoles, even if the generating site is other than a vacuole (6).This concept
can be expanded to the cell-cell interaction. The photoproduced H,O, may leak out from mesophyll cells and be scavenged
in epidermal cells that have a high flavonoid content (C).

oxidative stress and activates the production of active ox-
ygen species, including free radicals (Mount, 1996). There-
fore, it is likely that polyphenolic "sunscreen" pigments
protect cells from UV damage by indirect means such as
H,Oz scavenging in addition to their absorption properties.
Infuh and tt mutants UV irradiation increases lipid peroxi-
dation and APX activity (Landry et al., 1995).This strongly
suggests that active oxygens participate in the mechanism
of UV-B-induced injury (Foyer et al., 1994).
Flavonoids as Stress Protectants
Flavonoids are known to be induced not only by expo-
sure to UV-B but also by various type of stresses (Dixon
and Paiva, 1995; Shirley, 1996). They often accumulate in
response to wounding, pathogen infection, high light, chill-
ing, ozone, or nutrient deficiency (Dixon and Paiva, 1995).
Also, they are often abundant during senescence or shoot
growth even under favorable conditions. These conditions
are prone to produce H,Oz in cells. Recently, we have
demonstrated that anthocyanins (cyanidin-3-sophoroside)
can scavenge excess H,O, in conjunction with peroxidase
(Yamasaki, 1997). These results suggest that flavonoids
may contribute to the overall mechanism for protecting
cells from oxidative damage in addition to their action as
optical filters (Gould et al., 1995).
Figure 6 is a schematic diagram of a proposed function of
flavonoids in the H,O,-scavenging system in cells. In most
plants vacuoles dominate the cell volume, and peroxidases
may be localized in the inner surface of tonoplast mem-
branes (Thomas and Jen, 1980). This spatial distribution
readily enables vacuolar peroxidase to scavenge H,O,
leaked out from other organelles (Fig. 6B). The concept of
delocalized scavenging of H,O, by vacuoles can be applied
not only to organelle-organelle interactions but also to the
cell-cell interaction. The epidermal cells usually contain
much higher concentrations of flavonoids than mesophyll
cells (Hrazdina et al., 1982). The H,O, leaked out from
mesophyll cells under light stress can, according to this
scheme, be scavenged by the flavonoid-peroxidase system
in epidermal cells (Fig. 6C). Consistent with this idea,
blackening of the epidermis after severe light stress is
frequently observed in many species in the field, a phe-
nomenon that has been ascribed to the polymerization of
vacuolar phenolics as the result of the penetration of H,O,
into epidermal cells. In Pinaceae species hydrophilic flavo-
no1 glycosides are found in the cell wall (Strack et al., 1988),
where GuPXs are also present (Takahama, 1993). The pos-
sibility that the apoplastic flavonoid-GuPX may participate
in H,O, scavenging (Takahama and Oniki, 1992) cannot be
excluded from Figure 6.
CONCLUDING REMARKS
Because the generation and scavenging of active oxygen
is usually a localized event, the mechanism proposed here
(Fig. 6) is unlikely to function as a primary detoxification
system. However, it will be important when cellular H,O,
levels are increased under conditions of high stress or rapid
growth, or when ascorbate availability is limited, such as in
1412 Yamasaki et al.
juvenile leaves (Fig. 5) or in ascorbate-deficient mutants
(Yamasaki et al., 1995a, 1995b; Conklin et al., 1996). It is
plausible that flavonoids support the primary detoxifica-
tion system as a backup defense mechanism of vascular
plants. The negative correlation between foliar flavonoid
and ascorbate content (Fig. 5) may reflect a decrease of the
requirement for the flavonoid antioxidant system during
development. This functional dispensability might allow
chemical modifications that would produce a wide variety
of structures and new specific functions. It should be em-
phasized that the antioxidative function is not a specific
feature of flavonoids, but is a general feature of plant
phenolics (Takahama, 1988a; Castelluccio et al., 1995).This
view has been largely overlooked in plant stress research.
Although further evidence is required to confirm the fla-
vonoids’ role in vivo, it is clear that the antioxidative
function must be taken into consideration to assess the
physiological roles of those molecules.
ACKNOWLEDCMENTS
We gratefully acknowledge Dr. Umeo Takahama (Kyushu Den-
tal College, Kitakyushu, Japan) for his helpful suggestions and
comments. We also thank Dr. Stephen Grace (The Australian
National University, Canberra) for his critica1 reading of the manu-
script.
Received May 12, 1997; accepted August 28, 1997.
Copyright Clearance Center: 0032-0889/97/115/1405/08.
LITERATURE CITED
Amako K, Chen G-X, Asada K (1994) Separate assays specific for
ascorbate peroxidase and guaiacol peroxidase and for the chlo-
roplastic and cytosolic isozymes of ascorbate peroxidase in
plants. Plant Cell Physiol 3 5 497-504
Asada K (1992) Ascorbate peroxidase: a hydrogen peroxide-
scavenging enzyme in plants. Physiol Plant 8 5 235-241
Bors W, Heller W, Michel C, Saran M (1990) Flavonoids as
antioxidants: determination of radical-scavenging efficiencies.
Methods Enzymol 1 8 6 343-355
Bors W, Michel C, Saran M (1994) Flavonoid antioxidants: rate
constants for reactions with oxygen radicals. Methods Enzymol
234: 420-429
Castelluccio C, Paganga G , Melikian N, Bolwell GP, Pridham J,
Sampson J, Rice-Evans C (1995) Antioxidant potential of inter-
mediates in phenylpropanoid metabolism in higher plants. FEBS
Lett 368 188-192
Charriere-Ladreix Y, Tissut M (1981) Foliar flavonoid distribution
during Spinacia chloroplast isolation. Planta 151: 309-313
Conklin PL, Williams EH, Last R (1996) Environmental stress
sensitivity of an ascorbic acid-deficient Arabidopsis mutant.
Proc Natl Acad Sci USA 93: 9970-9974
Dixon RA, Paiva N (1995) Stress-induced phenylpropanoid me-
tabolism. Plant Cell 7: 1085-1097
Foyer CH (1993) Ascorbic acid. In RG Alscher, JL Hess, eds, Anti-
oxidants in Higher Plants. CRC Press, Boca Raton, FL, pp 31-58
Foyer CH, Lelandais M, Kunert KJ (1994) Photooxidative stress in
plants. Physiol Plant 92: 696-717
Gould KS, Kuhn DN, Lee DW, Oberbauer ST (1995) Why leaves
are sometimes red. Nature 378: 241-242
Grace SC, Logan BA (1996) Acclimation of foliar antioxidant
systems to growth irradiance in three broad-leaved evergreen
species. Plant Physiol112 1631-1640
Hrazdina G, Marx GA, Hoch HC (1982) Distribution of secondary
plant metabolites and their biosynthetic enzymes in pea (Pisum
sativum L.) leaves. Plant Physiol 70: 745-748
Plant Physiol. Vol. 11 5, 1 9 9 7
Jan CY, Takahama U, Kimura M (1991) Inhibition of photooxida-
tion of a-tocopherol by quercetin in human blood cell mem-
branes in the presence of hematoporphyrin as a photosensitizer.
Biochim Biophys Acta 1086 7-14
Koes RE, Quattrocchio F, Mo1 JNM (1994) The flavonoid biosyn-
thetic pathway in plants: function and evolution. BioEssays 1 6
123-132
Landry LG, Chapple CCS, Last R (1995) Arabidopsis mutant
lacking phenolic sunscreens exhibits cnhanced ultraviolet-B in-
jury and oxidative damage. Plant Physiol 109: 1159-1166
Lois R, Buchanan BB (1994) Severe sensitivity to ultraviolet radi-
ation in an Arubidopsis mutant deficient in flavonoid accumula-
tion 11. Mechanisms of UV resistance in Arabidopsis. Planta 194:
504-509
Markham KR (1993) Flavones, flavonols and their glycosides. In J
Harborne, ed, Methods in Plant Biochemistry, Vol 1. Academic
Press, San Diego, CAPpp 197-235
Mehlhorn H, Lelandais M, Korth HG, Foyer CH (1996) Ascorbate
is the natural substrate for plant peroxidase. FEBS Lett 378
203-206
Miller E, Schreier P (1985) Studies on flavonol degradation by
peroxidase (donor: H,O,-oxidoreductase, EC 1.11.1.7). Part 1:
kaempferol. Food Chem 17: 143-154
Mount DW (1996) Reprogramming transcription. Nature 383:
763-764
Schreier P, Miller E (1985) Studies on flavonol degradation by
peroxidase (donor: H,O,-oxidoreductase, EC 1.11.1.7). Part 2:
quercetin. Food Chem 18: 301-317
Shirley BW (1996) Flavonoid biosynthesis: ’new’ functions for an
’old’ pathway. Trends Plant Sci 1: 377-382
Stafford HA (1994) Anthocyanins and betalains: evolution of mu-
tually exclusive pathways. Plant Sci 101: 91-98
Strack D, Heilemann J, Momken M, Wray V (1988) Cell wall-
conjugated phenolics from Coniferae leaves. Phytochemistry 27:
3517-3521
Takahama U (1986) Spectrophotometric study on the oxidation of
rutin by horseradish peroxidase and characteristics of the oxi-
*
dized products. Biochim Biophys Acta 882 445451
Takahama U (1988a) Hydrogen peroxide-dependent oxidation of
flavonoids and hydroxycinnamic acid derivatives in epidermal
and guard cells of Tradescuntia virginiana L. Plant Cell Physiol29:
475481
Takahama U (198813)Oxidation of flavonols by hydrogen peroxide
in epidermal and guard cells of Vicia fuba L. Plant Cell Physiol
29: 433-438
Takahama U (1989) A role of hydrogen peroxide in the metabo-
lism of phenolics in mesophyll cells of Vicia fubu L. Plant Cell
Physiol 30: 295-301
Takahama U (1992) Hydrogen peroxide scavenging system in
vacuoles of mesophyll cells of Vicia fuba. Phytochemistry 31:
1127-1133
Takahama U (1993) Regulation of peroxidase-dependent oxidation
of phenolics by ascorbic acid: different effects of ascorbic acid on
the oxidation of coniferyl alcohol by the apoplastic soluble and
cell wall-bound peroxidases from epicotyls of V i g n u uizguluris.
Plant Cell Physiol. 34: 809-817
Takahama U, Egashira T (1991) Peroxidase in vacuoles of Vicia
faba leaves. Phytochemistry 30: 73-77
Takahama U, Oniki T (1992) Regulation of peroxidase-dependent
oxidation of phenolics in the apoplast of spinach leaves by
ascorbate. Plant Cell Physiol 33: 379-387
Thomas RL, Jen JJ (1980) The cytochemical localization of perox-
idase in tomato fruit cells. J Food Biochem 4: 247-259
Yamasaki H (1997) A function of colour. Trends Plant Sci 2: 7-8
Yamasaki H, Heshiki R, Ikehara N (1995a) Leaf-goldening in-
duced by high light in Ficus microcurpu L. f., a tropical fig. J Plant
Res 108: 171-180
Yamasaki H, Heshiki R, Yamasu T, Sakihama Y, Ikehara N
(199513) Physiological significance of the ascorbate regenerating
system for the high-light tolerance of chloroplasts. In P Mathis,
ed, Photosynthesis: From Light to Biosphere, Vol IV. Kluwer
Academic Publishers, Dordrecht, The Netherlands, pp 291-294
Yamasaki H, Uefuji H, Sakihama Y (1996) Bleaching of the red
anthocyanin induced by superoxide radical. Arch Biochem Bio-
phys 332: 183-186