J. Japan. Soc. Hort. Sci. 63 (2) : 439-444. 1994.
Effectiveness of Various Phenolic Compounds in Degradation of Chlorophyll
by In Vitro Peroxidase-Hydrogen Peroxide System1
Naoki Yamauchi and Alley E. Watada
"Himeji College of Hyogo, Shinzaike-honcho, Himeji, Hyogo 670
Horticultural Crops Quality Laboratory, Beltsville Agricultural Research Center,
Agricultural Research Service, USDA, MD 20705-2350 U.S.A.
Summary
The effectiveness of ethanol extracts from different leafy vegetables and commercial
phenolic compounds in degrading chlorophyll (Chi) in a model system using commercial
horseradish peroxidase was determined in vitro.
The amount of Chi degradation ranged widely among the leafy vegetables with parsley
and mitsuba ethanol extracts having 70 and 60 fold greater activity than that of the garland chrysanthemum extract respectively, which were the extremes among the eight leafy
vegetables.
Among sixteen commercial phenolic compounds studied, Chi degradation occurred only
with apigenin (flavone), apigetrin (apigenin-7-glucoside), naringenin (flavanone), p-coumaric acid (monophenol) and resorcinol (m-diphenol). These results indicate that Chi degradation in the peroxidase-hydrogen peroxide system is dependent on the type of phenol,
specifically those which have a hydroxy group at the p-position.
The only degraded Chi product noted in the HPLC chromatogram, when using pcoumaric acid, was 10-hydroxychlorophyll a (Chi a-1). The amount of Chi a-1 accumulated
was minimal, compared to the amount Chi degraded, and implies that most of the Chi was
degraded to a colorless product without the accumulation of Chi a-1 as an intermediate.
Introduction
Degradation of chlorophylls (Chls) is a regulated
process and various enzymes catalyzing the reactions have been identified. Three of these enzymes
are chlorophyllase (Holden, 1961), Chi oxidase
(Liithy et al., 1984), and peroxidase (Huff, 1982).
Chlorophyllase catalyzes the removal of phytol
from Chi to form chlorophyllide. Chi oxidase and
peroxidase degrade Chi indirectly by forming a
product that oxidizes Chi. In the Chi oxidase, Chi
a is oxidized to 10-hydroxychlorophyll a (Chi a-1)
with the oxidation of unsaturated or saturated fatty acids such as linolenic acid, linoleic acid and
stearic acid (Martinoia et al.. 1982; Schoch et al.,
1984). The involvement of a peroxidase-hydrogen
peroxide system in the mechanism of Chi degrada
tion, is poorly understood in vivo.
Received for publication 27 April 1993.
'Article No. VIII of Mechanism of chlorophyll degradation in harvested leafy vegetables.
The peroxidase-hydrogen peroxide system requires hydrogen peroxide and a phenolic compound for degradation of Chi (Huff, 1982; Kato
and Shimizu, 1985). Huff (1982) reported that
phenol, 2,4-dichlorophenol and resorcinol were
effective in degrading Chi and that the latter was
the most effective among the three compounds.
, Kato and Shimizu (1985) demonstrated 2,4-dichlorophenol, p-coumaric acid, phenol, p-hydroxybenzoic acid, p-hydroxyacetophenone, resorcinol and
umbelliferone, to be effective in the peroxidasehydrogen peroxide system, and indicate that most
of these are phenolic compounds with electron
attracting groups at the p-position.
In parsley, Chls were degraded by the
peroxidase-hydrogen peroxide model system which
required apigenin, a major flavone in the parsley
leaves, to catalyze the reaction (Yamauchi and
Minamide, 1985). We had shown that degradation
of spinach Chi appeared to be regulated by the
peroxidase-hydrogen peroxide system (Yamauchi
139
440
N. Yamauchi and A. E. Watada
and Watada, 1991), but the phenolic compound required for the reaction is not known. The phenolic
compound in spinach and other leafy vegetables is
suspected to be flavonoid pigments like an
apigenin, but this needs to be confirmed.
In this study, we examined the effectiveness of
phenolic compounds in the model system using
commercial horseradish peroxidase for degrading
Chi and determined the degradative product of Chi.
Materials and Methods
Ethanol extracts were obtained from mature
leaves of parsley (Petroselium crispum Nym. cv Paramaunto), spinach (Spinacia oleracea L. cv Atorasu), garland chrysanthemum (Chrysanthemum coronarium L. cv Banchu-kabuhari), mitsuba (Cryptotaenia japonica Hassk. cv Senkaku), Welsh onion
(Allium fistulosum L.), gynmigit (Allium tuberosum
Rottl.), chingensai (Brassica rapa var. chinensis) and
Osaka-shirona (Brassica rapa var. chinensis).
Extraction and purification of chlorophyll
Chls a and b were extracted and purified by the
method of Yoshiura and Iriyama (1979) with the
following modification. Pigments including Chls,
were extracted from spinach with cold acetone.
Three ml of 1,4-dioxane and 5 ml of distilled water were added to 25 ml of the extract; the mixture was allowed to stand at 5°C until a precipitate formed and then centrifuged at 12,000g for
10 min. The pellet, containing the Chls and a
small amount of carotenoids. was dissolved in
about 7 ml of ethanol. Aliquots of the ethanol solution was applied on a silica gel TLC plate and Chi
a was separated by developing with n-pentane :
acetone : t-butyl alcohol (90:5:5, v / v / v ) solvent
mixture. Chi a fraction was dissolved in ethanol
(150 / / g C h l a/ml).
CM degradation reaction
Chi degradation was determined as described by
Yamauchi and Minamide (1985). The reaction mixture contained 0.2 ml of Chi a ethanol solution (30
//g/0.2 ml), 0.4 ml of 70% ethanol extract from
leafy vegetable, 0.1 ml of 1% Triton X-100, 0.1
ml of 0.3% hydrogen peroxide, 4 units horseradish
peroxidase (20 jug, Sigma Chemical Type II), and
64 mM phosphate buffer (pH 6.0) in a total
volume of 2.5 ml. The reaction was allowed to
proceed for 5 min at 25°C and then stopped by
the addition of 2.5 ml ethanol and 5.0 ml hexane.
The hexane layer contained Chls that were not degraded, and its absorbance at 663 nm was subtracted from the blank (distilled water instead of
hydrogen peroxide) to calculate the amount of degraded Chi.
HPLC analysis of chlorophylls and their derivatives
Apparatus for HPLC analysis of Chls and their
derivatives consisted of Waters Model 6000
pumps with automated gradient controller and
Model 712 WISP interfaced into a Hewlett-Packard 1040A rapid-scanning UV-visible photodiode
array detector. The absorption spectra of the pigments were recorded between 200 and 600 nm at
the rate of 12 spectra/min. Pigments were separated by a Beckman Cis ultrasphere column, 4.6X
250 mm using two solvents "A". methanol:water
(80:20), and "B", ethyl acetate in a gradient. "A"
was added to "B" at a linear rate for a 20 min
period until a 50:50 mixture was attained at the
end of a 20 min period and the 50:50 mixture was
then used isocratically for an additional 25 min as
described by Eskin and Harris (1981). The flow
rate was 1 ml/min and injection volume was 200
f t l . Data from the photodiode array detector were
stored and processed by means of a Hewlett-Packard 9000/series 300 computing system which
was operated with a Hewlett-Packard Model 9153
disc drive, color display monitor 35741. Identifications of Chls and their derivatives were based
on retention time and by the visible absorption
spectra as described in the previous paper
(Yamauchi and Watada. 1991).
Results and Discussion
Chlorophyll degradation by peroxidase with phenolic
compounds
The amount of Chi that was degraded in the
peroxidase-hydrogen peroxide system differed extensively with the different sources of ethanol extracts used in the reaction media (Table 1). At the
high end, 181.7 and 152.9 /_tg Chi a were degraded when using ethanol extracts from parsley
and mitsuba, respectively. At the other extreme,
only 2.5 and 4.8 /ug Chi a were degraded when
using ethanol extracts from garland chrysanthemum and Osaka-shirona, respectively.
The differences noted among the leafy vege-
441
J. Japan. Soc. Hort. Sci. 63(2) : 439-444. 1994.
tables may be due to the form and amount of phenolic compouds present in the ethanol extract,
which probably differ in the effectiveness in the
Table 1. Chlorophyll degradation by using ethanol extracts from various leafy vegetables as substrate in
the peroxidase-hydrogen peroxide system.
Chlorophyll a degradation
Cug/g FW of leaves)
Leafy vegetables
Parsley
Mitsuba
Spinach
Welsh onion
Gynmigit
Chingensai
Osaka-shirona
Garland chrysanthemum
181.7
152.9
83.6
53.6
32.4
OO Q
^».y
4.8
2.5
reaction. In our earlier study, apigenin was found
to be the active phenolic compound in the ethanol
extract of parsley leaves for degrading Chi in the
model system using commercial horseradish peroxidase (Yamauchi and Minamide, 1985). Apigenin is
a major flavone in parsley. The form of phenolic
compounds present in leafy vegetables analyzed in
this study, except for parsley, is not known.
We determined the effectiveness of 16 different
commercial phenolic compounds in catalyzing the
peroxidase-hydrogen peroxide system to degrade
Chi (Table 2). Of these, only five degraded Chi,
with naringenin being the most effective. Naringenin degraded 72% of the Chi, apigetrin (apigenin7-glucoside) degraded 40%, and p-coumaric acid,
resorcinol and apigenin degraded 24 to 29%. Interestingly, apigetrin, glycoside of apigenin, was
Table 2. Chlorophyll degradation by using various phenolic compounds as substrate
in the peroxidase-hydrogen peroxide system.
Phenolic compounds (20 ,uM)
Monophenols
p-Coumaric acid
Tyrosine
o-Diphenols and their derivatives
c-( + )-Catechin
Dihydroxyphenyl-L-alanine (Dopa)
Caffeic acid
Chlorogenic acid
Ferulic acid
m-Diphenol
Resorcinol
Flavonoids
Flavones
Apigenin
Apigetrin (Apigenin-7-glucoside)
Chrysin
Flavonols
Kaempferol
Quercentin
Myricetin
Flavanone
Naringenin
Other
Gallic acid
Chlorophyll a degradation (%)
25
0
0
0
0
0
2
24
29
40
0
0
0
0
72
0
The reaction mixture contained 0.2 ml of chlorophyll a ethanol solution (30 ^g/0.2 m l ) ,
50 fj.1 of ethanol solution of phenolic compound. 0.1 ml o(l% Triton X-100, 0.1 ml of
0.3% hydrogen peroxide. 4 units peroxidase (20 /ug) and 80 mM phosphate buffer
( p H 6 . 0 ) in a total volume of 2.5ml.
442
N. Yamauchi and A. E. Watada
almost twice as effective as apigenin. Not any of
the o-diphenols, which included catechin, dopa,
caffeic acid, chlorogenic acid and ferulic acid degraded Chi.
Kato and Shimizu (1985) reported that Chi degradation by the peroxidase-hydrogen peroxide
system can proceed in the presence of many different kinds of phenolic compounds and that most of
them were phenols with electron attracting group
at the p-position. We found in this study that
apigenin (flavone) and its glycoside, naringenin
(flavanone) and p-coumaric acid (monophenol) were
effective in degrading Chi in the peroxidase-hydrogen peroxide system, whereas the o-diphenols
and flavonols were not effective (Table 2). In Satsuma mandarin fruits, flavonoid aglycones such
as hesperetin (flavanone), naringenin and apigenin,
degraded Chi, but naringenin and apigenin, which
have a hydroxyl group at the p-position, were the
most effective of the three flavonoids (Yamauchi
and Hashinaga, 1992). These results suggest that
the phenolic compounds such as p-coumaric acid,
apigenin, apigetrin and naringenin, which have a
hydroxyl group at the p-position, could be involved effectively in Chi degradation by the
peroxidase-hydrogen peroxide system, and that odiphenols are not involved. Apparently not all
phenols are able to degrade Chi in this sytem and
the effectiveness appears to depend on the molecular configuration. In addition, flavonoid glycoside
like apigetrin as well as its aglycone also was
effective in the Chi degradation by the peroxidasehydrogen peroxide system. It is well known that
flavonoids most frequently occur in plants bound
to sugar as glycosides (Harborne, 1973). These results suggest that the flavonoid glycosides could
be involved in the Chi degradation by the
peroxidase-hydrogen peroxide system in plants.
Chlorophyll degradation products
Chls and their degradative products in the
peroxidase-hydrogen peroxide system containing
p-coumaric acid, which is almost ubiquitous in
plants (Harborne, 1973). were determined with
HPLC (Fig. 1). Identification of all pigments including Chi a-1 was based on the retention time on
HPLC chromatogram and by the visible absorption
spectrum as described elsewhere (Yamauchi and
Watada, 1991). Quantitatively, Chi a decreased by
about 70%, Chi b decreased slightly and Chi a-1
oa
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REACTION TIME
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20
30
40
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60
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Fig. 1. HPLC chromatograms chlorophyll (Chi) a, a-1 and b after 0, 5 and 10-min reaction
time of Chls degraded in the peroxidase-hydrogen peroxide system. For the
peroxidase-hydrogen peroxide system, Chls (30 f i g Chi a. 11 to 12 ^g Chi b) in 0.1
ml ethanol were incubated with 50 n\ of 1% Triton X~100, 50 fj.\ ethanol solution of
1.25nM p coumaric acid, 50 fil of 0.3% hydrogen peroxide, 2 units peroxidase (10 /u
g) and 72 mM phosphate buffer. pH 6.0, in a volume of 1.25 ml for 10 min at 25°C;
stopping the peroxidase action by adding 5 ml cold acetone. The resulting reaction
mixture was filtered through a Millipore filter (0.22 ^m pore) and analyzed with a
HPLC. The reaction mixture contained 12 % of ethanol.
J. Japan. Soc. Hort. Sci. 63(2) : 439-444. 1994.
accumulated slightly after 10 min reaction (Figs.
1, 2). However, the amount of Chi a-1 that accumulated accounted for only a small amount of the degraded Chi.
A small amount of another Chi derivative,
showed as "unknown" on the chromatogram, .was
noted after 5 and 10 min reaction. The visible
absorption spectrum of the unknown was similar
to that of Chi a (data not shown).
The slight accumulation of Chi a-1 noted after 5
min reaction accounts for only a small amount of
Chi that was degraded. Chi a-1 is the product of
reaction catalyzed by Chi oxidase (Schoch et al.,
1984) and lipoxygenase (Yamauchi and Watada,
1989) and has been found to increase with senescence of excised barley and bean leaves (Maunders
et al., 1983). These results indicate that Chi a-1 is
an intermediate in a minor reaction associated
with the peroxidase-hydrogen peroxide system.
Apparently most of the Chi is degraded directly to
a colorless compounds and/or is degraded initially
to Chi a-1 by the system and subsequently is degraded immediately to a colorless compounds.
Chls and their derivatives with intact porphyrin
rings are green or brownish-green, whereas those
suspected of having open rings have pink pigments
or lipofuscin-like compounds, as noted with senescing leaves of fescue grass and barley (Matile et al.,
1987; Dugelin et al., 1988). In the peroxidase-hydrogen peroxide system, the Chi catabolite, as yet
accounted for, may have an open porphyrin ring.
Further studies are needed to answer this question.
Saunders and McClure (1976) reported that many
3000
z
Chi a
Chi a-1
B5 2000
X
K
^
<
1000
^
-
0
5
10
REACTION TIME (min)
Fig. 2. Changes in contents of chlorophyll a and a-1
in the peroxidase-hydrogen peroxide system.
Chl-Chlorophyll
0
443
kinds of flavonoid are present in the chloroplast of
many species of vascular plants. Peroxidase activity was also found in the chloroplasts of spinach
leaves (Takahama, 1984), cucumber cotyledons
(Abeles et al., 1989) and barley leaves (Kuroda et
al., 1990). Huff (1982) demonstrated that Chi de
rivatives, chlorophyllide and pheophytin, as well as
Chi were degraded by the peroxidase hydrogen peroxide system, inferring that Chls and their derivatives could be degraded by the system in the presence of the phenolic compounds, which have a hydroxyl group at the p-position, in the chloroplast.
Acknowledgement
We graciously thank Mr. Willard Douglas for
his excellent technical assistance.
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