Stimulatory effect of cyanidin 3-glycosides on the regeneration of rhodopsin

Article abstract of DOI:10.1021/jf034132y

Anthocyanins have been suggested to improve visual functions. This study examined the effect of four anthocyanins in black currant fruits on the regeneration of rhodopsin using frog rod outer segment (ROS) membranes. Cyanidin 3-glycosides, glucoside and rutinoside, stimulated the regeneration, but the corresponding delphinidins showed no significant effect. The formation of a regeneration intermediate was suggested to be accelerated by cyanidin 3-rutinoside. Their effects on the cGMP-phosphodiesterase activity in the ROS membranes were also investigated but found to be negligible. It was concluded that the major effect of anthocyanins in rod photoreceptors is on the regeneration of rhodopsin.

Full text of DOI:10.1021/jf034132y

3560 J. Agric. Food Chem. 2003, 51, 3560−3563
Stimulatory Effect of Cyanidin 3-Glycosides on the
Regeneration of Rhodopsin
,
†
†
‡
HITOSHI MATSUMOTO,* YUKO NAKAMURA, SHUJI TACHIBANAKI,
SATORU KAWAMURA,‡ AND MASAO HIRAYAMA†
Health and Bioscience Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1 Chiyoda,
Sakado-shi, Saitama 350-0289, Japan, and Department of Biology, Graduate School of Science,
Osaka University, Toyonaka 560-0043, Japan
Anthocyanins have been suggested to improve visual functions. This study examined the effect of
four anthocyanins in black currant fruits on the regeneration of rhodopsin using frog rod outer segment
(ROS) membranes. Cyanidin 3-glycosides, glucoside and rutinoside, stimulated the regeneration,
but the corresponding delphinidins showed no significant effect. The formation of a regeneration
intermediate was suggested to be accelerated by cyanidin 3-rutinoside. Their effects on the cGMP-
phosphodiesterase activity in the ROS membranes were also investigated but found to be negligible.
It was concluded that the major effect of anthocyanins in rod photoreceptors is on the regeneration
of rhodopsin.
KEYWORDS: Rhodopsin; regeneration; 11-cis-retinal; anthocyanin; black currant
INTRODUCTION
Rhodopsin, which is a member of the G-protein-coupled
receptor family, is localized on the lipid bilayers of disks
laminated in the retinal rod outer segment (ROS). On light
absorption, 11-cis-retinal, the chromophore of rhodopsin, is
isomerized to all-trans-retinal (1). Rhodopsin consequently
undergoes further conformational changes through a number
of intermediates and triggers a series of reactions in the
phototransduction cascade (2-4). One of the intermediates is
metarhodopsin II, which activates >500 transducin molecules
per second to amplify the light signal (5, 6). Transducin then
activates cGMP phosphodiesterase (PDE) to hydrolyze cGMP.
Because cGMP opens a cGMP-gated cation channel, hydrolysis
of cGMP by PDE induces the closure of the channel to
hyperpolarize the cell. All of the phototransduction components
recover to the original inactive state after light. Light-activated
rhodopsin is inactivated by phosphorylation, subsequently
dephosphorylated, and finally decomposed to all-trans-retinal
plus opsin, the protein moiety of rhodopsin. Rhodopsin is then
regenerated by binding of 11-cis-retinal to opsin.
Figure 1. Structure of the four anthocyanins in black currant.
suggest the effect of anthocyanins on the visual system, the
detailed site of the action is not known. In addition, the
molecular species responsible for the effects are not known.
Because the crude extract contains chemicals other than antho-
cyanins and there are 15 species of anthocyanins, it has been
difficult to identify the active species.
Black currant contains four anthocyanins (Figure 1): del-
phinidin 3-rutinoside (D3R), delphinidin 3-glucoside (D3G),
cyanidin 3-rutinoside (C3R), and cyanidin 3-glucoside (C3G).
In our previous studies, we successfully isolated and purified
the four anthocyanins from black currant as crystals (11) and
showed that they, as well as those from bilberry, have an
improving effect on dark adaptation in humans (12).
In the present study, using the four forms of the black currant
anthocyanins, first we examined the effect of anthocyanins on
the PDE activation. Second, we examined their effects on the
regeneration of rhodopsin.
Anthocyanins have been implicated to improve visual func-
tions. Cyanidin, an aglycon of anthocyanin, has been suggested
to have effects on the phototransduction cascade by activating
(7) or inhibiting (8) PDE. Anthocyanins are also suggested to
have effects on the regeneration of rhodopsin. A mixture of
anthocyanins extracted from bilberry is known to improve night
vision (9). The regeneration of rhodopsin has also been shown
to be stimulated by bilberry extract (10). Although these studies
*
Author to whom correspondence should be addressed (telephone 81-
MATERIALS AND METHODS
4
92-84-7591; fax 81-492-84-7598; e-mail hitoshi_matsumoto@meiji.co.jp.
†
Reagents. Four crystalline components of black currant, D3R, D3G,
C3R, and C3G, were purified according to the methods described
Meiji Seika Kaisha, Ltd.
Osaka University.
‡
1
0.1021/jf034132y CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/10/2003

Anthocyanin Stimulates the Regeneration of Rhodopsin
J. Agric. Food Chem., Vol. 51, No. 12, 2003 3561
previously (11). A standard buffer used in the following studies
contained 115 mM potassium gluconate, 2.5 mM KCl, 2 mM MgCl
2
,
1
mM dithiothreitol, 0.1 mM CaCl , 0.2 mM EGTA, and 10 mM
2
HEPES, pH 7.5 (K-gluconate buffer). Other chemicals were of reagent
grade.
Preparation of 11-cis-Retinal. Following the method of Shichida
et al. (13), 11-cis-retinal was prepared with several modifications.
Briefly, all-trans-retinal (Sigma) dissolved in acetonitrile (2 mg/mL)
was isomerized by fluorescent irradiation at 4 °C for >24 h. The
irradiated sample (500 µl) was loaded onto a normal-phase column
(
Supelcosil LC-Si, 250 × 21.2 mm, 5 µm, Supelco, Inc.) and eluted
with diethyl ether/n-hexane (15:85) at 10 mL/min. Absorbance at 254
nm was monitored. Referring to the retention time of 9,13-di-cis-retinal
(Sigma) as a standard (14), the eluate at 12.3 min was collected.
Measurement of the UV-vis absorption spectrum of this fraction
revealed two characteristic absorption maxima at 250 and 376 nm,
which are specific to 11-cis-retinal. The purification was repeated to
obtain a sufficient quantity of 11-cis-retinal. After the purification, the
solvent was removed by evaporation, and the sample was dissolved in
ethanol and stored at -80 °C until use. To avoid photoisomerization
of the retinal sample, the manipulations were performed in the dark.
The concentration of 11-cis-retinal was calculated on the basis of the
molar extinction coefficient at 376 nm (ꢀ ) 25000).
Figure 2. Effect of anthocyanins on PDE activation at low Ca2+
concentrations. PDE activities in the dark (a) and those elicited by a weak
light flash (with an ND 3.0 filter) (b) and by a saturating light (c) were
measured in the absence and presence of each of the four anthocyanins
(10 µM each). Dark activities are included in the results shown in (b) and
(c). The enzyme activity is expressed as a percentage of the maximum
activity of the control, which did not include the anthocyanins. Values are
the mean of four experiments, and vertical bars represent SEM.
Preparation of Frog ROS Membranes and Opsin Membranes.
Preparation of frog ROS was carried out under infrared light using an
infrared image converter (NVR 2015, NEC) (15). Frogs were dark-
adapted for at least 3 h and then sacrificed by pith. After the eyeballs
had been excised, the retinas were detached in the K-gluconate buffer
and the ROS was brushed off the retinas into the buffer. The ROS was
fragmented by passage through a syringe needle and suspended in the
K-gluconate buffer (ROS membranes).
RESULTS AND DISCUSSION
Effect of Anthocyanins on PDE Activity. In previous
publications, the effects of anthocyanins on ROS PDE were
controversial (7, 8). Therefore, first we examined the effect of
the four purified black currant anthocyanins on PDE. The PDE
activities elicited by both a weak light flash and a saturating
light were determined, and the relative activating efficiency by
a weak light flash was calculated (Figure 2). The result showed
that although the anthocyanins examined reduced slightly both
the maximum PDE activity (Figure 2c) and the relative
activating efficiency (Figure 2b), their effects were not so
significant.
The opsin membranes used for the regeneration of rhodopsin were
prepared from the ROS membranes. A 1 M NH OH solution (60 µL)
2
was added to 1.2 mL of the ROS membranes, and rhodopsin was
completely bleached on ice by white-light irradiation. To remove retinal-
oxime, the bleached membranes were washed three times by ultracen-
trifugation (100000g) at 4 °C for 30 min. The final precipitate was
then suspended in the K-gluconate buffer so that the opsin concentration
was 5 µM (opsin membranes). The opsin membranes thus obtained
were immediately frozen on liquid nitrogen and stored at -80 °C until
use.
2+
Because PDE activation is regulated by S-modulin in a Ca -
dependent manner (17), we then examined the effects of the
four anthocyanins on the PDE activity in the presence of
2
+
PDE Activity Measurement. Measurement of the PDE activity was
carried out according to the method described previously (15). Briefly,
a 200 µL portion of the ROS membranes was mixed with each of
S-modulin at a high (10 µM) Ca concentration. In agreement
with previous studies (18), S-modulin increased the efficiency
of PDE activation elicited by a weak light flash (compare the
difference in the PDE activities between low and high Ca
controls in Figure 3a) and prolonged the time required for 50%
recovery of the PDE activation (Figure 3b). The four antho-
cyanins slightly reduced the efficiency of the PDE activation
caused by a weak light flash (Figure 3a), but the maximum
PDE activity in addition to the dark PDE activity (not shown)
and the time required for 50% recovery (Figure 3b) were
indistinguishable from those of the high Ca controls. From
these results, we concluded that there might be a small effect
of the four anthocyanins on the PDE activation, but the effect
is very small even at concentrations of 10-50 µM. Because
anthocyanin concentrations are much lower in the plasma (19),
the in vivo effect of anthocyanins on PDE is negligible.
anthocyanin (final concentration ) 10-50 µM), ATP (0.1 mM), GTP
+
(
0.1 mM), and cGMP (6 mM) at a low (1 nM) or high (10 µM) Ca2
concentration. Rhodopsin concentration was 2-7 µM. When necessary,
S-modulin (10 µM) was added to the membranes. Measurements were
initiated by giving a weak light flash, and the pH decrease accompanied
by hydrolysis of cGMP was monitored with a micro pH electrode (MI-
4
10, Microelectrodes). After the pH decrease induced by a weak light
flash had been recorded, a strong light was given to measure the
maximum PDE activity. The data were digitized and stored in a hard
disk and analyzed by Chart software (ADI Instruments).
2
+
Regeneration of Rhodopsin. Rhodopsin was regenerated by incuba-
tion of purified 11-cis-retinal with the opsin membranes. The reaction
was initiated by the addition of 10 µL of 2 mM 11-cis-retinal to 1 mL
of the opsin membranes at 4 °C. After incubation, the reaction was
terminated by adding an equivolume of 40 mM cetyltrimethylammmo-
The above finding contrasts with the previous studies that
2
nium bromide solution containing 20 mM NH OH. The amount of
10 µM concentrations of anthocyanidins (aglycon of anthocya-
rhodopsin regenerated was determined by measuring the difference of
the absorbance at 500 nm before and after bleach. All of the
manipulations were carried out under infrared light. When necessary,
a 10 µL solution containing 2 mM anthocyanin was added to 1 mL of
the opsin membranes before the addition of 11-cis-retinal. Anthocyanins
are known to degrade at high pH (16), but its effect was negligible in
this study, which was carried out at 4 °C throughout. The absorption
spectrum of an anthocyanin solution did not change significantly (<5%)
for the period when the regeneration study was conducted (1 h).
nin) activate (7) or inhibit (8) this enzyme. One of the reasons
for this inconsistency may possibly be due to the difference in
the presence (this study) and absence (previous studies) of
saccharide modifications on anthocyanins. It is noteworthy that
anthocyanins in plasma and in any organs are usually glyco-
sylated. Another reason might be due to the difference in the
light condition used in the measurements. We measured the PDE
activity in real time after activation by a light flash. Previous

3562 J. Agric. Food Chem., Vol. 51, No. 12, 2003
Matsumoto et al.
Figure 3. Effect of anthocyanins on the sensitivity (a) and lifetime (b) of
PDE at high Ca2+ concentrations. PDE activities were measured in the
presence of S-modulin (10 µM) at a high Ca2 concentration (10 µM).
Control measurements were done in the absence of the anthocyanins
but in the presence of S-modulin at high (10 µM) and low (1 nM) Ca2
concentrations. Dark activities were subtracted to show the net effect of
the light. Values are the mean of two experiments, and vertical bars
represent the range of the variation.
Figure 5. Effect of C3R on the initial velocity of rhodopsin regeneration
at various concentrations of 11-cis-retinal. Initial velocity of rhodopsin
regeneration was determined for 40 s in the presence (O) and absence
(b) of C3R at various concentrations of 11-cis-retinal. The data points
were fitted with eq 2 to determine the rate constants in the regeneration
reaction (see text). Values are the mean of six experiments, and vertical
bars represent SEM.
+
+
Table 1. Kinetic Parameters in the Regeneration of Rhodopsin
controla
+ C3Ra
k2 (s-1)
Km (M)
1.0 × 10
-2
1.1 × 10
1.1 × 10
-2
-5
-
5
2.6 × 10
a
R 2 ) 0.989 (control); 0.995 (+ C3R).
The reaction scheme of the regeneration can be assumed as
in (20)
k1
k2
1
1-cis-retinal + opsin { } INT
98 rhodopsin
(1)
k-1
where k1, k-1, and k2 are the rate constants and INT is a
regeneration intermediate.
The solution of the above reaction under the condition of
excess 11-cis-retinal is
Figure 4. Effect of anthocyanins on the time course of rhodopsin
regeneration, which was measured in the absence and presence of each
of the four anthocyanins: (b) control; (]) D3G; ([) D3R; (4) C3G; (2)
C3R. The data points were fitted to an exponential function of 1 − exp-
Vobsd ) k [opsin][11-cis-retinal]/(K + [11-cis-retinal]) (2)
2
m
2
2
(
-t/τ): control, τ ) 114 s (R ) 0.997); D3G, τ ) 122 s (R ) 0.988);
where Vobsd is the observed velocity of the regeneration of
rhodopsin at a certain concentration of 11-cis-retinal and Km )
(k-1 + k2)/k1.
2
2
D3R, τ ) 119 s (R ) 0.993); C3G, τ ) 94 s (R ) 0.994); C3R, τ
)
2
89 s (R ) 0.994). Values are the mean of nine experiments, and
vertical bars represent SEM.
Equation 2 reveals that the observed velocity of the regenera-
tion is dependent on the concentration of 11-cis-retinal, and its
dependency is determined by k , k , and k . To estimate the
studies were done at several minutes after full bleaching of
rhodopsin, so this difference in the preparation may explain the
difference in the results.
Effect of Anthocyanins on Rhodopsin Regeneration.
Regeneration of rhodopsin was measured for 10 min after the
addition of 11-cis-retinal with or without purified anthocyanins.
Figure 4 shows the time course of the regeneration without
1
-1
2
values of these rate constants, we measured the initial velocities
of the regeneration of rhodopsin to determine Vobsd as a function
of the concentration of 11-cis-retinal. The measurement was
performed also in the presence of C3R, the major cyanidin
glycoside in black currant (Figure 5). We fitted the result with
eq 2 to obtain the values of k2 and Km in the presence and
absence of C3R (solid lines in Figure 5).
(control, b) or with anthocyanins (D3G, ]; D3R, [; C3G, 4;
C3R, 2). The rhodopsin regeneration was fitted to an expo-
nential function (solid lines). Figure 4 shows that D3G and
D3R (diamonds) showed no significant effects on rhodopsin
regeneration but that C3G and C3R (triangles) stimulated the
regeneration of rhodopsin. The result clearly demonstrated that
the cyanidin form of anthocyanin has the activity to accelerate
the regeneration of rhodopsin.
The result (Table 1) showed that C3R did not affect k2, but
instead decreased the Km value by 2.4-fold from that of the
control. The decrease in the Km value can be accounted for by
an increase in k1 or a decrease in k-1. Because of the scatter of
the data, it was difficult to determine which was the case.
However, it is evident that the production of a regeneration
intermediate (INT) is facilitated in the presence of C3R.

Anthocyanin Stimulates the Regeneration of Rhodopsin
J. Agric. Food Chem., Vol. 51, No. 12, 2003 3563
Effect of Anthocyanins on Dark Adaptation. Anthocyanins
first gained interest because they possess an improving effect
on visual acuity (21). Since then, several studies of anthocyanins
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1
17-151.
The improvement of dark adaptation (9) can be accounted for
by at least two reasons. Because dark adaptation accompanies
the increase in the light sensitivity of a rod, one possibility is
that there might be an effect of anthocyanins on the phototrans-
duction to increase the efficiency of cGMP hydrolysis. However,
our present study showed that they have only a limited effect
on PDE activation even in the presence of S-modulin (Figures
(
5) Emeis, D.; Kuhn, H.; Reichert, J.; Hofmann, K. P. Complex
formation between metarhodopsin II and GTP-binding protein
in bovine photoreceptor membranes leads to a shift of the
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activation. Biochemistry 1997, 36, 14173-14179.
(
2
and 3). Our result therefore indicated that the stimulatory effect
of the four anthocyanins on hydrolysis of cGMP does not seem
to be plausible.
The other possibility is the enhancement of the regeneration
of rhodopsin. This possibility was suggested in a previous study
(7) Virmaux, N.; Bizec, J. C.; Nullans, G.; Ehret, S.; Mandel, P.
Modulation of rod cyclic GMP-phosphodiesterase activity by
anthocyanin derivatives. Biochem. Soc. Trans. 1990, 18, 686-
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87.
(
8) Ferretti, C.; Magistretti, M. J.; Robotti, A.; Ghi, P.; Genazzani,
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(
10), but anthocyanins used in that study were partial compo-
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w/w). For this reason, the effective components of anthocyanins
(
2
0, 150.
were not certain. In the present study, we used purified
anthocyanins to show distinct effects of some of the anthocya-
nins on the regeneration of rhodopsin (Figure 4) and found that
the cyanidin form accelerates the rhodopsin regeneration most
probably through enhancement of the formation of an interme-
diate, INT (Figure 5 and Table 1).
(
9) Scialdone, D. L’Azione delle antocianine sul senso luminoso.
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(
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Recent studies have indicated that even after bleach, opsin
maintains its ability to activate the phototransduction mechanism
to a small extent and reduces the intracellular Ca2+ concentra-
tion, which results in the decrease in the light sensitivity of a
rod (4). Therefore, the acceleration of the regeneration of
rhodopsin by anthocyanins is expected to accelerate the recovery
of the light sensitivity of a rod to a high dark level. This
mechanism probably explains the improvement of night vision
by anthocyanins extracted from bilberry (9).
(
The anthocyanin concentration used in the regeneration study
(
(
20 µM) is considerably higher than that in the plasma (1 µM)
19), and thus the effect of the cyanidin form under in situ
(14) Knowles, A.; Priestley, A. The preparation of 11-cis-retinal.
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conditions might be smaller than that observed in the present
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of bleached visual pigments will affect the light sensitivity of
a rod significantly. This possibility, however, needs further
studies to be proven.
In the present study, the cyanidin form of anthocyanin was
found to be active but the delphinidin form was not. There is a
structural difference between cyanidin 3-glycosides and corre-
sponding delphinidin glycosides (Figure 1). Because 11-cis-
retinal is a nonpolar substance, the cyanidin form might be more
effective than the delphinidin form that is less hydrophobic than
cyanidin glycosides.
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001, 98, 14044-14049.
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(
(
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ABBREVIATIONS USED
2
001, 49, 1546-1551.
ROS, rod outer segment; PDE, phosphodiesterase; D3R,
delphinidin 3-rutinoside; D3G, delphinidin 3-glucoside; C3R,
cyanidin 3-rutinoside; C3G, cyanidin 3-glucoside; INT, inter-
mediate.
(
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(
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JF034132Y