244 Development of seed polyphenols Australian Journal of Grape and Wine Research 6, 244–254, 2000

Development of seed polyphenols in berries

from Vitis vinifera L. cv. Shiraz
JAMES A. KENNEDY1,2,4, GORDON J.TROUP3, JOHN R. PILBROW3, DONALD R. HUTTON3,
DAVID HEWITT3, CHARLES R. HUNTER3, RENATA RISTIC1,2,
PATRICK G. ILAND1,2 and GRAHAM P. JONES1,2
1
Department of Horticulture,Viticulture and Oenology,Adelaide University,Waite Campus,
PMB 1, Glen Osmond, SA 5064, Australia
2
Cooperative Research Centre for Viticulture, PO Box 154, Glen Osmond, SA 5064, Australia
3
Department of Physics, Monash University, Clayton,Vic. 3168,Australia
4
Corresponding author: Dr James A. Kennedy, facsimile +61 8 8303 7116, email jim.kennedy@adelaide.edu.au

Abstract
Polyphenols extracted from the seeds of Vitis vinifera L. cv. Shiraz berries were monitored during berry
development. Initially seeds were green, plump and had pliable seed coats, but beginning at veraison the
seeds browned in colour, became desiccated and the seed coats hardened. Isolated polyphenols consisted
of flavan-3-ol monomers ((+)-catechin, (–)-epicatechin and (–)-epicatechin-3-O-gallate) and procyanidins.
The procyanidins were maximal in the 3 weeks prior to veraison, increasing little during this period. The
amounts of flavan-3-ol monomers increased 5-fold during this same period of time, indicating that the
procyanidins and the flavan-3-ol monomers accumulate at different stages. Beginning at veraison,
amounts of all polyphenols declined and changed in composition. The decrease in amount followed
second-order kinetics. Polyphenol changes after veraison could be explained by oxidation and therefore,
electron paramagnetic resonance (EPR) spectroscopy was used to follow the potential development of
radical species in the developing seeds. Spectra consistent with a phenoxyl radical were observed in the
developing seeds. The concentration of radicals remained low until veraison but then increased, reaching
a maximum three weeks later, declining slowly thereafter. Changes in radical intensity together with
other documented changes in the seed are consistent with an oxidative event occurring during fruit
ripening.
Abbreviations
C (+)-catechin; EC (–)-epicatechin; ECG (–)-epicatechin-3-O-gallate; EPR electron paramagnetic resonance

Keywords: Vitis vinifera L. cv. Shiraz, grape seed, development, electron paramagnetic resonance, oxidation,
procyanidin, flavan-3-ol, polyphenol

Introduction ship between grape production practices and the quantity

Perhaps the most ignored components of grapes, relative
to their importance in red wine, are the polyphenolic
flavan-3-ol monomers and proanthocyanidins. These
compounds provide red wine with its bitter and astrin-
gent properties (Robichaud and Noble 1990, Gawel
1998).
Concentrated in the seed coat, grape seed contains
high amounts of these polyphenols (Thorngate and
Singleton 1995). The major identified polyphenols
include the C, EC and ECG flavan-3-ol monomers, as well
as the polymeric procyanidins (Figure 1). A significant
proportion of these polyphenols is extracted from seeds
during red wine production (Sun et al. 1999), and wine
production practices such as maceration time, pressing,
maturation and fining probably affect their eventual
concentrations in red wine.
Unfortunately very little is known about the relation-
and composition of seed polyphenols found in red wine.
In order to understand this relationship, it is important
to first understand the pattern of accumulation and
modification of these polyphenols during grape berry
development.
Several studies have investigated seed polyphenol
changes during fruit ripening and have found that the
amount of extracted polyphenols declines with maturity
(Czochanska et al. 1979, Romeyer et al. 1986, Iland et al.
1998). Kennedy et al. (2000) suggest that this decline in
polyphenol extractability and the associated composi-
tional changes that occur are consistent with an oxidative
process, although no direct evidence was provided.
The purpose of this investigation was to monitor the
pre-veraison accumulation of seed polyphenols to better
understand at what stage during berry development
biosynthesis occurs. Additionally, EPR spectroscopy was
Kennedy et al. Development of seed polyphenols 245

Flavan-3-ol monomers out damage. A random 150-berry sample was collected

from each replicate at regular intervals (approximately
6’
4’ OH OH
weekly) beginning 5 weeks after fruit set and ending
8 1 B
HO O 2
OH
HO O
OH
when berries reached 24°Brix.
A C 3 2’
6
OH OH Chemicals
4
OH OH
(+)-catechin
All organic solvents and phosphoric acid were LC grade,
(–)-epicatechin
OH and in addition to L-ascorbic acid, sodium acetate and
hydrochloric acid, were obtained from Merck Pty Ltd.
HO O
OH Flavan-3-ol monomers (C, EC and ECG) and phloro-
O glucinol were purchased from Sigma-Aldrich. Phloro-
OH OH glucinol-flavan-3-ol adducts were prepared from
O
proanthocyanidins isolated from Vitis vinifera L. cv.
OH Chardonnay skin and seeds, and were characterised by
OH UV-Vis, MS and 1H NMR (Kennedy and Jones 2000).
(–)-epicatechin-3-O-gallate
A procyanidin extract was isolated from seeds collect-
ed from commercially ripe grapes (Vitis vinifera L. cv.
Shiraz) as follows. Isolated seeds were rinsed with
General procyanidin structure
distilled-deionised water, and then extracted with 2:1
OH acetone:water for 24 hrs. The extract was concentrated
HO O under reduced pressure at 35°C to remove acetone, and
OH
then lyophilised to a dry powder. The dried procyanidin
OR powder was then purified by gel filtration using Toyopearl
OH OH
extension
TSK HW 40-F size exclusion media (Sigma). The column
subunits HO O
OH (270 × 28 mm) was equilibrated with 1:1 methanol:water
n containing 0.1 % v/v trifluoroacetic acid. The procyanidin
OR
OH OH powder was dissolved in a minimum amount of this
terminal mobile phase, applied to the column, rinsed with 5 column
subunit HO O
OH volumes of the methanolic mobile phase followed by 3
OR column volumes of 2:1 acetone:water containing 0.1%
OH v/v trifluoroacetic acid to elute the procyanidins. The
eluent was concentrated under reduced pressure at 35°C
Figure 1. Polyphenols found in Vitis vinifera L. cv. Shiraz seeds
consist of flavan-3-ol monomers ((+)-catechin (C), (–)-epicatechin
to remove acetone, and then lyophilised to a dry powder.
(EC) and (–)-epicatechin-3-O-gallate (ECG)) and procyanidins
(containing C, EC and ECG extension and terminal sub-units). Sample preparation
Sample preparation was as follows. Each 150-berry sample
used to monitor seed development, based on the sug- was weighed and then divided into 3 sub-samples.
gestion that during fruit ripening an event occurs that Berries from one sub-sample (~30 berries) were crushed
leads to the oxidation of seed polyphenols. Since the and the juice total soluble solids measured using a hand-
oxidation of seed polyphenols would likely go through held refractometer. Seeds from the second sub-sample
an observable radical intermediate, EPR spectroscopy (~60 berries) were removed, and kept at –20°C for
(Borg 1976) provides a means of observing polyphenol photographic documentation. Seeds were removed from
oxidation. the last sub-sample (60 berries), weighed, freeze-dried,
and then re-weighed, prior to polyphenol analysis.
Materials and methods For polyphenol analysis, two-thirds of the freeze-dried
Site description and sample collection seeds were counted (~65 seeds) and then extracted in 50
The experiment was conducted on vines (Vitis vinifera L. mL 2:1 acetone:water at 20°C for 24 hr. Extracts were
cv. Shiraz (clone NSW 15)) located within the Barossa filtered (Millipore #1 filters), acetone removed under
Valley at Nuriootpa, South Australia. Row and vine reduced pressure at 35°C, and residual aqueous solution
spacing was 3 × 2.25 m respectively, with rows oriented diluted to 25 mL with distilled-deionised water. Ten mL
in an east-west direction. Fertiliser addition, pest control of this solution was transferred to a sample tube, frozen,
and other vineyard operations were consistent with com- and then freeze-dried. The solid residue was weighed and
mercially accepted practice. Three replicates, consisting then dissolved in methanol (1 mL methanol/10 mg solid).
of 6 vines each, were established within a single vineyard For EPR analysis, the remaining freeze-dried seeds
row. (~33 seeds) were ground to a powder under liquid nitro-
Sample collection began when berries were pepper- gen, washed with 15 mL petroleum ether to remove
corn size (approximately 3–4 mm in diameter), corre- lipophilic material, and then dried with dry nitrogen.
sponding to Eichhorn and Lorenz (E-L) growth stage
29 (Coombe 1995). Before this time, it was difficult to Polyphenol analysis
separate the seed from the remainder of the berry with- Flavan-3-ol monomers were analysed according to the
246 Development of seed polyphenols
method of Peng et al. (2000). One part of the poly-
phenol/methanol solution was first diluted with 9 parts of
water, filtered and then analysed by reversed-phase
HPLC. The column consisted of an Alltima C18 analytical
column (particle size 5 µm, 250 × 4.6 mm) purchased
from Alltech Associates, Pty Ltd, protected by a guard
column (10 × 4.6 mm) containing the same material. The
method utilised a binary gradient with mobile phases
containing 0.2% v/v aqueous phosphoric acid (mobile
phase A) and 1:4 v/v mobile phase A:acetonitrile (mobile
phase B). The elution conditions were as follows: 1.0
mL/min; linear gradients from 0 to 15% B in 15 min, 15
to 16% B in 25 min, 16 to 25% B in 1 min, 25% B for 5
min, linear gradients from 25 to 60% B in 5 min, 60 to
100% B in 5 min, 100% B for 3 min, and a linear gradi-
ent from 100 to 0% B in 1 min. The column was then
equilibrated for 5 min before the next injection. Eluting
peaks were monitored at 280 nm. Peak identification was
made by comparison with authentic standards.
Monomers were quantified using their response factors
relative to C, which was used as the quantitative stan-
dard.
Procyanidins were analysed while intact and after
acid-catalysed cleavage which generated their constitutive
sub-units. To determine intact procyanidins, the filtered
polyphenol/methanol solution was analysed directly by
normal-phase HPLC using a previously described method
(Kennedy and Waterhouse 2000), but without ion-pair
reagent.
Procyanidin sub-units were generated as follows. The
polyphenol/methanol solutions were diluted (1:1) with a
solution containing 0.2 M HCl, 100 g/L phloroglucinol
and 20 g/L ascorbic acid in methanol. The mixture was
reacted at 50°C for 20 min, and then combined with
5 volumes of 40 mM aqueous sodium acetate before
analysis by reversed-phase HPLC.
Generated procyanidin sub-units were analysed as fol-
lows. The column consisted of a Wakosil II 5C18 analyti-
cal column (particle size 5 mm, 250 × 4.6 mm) purchased
from SGE, protected by a guard column containing the
same material. The method utilised a binary gradient
with mobile phases containing 1% v/v aqueous acetic
acid (mobile phase A) and methanol (mobile phase B).
Elution conditions were as follows: 1.0 mL/min; 5% B for
10 min, linear gradients from 5 to 20% B in 20 min, and
20 to 40 % B in 25 min. The column was washed with
90% B for 10 min and re-equilibrated with 5% B for
5 min before the next injection. Eluting peaks were
monitored at 280 nm, and identified using characterised
standards. Products were quantified using their response
factors relative to C, which was used as the quantitative
standard.
EPR spectroscopy
EPR measurements were made using Bruker (~9.3 GHz)
and Varian E-12 X-band (~9.1 GHz) spectrometers in CW
mode at room temperature. Saturation measurements
were made (Bruker system), and from this, a microwave
power of 0.2 mW was selected, well below the onset of
saturation. Measurements were made (Varian system)
Australian Journal of Grape and Wine Research 6, 244–254, 2000
under the following conditions: centre magnetic field,
3250 g; total field sweep, 200 g; modulation field (p-p) 4
g, well below the modulation broadening level. Quartz
sample tubes gave no detectable free radical signal over
the range of the swept magnetic field.
After matching sample tubes for size (~2 mm), ground
seed samples were added and shaken down without
tamping (~2.5 cm sample length). The overfilled tubes
were then placed into the spectrometer (0.15 mL active
volume in the microwave cavity) for data acquisition.
Repeat measurements were made at different sample
heights to check for sample homogeneity. All samples
were similar in line shape, except for the peak-to-peak
heights. The total intensity, proportional to the number of
‘spins’, has been shown to be proportional to the peak-to-
peak heights of the recorded (single-line) spectrum, and
to the square of the peak-to-peak width thereof (Poole
1967). Since the line widths were indistinguishable, it is
only the peak-peak heights that needed to be measured.
Individual sample peak-to-peak heights (measured to the
nearest 0.5 mm) were multiplied by the corresponding
dry seed weight to arrive at a sample EPR intensity.
Spectrometer sensitivity was measured regularly using a
gamma-irradiated alanine standard.
Results
Berry development
Fruit set for this experiment occurred on 1 December
1999, and sample collection commenced on 5 January
2000. Initially, berries were green, but by the second
sampling they had begun to colour (3% coloured on 18
January). By 2 February, essentially all berries were
coloured.
Berry mass on 5 January was 0.49 g increasing slowly
through the first 3 sampling dates to 0.70 g on 24
January (Figure 2). Berry mass then increased rapidly,
reaching a maximum of 1.26 g on 22 February, 84 days
after fruit set. Thereafter, berry mass declined to 1.09 g on
28 March. This growth pattern is consistent with that
observed for Shiraz berries by McCarthy (1999a).
Initially, accumulation of total soluble solids was slow
(4.9 to 8.1°Brix from 5 January to 24 January), but then
1.3 30
28
1.2
26
1.1
24
Total soluble solids (°Brix)
1.0
22 veraison
0.9
Berry weight (g)
20
0.8 18 commercial
0.7 16 ripeness
0.6 14
0.5
12
10
0.4
8
0.3
6
0.2 4
0.1 2
03-Jan-00 17-Jan-00 31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00
Date
●), and soluble solids (●) during
Figure 2. Change in berry weight (●
cv. Shiraz berry development. Error bars represent ± one standard
error of the mean (n = 3).
Kennedy et al. Development of seed polyphenols 247

Table 1. Change in total soluble solids, the total seed polyphenols and their proportional composition during Vitis
vinifera L. cv. Shiraz berry development.

5 Jan 18 Jan 24 Jana 2 Feb 9 Feb 22 Feb 29 Feb 7 Mar 14 Mar 21 Marb 28 Mar

Total soluble solidsc

4.9 6.9 8.1 15.7 18.0 20.0 22.3 22.2 23.2 23.7 24.1

Total amountd
Monomere 367.2 1226.0 1845.5 1576.7 985.5 588.4 479.0 446.8 388.2 410.7 380.0
Procyanidin
Extensionf 3651.9 3871.1 3633.0 3563.4 3338.6 2264.9 2067.5 2012.9 1885.5 2020.1 1856.2
Terminalg 448.3 616.8 559.2 564.5 651.4 455.4 419.9 434.2 410.7 426.1 399.3
Totalh 4100.2 4486.9 4192.2 4127.9 3990.0 2720.3 2487.4 2447.1 2296.2 2446.2 2255.5

Percent compositioni
Monomer
C 36.3 43.3 48.6 34.0 25.9 24.6 25.4 26.0 26.4 26.7 28.0
EC 46.5 43.9 41.7 58.8 68.4 71.0 72.3 72.0 72.7 73.0 71.7
ECG 17.2 12.8 9.7 7.2 5.7 4.4 2.3 2.0 0.9 0.3 0.3
Procyanidin
extension
C 6.8 7.5 7.6 7.4 7.6 8.2 8.2 8.6 8.1 8.2 8.2
EC 74.5 74.7 74.9 75.6 75.4 75.0 74.9 74.5 74.6 74.5 74.6
ECG 18.7 17.8 17.5 17.1 17.0 16.8 16.9 16.9 17.3 17.3 17.2
terminal
C 41.3 33.8 35.0 39.9 39.3 41.3 37.9 37.7 37.5 36.4 36.6
EC 15.3 20.3 20.7 25.0 28.5 32.1 35.2 36.2 36.3 36.2 36.6
ECG 43.4 45.9 44.3 35.1 32.2 26.6 26.9 26.1 26.2 27.4 26.8
a véraison
b commercial ripeness
c °Brix
d nmol/seed
e flavan-3-ol monomer
f extension subunit
g terminal subunit
h total procyanidin subunit
i mole per cent composition within each class (i.e. flavan-3-ol monomer, extension subunit and terminal subunit)

increased rapidly to 15.7°Brix on 2 February (Figure 2).
Thereafter total soluble solids accumulation increased,
almost linearly, until the end of the experiment on 28
March (24.1°Brix). Based on berry colouring, berry mass
and total soluble solids accumulation, veraison occurred
on 24 January. Berries were considered commercially
ripe on 21 March (23.7°Brix).
Seed number per berry changed little with respect to
replicate and time, with an average of 1.65 ± 0.03 over all
samples. Seeds underwent distinct visual changes during
development (Figure 3). Initially, seeds were plump,
bright green and pliable, but from veraison they changed,
shrinking and becoming hard and brown. After 22
February the seed appearance changed very little.
Fresh seed mass was almost maximal on the first
sample date (42.0 mg/seed, 96% of maximum), and
reached a maximum on the second sample date (43.8
mg/seed)(Figure 4). Thereafter, fresh seed mass declined
steadily, reaching 30.1 mg/seed (69% of maximum) on
21 March.
The time trend in dry seed mass trend was opposite to
that for fresh seed mass. The lowest value for dry seed
mass was measured on 5 January (14.4 mg/seed, 56% of
maximum), increasing steadily to an apparent plateau on
22 February (25.9 mg/seed). The increase in dry seed
mass before 22 February suggests that the seeds were still
developing during this time.
Seed desiccation status was determined by subtracting
the dry seed mass from the fresh seed mass (Figure 4).
Initially, fresh seed lost 66% of its fresh mass upon
drying. Mass loss reached a minimum on or around 22
February (23% mass loss). These data indicate that seed
desiccation was complete by 22 February.
Polyphenol development: flavan-3-ol monomers
The flavan-3-ol monomers observed in this study included
C, EC and ECG. These constituents are consistent with
previous studies (Su and Singleton 1969, Prieur et al.
1994, Kennedy et al. 2000).
Flavan-3-ol monomers increased up to veraison, but
declined thereafter (Table 1, Figure 5). Beginning on 5
January, and for C, EC and ECG respectively, flavan-3-ol
monomers increased from 133, 171 and 63 to a maxi-
mum of 897, 769 and 179 nmol/seed on 24 January
(veraison), a 5-fold increase. The rate of increase was
similar for C and EC during this time, but slower for ECG.
While C and ECG were maximal at about veraison, EC
continued to increase, reaching a maximum (928
248 Development of seed polyphenols Australian Journal of Grape and Wine Research 6, 244–254, 2000

a b

c d

Figure 3. Appearance of seeds from Vitis vinifera L. cv. Shiraz berries on (a) 18 January, (b) 24 January, (c) 2 February and (d) 22 February;
indicating the visible transformation which occurred during maximal polyphenol change.

30 1000
45
Fresh weight
Flavan-3-ol monomers (nmol/seed)

(+)-catechin
Dry weight (–)-epicatechin
40
25 Difference 800 (–)-epicatechin-3 -O-gallate
Seed weight (mg)

Difference (mg)

35

20 600
30

25 15 400

20
10 200
15

10 5 0
03-Jan-00 17-Jan-00 31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00
03-Jan-00 17-Jan-00 31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00
Date
Date

●) seed weight from berries

Figure 4. Change in fresh (●) and dry (● Figure 5. Change in extractable flavan-3-ol monomer during cv.
of cv. Shiraz with their difference (▼) during the period of study. Shiraz berry development. Error bars represent ± one standard error
Error bars represent ± one standard error around the mean (n = 3). around the mean (n = 3).

nmol/seed) the following week. Assuming a similar veraison to approximately 24°Brix, the total flavan-3-ol
pattern of increase, the flavan-3-ol monomer amounts monomers declined by 80% from a high of 1846
peaked between 24 January and 2 February. After nmol/seed at veraison.
veraison, C, EC and ECG declined, reaching 106, 272 and The composition of flavan-3-ols also changed, and this
1 nmol/seed respectively on 28 March. Overall, from change can be divided into two periods separated by
Kennedy et al. Development of seed polyphenols 249

2.3
3000
2.2 (+)-catechin

Extension subunits (nmol/seed)

(–)-epicatechin
Extracted procyanidin (mg/seed)

2.1 (–)-epicatechin-3-O-gallate
2.0 2500

1.9

1.8
2000
1.7

1.6

1.5 1500
1.4

1.3
500
1.2

1.1 0
03-Jan-00 17-Jan-00 31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00 03-Jan-00 17-Jan-00 31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00

Date Date

Figure 6. Change in extractable procyanidin during cv. Shiraz berry Figure 7. Change in extractable procyanidin extension sub-units
development (measured while still intact). Error bars represent ± during cv. Shiraz berry development. Error bars represent ± one
one standard error around the mean (n = 3). standard error around the mean (n = 3).

320
veraison (Table 1). Initially, the ratio of C:EC:ECG
(+)-catechin
changed from 36:47:17 on 5 January, to 48:42:10 at 300
Terminal subunits (nmol/seed)
A
veraison, an increase in C relative to the other flavan-3-ol 280

monomers. After veraison this trend changed, with EC 260

increasing in proportion relative to other flavan-3-ol 240
monomers. The final ratio of C:EC:ECG was 28:72:~0.
220
The change in proportion after veraison was due to
200
different rates of decline for the individual flavan-3-ol
monomers. Those rates closely followed second-order 180

kinetics (assuming constant compartment volume), and 160

were 2.1, 0.7 and 34.3/mole.s for C, EC and ECG respec- 140
tively. These rate differences are consistent with expected 120
differences when C, EC and ECG are exposed to radical 220
induced oxidation under aqueous conditions (Plumb et (–)-epicatechin B
Terminal subunits (nmol/seed)

200
al. 1998). After 22 February (20.0°Brix) the rates of
decline for all flavan-3-ol monomers slowed, and there- 180

fore, the composition changed very little, with EC making 160

up ~75% of the pool size, followed by C (~25%) and an 140
insignificant amount of ECG.
120
Results obtained for the flavan-3-ol monomers can
be summarised as follows. Extracted flavan-3-ol monomers 100

can be divided into a period of accumulation and a period 80

of decline. Separation of these periods occurs near
60
veraison. The majority of the extracted flavan-3-ol
monomers accumulated in the 3 weeks prior to veraison. 40
350
The pattern of decline for the individual flavan-3-ol
(–)-epicatechin-3-O-gallate
monomers after veraison (i.e. ECG >> C > EC) is similar C
Terminal subunits (nmol/seed)

300
to that reported by Kennedy et al. (2000).
250
Polyphenol development: procyanidins
Procyanidins were analysed while still intact and after
200
acid-catalysed hydrolysis in the presence of excess
phloroglucinol. When analysed intact, and from 5
150
January until 2 February, the amount of extracted pro-
cyanidins changed very little, averaging 2.00 mg/seed
100
(Figure 6). The amount declined thereafter, falling rapidly
to 1.37 mg/seed by 22 February (69% of initial amount),
50
and slowing thereafter. On 28 March (24.1°Brix), the 03-Jan-00 17-Jan-00 31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00

amount had fallen to 1.27 mg/seed (64% of initial Date

amount). Figure 8. Change in extractable procyanidin terminal sub-units
When analysed after acid-catalysis in the presence of during cv. Shiraz berry development. Error bars represent ± one
excess phloroglucinol, information on the amount of standard error around the mean (n = 3).
250 Development of seed polyphenols
Mean degree of polymerisation (# sub-units)
9
8
7 an overall decline in the apparent mDP of the pro-
6 mDP declined more slowly, reaching 5.7 on the final
03-Jan-00 17-Jan-00
Figure 9. Change in procyanidin mean degree of polymerisation
during cv. Shiraz berry development. Error bars represent ± one
standard error around the mean (n = 3).
extractable sub-units, as well as the sub-unit composi-
tion, apparent mean degree of polymerisation (mDP),
and the susceptibility of the procyanidins to acid-catalysis,
was obtained (Table 1, Figures 7–9). Following this treat-
ment, the identifiable procyanidin sub-units included the
terminal sub-units C, EC and ECG along with their cor-
responding extension sub-units, consistent with previous
studies (Prieur et al. 1994, Kennedy et al. 2000).
The total concentration of sub-units increased from
4100 nmol/seed on 5 January, to a maximum of 4487
nmol/seed on 18 January. The sub-unit amount declined
thereafter. Initially, the rate of decline was slow (3990
nmol/seed on 9 February), but then increased, reaching
2720 nmol/seed on 22 February (61% of maximum).
The rate of decline then slowed through the remainder of
the experiment, falling to 2256 nmol/seed (50% of
maximum) on 28 March.
Individually, the extension and terminal sub-units
behaved differently (Table 1, Figures 7-8). The extension
sub-unit amount was close to the maximal amount on 5
January (3652 nmol/seed, 94% of maximum), and
reached a maximum by 18 January (3871 nmol/seed).
The extension sub-unit composition was constant
throughout the study with the ratio of C:EC:ECG
averaging 8:75:17.
On 5 January the terminal sub-unit amount was 448
nmol/seed, rising to an apparent plateau on 18 January
(617 nmol/seed), where it remained until 9 February
(651 nmol/seed). The extension sub-units did not have
this plateau. Thereafter the terminal sub-unit amount
declined to 400 nmol/seed (61% of maximum) on 28
March.
The individual terminal sub-units behaved differently.
C changed very little, decreasing from 185 nmol/seed on
5 January to 146 nmol/seed on 28 March, with a peak
amount of 256 nmol/seed on 9 February (Figure 8A). EC
increased from 69 nmol/seed on 5 January to 146
nmol/seed on 28 March, also with a peak amount on 9
February of 185 nmol/seed (Figure 8B). ECG decreased
from 195 nmol/seed on 5 January to 107 nmol/seed on
Australian Journal of Grape and Wine Research 6, 244–254, 2000
28 March, without a discernible peak (Figure 8C). When
expressed proportionally, C varied from 34 to 41%, EC
increased from 15 to 37%, and ECG decreased from 46 to
27% during the study (Table 1).
The apparent mDP of the procyanidins was deter-
mined by dividing the total procyanidin sub-unit amount
by the terminal sub-unit amount (Figure 9). There was
cyanidins through seed development, from 9.2 on 5
January to 6.0 on 22 February. Thereafter, the apparent
sampling date. This decline in apparent mDP, while sug-
gesting a decrease in molecular size could indicate that
31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00 the procyanidins are being modified, leading to an actual
Date increase in molecular size (Kennedy et al. 2000).
To investigate the possibility that the procyanidins are
being modified, the extent to which the procyanidins
could be converted to known sub-units was determined
by dividing the total known sub-unit concentration
(Figures 7 and 8 converted to C equivalents) by the total
concentration of intact material (C equivalents Figure 6)
to arrive at a percentage conversion (Figure 10). Initially,
the extracted material was converted to a large extent to
known sub-units, averaging 88% conversion during the
first two sampling dates. The conversion increased to
92% at veraison but then declined rapidly to 76% on 9
February. Thereafter, the conversion of procyanidins to
known sub-units declined more slowly, reaching 69% on
the final sampling date.
Conversion of tannin into known sub-units (%
95
90
85
80
75
70
65
03-Jan-00 17-Jan-00 31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00
Date
Figure 10. Change in procyanidin conversion to known sub-units
during cv. Shiraz berry development. Error bars represent ± one
standard error around the mean (n = 3).
The results obtained for the procyanidins can be sum-
marised as follows. As with the flavan-3-ol monomers,
extracted procyanidins can be divided into a period of
accumulation and a period of decline separated by
veraison. The majority of the extracted procyanidins had
accumulated prior to the beginning of the investigation.
The pattern of decline for the procyanidins after veraison
as suggested by Kennedy et al. (2000) could be explained
by oxidation.
Kennedy et al.
Figure 11. EPR spectrum of a purified procyanidin isolated from
Vitis vinifera L. cv. Shiraz.
EPR measurements
EPR spectroscopy was used to investigate the suggestion
that the polyphenols are becoming oxidised during seed
development. Preliminary EPR spectra of a purified
procyanidin extract (Figure 11) were consistent with a
phenoxyl radical signal and were similar to the EPR
spectra of an oxidised procyanidin extract isolated from
Pinus maritima (Guo et al. 1999). No interfering EPR
signals (Cu2+, Fe3+, Mn2+) were observed in the free radical
region at room temperature. For the procyanidin extract
at 77K, a Cu2+ signal was observed, but this was motion-
ally broadened and unobservable at room temperature.
50
45
EPR signal intensity
40
35
30
25
20
15
10
03-Jan-00 17-Jan-00 31-Jan-00 14-Feb-00 28-Feb-00 13-Mar-00 27-Mar-00
Date
Figure 12. Change in grape seed EPR signal intensity during cv.
Shiraz berry development. Error bars represent ± one standard error
around the mean (n = 3).
Based on the presence of a radical signal in the pro-
cyanidin extract, the radical signal intensity of ground
seeds was measured (Figure 12). The EPR signal of de-
fatted seeds was similar to the procyanidin extract signal,
but showed clear temporal changes in intensity. Prior to
veraison, the EPR signal intensity remained low, averag-
ing 14.5, indicating a relatively low radical intensity. On 2
February, the EPR signal intensity had increased to 27.3,
and increased to a maximum of 43.3 on 7 March,
although the signal decreased to 35.6 on 29 February.
Thereafter, the signal intensity declined slowly, although
the final signal intensity remained much higher (38.1)
than pre-veraison samples.
Discussion
The major goal of this study was to monitor the develop-
ment of polyphenols in Vitis vinifera L. cv. Shiraz seeds,
Development of seed polyphenols 251
and by so doing to provide a preliminary step for under-
standing how grape production practices can be used for
their modification. Results from our study are consistent
with two distinct periods of polyphenol development,
where initially there is a period of polyphenol biosynthesis,
followed by a period of polyphenol modification leading
to a decline in their extractability. Chemical evidence
along with the visual changes in seed colour that
occurred after veraison are consistent with polyphenol
oxidation in the seed, which leads to a decline in their
extractability.
The biosynthesis of seed polyphenols in the seed
coat is consistent with their hypothesised role in seed
dormancy and longevity (Werker 1980/81). There is also
evidence that while the presence of polyphenols is impor-
tant in seed dormancy and longevity, the mere presence
of the polyphenols may not be sufficient in that an addi-
tional oxidative step may be important for rendering the
seed coats impermeable to water (Marbach and Mayer
1974, 1975).
Some generalised outcomes from this investigation
are summarised in Figure 13. The two periods of
polyphenol development in seeds can be further divided
into four distinct stages where each developmental period
is divided into two stages. In temporal order, these stages
have been classified as follows.
Stage 1: procyanidin biosynthesis
During the first stage of development, probably begin-
ning at anthesis and continuing through the first month
of berry development, the biosynthesis of procyanidins
occurred. While this portion of berry development was
for the most part not monitored, it is clear that the
procyanidins were, in a large part (approximately 91%),
present 5 weeks after anthesis. The biosynthesis of pro-
cyanidins appears to coincide with the initial rapid period
of berry growth (Coombe 1973).
Stage 2: flavan-3-ol monomer biosynthesis
One of the results not expected in this study was
the biosynthesis at different times of the flavan-3-ol
monomers and the procyanidins. In contrast to 91% of
the procyanidins being present when the first sample was
taken, only 15% of the flavan-3-ol monomers were
present at this stage. After the first sample was taken,
but prior to veraison, the rate of flavan-3-ol monomer
biosynthesis increased with a concomitant decline in the
rate of procyanidin biosynthesis; therefore, the sub-unit
increases during the two weeks preceding veraison were
almost exclusively made up of flavan-3-ol monomers.
The decline in the rate of flavan-3-ol monomer bio-
synthesis coincided roughly with veraison.
Our observations during the period of polyphenol
biosynthesis are of interest from a biochemical standpoint
because the enzyme that would be responsible for the
final condensation of flavan-3-ol sub-units into pro-
cyanidin molecules has not been characterised (Figure
14). Procyanidins and flavan-3-ol monomers are derived
from the same metabolic precursor, leucocyanidin
(Stafford 1989). Moreover, the flavan-3-ol monomers
 252 Development of seed polyphenols Australian Journal of Grape and Wine Research 6, 244–254, 2000

Polyphenol development per seed

sub-unit amount

Metabolic
Total

precursors Abbreviations
DFR = dihydroflavonol-4-reductase
LAR = leucoanthocyanidin-4-reductase

Polyphenol Biosynthesis Polyphenol Oxidation

Stage 1 Stage 2 Stage 3 Stage 4 Dihydroquercetin
procyanidin flavan-3-ol
synthesis monomer programmed non-programmed
synthesis oxidation oxidation
DFR
Rate

LAR
Flavan-3-ol
Leucocyanidin
monomer

Condensing
enzyme
? ?

Solute accumulation per berry

organic sugars and non-anthocyanin
acids anthocyanins glucosides Vacuole
Procyanidin
Berry weight

and
Flavan-3-ol monomer
Col 3 vs Col 6

01-Dec-99 01-Jan-00 01-Feb-00 01-Mar-00 01-Apr-00

01-Dec-99 24-Jan-00 22-Feb-00
Fruit Veraison Maximum berry
set weight
Figure 13. Schematic representation summarising the observed
changes in Vitis vinifera L. cv. Shiraz seed polyphenols (top) in
relation to the development of the whole berry (bottom). For seed
polyphenol development, there was an increase in the total sub-unit
amount (procyanidin sub-unit + flavan-3-ol monomer) until
approximately veraison. Beginning at veraison the amount of
extracted polyphenols declined and became modified. Polyphenol
modification is consistent with an oxidative process. Very little
change in the polyphenols occurred after the berry had attained
maximum berry weight. Dashed lines are predicted.
and the procyanidin sub-units appear at different times in
berry development. Nevertheless, the overall rate of
polyphenol biosynthesis appears to be nearly constant
from fruit set until veraison. Presumably, there is com-
petition for this precursor. Low amounts of flavan-3-ol
monomers relative to procyanidins at the beginning of
stage 2 are consistent with there being a low leucoantho-
cyanidin-4-reductase (LAR) activity. If the rate of leuco-
cyanidin biosynthesis were constant at this time, a lack of
LAR activity would lead to an accumulation of leuco-
cyanidin. Conceivably, an enzyme is not involved in this
final condensation step (Delcour et al. 1983). Instead,
leucocyanidin is directly transferred into the vacuole,
resulting in the formation of procyanidin. Additional
studies where enzyme activities (LAR and dihydro-
flavonol-4-reductase (DFR)) are monitored (Joseph et al.
1998) in addition to the appearance of these polyphenols,
could increase our understanding of this portion of the
flavonoid biosynthetic pathway.
Figure 14. A simplified schematic showing the final stages of flavan-
3-ol monomer and procyanidin biosynthesis (discounting enzyme
21-Mar-00
Commercial localisation). The question-marked region has not been characterised
ripeness with respect to procyanidin biosynthesis. We have assumed that
flavan-3-ol monomers and procyanidins occupy the same vacuolar
space.
Stage 3: programmed oxidation
Beginning at veraison, seed colour changed from green to
brown, with a concomitant hardening of the seed coat.
These visible changes coincided with a decreased rate of
flavan-3-ol monomer biosynthesis, and a rapid increase
in EPR signal intensity consistent with phenoxyl radical
generation. A rapid increase in EPR signal intensity at
this time suggests that the seeds underwent a pro-
grammed change in development, which then led to the
oxidation of flavan-3-ol monomers and procyanidins.
Overlapping patterns of flavan-3-ol monomer accumula-
tion and EPR signal intensity suggest that there was some
overlap in the biosynthesis of flavan-3-ol monomers and
their oxidation. The flavan-3-ol monomers decreased
more than the procyanidins when normalised to moles of
sub-units (Table 1). This is consistent with previous
observations (Kennedy et al. 2000, Plumb et al. 1998).
The cessation of the third stage of polyphenol develop-
ment is very closely associated with maximum berry
weight and the completion of seed desiccation.
Stage 4: non-programmed oxidation
During Stage 4, there was little change in the amount
and composition of extracted polyphenols. The EPR
signal intensity remained relatively high during Stage 4
suggesting that phenoxyl radicals are still present in the
Kennedy et al.
system. Stable procyanidin phenoxyl radicals have
recently been observed (Guo et al. 1999), and the relative
stability of the phenoxyl radical over free flavan-3-ol
monomers may be due to π-π interactions between
adjacent sub-unit B-rings (Hatano and Hemingway
1997).
In this study and in others, the inception of Stage 4 in
cv. Shiraz berries seems to be characterised by several
events, including maximal berry weight, accumulation
of non-anthocyanin glucosides, complete seed desiccation
and a levelling in phenolic extraction and composition
(Coombe and McCarthy 1997, 2000).
Significance
Present results indicate that the amounts and composition
of seed polyphenols change during berry development. In
cv. Shiraz it is clear that much of the change has occurred
by the time the grape berry reaches maximum berry
weight (approximately 20°Brix). Changes after this time
would likely be due to residual radical presence in the
seed.
The presence of oxidised seed polyphenols containing
phenoxyl radicals may have implications in winemaking.
Stable radicals have been observed in red wine, and are
consistent with the radicals observed in this study (Troup
et al. 1994). These radicals are likely to exist on the B-
ring of procyanidins (Kondo et al. 2000), and since B-ring
phenolic groups are considered to be important in protein-
tannin interactions (Hagerman et al. 1998) subsequent
radical-induced reactions that occur at this site could lead
to a modification of astringency in red wine.
Acknowledgements
We gratefully acknowledge the Grape and Wine Research
and Development Corporation of Australia and the
Commonwealth Cooperative Research Centres Program
(conducted by the CRC for Viticulture) for financial sup-
port. We are also indebted to Dr M. McCarthy and staff of
the Nuriootpa Viticultural Research Station for assistance
as well as the use of a section of their vineyard.
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Manuscript received: 18 October 2000
Amended version received: 9 November 2000