Isolation and structure elucidation of tetrameric procyanidins from unripe apples (Malus pumila cv. Fuji) by NMR spectroscopy

Article abstract of DOI:10.1016/j.phytochem.2012.07.011

Procyanidins are plant secondary metabolites widely consumed and known to have various physiological functions, but their bioavailability and mechanism of action are still unclear especially for larger oligomers. One of the reasons is scarce information about the detailed structure of oligomeric procyanidins. As for apple, structures of procyanidin components larger than trimers are scarcely known. In this study, 11 tetrameric procyanidins including two known compounds were isolated from unripe apples (Malus pumila cv. Fuji) and identified by NMR spectroscopic analysis and phloroglucinol degradation. As a result, the detailed structural diversity of tetrameric procyanidins in apple was established.

Full text of DOI:10.1016/j.phytochem.2012.07.011

Phytochemistry 83 (2012) 144–152
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Isolation and structure elucidation of tetrameric procyanidins from unripe
apples (Malus pumila cv. Fuji) by NMR spectroscopy
⇑
Shohei Nakashima , Chihiro Oda, Susumu Masuda, Motoyuki Tagashira, Tomomasa Kanda
Asahi Group Holdings, Ltd., 1-21, Midori 1, Moriya, Ibaraki 302-0106, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 April 2012
Received in revised form 5 July 2012
Available online 11 August 2012
Procyanidins are plant secondary metabolites widely consumed and known to have various physiological
functions, but their bioavailability and mechanism of action are still unclear especially for larger oligo-
mers. One of the reasons is scarce information about the detailed structure of oligomeric procyanidins.
As for apple, structures of procyanidin components larger than trimers are scarcely known. In this study,
1
1 tetrameric procyanidins including two known compounds were isolated from unripe apples (Malus
Keywords:
Apple
Malus pumila cv. Fuji
Rosaceae
pumila cv. Fuji) and identified by NMR spectroscopic analysis and phloroglucinol degradation. As a result,
the detailed structural diversity of tetrameric procyanidins in apple was established.
Ó 2012 Elsevier Ltd. All rights reserved.
Proanthocyanidin
Procyanidin tetramer
NMR
Phloroglucinol degradation
1
. Introduction
Polyphenols extracted from apple were found to show a wide
absorption of simple procyanidins has progressed along with anal-
ysis of catabolism by human gut microflora (Appeldoorn et al.,
2009; Déprez et al., 2000; Stoupi et al., 2010), but there have been
few studies on higher oligomeric procyanidins. This is associated
with the difficulty of preparing structurally characterized procyani-
dins as a subject of research.
The general approach to obtain procyanidins is extraction and
purification from natural resources. Natural oligomeric procyani-
dins are easily fractionated according to their degree of polymeri-
zation by well-established methods (Kelm et al., 2006; Shoji et al.,
2003); however, identification of components is difficult owing to
their complexity. Procyanidins are either oligomeric or polymeric
compounds of catechin (Cat) or epicatechin (EC) through their
C4 ? C6 or C4 ? C8 interflavonoid linkages (Fig. 1). This difference
in position of the interflavonoid linkage and constitutive units
gives structural diversity to the higher oligomers; thus, the number
of isomers increases along with degree of polymerization (DP).
As another approach for preparation of structurally character-
ized procyanidins, the total synthesis (Kozikowski et al., 2003;
Saito et al., 2009; Tarascou et al., 2006) or semi-synthesis
variety of physiological functions, such as reduction of triglyceride
absorption (Sugiyama et al., 2007), inhibition of melanogenesis
(
(
Shoji et al., 2005), protective effects against muscle injury
Nakazato et al., 2010), and anti-inflammatory (Yoshioka et al.,
2
008), anti-cancer (Miura et al., 2008), and anti-allergic activities
(Akiyama et al., 2000, 2005; Tokura et al., 2005). The main compo-
nents of apple polyphenols are procyanidins, which have attracted
attention especially due to their positive effects on vascular health
(Corder et al., 2006; García-Conesa et al., 2009). Apple procyanidins
have also been confirmed to have activities on the vascular endo-
thelium in vitro and in vivo (Caton et al., 2010; Matsui et al.,
2
009; Nishizuka et al., 2011). On the other hand, their mechanisms
of action are still unclear. Some reports have suggested a relation-
ship between their activity and the degree of polymerization
(Caton et al., 2010; Sugiyama et al., 2007); however, the influence
of differences in detailed structure has rarely been reported,
especially for larger oligomers. Furthermore, investigation of the
(Esatbeyoglu et al., 2011) of oligomeric procyanidins can be con-
ducted. At this time, however, the synthetic technique is limited
to extension of the unit only through the C4 ? C8 interflavonoid
connection; the creation of various structures with C4 ? C6 bonds,
which exist in natural resources, remains to be achieved. Even in
the case of the semi-synthetic method, precursor compounds that
have C4 ? C6 bonds must be prepared from natural resources.
Abbreviations: LC-ESI-TOFMS, liquid chromatography electrospray ionization
time of flight mass spectrometry; RP-HPLC, reversed-phase high-performance
liquid chromatography; COSY, correlation spectroscopy; HSQC, hetero-nuclear
single quantum coherence; HMBC, hetero-nuclear multiple-bond connectivity.
⇑
Corresponding author. Tel.: +81 297 46 9352; fax: +81 297 46 1506.
E-mail address: syohei.nakashima@asahigroup-holdings.com (S. Nakashima).
0
031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.07.011

S. Nakashima et al. / Phytochemistry 83 (2012) 144–152
145
Fig. 1. General structure of procyanidins.
In consideration of this background, the investigation of the
and 158.12 in methanol-d
in methanol-d
unit. Next, the A-ring and C-ring protons in each unit were as-
signed as follows. The H4 of the terminal unit T (represented as
H4-unit T ) were only methylene protons in the procyanidins
and were easily assigned according to their own coupling. The
H4 of the extension units (M , M , U ) were recognized by a HSQC
correlation with C4, which showed characteristic chemical shifts in
the range of d of 30–40. The remaining non-aromatic protons in
C-rings were assigned on the basis of COSY correlation with H4.
Each A-ring proton in the d range of 5.8–6.2 was linked with H4
4
) gave no isotopic shift when measured
3
; thus, they were determined to be C8a for each
detailed structure of oligomeric procyanidins extracted from natu-
ral resources is important. In this study, 11 tetramers detected in-
apple polyphenolic extract were isolated, and the structures of
nine unknown compounds were determined by NMR spectroscopic
and phloroglucinol degradation analyses. Combined with our previ-
ously reported studies (Abe et al., 2008; Shoji et al., 2003), this is the
first study on the detailed profile of tetrameric procyanidins in
apple.
1
1
1
2
1
C
H
2
. Results and discussion
in the same unit on the basis of the HMBC correlation to C4a.
The positions of the interflavonoid bonds were investigated on
the basis of HMBC correlations from protons in the A-ring or
Compounds 7 and 10 were identified as EC-(4b ? 6)-EC-
(
8
4b ? 8)-EC-(4b ? 8)-EC and EC-(4b ? 8)-EC-(4b ? 8)-EC-(4b ?
)-EC with reference to previous reports (Abe et al., 2008; Shoji
C-ring. The C8a-unit T
lation with H2-unit T
the H4-unit M , which indicated that unit T
through a C4 ? C8 interflavonoid bond. The connection be-
tween unit M and unit M was determined in the same way using
the correlation from both the H4-unit M and the H4-unit M to
C8a-unit M . In unit M , both H8-unit M and H4-unit M showed
correlations to C8a-unit M , which suggested that unit M was con-
nected to unit U through a C4 ? C6 interflavonoid bond. This con-
nection was confirmed by correlation from both H4-unit M and
H4-unit U to C5-unit M . As a result of these observations, the
1
was assigned on the basis of a HMBC corre-
. The C8a-unit T was also correlated with
was connected to unit
1
1
et al., 2003), respectively. The structures of other isolated com-
pounds were elucidated by NMR spectroscopic and phloroglucinol
degradation analyses. The H and C chemical shifts of tetrameric
procyanidins are summarized in Tables 1 and 2, and determined
structures are depicted in Fig. 2.
Compound 1 was established to consist of one catechin as a
terminal unit and three epicatechins as extension units by phloro-
glucinol degradation analysis. Structure 1 was elucidated on the
basis of 1D and 2D NMR spectroscopic analysis. All NMR spectra
were measured at 243 K to avoid signal broadening due to atrop-
isomerism. First, an H-D exchange experiment was conducted with
1
1
M
1
1
13
1
2
1
2
2
2
2
2
2
2
1
2
1
2
structure of compound 1 was elucidated as EC-(4b ? 6)-EC-
(4b ? 8)-EC-(4b ? 8)-Cat.
methanol-d
C7. Among aromatic carbon signals of the A-ring in the range of
of 150–160, only four resonances (d 153.66, 154.71, 155.49
4
and methanol-d
3
to distinguish the C8a from C5 and
As for compounds 2–11, only the characteristic points are
explained here because the basic strategy matched that for 1. Com-
pound 2 consisted of one catechin as the terminal unit and three
d
C
C

1
46
S. Nakashima et al. / Phytochemistry 83 (2012) 144–152
Table 1
1
4 4
H NMR spectroscopic data of compounds 1, 3–6, 8, 9, 11 in methanol-d at 243 K and 2 in methanol-d /deuterium oxide (25:15) at 278 K.
Unit
Position d (multiplicity, J)
1
2
3
4
5
6
8
9
11
T1
2
3
5.01 (d, 4.0) 3.94 (d, 9.1)
4.29
3.74
5.09
4.23 (q,
4.4)
2.54 (d,
12.9)
2.64 (d,
13.1)
3.73 (d, 8.1)
3.68 (q, 8.1)
4.94
4.34
4.99
4.24
4.59 (d, 7.3)
3.99
4.83
4.15
4.20 (qbr,
.7)
2.52 (d,
3.86 (dd, 8.8,
15.6)
2.15 (dd, 10.4,
15.7)
4
4
1.90
2.24
2.28 (dd, 9.1,
17.2)
2.92 (dd, 7.1,
17.2)
2.88 (d,
16.8)
2.97 (d,
13.0)
2.83 (d,
17.3)
2.98 (d,
13.1)
2.50 (dd, 8.0,
15.4)
2.78 (d, 15.8)
2.75 (d,
16.1)
2.90 (d,
13.6)
1
2
1
3.3)
.61 (d,
2.4)
2.65 (dd, 6.0,
15.5)
6
8
2
5
5.89
5.91
6.09
5.91
5.95
6.91
6.87
5.97
7.00
6.76 (d,
6.04i
6.83
6.76
6.11
0
0
6.82
6.69
6.87
6.73–6.75
6.55
6.65
7.16
6.73
7.10
6.72–6.74
6.97
e
h
6.65–6.80j
7
6.86
.7)
0
6.87 (d, 7.7) 6.84d
6
6.95
6.20 (d, 8.3)
6.84
6.85 (d,
6.72
6.79
8.6)
M1
2
3
4
6
8
2
5
5.26
4.04
4.70
5.91
4.53
3.92
4.45
6.18
4.67
3.86
4.50
6.12
5.37
4.14
4.80
5.95
5.22
3.80
4.48
5.10
3.81
4.63
4.97
4.03
4.50
5.99
5.05
4.13
4.63
6.04i
5.07
4.14
4.64
6.03
5.52
6.91
6.73 (d, 8.3)
6.18
6.87
6.70
0
0
7.09
6.67
6.56 (d, 1.4)
6.54 (d, 8.4)
6.59
6.55 (d,
7.06
6.73–6.75
7.14
6.70
7.04
6.73
7.04
a
e
6.65–6.80j
8
.3)
6.33 (d,
.0)
0
6.67a
6
6.01 (d, 8.3)
6.85
6.69
6.65 (d,
9.6)
6.56 (d,
7.7)
6.75
6.75
8
M2
(
8
2
3
4
6
4.94
4.05
4.58
5.32
3.98
4.34
5.45
3.90
4.48
5.29
4.11
4.76
5.94
4.98
4.09
4.51
5.90
4.99
3.95
4.59
5.96
4.88
3.85
4.78
6.11 (d,
5.24
4.00
4.73
5.92
5.25
4.00
4.74
5.92
U2 of
)
2
.2)
6.08 (d,
.3)
6.90
6.72–6.74
6.52 (d,
8
6.20
5.88
5.84
2
0
0
0
6.90b
6.70
6.74
2
5
6
7.04 (d, 1.6)
6.84d
6.90
7.05
6.74
6.30
7.12
6.73–6.75
6.79
7.13
6.79 (d, 8.4)
6.90
7.03
7.14
6.72
6.67
7.13
e
6.67g
6.67g
h
6.65–6.80j
6.72
7.7)
U1
2
3
4
6
4.94
3.83
4.57
5.92c
4.75
3.82
4.31
6.04 (d, 2.3)
4.86
3.76
4.36
5.95
5.11
4.00
4.74
5.99
5.04
3.83
4.55
5.17
3.95
4.69
5.90 (d,
5.04
3.88
4.68
5.98
5.10
3.98
4.72
5.98 (d, 2.1)
5.11
3.98
4.72
5.99
f
5.91
2
.4)
5.94 (d,
.5)
8
2
5
6
5.92c
6.91b
6.72
6.69
6.13 (d, 2.2)
7.10 (d, 1.6)
6.33 (d, 8.1)
6.09 (bd, 8.3)
6.04
6.02
6.92
5.96f
5.94 (d,
3.2)
6.92 (d,
6.01 (d, 1.9)
6.92
6.01
2
0
0
0
7.09
6.87
6.77
6.69
6.82
6.93
1.4)
6.73–6.75e
6.71
6.85 (d, 8.4)
6.57
6.72–6.74
h
6.74
6.65–6.80j
6.70
6.39 (d,
7
.7)
6.15 (d,
.7)
6.64 (d,
8.6)
6.69
7
a,c–e,g–j
b,f
Overlapped with each other.
Values with the same superscript letters are not significantly different.
epicatechins as extension units, as established by phloroglucinol
degradation analysis. In the NMR spectroscopic analysis, 2 showed
a more complicated spectrum than 1, even when measured under
the same conditions, which indicated that 2 existed in two confor-
mations. This was resolved according to the method of Cui et al.
Compound 3 consisted of four epicatechin units, as again estab-
lished by phloroglucinol degradation analysis. It also showed a
1
complicated H NMR spectrum, but the signals were fairly well
separated for them to be assigned. Here the assignment of the ma-
jor conformer is given. The H8-unit T
HMBC correlation with C4a-unit T , which was correlated with
the H3-unit T . The H8-unit T also correlated with C8a-unit T
by which the connection between units T
as C4 ? C6. The connection between units M
1
was identified from the
(
1992). By addition of deuterium oxide with three-fifths volume
of methanol-d , the conformers of 2 converged to a single confor-
mation, and showed a simple H NMR spectrum at 278 K (Fig. 3).
In the HMBC spectrum, the H8-unit T was correlated with the
C8a-unit T , determining that the connection between units T
and M was C4 ? C6. The connection between units M and M
1
4
1
1
1
,
1
1
and M
1
was determined
1
1
2
and M was deter-
1
1
2
mined to be C4 ? C8 on the basis of the correlation from both
H4-unit M and H4-unit M to C8a-unit M . The connection be-
tween units M and U was also determined to be C4 ? C6 on
the basis of the correlation from both H4-unit M and H4-unit U
to C5-unit M . Therefore, structure 3 was elucidated as EC-
1
1
1
2
2
was determined to be C4 ? C8 on the basis of the correlation from
H4-unit M and H4-unit M to C8a-unit M . The connection be-
tween units M and U was determined to be C4 ? C6 on the basis
of the correlation from H4-unit M and H4-unit U to C5-unit M
2
1
1
2
1
2
1
2
1
2
2
1
2
.
(4b ? 6)-EC-(4b ? 8)-EC-(4b ? 6)-EC.
Therefore, structure 2 was elucidated as EC-(4b ? 6)-EC-(4b ? 8)-
EC-(4b ? 6)-Cat.
Compound 4 consisted of four epicatechin units, as determined
by phloroglucinol degradation analysis. The C8a-unit T was as-
1

S. Nakashima et al. / Phytochemistry 83 (2012) 144–152
147
Table 2
1
3C NMR spectroscopic data of compounds 1, 3–6, 8, 9, 11 in methanol-d
4
4
at 243 K and 2 in methanol-d /deuterium oxide (25:15) at 278 K.
Unit
T1
posItion
d
1
2
3
4
5
6
8
9
11
2
3
4
81.40
67.93
26.05
99.99
155.75
96.88
156.39
107.99
153.66
132.18
113.68
146.02
145.66
115.91
119.12
82.64
68.86
24.31
79.37
68.06
29.45
81.38
68.01
26.00
99.93
155.69
96.90
156.38
108.16
153.06
132.33
113.69
83.48
69.32
30.62
101.52
155.21
95.89
79.21
67.02
30.26
99.40
157.17
96.82
79.49
67.15
30.04
101.45
155.33
108.54
153.89
107.27
152.58
132.07
82.47
68.81
28.42
79.56
67.51
29.68
a
4
102.87
155.41
107.31
154.78
95.46
154.55
131.33
116.30
145.49
145.92
116.54
121.46
101.37
154.98
107.60
156.21
95.65
155.09
132.66
115.35
145.59
145.53
115.57
119.66
101.21
100.44
156.37
108.08
155.71
96.15
155.50
132.30
114.98
5
6
7
8
8
1
155.85z
aa
107.94
155.85z
97.54
154.86
131.98
114.88
146.16
146.16
115.7–116.0
119.87
e
f
155.32p
108.81
155.92
130.93
115.23
145.30
156.89
107.04
154.61
131.96
114.87
145.0–146.0
145.0–146.0
a
0
0
ag
2
114.89
0
0
0
0
j
u
u
145.94w
ab
ah
3
4
5
6
146.02
145.2–146.1
145.2–146.1
115.5–116.0
145.5–145.7l
q
ab
ah
ai
145.4–146.1
145.65
115.0–116.0
118.77
m
115.35r
115.0–116.0v
118.55
x
ac
115.6–116.0
119.08
aj
121.11
119.12
M1
2
3
4
4
5
6
7
8
8
1
2
3
4
5
6
76.60
72.33
36.78
102.70
157.16
96.63
77.02
71.46
37.81
98.85
156.64
96.83
156.89
109.29
155.62
131.39
115.03
144.52
144.49
115.60
119.90
77.38
72.53
37.85
98.61
157.40
96.26
157.46
109.44
156.17
131.68
114.60
144.88
144.96
115.21
119.74
76.84
72.07
37.10
102.73
76.48
73.35
36.85
103.73
155.32p
106.72
154.75
95.76s
155.39
76.80
73.39
36.48
77.06
72.45
38.02
98.55
157.99
97.35
158.91
107.82
154.98
131.77
114.87
146.04
145.52
115.0–116.0
117.99
77.13
71.54
37.87
99.25
157.83
95.69
77.16
71.58
37.84
99.13
157.98
97.43
158.70
107.89
154.95
131.77
k
a
102.34
155.38
107.81
155.69
96.37
155.91
132.45
115.04
n
y
ad
ak
156.98
o
96.90
156.84
105.22
154.71
132.71
114.62
145.69
144.98
156.72
106.67
154.94
132.70
114.83
145.78
145.19
115.6–116.0
118.64
158.65
107.94
aa
a
0
0
0
0
154.96
131.76
114.98
145.62
145.99
130.05
115.35r
ae
af
af
ag
114.98
a
a
q
u
ah
ah
ai
145.4–146.1
145.0–146.0
145.2–146.1
145.2–146.1
115.5–116.0
118.58
145.4–146.1q
u
145.0–146.0
0
0
115.81c
118.19
g
m
115.0–116.0v
x
ac
115.63
119.39
115.7–116.0
118.62
118.55
M2
(
2
3
4
4
5
6
7
8
8
1
2
3
4
5
6
2
3
4
4
5
6
7
8
8
1
2
3
4
5
6
76.72
73.31
37.41
76.72
73.52
38.15
76.84
73.78
37.90
76.58
73.09
37.30
102.12
156.26
77.06
71.11
37.69
99.49
157.94
97.09
158.26
107.27
154.78
131.83
115.88
77.68
73.22
38.25
98.33
158.26
97.02
158.75
107.87
155.91
132.04
115.09
77.78
73.22
37.39
97.88
159.41
96.78
160.44
96.32
158.22
131.62
115.08
76.65
73.42
37.43
102.03
156.28
96.85
76.69
73.51
37.43
102.03
156.27
96.83
156.95
107.03
154.88
132.58
114.81
145.2–146.1
145.2–146.1
115.5–116.0
118.31
76.82
73.51
36.97
102.03
157.80
95.83
157.98
95.88
157.90
132.58
114.98
145.2–146.1
145.2–146.1
115.5–116.0
al
U2 of 8)
k
a
am
101.09
156.87
108.47
156.59
96.18
155.49
132.30
114.76
145.0–146.0
103.89
155.78
105.90
154.38
96.18
154.78
132.80
115.60
145.23
145.12
116.35
120.16
76.97
103.57
156.04
106.16
154.78
95.97
155.04
133.14
115.68
145.59
145.59
115.56
119.99
77.16
o
96.90
156.98
n
156.96
107.03
154.92
132.52
114.80
145.83
145.21
115.7–116.0
118.24
76.82
73.51
36.99
101.99
157.98
95.89
157.83
95.89
157.89
132.57
114.98
145.62
145.33
115.7–116.0
119.08
107.07
154.79
132.61
114.70
a
0
0
0
0
e
an
g
h
i
b
j
j
145.5–145.7l
q
u
u
ah
ah
ai
145.4–146.1
145.0–146.0
145.0–146.0
146.09
145.4–146.1q
145.94w
145.34
145.06
0
0
115.81c
f
m
115.0–116.0v
x
ac
115.6–116.0
118.50
76.78
73.53
36.98
102.00
157.79
95.82
157.92
95.86
115.76
119.76
76.65
73.76
36.61
101.96
157.6–157.9
95.89
157.6–157.9
115.0–116.0
119.24
76.89
73.89
37.06
101.70
157.91
95.78
157.99
95.81
157.93
132.45
114.99
145.83
145.48
115.0–116.0
118.84
118.95
77.34
73.20
118.83
76.95
72.41
U1
al
71.80
37.90
99.00
159.03
96.75
72.74
37.61
98.55
159.83
96.16
37.34
35.95
a
am
100.35
159.15
95.65d
102.45
157.03
95.69
157.32
95.54
158.00
132.52
114.29
t
t
y
ad
ak
158.86
158.63
96.52
159.54
95.94
95.87d
95.76s
157.76
132.75
114.52
145.25
144.91
116.18
119.06
a
0
0
0
0
158.12
132.62
115.26
145.0–146.0
145.0–146.0
115.50
119.54
157.65
131.88
116.14
145.12
145.23
115.49
120.96
158.08
132.95
115.51
145.59
145.22
115.12
119.94
157.85
132.54
an
ae
ag
114.87
b
b
i
j
145.5–145.7l
u
u
ah
ah
ai
145.0–146.0
145.0–146.0
h
145.29
115.6–116.0
119.01
0
0
m
115.0–116.0v
x
ac
aj
119.66
119.12
b,c,e,g–j,l–z,aa–ad,ag–an
a,d,f,k,af
Overlapped with each other.
Values with the same superscript letters are not significantly different.
signed on the basis of the HMBC correlation with H2-unit T
interflavonoid bonds were all determined as C4 ? C8 because the
C8a of units T , M and M were correlated with H4 of units M
and U in HMBC, respectively. Thus, structure 4 was elucidated
as EC-(4b ? 8)-EC-(4b ? 8)-EC-(4b ? 8)-Cat.
1
, and the
Compound 5 consisted of one catechin as the terminal unit and
three epicatechins as extension units, as indicated by phloroglu-
1
1
2
1
,
cinol degradation analysis. In the HMBC spectrum, the H6-unit T
showed correlations with C5 and C7, suggesting that unit M was
attached to the C8-unit T . The connection between units M and
1
M
2
1
1
1
1

1
48
S. Nakashima et al. / Phytochemistry 83 (2012) 144–152
Fig. 2. Structures of isolated compounds: EC-(4b ? 6)-EC-(4b ? 8)-EC-(4b ? 8)-Cat (1); EC-(4b ? 6)-EC-(4b ? 8)-EC-(4b ? 6)-Cat (2); EC-(4b ? 6)-EC-(4b ? 8)-EC-
4b ? 6)-EC (3); EC-(4b ? 8)-EC-(4b ? 8)-EC-(4b ? 8)-Cat (4); EC-(4b ? 8)-EC-(4b ? 6)-EC-(4b ? 8)-Cat (5); EC-(4b ? 8)-EC-(4b ? 6)-EC-(4b ? 8)-EC (6); EC-(4b ? 6)-
EC-(4b ? 8)-EC-(4b ? 8)-EC (7); EC-(4b ? 8)-EC-(4b ? 6)-[EC-(4b ? 8)]-EC (8); EC-(4b ? 8)-EC-(4b ? 8)-EC-(4b ? 6)-Cat (9); EC-(4b ? 8)-EC-(4b ? 8)-EC-(4b ? 8)-EC
10); EC-(4b ? 8)-EC-(4b ? 8)-EC-(4b ? 6)-EC (11).
(
(
M
2
was determined to be C4 ? C6 on the basis of the correlation
between the H8-unit M and the C8a-unit M . In the unit M , the
C5-unit M was correlated with both H4-unit M and H6-unit M
indicating that the connection between units M and U was
C4 ? C8. Therefore, structure 5 was elucidated as EC-(4b ? 8)-EC-
C8a-unit T
determined to be C4 ? C6 on the basis of the correlation from both
H4-unit M and H4-unit M to C5-unit M . The connection be-
tween units M and U was determined to be C4 ? C8 on the basis
of the correlation from both H4-unit M and H6-unit M to C5-unit
. Therefore, structure 6 was elucidated as EC-(4b ? 8)-EC-
(4b ? 6)-EC-(4b ? 8)-EC.
1 1 2
. The connection between units M and M was also
1
1
2
2
2
2
,
1
2
1
2
1
2
1
2
2
(
4b ? 6)-EC-(4b ? 8)-Cat.
M
2
Compound 6 consisted of four epicatechins, as established by
phloroglucinol degradation analysis. Only three carbon signals
showed no isotopic shift in the H-D exchange experiment; thus,
overlapping of the C8a resonances seemed to occur. In the HMBC
Compound 8 consisted of four epicatechins, as demonstrated by
phloroglucinol degradation analysis. Different from other tetra-
mers, it seemed to include two ends of extension, as indicated by
spectrum, the H6-unit T
cating that the connection between units T
This was also confirmed by the correlation from H4-unit M
1
showed correlations with C5 and C7, indi-
and M was C4 ? C8.
to
two sets of coupled A-ring protons (d
H
5.94, 5.98 and d
6.11) in the H NMR spectrum. The HMBC correlation from both
H4-unit U and H4-unit M to C8a-unit M indicated a C4 ? C8
H
6.08,
1
1
1
1
1
1
1

S. Nakashima et al. / Phytochemistry 83 (2012) 144–152
149
Fig. 3. Effect of solvent on the 1H NMR spectrum of 2. (a) in methanol-d
at 243 K, (b) in methanol-d /deuterium oxide (25:15) at 278 K.
4 4
connection between these units. The H4-unit M
tion to another three oxygen-attached aromatic carbons in the d
range of 153–158, which showed isotopic shifts in the H-D
exchange experiment. This observation indicated that unit M
was never attached to C8 of the next unit. Meanwhile, the H4-unit
showed an HMBC correlation to C8a-unit T , by which the con-
nection between units T and U was determined to be C4 ? C8.
Moreover, both the H4-unit M and the H4-unit U showed a cor-
relation to the C7-unit T ; thus, it was confirmed that unit T was
attached to two extension units (units M –U and unit U ), and
1
showed a correla-
finally structure 8 was elucidated as EC-(4b ? 8)-EC-(4b ? 6)-
[EC-(4b ? 8)]-EC. Key HMBC correlations are depicted in Fig. 4.
Compound 9 consisted of one catechin as the terminal unit and
three epicatechins as extension units, as shown by phloroglucinol
C
1
degradation analysis. In the HMBC spectrum, the C8a-unit T
correlated with both H2-unit T and H8-unit T , indicating that
the unit T was connected to unit M through a C4 ? C6 interflavo-
noid bond. The H8-unit T was severely overlapped with the
H6-unit M and their HMBC cross-peaks were hard to distinguish,
but they were discriminable in methanol-d . In unit M , both
H4-unit M and H6-unit M were correlated with the C5-unit M
thus, the connection between units M and M
be C4 ? C8. Likewise, the connection between units M
1
was
U
2
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
2
3
1
1
1
1
;
1
2
was suggested to
2
and U
1
was determined to be C4 ? C8 on the basis of the correlation from
both H4-unit M and H6-unit M to C5-unit M . Therefore, structure
was elucidated as EC-(4b ? 8)-EC-(4b ? 8)-EC-(4b ? 6)-Cat.
Compound 11 consisted of four epicatechins, as demonstrated
by phloroglucinol degradation analysis. In the HMBC, the H8-unit
showed a correlation with the C8a-unit T , indicating that the
connection between units T and M was C4 ? C6. The connection
between units M and M was also determined to be C4 ? C8 on the
basis of the correlation from both H4-unit M and H4-unit M to
C8a-unit M . The connection between units M and U was then
determined to be C4 ? C8 on the basis of the correlation from both
H4-unit M and H6-unit M to C5-unit M . Therefore, structure 11
was elucidated as EC-(4b ? 8)-EC-(4b ? 8)-EC-(4b ? 6)-EC.
2
2
2
9
T
1
1
1
1
1
2
1
2
1
2
1
2
2
2
3
. Conclusion
To determine the position of interflavonoid linkages in the olig-
omeric procyanidins by NMR spectroscopic analysis, key observa-
tions for unambiguous elucidation are: (1) discrimination of C8a
Fig. 4. Key HMBC correlations (arrow) of 8.

1
50
S. Nakashima et al. / Phytochemistry 83 (2012) 144–152
from C5 and C7; (2) HMBC correlation from A-ring proton (H6 or
H8); (3) HMBC correlation from H4 in upper and lower units. In
the case of a C4 ? C8 connected dimeric structure, the H4 in both
upper and lower units show a HMBC correlation to C8a in the low-
er unit, theoretically. The clear correlations from H6 to both C5 and
C7, or from both H4 and H6 to C5 in the lower unit, also indicate
the connection through C8. As for the C4 ? C6 connected dimer,
the H4 in both upper and lower units show a HMBC correlation
to C5 in the lower unit. HMBC correlation from H8 to C8a indicates
the connection through C6. In the practical case of higher oligo-
mers, signal overlapping sometimes occurs at H4, or A-ring protons
and carbons. The most important thing is to pick out the evident
correlation, which enables avoidance of misreading along with
the theory described above.
Taken together, the detectable tetrameric procyanidins in an
unripe apple extract were isolated in detail and the compounds
were unambiguously identified by NMR spectroscopic and phloro-
glucinol degradation analyses. To our knowledge, this is the first
report of the structural diversity of tetrameric procyanidins in
apple. Additionally, the isolation and identification of compounds
(+)-Catechin, (ꢀ)-epicatechin, and (+)-taxifolin were purchased
from Funakoshi Co., Ltd. (Tokyo, Japan). Phloroglucinol was pur-
chased from Sigma–Aldrich Co. (St. Louis, MO). All other reagents
and chemicals were of special grade of Japanese Industrial Stan-
dards unless otherwise stated.
4.2. Materials
The apple procyanidin fraction was prepared from unripe apples
(Malus pumila cv. Fuji) in the factory scale, according to the method
of Shoji et al. (2003) with slight modification. Briefly, the crude
apple polyphenol extract was obtained from unripe apple juice by
solid phase extraction with SEPABEADSÒ SP-70 (Mitsubishi Chem-
ical Corporation, Tokyo, Japan). Next, the extract was adjusted to
pH 7.0 and applied to a column filled with DiaionÒ HP-20 (Mitsubi-
shi Chemical Corporation, Tokyo, Japan). Adsorbed apple procyani-
dins were rinsed with distilled H
2 2
O, followed by elution EtOH-H O
(
21:79, w/w). The corresponding eluate was concentrated and
spray-dried to obtain the powdered apple procyanidin fraction.
1, 2, 5, 6, and 8 were reported for the first time. To obtain new in-
4.3. Isolation
sights on structure–activity relationship of oligomeric procyani-
dins, further investigations with these procyanidins are needed.
The apple procyanidin fraction (12 g) was further fractionated
according to the DP by semi-preparative HPLC with a Deverosil
1
00diol-5 column (i.d. 20 ꢁ 250 mm, 5
Torrance, CA) (Kelm et al., 2006). The binary mobile phase con-
sisted of (A) CH CN and (B) MeOH/H O (95:5, v/v). The gradient
program was as follows: 0–3 min, 5% B; 3–60 min, 5–30% B; 60–
3 min, 30–100% B; 63–70 min, 100% B; 70–76 min, 100–5% B;
lm) (Phenomenex Inc.,
4
. Experimental
3
2
4.1. General experimental procedures
6
1H (600 MHz), 13C (150 MHz), and 2D NMR spectra were mea-
76–86 min, 5% B. The column temperature was 35 °C and the flow
sured using a Bruker AV600 equipped with a Polycold refrigerant
system. LC-ESI-TOFMS analysis was conducted with an Applied
Biosystems QSTAR Elite equipped with two Hitachi L-2100 pumps,
a Hitachi L-2300 column oven, and a Hitachi L-2200 autosampler.
Semi-preparative HPLC was conducted with a GL Science PLC761
LC system, which consists of two PU714 pumps, an UV702 UV–
VIS detector, a CO705 column oven, an FC204 fraction collector,
and an SC762 system controller. Optical rotations were measured
in MeOH with a JASCO-DIP1000 Perkin Elmer 341 digital polarim-
rate was 12.0 mL/min. The apple procyanidin fraction was dis-
solved in MeOH (0.5 g/mL) and applied after filtration (0.45 lm)
(GL Science, Tokyo, Japan). The eluate was monitored by absor-
bance at 280 nm and collected as follows: 6–10 min, as Fr.1;
15–20 min, as Fr.2; 27–32 min, as Fr.3; 37–42 min, as Fr.4; 45–
50 min, as Fr.5; 52–56 min, as Fr.6; 58–62 min, as Fr.7; 63–
66 min, as Fr.8; and 68–71 min, as Fr.9. The obtained amounts of
nine fractions were 1.66, 1.42, 1.05, 1.02, 0.70, 0.61, 0.35, 0.25,
and 1.24 g, respectively.
eter. UV spectra were recorded in H
photometer. IR (Film) spectra were recorded on a Shimazu
IRPrestige-21.
2
O on a Hitachi U-2900 spectro-
The Fr.4 was confirmed to include 11 tetrameric procyanidins
by LC-ESI-TOFMS (Fig. 5), and an aliquot of this fraction (593 mg)
was further purified to obtain isolated compounds by RP-HPLC
Fig. 5. Extracted ion chromatogram (m/z 1153 to 1154) of tetrameric procyanidin fraction by negative ESI-TOFMS analysis.

S. Nakashima et al. / Phytochemistry 83 (2012) 144–152
151
using Inertsil ODS-3 column (i.d. 20 ꢁ 250 mm, 4
Inc., Japan). Compounds 1, 6, 7, 9, and 10 were isolated by isocratic
elution with CH CN-H O (12:88, v/v) at 12.0 mL/min and by peak
collection, but others were obtained as mixed fractions. Therefore,
these fractions were subjected to recycling HPLC and every
remaining compound was completely isolated. The amounts of
isolated compounds were described as follows: 1, 11.4 mg; 2,
l
m) (GL Science
4.3.8. Epicatechin-(4b ? 8)-epicatechin-(4b ? 8)-epicatechin-
(4b ? 6)-catechin (9)
D
3
2
Pale brown amorphous solid; ½
(water) kmax (log
1522, 1447, 1283, 1148, 1109, 1063 cm ; for H and C NMR
a
ꢂ
2
7
= +130 (c 0.10, MeOH); UV
e) 278 (4.13) nm; IR (film) cmax 3422, 1609,
ꢀ1
1
13
spectroscopic data, see Tables
1155.2779 ([M + H] , calcd for C60
1
and 2; HRESITOFMS m/z
24, 1155.2692).
+
H
51
O
1
.2 mg; 3, 3.1 mg; 4, 28.9 mg; 5, 13.6 mg; 6, 27.4 mg; 7, 23.9 mg;
8, 5.7 mg; 9, 3.7 mg; 10, 94.8 mg; and 11, 9.9 mg.
4.3.9. Epicatechin-(4b ? 8)-epicatechin-(4b ? 8)-epicatechin-
(
4b ? 6)-epicatechin (11)
D
4.3.1. Epicatechin-(4b ? 6)-epicatechin-(4b ? 8)-epicatechin-
Pale brown amorphous solid; ½
a
ꢂ
= +120 (c 0.10, MeOH); UV
27
(
4b ? 8)-catechin (1)
(water) kmax (log
1518, 1445, 1285, 1148, 1111, 1061 cm ; for H and C NMR
e
) 278 (4.13) nm; IR (film)
c
max 3422, 1609,
D
2
ꢀ1
1
13
Pale brown amorphous solid; ½
a
ꢂ
= + 110 (c 0.10, MeOH); UV
4
(
1
water) kmax (log
e
) 278 (4.12) nm; IR (film)
c
max 3421, 1609,
spectroscopic data, see Tables
1155.2677 ([M + H] , calcd for C60
1
and 2; HRESITOFMS m/z
24, 1155.2692).
522, 1445, 1283, 1109, 1055 cm ; for 1H and C NMR spectro-
ꢀ1
13
+
H
51
O
scopic data, see Tables 1 and 2; HRESITOFMS m/z 1155.2736
+
(
[M + H] , calcd for C60
H
51
O
24, 1155.2692).
4.4. Phloroglucinol degradation analysis
4
.3.2. Epicatechin-(4b ? 6)-epicatechin-(4b ? 8)-epicatechin-
To investigate the constitutive flavan-3-ol units of the isolated
tetrameric procyanidins, acid-catalyzed degradation in the pres-
ence of phloroglucinol was conducted according to the method of
Kennedy and Jones (2001). Phloroglucinol (1 g) and ascorbic acid
(200 mg) were dissolved in MeOH (20 mL) containing 0.1 N HCl.
Each procyanidin was reacted in this solution (5 g/L) at 45 °C for
30 min, followed by addition of a 5-fold volume of 40 mM NaOAc
to stop the reaction.
(
4b ? 6)-catechin (2)
D
Pale brown amorphous solid; ½
a
ꢂ
= + 160 (c 0.10, MeOH); UV
25
(
water) kmax (log e) 278 (4.15) nm; IR (film) cmax 3414, 1609,
522, 1445, 1285, 1115, 1063 cm ; for 1H and C NMR spectro-
ꢀ
1
13
1
scopic data, see Tables 1 and 2; HRESITOFMS m/z 1155.2721
+
(
[M + H] , calcd for C60
51
H O24, 1155.2692).
Reaction products were analyzed by RP-HPLC using an Inertsil
4
.3.3. Epicatechin-(4b ? 6)-epicatechin-(4b ? 8)-epicatechin-
ODS-3 column (i.d. 4.6 ꢁ 250 mm, 4
l
m). The binary mobile phase
(
4b ? 6)-epicatechin (3)
D
consisted of (A) 0.1% HCO H and (B) CH
2
3
CN containing 0.1% HCO H.
2
Pale brown amorphous solid; ½
a
ꢂ
= + 130 (c 0.10, MeOH); UV
25
The gradient program was as follows: 0–10 min, 5% B; 10–40 min,
5–20% B; 40–40.1 min, 20–80% B; 40.1–50 min, 80% B; 50–
0.1 min, 80–5% B; 50.1–65 min, 5% B. The column temperature
was 35 °C and the flow rate was 1.0 mL/min. Eluate was monitored
at 280 nm. Detected peaks were identified by comparing their
retention times to standard compounds, such as (+)-catechin and
(
water) kmax (log e) 278 (4.17) nm; IR (film) cmax 3422, 1611,
522, 1445, 1285, 1117, 1059 cm ; for 1H and C NMR spectro-
ꢀ1
13
1
5
scopic data, see Tables 1 and 2; HRESITOFMS m/z 1155.2688
+
(
[M + H] , calcd for C60
51
H O24, 1155.2692).
4.3.4. Epicatechin-(4b ? 8)-epicatechin-(4b ? 8)-epicatechin-
(
ꢀ)-epicatechin. Catechin-(4
from (+)-taxifolin according to the method of Kennedy and Jones
2001). Briefly, (+)-taxifolin (51.2 mg) and NaBH (24.5 mg) were
a ? 2)-phloroglucinol was prepared
(
4b ? 8)-catechin (4)
D
Pale brown amorphous solid; ½
aꢂ
= +84 (c 0.10, MeOH); UV
25
(
4
(
water) kmax (log e) 278 (4.09) nm; IR (film) cmax 3377, 1609,
522, 1447, 1285, 1107, 1061 cm ; for 1H and C NMR spectro-
ꢀ1
13
stirred in absolute EtOH (10 mL) at room temperature for 1 h.
Phloroglucinol (175.5 mg) was dissolved in 0.1 N HCl (10 mL) and
then combined with the reaction mixture and stirred for 30 min,
1
scopic data, see Tables 1 and 2; HRESITOFMS m/z 1155.2751
+
(
[M + H] , calcd for C60
51
H O24, 1155.2692).
2
followed by addition of 10 mL H O and extraction with EtOAc
(
3 ꢁ 10 mL). The extract was lyophilized to dry powder. The pro-
4
.3.5. Epicatechin-(4b ? 8)-epicatechin-(4b ? 6)-epicatechin-
duction of the desired compound was confirmed by LC-ESI-TOFMS
analysis. The retention time of epicatechin-(4b ? 2)-phloroglucinol
was confirmed by phloroglucinol degradation of procyanidin B2,
which had been previously isolated.
(
4b ? 8)-catechin (5)
D
Pale brown amorphous solid; ½
aꢂ
= +150 (c 0.10, MeOH); UV
26
(
water) kmax (log e) 278 (4.13) nm; IR (film) cmax 3422, 1616,
ꢀ1
1
13
1
522, 1449, 1285, 1211, 1090, 1057 cm ; for H and C NMR
spectroscopic data, see Tables and 2; HRESITOFMS m/z
24, 1155.2692).
1
+
Acknowledgements
1
51
155.2715 ([M + H] , calcd for C60H O
We thank Ms. Kakuguchi and Ms. Ikuine for their great techni-
cal support, Prof. Matsunaga and Mr. Hitora for measurement of
optical rotations, and Mr. Sakai for measurement of IR spectra.
4
.3.6. Epicatechin-(4b ? 8)-epicatechin-(4b ? 6)-epicatechin-
(
4b ? 8)-epicatechin (6)
D
Pale brown amorphous solid; ½
aꢂ
= +89 (c 0.10, MeOH); UV
26
(
water) kmax (log e) 278 (4.08) nm; IR (film) cmax 3422, 1609,
522, 1447, 1285, 1109, 1057 cm ; for 1H and C NMR spectro-
ꢀ1
13
Appendix A. Supplementary data
1
scopic data, see Tables 1 and 2; HRESITOFMS m/z 1155.2705
+
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
(
[M + H] , calcd for C60
H
51
O
24, 1155.2692).
0
7.011. These data include MOL files and InChiKeys of the most
4
.3.7. Epicatechin-(4b ? 8)-epicatechin-(4b ? 6)-[epicatechin-
important compounds described in this article.
(
4b ? 8)-]epicatechin (8)
Pale brown amorphous solid; ½
water) kmax (log
518, 1449, 1285, 1148, 1111, 1061 cm ; for H and C NMR
spectroscopic data, see Tables and 2; HRESITOFMS m/z
24, 1155.2692).
D
aꢂ
= +110 (c 0.10, MeOH); UV
26
References
(
1
e) 278 (4.11) nm; IR (film) cmax 3422, 1609,
ꢀ1
1
13
Abe, Y., Shoji, T., Kawahara, N., Kamakura, H., Kanda, T., Goda, Y., Ozeki, Y., 2008.
Structural characterization of a procyanidin tetramer and pentamer from the
apple by low-temperature NMR analysis. Tetrahedron Lett. 49, 6413–6418.
1
+
1
51
155.2755 ([M + H] , calcd for C60H O

1
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