Antioxidant lignoids from leaves of Ribes nigrum

Article abstract of DOI:10.1016/j.phytochem.2013.07.022

Phytochemical investigation of the leaves of Ribes nigrum resulted in the isolation of fourteen compounds, including four 7,7′-epoxylignans, three tetrahydrofuran-type sesquilignans, and a spirocyclic dilignan. Their structures were elucidated by extensive spectroscopic analyses and by chemical transformations. The isolated compounds were evaluated for their antioxidant activities using superoxide anion scavenging assay and DPPH free radical scavenging assay. Ribesin D and ribesin G showed the most potent superoxide anion scavenging activity with EC₅₀ values of 1.24 and 1.12 μM, respectively, and the structure-activity relationship was discussed.

Full text of DOI:10.1016/j.phytochem.2013.07.022

Phytochemistry 95 (2013) 333–340
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Antioxidant lignoids from leaves of Ribes nigrum
a
b
c
d
Tatsunori Sasaki a, Wei Li a, , Shinnosuke Zaike , Yoshihisa Asada , Qin Li , Fenghua Ma ,
⇑
Qingbo Zhang d, Kazuo Koike a
a Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
b Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
c Laboratory Center, The Fourth Affiliated Hospital, China Medical University, Shenyang 110032, People’s Republic of China
d Heilongjang Institute for Food and Drug Control, Harbin 150001, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytochemical investigation of the leaves of Ribes nigrum resulted in the isolation of fourteen compounds,
including four 7,70-epoxylignans, three tetrahydrofuran-type sesquilignans, and a spirocyclic dilignan.
Their structures were elucidated by extensive spectroscopic analyses and by chemical transformations.
The isolated compounds were evaluated for their antioxidant activities using superoxide anion scaveng-
ing assay and DPPH free radical scavenging assay. Ribesin D and ribesin G showed the most potent
Received 21 September 2012
Received in revised form 15 February 2013
Available online 16 August 2013
Keywords:
superoxide anion scavenging activity with EC50 values of 1.24 and 1.12
ture–activity relationship was discussed.
lM, respectively, and the struc-
Ribes nigrum
Grossulariaceae
Lignan
Ó 2013 Elsevier Ltd. All rights reserved.
Sesquilignan
Dilignan
Antioxidant activity
1. Introduction
compounds were evaluated by superoxide anion scavenging assay
and DPPH free radical scavenging assay.
The genus Ribes belongs to the family of Grossulariaceae and has
about 150 different species. Ribes nigrum, known as blackcurrant, is
a perennial small shrub, which is widely distributed in Europe and
North Asia, and is cultivated in many countries for its usage of the
fruits in the food industry (Slimestad and Solheim, 2002; Castillo
et al., 2004; Zheng et al., 2009). The leaves of R. nigrum have been
used as a traditional medicine for treatment of rheumatic disease
in Europe (Garbacki et al., 2004), and have been shown to exhibit
antioxidant and anti-inflammatory effects (Garbacki et al., 2005,
2002). Although the finding of prodelphinidins and flavonoids
has been previously reported (Tits et al., 1992; He et al., 2010;
Tabart et al., 2011), knowledge on the chemical composition of
their leaves is still quite limited. As a part of ongoing studies on
bioactive compounds from traditional medicinal plants (Li et al.,
2012; Bi et al., 2011), a phytochemical investigation was carried
out herein on the leaves of R. nigrum. This work resulted in the
isolation of fourteen compounds, including four novel 7,70-epoxy-
lignans (1–4), three novel tetrahydrofuran-type sesquilignans
(7–9) and one novel spirocyclic dilignan (10) (Fig. 1). Their struc-
tures were elucidated by extensive spectroscopic analyses and
chemical transformations. The antioxidant activities of the isolated
2. Results and discussion
The air-dried leaves of R.nigrum were extracted with 70% etha-
nol. The extract was separated by Diaion HP-20, silica gel, and ODS
column chromatography, and further purification was carried out
by reversed phase preparative HPLC to afford eight novel com-
pounds (1–4, 7–10) and six known compounds.
The known compounds were identified as (ꢀ)-larreatricin (5)
(Moinuddin et al., 2003), 3,30-didemethoxynectandrin B (6) (Xorge
et al., 1990), roseoside (11) (Otsuka et al., 1995), icariside B1 (12)
(Hisamoto et al., 2004), kaempferol (13) (Hadizadeh et al., 2003),
and methyl p-coumarate (14) (Kwon and Kim, 2003), by detailed
NMR, MS, optical rotation and CD spectroscopic analyses, as well
as by comparison with literature data. The previously unknown
lignoids (1–4, 7–10) were isolated as colorless solids, and their
structures were determined as follows.
The molecular formulas of ribesin A (1) and ribesin C (3) were
C
18H18O3, and ribesin B (2) and ribesin D (4) were C20H22O5, by
analyses of the negative-ion HRESIMS data. In the 13C NMR spectra
of 1 and 2, the number of carbon resonances was observed by half,
suggesting their symmetrical nature.
As shown in Table 1, the 1H and 13C NMR spectroscopic data of 1
indicated characteristic A2X2-type aromatic proton resonances at
dH 7.15 and 6.82, as well as carbon resonances at dC 133.9, 128.5,
⇑
Corresponding author. Tel.: +81 47 4721161; fax: +81 47 4721404.
E-mail address: liwei@phar.toho-u.ac.jp (W. Li).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.07.022

334
T. Sasaki et al. / Phytochemistry 95 (2013) 333–340
Fig. 1. Chemical structures.
Table 1
1H and 13C NMR spectroscopic data (d) for compounds 1–4 in CD3OD.
Position
1
2
3
4
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
1
2
3
133.9
128.5
115.2
135.9
114.9
149.1
56.5
132.3
129.2
115.9
134.5
115.0
148.4
56.5
7.15 (d, 8.5)
6.82 (d, 8.5)
6.74 (d, 2.0)
3.84 (s)
7.10 (d, 8.5)
6.75 (d, 8.5)
6.76 (d, 2.3)
3.83 (s)
3-OCH3
4
5
6
7
157.2
115.2
128.5
91.2
131.2
9.8
133.9
128.5
115.2
147.8
112.5
119.9
92.7
132.0
10.3
135.9
114.9
149.1
56.5
147.8
112.5
119.9
92.7
158.0
115.9
129.2
85.3
43.4
14.6
135.0
129.0
116.2
157.2
158.3
116.2
129.0
84.4
147.3
112.4
119.4
85.3
43.2
14.4
137.1
114.6
148.7
56.5
147.6
112.6
119.0
84.4
6.82 (d, 8.5)
7.15 (d, 8.5)
5.63 (s)
6.89 (d, 8.5)
6.76 (dd, 8.5, 2.0)
5.59 (s)
6.75 (d, 8.5)
7.10 (d, 8.5)
5.28 (d, 7.4)
3.15 (m)
6.86 (d, 8.3)
6.71 (dd, 8.3, 2.3)
5.23 (d, 7.6)
3.13 (m)
8
9
1.48 (s)
1.50 (s)
0.78 (d, 6.9)
0.77 (d, 7.1)
10
20
7.15 (d, 8.5)
6.82 (d, 8.5)
6.74 (d, 2.0)
3.84 (s)
7.23 (d, 8.5)
6.77 (d, 8.5)
6.88 (d, 1.8)
3.83 (s)
30
30-OCH3
40
157.2
115.2
128.5
91.2
131.2
9.8
50
6.82 (d, 8.5)
7.15 (d, 8.5)
5.63 (s)
6.89 (d, 8.5)
6.76 (dd, 8.5, 2.0)
5.59 (s)
6.77 (d, 8.5)
7.23 (d, 8.5)
5.63 (br s)
6.89 (d, 8.0)
6.86 (dd, 8.0, 1.8)
5.60 (d, 2.1)
60
70
80
132.0
10.3
157.9
106.9
157.5
106.9
90a
90b
1.48 (s)
1.50 (s)
4.92 (t, 2.1)
4.99 (t, 2.1)
4.95 (t, 2.1)
4.99 (t, 2.1)
115.2 and 157.2, for a 4-hydroxyphenyl moiety. The 1H NMR spec-
troscopic data also showed two singlet proton resonances, one for
a quaternary methyl at dH 1.48, and the other for an oxymethine at
dH 5.63, and their corresponding carbon resonances were assigned
to dC 9.8 and 91.2, respectively, as indicated by the HMQC spectro-
scopic data. In the 13C NMR spectrum, in addition to the aforemen-
tioned carbon resonances, the remaining resonance was an olefinic
quaternary carbon at dC 131.2. The connection of these moieties in
the sequence of 4-hydroxyphenyl, oxymethine, olefinic quaternary
carbon, and quaternary methyl, was determined by detailed anal-
ysis of the HMBC spectroscopic data. Namely, in the HMBC spec-
3
trum, the JCH correlations were observed from dH 7.15 to dC 91.2,
2
dH 5.63 to dC 9.8, and dH 1.48 to dC 91.2, and the JCH correlations
were from dH 5.63 and 1.48 to dC 131.2 (Fig. 2). Taking account of
the symmetrical structure and the chemical formula, the presence
of 2,5-dihydrofuran ring moiety in 1 was elucidated.
Comparison of the 1H and 13C NMR spectroscopic data of 2 and 1,
suggested that 2 is only structurally different from 1 by its aromatic
constituents. Namely, the presence of 4-hydroxy-3-methoxyphenyl
moieties in 2 was indicated by the ABX-type aromatic proton reso-

T. Sasaki et al. / Phytochemistry 95 (2013) 333–340
335
Fig. 2. Key HMBC correlations for compounds 1–4, 7 and 10.
nances at dH 6.89, 6.76 and 6.74, and the methoxy resonance at dH
3.84. The position of the methoxy group was confirmed by the
NOESY correlation between dH 3.84 and 6.74.
To determine the absolute configurations of compounds 1–4,
Pd/C catalyzed reductions were carried out. From 1 and 3, the same
product was obtained with identical spectroscopic data as for (ꢀ)-
larreatricin (5), which was also isolated in this study. From 2 and 4,
was obtained the same product 15, whose structure was indenti-
The 1H NMR spectroscopic data of 3 indicated the presence of
two 4-hydroxyphenyl moieties by two groups of A2X2-type aro-
matic proton resonances at dH 6.77 and 7.23, and dH 6.75 and
7.10. Further resonances were observed for a methyl at dH 0.78
(H-9), a methine at dH 3.15 (H-8), a terminal methylene at dH
4.92 and 4.99 (Ha,b-90), and two oxymethine protons at dH 5.28
and 5.63 (H-7, 70). In the 13C NMR spectrum, except for the carbon
resonances assignable to the above-mentioned moieties, it also
showed two olefinic quaternary carbon resonances at dC 157.9
and 106.9 (C-80 and C-90). The presence of a methyl–methine–oxy-
methine moiety (CH3-9–CH-8–CH(O)-7) was deduced from the
correlations between their protons in the 1H–1H COSY spectrum,
fied to be 3,30-dimethoxylarreatricin by detailed spectroscopic
25
analyses. Both optical rotation values of compounds 5 {[
a
]
:
D
ꢀ20.3 (c 0.40, MeOH)} and 15 {[
a]
25: ꢀ78.0 (c 0.08, MeOH)} were
D
similar to that, with previously reported natural compounds for
(ꢀ)-larreatricin {[
a]
20: ꢀ19.3 (c 0.0145, MeOH)} from Larrea
D
tridentata (Moinuddin et al., 2003) and 3,30-dimethoxylarreatricin
{[a]
25: ꢀ85.7 (c 0.096, CHCl3)} from Castanea mollissima (Long
D
et al., 2008). However, their absolute configurations were not yet
determined. Fortunately, the absolute configuration of (ꢀ)-chica-
nine with a very similar structure to that of 15 was recently estab-
lished by its asymmetric total synthesis (Harada et al., 2011).
Therefore, the absolute configuration of 15 was assigned by com-
parison of the CD data and optical rotation data with those of
(ꢀ)-chicanine. Namely, the CD spectrum of 15 had the same nega-
tive Cotton effects at both 231 nm (K absorption band) and 286 nm
0
0
and the D8 ,9 double bond connecting to C-70 was confirmed by
the HMBC correlations from dH 4.92 and 4.99 (Ha,b-90) to dC 84.4
(C-70) and 157.9 (C-80). Furthermore, the HMBC correlations from
dH 0.78 (H3-9) to dC 157.9 (C-80), dH 4.92, 4.99 (Ha,b-90) to dC 43.4
(C-8) and 157.9 (C-80), dH 5.28 (H-7) to dC 157.9 (C-80), and dH
5.63 (H-70) to dC 43.4 (C-8) and 157.9 (C-80) (Fig. 2), indicated the
presence of a tetrahydrofuran ring in 3. Two 4-hydroxyphenyl moi-
eties connecting to C-7 and C-70 oxymethines were deduced from
the HMBC correlations from dH 7.10 (H-2, 6) to dC 85.3 (C-7), and
dH 7.23 (H-20, 60) to dC 84.4 (C-70) (Fig. 2). By these analyses, the
gross structure of 3 was elucidated. The relative configurations in
3 were assigned by NOESY spectroscopic data analysis. The NOESY
correlations between dH 7.10 (H-2,6)/0.78 (H3-9), 7.10 (H-2,6)/5.63
(H-70), 5.28 (H-7)/3.15 (H-8) and 5.28 (H-7)/7.23 (H-20,60), indi-
cated that H-7 and H-70 are trans-, and H-7 and H-8 are cis-rela-
tionship (Fig. 3).
(B absorption band) as (ꢀ)-chicanine due to
p–
p⁄ transitions, sug-
gesting that they have same absolute configurations (2S,3R,4S,5S).
Although the Cotton effects at B band (ca 280 nm) were too weak
to be observed for 5, the same negative Cotton effect at 232 nm
in the CD spectrum, and the same negative value in optical rotation
with those of 15, indicated 15 had also the same absolute
configurations.
Conclusively, ribesin A (1) was determined to be (7R,70R)-8(80)-
ene-4,40-dihydroxy-7,70-epoxylignan, ribesin B (2) to be (7R,70R)-
8(80)-ene-4,40-dihydroxy-3,30-dimethoxy-7,70-epoxylignan, ribesin
C (3) was identified to be (7S,70S,8R)-80(90)-ene-4,40-dihydroxy-
7,70-epoxylignan, ribesin D (4) was determined as (7S,70S,8R)-80
(90)-ene-4,40-dihydroxy-3,30-dimethoxy-7,70-epoxylignan, and the
known compounds of (ꢀ)-larreatricin (5) to be (7S,70S,8R,80S)-4,40-
dihydroxy-7,70-epoxylignan, and 3,30-dimethoxylarreatricin (15)
to be (7S,70S,8R,80S)-4,40-dihydroxy-3,30-dimethoxy-7,70-epoxylig-
nan (Fig. 1).
Comparison of the 1H and 13C NMR spectroscopic data of 4 and
3 suggested that 4 is only structurally different from 3 by its aro-
matic constituents. Namely, the presence of two 4-hydroxy-3-
methoxyphenyl moieties in 4 was indicated by two set of ABX-type
aromatic proton resonances at dH 6.89, 6.86, 6.88, and dH 6.86, 6.71,
6.76, as well as from the methoxy proton resonance at dH 3.83. Fur-
thermore, the relative configurations in 4 is also the same as 3 by
their similar correlations in the NOESY spectra.
Ribesin E (7) and ribesin F (8) were isomers with the same
molecular formula of C27H30O4 as determined by the positive-ion
HRESIMS data. The 13C NMR spectra of both 7 and 8 showed

336
T. Sasaki et al. / Phytochemistry 95 (2013) 333–340
Fig. 3. Key NOESY correlations for compounds 3, 7–9 and 10.
resonances for 27 carbons, among which 18 were assignable to the
aromatic carbons and 9 assignable to the aliphatic carbons,
suggesting a sesquilignan structure (Table 2). Further comparison
of the 1H NMR spectroscopic data of 7 and 8 indicated quite similar
resonances for the aromatic constituents, including two set of
A2X2-type aromatic proton resonances and a set of ABX-type
aromatic proton resonances assignable to three hydroxylated aro-
matic rings (A-, B- and C-ring) (Fig. 1).
In the 1H NMR spectrum of 7, the highfield resonances for two
methine groups at dH 2.62 (H-8 and H-80) were coupled with two
oxymethine groups at dH 5.06 and 5.04 as well as with two nearly
equivalent methyl groups at dH 0.55 and 0.50, suggesting the
Table 2
1H and 13C NMR spectroscopic data (d) for compounds 7–9 in CD3OD.
Position
7
8
9
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
1
2
3
4
5
6
7
8
132.5
128.7
115.8
157.5
115.8
128.7
84.3
134.0
129.0
116.2
158.2
116.2
129.0
88.8
133.2
115.4
146.5
146.3
116.3
119.7
89.1
7.23 (d, 8.5)
6.79 (d, 8.5)
7.23 (d, 8.5)
6.79 (d, 8.5)
6.97 (d, 1.8)
6.79 (d, 8.5)
7.23 (d, 8.5)
5.06 (d, 6.6)
2.62 (m)
6.79 (d, 8.5)
7.23 (d, 8.5)
4.42 (d, 6.9)
2.25 (m)
6.78 (d, 8.2)
6.81 (dd, 8.2, 1.8)
4.26 (d, 9.4)
1.69 (m)
42.9
45.4
49.9
9
0.55 (d, 7.1)
12.2
0.98 (d, 7.1)
13.0
0.97 (d, 6.6)
14.8
10
20
30
40
50
60
70
80
90
100
200
300
400
500
600
700
132.1
127.0
133.8
154.9
115.6
125.8
84.6
133.7
126.7
134.2
155.5
115.7
125.8
89.2
132.9
127.7
133.7
155.2
115.6
126.4
84.9
7.10 (d, 2.0)
7.08 (d, 2.0)
7.02 (d, 2.1)
6.73 (d, 8.4)
7.03 (dd, 8.4, 2.0)
5.04 (d, 7.1)
2.62 (m)
6.74 (d, 8.2)
7.02 (dd, 8.2, 2.0)
4.39 (d, 7.1)
2.00 (m)
6.72 (d, 8.6)
6.98 (dd, 8.6, 2.1)
5.03 (d, 8.6)
2.17 (m)
42.8
12.4
45.9
13.0
47.2
15.3
0.50 (d, 6.9)
0.94 (d, 6.9)
0.55 (d, 6.9)
133.6
131.2
115.8
156.3
115.8
131.2
43.2
133.2
131.3
115.6
156.3
115.6
131.3
43.0
133.9
131.1
115.8
156.2
115.8
131.1
43.2
6.93 (d, 8.5)
6.61 (d, 8.5)
6.88 (d, 8.4)
6.62 (d, 8.4)
6.93 (d, 8.5)
6.60 (d, 8.5)
6.61 (d, 8.5)
6.93 (d, 8.5)
2.94 (dd, 13.5, 6.6)
2.64 (m)
3.37 (m)
1.19 (d, 7.1)
6.62 (d, 8.4)
6.88 (d, 8.4)
2.89 (dd, 13.3, 5.9)
2.64 (dd, 13.3, 5.5)
3.41 (m)
6.60 (d, 8.5)
6.93 (d, 8.5)
2.92 (dd, 13.5, 6.7)
2.62 (dd, 13.5, 8.0)
3.35 (m)
800
900
36.3
20.2
35.5
19.8
36.5
20.3
1.16 (d, 6.9)
1.18 (d, 7.1)

T. Sasaki et al. / Phytochemistry 95 (2013) 333–340
337
Table 3
presence of a substituted tetrahydrofuran moiety (Toferna et al.,
1H and 13C NMR spectroscopic data (d) for compound 10 in CD3OD.
2000). Moreover, the methine proton resonance at dH 3.37 was
coupled with the protons for a methylene at dH 2.94, 2.64 and a
methyl at dH 1.19, suggesting the presence of an 1,2-diphenylpro-
pyl group. The connection of these partial structures as shown in
Fig. 2 was determined by analyses of the HMBC spectroscopic data.
Namely, The HMBC correlations from dH 2.94, 2.64 (Ha,b-700) to dC
131.2 (C-200 and C-600), dH 7.23 (H-2, 6) to dC 84.3 (C-7), dH 7.10
(H-20), 7.03 (H-60) to dC 84.6 (C-70), dH 1.19 (H3-900) to dC 133.8
(C-30), dH 3.37 (H-800) to dC 127.0 (C-20), 154.9 (C-40), and dH 6.93
(H-200, 600) to dC 43.2 (C-700), suggested that A-ring was connected
at C-1 to C-7, B-ring was connected at C-10 to C-70 and at C-30 to
C-800, and C-ring was connected at C-100 to C-700, respectively. The
relative configurations in the tetrahydrofuran ring were
determined by the NOESY spectroscopic data (Fig. 3). Namely,
the NOESY correlations between dH 0.55 (H3-9)/7.23 (H-2), 0.55
(H3-9)/0.50 (H3-90), 0.50 (H3-90)/7.10 (H-20) and 7.23 (H-6)/7.03
(H-60), suggested the cis-relationship between H-7, H-8, H-70 and
H-80. Thus, the structure of ribesin E (7) was determined as
rel-(7R,8S,70S,80R)-4,40-dihydroxy-30-[1-methyl-2-(4-hydroxyphenyl)]-
ethyl-7,70-epoxylignan.
Position
dH (J in Hz)
dC
Position
dH (J in Hz)
dC
1
2
3
4
5
6
7
8
9a
9b
10
20
30
40
50
60
70
80
90a
90b
133.5
129.1
116.4
158.5
116.4
129.1
90.8
100
134.2
130.3
116.2
158.5
116.2
130.3
83.4
7.18 (d, 8.5)
6.80 (d, 8.5)
200
7.11 (d, 8.7)
6.73 (d, 8.7)
300
400
6.80 (d, 8.5)
7.18 (d, 8.5)
5.67 (s)
500
6.73 (d, 8.7)
7.11 (d, 8.7)
5.57 (s)
600
700
135.0
19.8
800
159.4
108.6
1.55 (m)
1.94 (m)
900a
900b
1000
2000
3000
4000
5000
6000
7000
8000
4.50 (d, 2.1)
4.81 (d, 2.1)
133.9
129.5
115.8
158.6
115.8
129.5
91.0
129.6
130.1
116.4
158.3
116.4
130.1
89.4
7.05 (d, 8.5)
6.76 (d, 8.5)
7.15 (d, 8.7)
6.75 (d, 8.7)
6.76 (d, 8.5)
7.05 (d, 8.5)
5.71 (s)
6.75 (d, 8.7)
7.15 (d, 8.7)
4.75 (s)
135.7
30.6
49.5
27.7
1.68 (d, 17.2)
2.30 (d, 17.2)
9000
9000
a
b
0.95 (m)
1.74 (m)
Detailed analyses of the 2D NMR spectroscopic data led to iden-
tical gross structures of 7 and 8. When the tetrahydrofuran ring res-
onances of 8 were compared to those of 7, the methyl protons at dH
0.98 (H3-9) and 0.94 (H3-90) were shifted downfield and the oxyme-
thine protons at dH 4.42 (H-7) and 4.39 (H-70) were shifted upfield.
These observations indicated that H-7 and H-70 have trans-relation-
ship with the adjacent protons of H-8 and H-80 (Nguyen et al.,
2010). The relative configurations in the tetrahydrofuran moiety
were also supported by the NOESY spectroscopic data (Fig. 3).
Namely, NOESY correlations were observed between dH 0.98 (H3-
9)/0.94 (H3-90), 2.25 (H-8)/2.00 (H-80), 2.25 (H-8)/7.23 (H-2), 2.00
(H-80)/7.08 (H-20), and 7.23 (H-6)/7.02 (H-60). Thus, the structure
of ribesin F (8) was determined as rel-(7R,8R,70S,80S)-4,40-dihy-
droxy-30-[1-methyl-2-(4-hydroxyphenyl)]ethyl-7,70-epoxylignan.
Ribesin G (9) was also a structurally related sesquilignan to 7
and 8. Its molecular formula was C27H30O5 as determined by the
positive-ion HRESIMS data, which was one more oxygen atom than
7 and 8. The presence of a 3,4-dihydroxyphenyl moiety in 9 was
deduced from the characteristic ABX type aromatic proton reso-
nances at dH 6.97, 6.78, 6.81 in the 1H NMR spectrum, and ortho-
dihydroxylated aromatic carbon resonances at dC 146.5 and 146.3
in the 13C NMR spectrum. This 3,4-dihydroxyphenyl moiety was
determined as A-ring at C-7 by the HMBC correlations from dH
6.97 (H-2) and dH 6.81 (H-6) to dC 89.1 (C-7), dH 4.26 (H-7) to dC
115.4 (C-2) and dC 119.7 (C-6), and dH 1.69 (H-8) to dC 133.2 (C-
1). The relative configurations in the tetrahydrofuran moiety of 9
was determined by the NOESY correlations between dH 6.81 (H-
6)/1.69 (H-8), 1.69 (H-8)/0.55 (H3-90), 0.55 (H3-90)/7.02 (H-20),
4.26 (H-7)/5.03 (H-70), 0.97 (H3-9)/2.17 (H-80) and 2.17 (H-80)/
5.03 (H-70) (Fig. 3). Thus, the structure of ribesin G (9) was deter-
mined as rel-(7R,8R,70S,80R)-3,4,40-trihydroxy-30-[1-methyl-2-(4-
hydroxyphenyl)]ethyl-7,70-epoxylignan.
(m), 1.74 (m), 1.68 (d, 17.2), 2.30 (d, 17.2)], and a quaternary carbon
[dc 49.5]. Detailed analyses of the 1H–1H COSY and HMBC spectro-
scopic data deduced the connection of these structural moieties.
The HMBC correlations from the oxymethine proton at dH 5.71
(H-70) to dC 30.6 (C-90), 135.0 (C-8), 135.7 (C-80) and from the oxy-
methine proton at dH 5.67 (H-7) to dC 19.8 (C-9), 135.0 (C-8),
135.7 (C-80) determined the presence of a tetrahydrofuran ring with
the C8–C80 double bond (Fig. 2). Furthermore, the 1H–1H COSY cor-
relations between H-9 and H-9000, as well as the HMBC correlations
from methylene protons at dH 1.68, 2.30 (Ha,b-90) and at dH 0.95,
1.74 (Ha,b-9000) to dC 49.5 (C-8000), suggested that the furano moiety
was annulated. The HMBC correlations from the terminal olefinic
protons at dH 4.50, 4.81 (Ha,b-900) to dC 83.4 (C-700), 159.4 (C-800) indi-
0
0
cated the D8 ,9 double bond connecting to the oxymethine carbon
C-700, and the HMBC correlations from the methylene protons at
dH 1.68, 2.30 (Ha,b-90) to the nonprotonated olefinic carbon at dH
159.4 (C-800) and the oxymethine carbon at dC 89.4 (C-7000) enabled
assembly of the spirocyclic portion in the molecule. Finally, the
connections of four 4-hydroxyphenyl moieties (A, B, C and D-ring)
to C-7 and C-70 in the annulated furan moiety and C-700 and C-7000
in the spirocycle moiety, were determined by the HMBC correla-
tions from four oxymethine protons (H-7, H-70, H-700 and H-7000) to
four aromatic carbons (C-2, C-20, C-200 and C-2000), respectively. The
NOESY correlations between dH 5.71 (H-70)/5.67 (H-7), 1.68 (H-90)
and 4.75 (H-7000)/1.68 (H-90), 5.57 (H-700), suggested syn-orientation
for C-7, 70, 700, 7000 aromatic substituent and b-orientation for C-90
methylene (Fig. 3). On the basis of this evidence, the structure of
ribesin H (10) was concluded to be as shown in Fig. 1.
Since the 70% ethanol extract had significant superoxide anion
scavenging activity and DPPH free radical scavenging activity
The molecular formula of ribesin H (10) was determined as
(EC50 = 1.84 and 34.07 lg/mL, respectively), the antioxidative ef-
C
36H32O6 by the negative-ion HRESIMS. The 13C NMR spectrum
fects of all isolated compounds were evaluated. Six lignoids (2, 3,
4, 6, 9, 10) and a flavonol (13) showed potent superoxide anion
scavenging activity with EC50 values ranging from 1.12 to
showed resonances for 36 carbons, of which 24 assignable to the
aromatic carbons, suggesting the dimeric lignan structure of 10 (Ta-
ble 3). The presence of four 4-hydroxyphenyl moieties was deduced
from the four sets of typical A2X2 type aromatic proton resonances
in the 1H NMR spectrum. The 13C resonances corresponding to these
aromatic rings were then assigned by analyzing the HMQC and
HMBC correlations. The remaining 1H and 13C resonances were
for one internal olefin [dC 135.0, 135.7]; one terminal olefin [dC
108.6, 159.4; dH 4.50 (d, 2.1), 4.81 (d, 2.1)], four oxymethines [dc
83.4, 89.4, 90.8, 91.0; dH 5.57 (s), 4.75 (s), 5.67 (s), 5.71 (s)], three
methylenes [dc 19.8, 27.7, 30.6; dH 1.55 (d, 17.2), 1.94 (m), 0.95
6.09 lM, and the lignan (4) and the flavonol (13) showed moderate
DPPH free radical scavenging activity, which were comparable to
those of the positive control, butylated hydroxyanisole (BHA) (Ta-
ble 4). The effectiveness level of superoxide anion scavenging activ-
ity between test compounds could be due to their structural
differences. Among the 7,70-epoxylignans, compounds 3 and 4 have
shown the most potent superoxide anion scavenging activity with
EC50 values of 2.05 and 1.24
l
M, indicating that the presence of
0
0
D8 ,9 double bond might be crucial for higher activity. A comparison

338
T. Sasaki et al. / Phytochemistry 95 (2013) 333–340
Table 4
J-720 W. The IR spectra were obtained on a JASCO FT/IR-4100 fou-
rier transform infrared spectrometer by the KBr disk method. The
UV spectra were acquired with a Shimadzu UV-160 spectropho-
tometer. The 1H and 13C NMR spectra were measured on a JEOL
ECP-500 spectrometer with TMS as the internal reference, and
the chemical shifts are expressed in d (ppm). ESIMS and HRESIMS
were conducted using a JEOL JMS-T100LP AccuTOF LC-plus mass
spectrometer. For HPLC, a JASCO PU-2086 HPLC system, equipped
with a JASCO RI-2301 Differential Refractometer detector, was
used. RP-C18 silica gel column (YMC-Pack ODS-A, 20 ꢁ 150 mm,
YMC CO., Ltd. Kyoto, Japan) was used for HPLC at a flow rate of
5.0 mL/min. Diaion HP-20 (Mitsubishi Chemical Corporation,
Tokyo, Japan), silica gel (silica gel 60 N, Kanto Chemical Co., Inc.,
Tokyo, Japan), and ODS (100–200 mesh, Chromatorex DM1020T
ODS, Fuji Silysia Chemical Co., Ltd., Aichi, Japan) were used for
column chromatography (CC). TLC was conducted using silica gel
60 F254 plates (E. Merck). Absorbance was measured using Immu-
noMini NJ-2300 microplate reader.
Superoxide anion, DPPH radical scavenging activities of isolated compounds from the
leaves of Ribes nigrum (mean value SD).
a
Sample
EC50
Superoxide anion
DPPH
70% EtOH extract
1
2
3
4
5
6
7
1.84 0.27b
NA
6.09 0.68
2.05 0.17
1.24 0.33
NA
34.07 0.60b
NA
NA
NA
32.33 1.42
NA
NA
NA
NA
NA
NA
NA
3.05 0.50
NA
8
9
10
11
12
13
14
NA
1.12 0.11
3.26 0.13
NA
NA
NA
4.85 0.92
NA
17.02 0.95
31.52 5.50
NA
26.71 1.34
Butylated hydroxyanisolec
NA: No activity >50 lM.
a
4.2. Plant material
EC50 in
lM.
b
c
EC50 in
lg/mL.
Positive control.
Leaves of R. nigrum were collected at Shangzhi City, Heilongji-
ang Province, People0s Republic of China in September, 2006, and
identified by one of the authors (FM). A voucher specimen
(TH315) has been deposited at Department of Pharmacognosy,
Faculty of Pharmaceutical Sciences, Toho University.
of the structures between 4 and 3, and 2 and 1, indicated that the
presence of 3- and 30-methoxy moieties enhanced the activity.
Among the closely structural related sesquilignans (7, 8, 9), only
ribesin G (9) has shown potent superoxide anion scavenging activ-
ity with EC50 value of 1.12 lM, but the others (7 and 8) did not, sug-
4.3. Extraction and isolation
gesting that the ortho-hydroxyphenyl group or/and the stereo
configurations in the tetrahydrofuran ring is important to exhibit
the activity.
Dried leaves of R.nigrum (3.0 kg) were extracted with EtOH–H2O
(7 L ꢁ 3, 70:30, v/v), and then the combined extracts were evapo-
rated in vacuo to give an extract (695 g). This was then subjected
to Diaion HP-20 CC, eluted sequentially with H2O–MeOH, (100:0,
70:30, 30:70, 0:100, v/v) and acetone to give five fractions (1–5).
Further separation was carried out by silica gel and ODS CC as well
as preparative reversed-phase HPLC, from fraction 2 (9.9 g) to
afford 11 (2 mg), 12 (6 mg) and 14 (340 mg), from fraction 3
(19.8 g) to afford 1 (41 mg), 2 (5 mg), 3 (16 mg), 4 (44 mg), 5
(115 mg), 6 (12 mg), 9 (3 mg), 13 (22 mg) and 10 (26 mg), and from
fraction 4 (21.5 g) to afford 7 (6 mg) and 8 (10 mg), respectively.
3. Conclusion
As a conclusion, fourteen compounds including eight novel
compounds were isolated from leaves of R. nigrum. These
compounds are classified into 7,70-epoxylignans (1–6), tetrahydro-
furan-type sesquilignans (7–9), spirocyclic dilignan (10), norses-
quiterpene glucosides (11 and 12), flavonol-type flavonoid (13),
and the phenylpropanoid (14). To the best of our knowledge, this
is the first report of the occurrence of 7,70-epoxylignans, sesqui-
lignans, dilignan and norsesquiterpene glucosides in the plant fam-
ily Grossulariaceae. The spirocyclic dilignan (10) has a unique
furano-2-oxaspiro[4,5]decane core in its structure. This type of
compounds have only been previously reported from Zygophyllales
spp. (Zygophyllaceae), and were proposed to be biosynthesized
from the corresponding monomeric 8(9),80(90)-diene-7,70-epoxy-
lignan through an inter [4 + 2] Diels–Alder reaction (Chavez
et al., 2011). Seven compounds including four novel compounds
showed potent superoxide anion scavenging activity. It is also
worth noting that the present study demonstrated the superoxide
anion scavenging activity of sesquilignans and dilignans for the
first time. The leaves ofR. nigrum may provide a beneficial effect
for oxidative stress-induced diseases and have potential for the
development of raw material of functional foods with antioxidant
effects. Naturally, further investigations of the quality assessment
were also demanded for promotion of utilization.
4.4. Ribesin A (1): (7R,70R)-8(80)-ene-4,40-dihydroxy-7,70-epoxylignan
Colorless solid. [
a
]
25: ꢀ88.0 (c 0.4, MeOH). UV (MeOH): kmax
D
nm (log
e
) 232 (4.39), 276 (3.59). IR (KBr): mmax 3389, 3019, 2914,
2839, 2366, 2345, 1613, 1598, 1513, 1365, 1261, 1226, 1189,
1103, 1022, 1008 cmꢀ1. For 1H NMR (CD3OD, 500 MHz) and 13C
NMR (CD3OD, 125 MHz) spectroscopic data, see Table 1. ESIMS
(positive) m/z: 305.1 [M+Na]+. HRESIMS (negative) m/z: 281.1177
[MꢀH]ꢀ (Calc. for C18H17O3 281.1178). CD (MeOH): [h]25 (nm)
ꢀ188,768 (234).
4.5. Ribesin B (2): (7R,70R)-8(80)-ene-4,40-dihydroxy-3,30-dimethoxy-
7,70-epoxylignan
Colorless solid. [
nm (log
a
]
25: ꢀ45.2 (c 0.25, MeOH). UV (MeOH): kmax
D
e
) 232 (4.21), 282 (3.84). IR (KBr): mmax 3427, 1595, 1509,
4. Experimental
1441, 1383, 1364, 1270, 1238, 1218, 1129, 1176, 1160, 1028,
972 cmꢀ1. For 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD,
125 MHz) spectroscopic data, see Table 1. ESIMS (positive) m/z:
365.1 [M+Na]+. HRESIMS (negative) m/z: 341.1384 [MꢀH]ꢀ (Calc.
for C20H21O5 341.1390). CD (MeOH): [h]25 (nm) ꢀ37,685 (236),
ꢀ7877 (280).
4.1. General
Optical rotations were measured on a JASCO P-2200 polarime-
ter in a 0.5-dm cell. The CD spectra were measured on a JASCO

T. Sasaki et al. / Phytochemistry 95 (2013) 333–340
339
4.6. Ribesin C (3): (7S,70S,8R)-80(90)-ene-4,40-dihydroxy-7,70-
epoxylignan
scopic data, see Table 3. ESIMS (positive) m/z: 583.3 [M+Na]+. HRE-
SIMS (negative) m/z: 559.2140 [MꢀH]ꢀ (Calc. for C36H31O6
559.2137). CD (MeOH): [h]25 (nm) ꢀ64,824 (208), 5802 (223),
ꢀ212,364 (235).
Colorless solid. [
nm (log ) 229 (4.41), 277 (3.60). IR (KBr): mmax 3348, 3026, 2968,
a]
25:+33.0 (c 1.60, MeOH). UV (MeOH): kmax
D
e
2931, 2874, 1614, 1598, 1515, 1451, 1366, 1230, 1171, 1106,
1069 cmꢀ1. For 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD,
125 MHz) spectroscopic data, see Table 1. ESIMS (positive) m/z:
305.1 [M+Na]+. HRESIMS (negative) m/z: 281.1177 [MꢀH]ꢀ (Calc.
for C18H17O3 281.1178). CD (MeOH): [h]25 (nm) ꢀ21,222 (235).
4.12. Catalytic reduction using Pd/C of 1–4
A solution of 1 (10.0 mg), 2 (1.0 mg), 3 (5.0 mg), and 4 (0.8 mg)
in MeOH was separately treated with palladium-activated carbon
(Pd 10%) (Wako, Japan, 1 mg) and then the reaction mixture was
stirred at room temperature for overnight under H2 atmosphere.
The reaction mixture was passed through a Sep-pak C18 cartridge
using H2O and MeOH and further purified by reversed-phase HPLC
with MeOH–H2O (70:30, v/v) to give 5 (1.5 mg from 1, and 4.0 mg
from 3), and 15 (0.5 mg from 2, and 0.5 mg from 4), respectively.
(7S,70S,8R,80S)-4,40-Dihydroxy-7,70-epoxylignan (5): colorless
4.7. Ribesin D (4): (7S,70S,8R)-80(90)-ene-4,40-dihydroxy-3,30-
dimethoxy-7,70-epoxylignan
Colorless solid. [
nm (log
a]
25:+20.8 (c 0.05, MeOH). UV (MeOH): kmax
D
e
) 229 (4.13), 280 (3.83). IR (KBr): mmax 3434, 1593, 1510,
1442, 1377, 1274, 1128, 1028, 957 cmꢀ1. For 1H NMR (CD3OD,
500 MHz) and 13C NMR (CD3OD, 125 MHz) spectroscopic data,
see Table 1. ESIMS (positive) m/z: 365.1 [M+Na]+. HRESIMS (nega-
tive) m/z: 341.1394 [MꢀH]ꢀ (Calc. for C20H21O5 341.1390). CD
(MeOH): [h]25 (nm) ꢀ15,381 (237), ꢀ8905 (287).
solid. [
a
]
25: ꢀ20.3 (c 0.40, MeOH). 1H NMR (CD3OD, 500 MHz):
D
d 7.24 (2H, d, J = 8.0 Hz, H-2,6), 7.17 (2H, d, J = 8.0 Hz, H-20,60),
6.80–6.83 (4H, aromatic protons), 5.42 (1H, d, J = 4.6 Hz, H-7),
4.60 (1H, d, J = 9.3 Hz, H-70), 2.42 (2H, m, H-8,80), 0.97 (3H, d,
J = 7.3 Hz, H-90), 0.58 (3H, d, J = 6.7 Hz, H-9). 13C NMR (CD3OD,
125 MHz): d 157.6 (C-4, 40), 135.4 (C-1), 132.8 (C-10), 128.3 (C-2,
6), 128.0 (C-20, 60), 115.8 (C-3,5), 115.5 (C-30,50), 86.2 (C-7), 85.2
(C-70), 48.5 (C-8), 44.1 (C-80), 12.1 (C-9), 9.8 (C-90). CD (MeOH):
[h]25 (nm) ꢀ51,146 (232).
4.8. Ribesin E (7): rel-(7R,8S,70S,80R)-4,40-dihydroxy-30-[1-methyl-2-
(4-hydroxyphenyl)]ethyl-7,70-epoxylignan
25: ꢀ35.0 (c 0.56, MeOH). UV (MeOH): kmax
(7S,70S,8R,80S)-4,40-Dihydroxy-3,30-dimethoxy-7,70-epoxylignan
Colorless solid. [
a
]
D
(15): colorless solid. [
500 MHz):
a
]
25: ꢀ78.0 (c 0.08, MeOH). 1H NMR (CD3OD,
nm (log
e) 226 (4.50), 279 (3.87). IR (KBr): mmax 3398, 3017, 2961,
D
2925, 2854, 1614, 1514, 1456, 1377, 1238, 1171, 1092,
1005 cmꢀ1. For 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD,
125 MHz) spectroscopic data, see Table 2. ESIMS (positive) m/z:
441.1 [M+Na]+. HRESIMS (positive) m/z: 441.2044 [M+Na]+ (Calc.
for C27H30O4Na 441.2041). CD (MeOH): [h]25 (nm) ꢀ9565 (221),
3267 (237).
d
6.82–6.99 (6H, aromatic protons), 5.39 (1H, d,
J = 7.8 Hz, H-70), 4.59 (1H, d, J = 8.9 Hz, H-7), 3.88 (6H, s, OCH3),
2.42 (2H, m, H-8,80), 0.99 (3H, d, J = 5.7 Hz, H-9), 0.62 (3H, d,
J = 6.4 Hz, H-90). 13C NMR (CD3OD, 125 MHz): d 146.0 (C-30),
145.7 (C-3), 145.4 (C-40), 145.3 (C-4), 136.6 (C-10), 134.0 (C-1),
117.8 (C-60), 117.6 (C-6), 112.5 (C-50), 112.4 (C-5), 110.5 (C-20),
110.2 (C-2), 85.4 (C-7), 84.5(C-70), 56.1 (3-OCH3), 56.0 (30-OCH3),
47.5 (C-8), 43.3 (C-80), 11.9 (C-9), 9.4 (C-90). CD (MeOH): [h]25
(nm) ꢀ86,847 (231), ꢀ16,201 (286).
4.9. Ribesin F (8): rel-(7R, 8R, 70S, 80S)-4,40-dihydroxy-30-[1-methyl-2-
(4-hydroxyphenyl)]ethyl-7,70-epoxylignan
Colorless solid. [
a
]
25: ꢀ15.7 (c 0.40, MeOH). UV (MeOH): kmax
D
4.13. Superoxide anion scavenging assay and the DPPH radical
scavenging assay
nm (log
e) 226 (2.89), 278 (2.26). IR (KBr): mmax 3387, 3025, 2959,
2325, 1613, 1514, 1450, 1377, 1238, 1171, 1120, 1101, 1028,
1005 cmꢀ1. For 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD,
125 MHz) spectroscopic data, see Table 2. ESIMS (positive) m/z:
441.1 [M+Na]+. HRESIMS (positive) m/z: 441.2039 [M+Na]+ (Calc.
for C27H30O4Na 441.2041). CD (MeOH): [h]25 (nm) 3185 (224),
ꢀ4950 (236).
The assay was carried out as reported previously (Bi et al.,
2011).
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