Giffonins A-I, antioxidant cyclized diarylheptanoids from the leaves of the hazelnut tree (corylus avellana), source of the italian PGI product nocciola di Giffoni

Article abstract of DOI:10.1021/np5004966

Eight new diaryl ether heptanoids, giffonins A-H (1-8), and one diaryl heptanoid, giffonin I (9), were isolated from the methanol extract of the leaves of Corylus avellana. Its hazelnut is the PGI product of the Campania region (Italy) known as Nocciola di Giffoni . The MeOH extract of C. avellana leaves and giffonins A-I (1-9) were evaluated for their inhibitory effects on human plasma lipid peroxidation induced by H₂O₂ and H₂O₂/Fe²⁺, by measuring the concentration of TBARS (thiobarbituric acid reactive substances). Compounds 4 and 8 at 10 μM reduced both H₂O₂- and H₂O₂/Fe²⁺-induced lipid peroxidation by more than 60% and 50%, respectively, indicating higher activity than curcumin used as reference compound.

Full text of DOI:10.1021/np5004966

Article
pubs.acs.org/jnp
Giffonins A−I, Antioxidant Cyclized Diarylheptanoids from the
Leaves of the Hazelnut Tree (Corylus avellana), Source of the Italian
PGI Product “Nocciola di Giffoni”
Milena Masullo,† Antonietta Cerulli,† Beata Olas,‡ Cosimo Pizza,† and Sonia Piacente*,†
†Dipartimento di Farmacia, Universita
̀
degli Studi di Salerno, Via Giovanni Paolo II n. 132, 84084 Fisciano, Salerno, Italy
‡Department of General Biochemistry, Institute of Biochemistry, Faculty of Biology and Environmental Protection, University of
Lodz, Pomorska 141/3, 90-236, Lodz, Poland
S
* Supporting Information
ABSTRACT: Eight new diaryl ether heptanoids, giffonins A−H (1−8), and one diaryl heptanoid, giffonin I (9), were isolated
from the methanol extract of the leaves of Corylus avellana. Its hazelnut is the PGI product of the Campania region (Italy) known
as “Nocciola di Giffoni”. The MeOH extract of C. avellana leaves and giffonins A−I (1−9) were evaluated for their inhibitory
effects on human plasma lipid peroxidation induced by H2O2 and H2O2/Fe2+, by measuring the concentration of TBARS
(thiobarbituric acid reactive substances). Compounds 4 and 8 at 10 μM reduced both H2O2- and H2O2/Fe2+-induced lipid
peroxidation by more than 60% and 50%, respectively, indicating higher activity than curcumin used as reference compound.
azelnut (Corylus avellana L.), belonging to the Betulaceae
heptanoids (1−8) along with a new diaryl heptanoid (9) are
Hfamily, is one of the most popular tree nuts on a described as well as their effects on oxidative damage of human
worldwide basis and ranks second in tree nut production after
almond. Italy is the second largest producer of hazelnut (13%)
after Turkey. The main products of C. avellana are kernels,
nutritious food with a high content of healthy lipids,1 used by
the confectionary industry, consumed raw (with skin) or
preferably roasted (without skin). The leaves of C. avellana are
used in traditional medicine for the treatment of varicose veins
and hemorrhoidal symptoms and also for their mild
antimicrobial effects.2 Antioxidant activity was reported for
hazelnuts and leaves of C. avellana.1 Previous phytochemical
investigations on the leaves resulted in the isolation of phenolic
constituents, such as flavonoids, caffeic acid, and diary-
lheptanoid derivatives.3
plasma lipids, induced by H2O2 and H2O2/Fe2+.
RESULTS AND DISCUSSION
■
The MeOH extract of the leaves of C. avellana was fractionated
on a Sephadex LH-20 column, and the fractions were purified
by semipreparative HPLC to afford giffonins A−I (1−9).
The HR-MALDI-TOF-MS of 1 (m/z 377.1369 [M + Na]+,
calcd for C21H22O5Na, 377.1365) and the 13C NMR data
supported a molecular formula of C21H22O5. The IR spectrum
showed a band at 1705 cm−1 for the presence of a ketocarbonyl
group.
1
The H NMR spectrum displayed signals for five aromatic
protons ascribable to two aromatic rings: a signal at δ 4.41 (s),
typical of the proton of a pentasubstituted aromatic ring and
signals at δ 7.41 (2H, d, J = 8.3 Hz) and 7.01 (2H, d, J = 8.3
Hz), due to the proton of a 1,4-disubstituted aromatic ring. The
1H NMR data displayed further signals due to a disubstituted
trans-olefinic group at δ 6.34 (d, J = 15.7 Hz) and 5.20 (dt, J =
8.0, 15.7 Hz) and four methylene groups at δ 2.32 (2H, m),
The Italian “Nocciola di Giffoni”, also known as “tonda di
Giffoni”, is a labeled PGI (protected geographical indication)
product of the Campania region, representing an important
economic resource. Although the nutritive features of the PGI
“Nocciola di Giffoni” hazelnut are well known,4,5 no studies are
reported on the chemical composition of the leaves of C.
avellana, the source of PGI hazelnut. As part of an ongoing
effort to search for new bioactive compounds,6−8 the MeOH
extract of the leaves of C. avellana was investigated. Herein, the
isolation and the structural elucidation of eight new diaryl ether
Received: June 18, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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H-6 (δ 4.41) with the 13C NMR resonances at δ 152.7 (C-1),
136.9 (C-2), 140.4 (C-4), 127.8 (C-5), and 125.5 (C-7)
suggested the 1,2,3,4-tetrahydroxylation of the A ring. The
disposition of the heptanoid chain and the position of the keto
group were determined by HSQC, HMBC, and COSY
experiments. In the HMBC spectrum of 1, the methylene
protons at δ 3.03 were assigned to C-13 on the basis of their
correlations with the 13C NMR resonances at δ 132.2 (C-15
and C-19), 141.0 (C-14), 46.2 (C-12), and 211.1 (C-11). The
COSY correlation between the methylene protons at δ 3.03 and
2.86 allowed the latter methylene group to be located at C-12,
and therefore the keto group to be located at C-11. The HMBC
correlations of the proton at δ 6.34 (H-7) with the 13C NMR
resonances of the B ring at δ 140.4 (C-4), 127.8 (C-5), and
109.0 (C-6) and the linear connectivity observed in the COSY
spectrum from H-7 to H-10 were used to assign the heptanoid
chain. Finally, the HMBC correlations between the protons at δ
4.00 and 3.67 with the 13C NMR resonances at δ 136.9 (C-2)
and 140.4 (C-4), respectively, allowed the methoxy groups to
be located at C-2 and C-4 of the A ring. The foregoing
spectroscopic data allowed the structure of compound 1,
named giffonin A, to be assigned as shown.
2.44 (2H, t, J = 5.5 Hz), 2.86 (2H, t, J = 5.5 Hz), and 3.03 (2H,
t, J = 5.5 Hz) (Table 1). Moreover, two signals for two methoxy
groups at δ 4.00 and 3.67 were evident. The 13C NMR
spectrum of 1 showed 21 carbon signals (Table 1), typical of
diaryl ether heptanoid derivatives,9−11 comprising a signal at δ
211.1, ascribable to a ketocarbonyl group. The signal at δ 4.41
was attributed to H-6, which generally resonates at an
abnormally high field, due to the anisotropic effect of the A
ring in diaryl ether heptanoids.10 This observation together
with the ROESY correlations of H-6 at δ 4.41 with H-18 (δ
7.01) and H.16 (δ 5.20) suggested the ether linkage between
C-1 and C-17 of the aryl moieties. The HMBC correlations of
The HR-MALDI-TOF-MS of 2 (m/z 393.1318 [M + Na]+,
calcd for C21H22O6Na, 393.1314) and the 13C NMR data
1
supported a molecular formula of C21H22O6. The H NMR
spectrum displayed five aromatic proton signals, which
suggested the occurrence of a pentasubstituted [δ 4.39 (s)]
and a 1,4-disubstituted aromatic ring [δ 7.70 (dd, J = 8.3, 1.9
Hz), 7.45 (dd, J = 8.3, 1.9 Hz), 7.16 (dd, J = 8.3, 1.9 Hz), 7.00
(dd, J = 8.3, 1.9 Hz)] (Table 1), as in 1. The NMR data of 2
revealed that it differed from 1 by the presence of a secondary
hydroxy group suggested by a signal at δ 5.23 (dd, J = 5.4, 8.9
Hz) (Table 1). The HMBC correlations of the proton at δ 5.23
1
Table 1. 13C and H NMR Data (J in Hz) of Compounds 1−4 (600 Mz, δ ppm, in Methanol-d4)
1
2
3
4
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
4
5
6
7
8
152.7
136.9
144.3
140.4
127.8
152.7
151.8
151.8
137.6
145.0
141.9
125.7
137.0
137.5
144.1
144.7
140.6
141.6
128.0
124.7
109.0 4.41, s
109.2 4.39, s
108.3 5.34, s
108.0 5.18, s
125.5 6.34, d (15.7)
125.6 6.36, d (15.7)
124.6 6.35, d (11.4)
125.9 6.40, d (11.4)
135.5 5.20, dt (8.0,
15.7)
135.5 5.14, ddd (15.7, 10.8, 4.0)
132.5 5.51, ddd (11.4, 7.3, 7.3)
131.0 5.48, dt (11.4,
8.0)
9
26.7 2.32, (2H) m
26.1 2.52, m; 2.00, m
27.0 1.90, m; 1.78, m
40.1 1.05, m; 0.43, m
23.0 1.94, (2H) m
46.5 1.94, (2H) m
10
44.3 2.44, (2H) t
(5.5)
45.2 2.57, dt (17.0, 3.0); 2.29, ddd (3.0,
11.0, 12.0)
11
12
211.1
209.5
72.6 3.31, overlapped
40.9 1.96, m; 1.72, m
213.9
46.2 2.86, (2H) t
(5.5)
54.2 3.04, (2H) t (5.5)
45.0 2.66, (2H) t
(6.8)
13
29.8 3.03, (2H) t
(5.5)
71.2 5.23, dd (5.4, 8.9)
34.2 3.04, dt (12.8, 3.7); 2.74, td
(12.8, 5.0)
33.6 3.03, (2H) t
(6.8)
14
141.0
143.4
140.0
138.6
15
132.2 7.41, d (8.3)
125.2 7.01, d (8.3)
155.8
131.4 7.45, dd (8.3, 1.9)
124.4 7.00, dd (8.3, 1.9)
158.5
130.8 7.37, dd (8.3, 1.9)
124.9 7.17, dd (8.3, 1.9)
156.3
131.8 7.26, d (8.3)
124.5 7.06, d (8.3)
157.0
16
17
18
125.2 7.01, d (8.3)
132.2 7.41, d (8.3)
61.7 4.00, s
125.8 7.16, dd (8.3, 1.9)
128.4 7.70, dd (8.3, 1.9)
61.6 4.00, s
123.8 6.98, dd (8.3, 1.9)
132.8 7.30, dd (8.3, 1.9)
61.3 4.03, s
124.5 7.06, d (8.3)
131.8 7.26, d (8.3)
61.4 4.02, s
19
2-OCH3
4-OCH3
61.2 3.67, s
61.0 3.66, s
61.0 3.68, s
61.2 3.69, s
B
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Figure 1. Δδ (δS − δR) values (in ppm) for the MTPA esters of 2, 3, and 5−8.
with the 13C NMR resonances at δ 131.4 (C-15), 128.4 (C-19),
The positive-ion HR-MALDI-TOF-MS data of 3 showed a
143.4 (C-14), 54.2 (C-12), and 209.5 (C-11) suggested the
secondary hydroxy group to be located at C-13. The presence
of hydroxy groups on the heptane moiety causes the different
chemical shifts for the corresponding protons on the aromatic
ring (H-15 and H-19; H-16 and H-18).11 The ROESY
spectrum showed correlations between H-6 at δ 4.39 and H-
8 (δ 5.14). The aromatic proton H-19 at δ 7.70 showed a
correlation with the methylene protons H-9 (δ 2.00), and H-13
at δ 5.23 showed a strong correlation with H-15 (δ 7.45). The
absolute configuration at C-13 of compound 2 was determined
through the application of the modified Mosher’s method.12
Mosher derivatization was performed on compound 2, which
was treated with (R)- and (S)-MTPA chloride to form (S)-
MTPA and (R)-MTPA esters, respectively. To determine the
absolute configuration of C-13 in 2, the Δδ (δS− δR) values
were observed for signals of the protons close to C-13,
revealing a 13S configuration for 2. Thus, on the basis of the
above data the structure of 2, named giffonin B, was determined
as reported.
pseudomolecular ion at m/z 379.1524 [M + Na]+ (calcd for
C21H24O5Na, 379.1521), which in combination with the 13C
NMR data supported a molecular formula of C21H24O5.
1
Comparison of the H NMR spectrum of 3 in the aromatic
region with that of giffonin B (2) suggested that they share the
same aromatic substitution patterns (Table 1). Moreover, the
1H NMR spectrum displayed signals due to a disubstituted cis-
olefinic function at δ 6.35 (d, J = 11.4 Hz) and 5.51 (ddd, J =
11.4, 7.3, 7.3 Hz), a proton linked to an oxymethine carbon at δ
3.31, and two methoxy groups at δ 4.03 (s) and 3.68 (s) (Table
1). On the basis of the COSY experiment the connectivity from
H-7 to H-13 was established and the hydroxy group was located
at C-11 (δ 72.6). The methoxy groups were placed at C-2 and
C-4 via the HMBC correlations between the protons at δ 4.03
and 3.68 with the 13C NMR resonances at δ 137.5 and 141.6,
respectively. The hydroxy group at C-11, bulkier than a
hydrogen atom, should be directed away from the inside of the
macrocyclic ring.11 The ROE correlations between H-15 (δ
7.37) and H-13β (δ 3.04) and between H-19 (δ 7.30) and H-
C
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1
Table 2. 13C and H NMR Data (J in Hz) of Compounds 5−8 (600 Mz, δ ppm, in Methanol-d4)
5
6
7
8
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
4
5
6
7
8
9
151.9
137.6
144.5
141.6
125.7
152.1
138.3
145.2
142.4
124.7
150.4
155.6
136.9
151.7
136.7
144.2
140.4
110.8 6.30, d (1.7)
133.5
125.1
108.5 5.11, s
108.3 4.89, s
110.0 5.46, s
109.6 5.51, d (1.7)
129.9 6.09, d (11.2)
128.8 5.94, t (11.2)
126.8 5.68, dd (15.4, 11.2)
123.0 6.33, d (11.6)
136.8 5.32, dd (11.6, 8.6)
124.9 6.46, d (12.0)
132.2 5.31, dd (12.0, 8.8)
65.3 4.33, t (9.8)
124.5 6.36, d (11.4)
129.0 6.01, dd (11.4, 9.2)
126.6 5.67, dd (15.4, 9.2)
69.5 3.86 ddd (12.1, 8.6,
3.6)
10
39.0 1.32, m; 0.31, br t
(12.1)
51.3 2.49, m; 1.74, br s
139.9 5.64, dd (15.4, 8.6)
140.1 5.63, dd (15.4, 8.6)
11
12
22.9 1.78, q (10.8); 1.08, m 215.4
73.2 4.07, dt (8.6, 3.3)
73.2 4.09, dt (8.6, 3.4)
30.5 2.11, m; 1.30, m
44.7 2.84, td (12.5, 5.6);
42.3 2.05, dq (14.2, 3.3); 1.66, tt
(14.2, 3.3)
42.5 2.05, dq (14.2, 3.4); 1.67, tt
(14.2, 3.4)
2.51, m
13
36.3 3.05, m; 2.47, td
(12.7, 5.4)
32.9 3.08, m; 3.03, td
(12.1, 5.6)
34.3 3.04, dt (12.8, 3.0); 2.71, td
(12.8, 3.0)
34.6 3.04, dt (13.0, 3.3); 2.72, td
(13.0, 3.3)
14
15
16
17
18
19
2-
140.1
138.6
139.3
140.9
130.9 7.40, dd (8.3, 1.9)
124.9 7.15, dd (8.3, 1.9)
155.8
130.5 7.52, dd (8.3, 1.9)
125.0 7.20, dd (8.3, 1.9)
156.9
132.8 7.33, dd (8.3, 1.9)
122.8 7.20, dd (8.3, 1.9)
155.2
133.2 7.36, dd (8.3, 1.9)
123.0 7.19, dd (8.3, 1.9)
156.7
123.2 7.00, dd (8.3, 1.9)
134.0 7.39, dd (8.3, 1.9)
61.4 4.03, s
123.8 6.98, dd (8.3, 1.9)
134.0 7.12, dd (8.3, 1.9)
61.2 4.03, s
125.6 7.01, dd (8.3, 1.9)
130.5 7.42, dd (8.3, 1.9)
61.2 4.05, s
126.3 7.03, dd (8.3, 1.9)
130.7 7.44, dd (8.3, 1.9)
60.0 4.03, s
OCH3
4-
OCH3
61.2 3.71, s
61.2 3.71, s
61.0 3.69, s
13α (δ 2.74) and H-11 (δ 3.31) were observed. Mosher
derivatization was performed on compound 3, which was
treated with (R)- and (S)-MTPA chloride to form (S)-MTPA
and (R)-MTPA esters, respectively. Analysis of the Δδ(S − R)
values of the protons close to the oxygenated methine
according to the Mosher model12 (Figure 1) allowed the
assignment of the 11R absolute configuration of 3. Thus, the
structure of compound 3, named giffonin C, was elucidated as
depicted.
12.1, 8.6, 3.6 Hz) (Table 2) were observed. The position of the
hydroxy group was determined by COSY and HMBC
correlations. In the HMBC spectrum, correlations between
the proton signals at δ 6.33 (H-7) and the 13C NMR
resonances at δ 141.6 (C-4), 125.7 (C-5), 108.5 (C-6), 136.8
(C-8), and 69.5 (C-9) were observed. In the COSY spectrum
the correlation between the proton at δ 6.33 (H-7) and the
proton at δ 5.32 (H-8), which in turn correlated with the
proton at δ 3.86 (H-9), allowed the hydroxy group to be
The 13C NMR and HR-MALDI-TOF-MS data of 4 (m/z
377.1367 [M + Na]+, calcd for C21H22O5Na, 377.1365)
supported a molecular formula of C21H22O5. The IR spectrum
showed a band at 1713 cm−1 for the presence of a ketocarbonyl
group. The NMR data of compound 4 were comparable to
those of giffonin C (3) except for the presence of a carbonyl
group replacing the secondary hydroxy group in 3. Analyses of
HMBC and COSY experiments confirmed the presence of a
carbonyl group at C-11 (δ 213.9). Notably, the absence of the
hydroxy group on the heptanoid chain induced similar chemical
shifts of H-15 and H-19 (each, δ 7.26, d, J = 8.3 Hz) and H-16
and H-18 (each, δ 7.06, d, J = 8.3 Hz) (Table 1). On the basis
of the reported data the structure of 4, named giffonin D, was
deduced as depicted.
located at C-9. The H and 13C NMR chemical shifts of the
1
heptene moiety and the coupling constant of the proton of the
secondary hydroxy function (δ 3.86, ddd, J = 12.1, 8.6, 3.6 Hz)
of compound 5 were almost superimposable to those of 3,5′-
dihydroxy-4′-methoxy-3′,4″-oxy-1,7-diphenyl-1-heptene, iso-
lated from Betula platyphylla var. japonica.13 The absolute
configuration of compound 5 was determined through the
application of the Mosher methodology.12 The Δδ (δS − δR)
values observed for signals of the protons close to C-9 in
compounds 5a and 5b revealed a 9S configuration for 5. On the
basis of these observation, the structure of 5, named giffonin E,
was determined as depicted.
The 13C NMR and HR-MALDI-TOF-MS data of 6 (m/z
393.1319 [M + Na]+, calcd for C21H22O6Na, 393.1314)
supported a molecular formula of C21H22O6. The IR spectrum
showed a band at 1710 cm−1 for the presence of a carbonyl
group. The NMR data of compound 6 were similar to those of
giffonin E (5) except for the presence of the resonance of a
carbonyl group (δ 215.4). The location of the carbonyl group at
C-11 was confirmed by the HMBC correlations of the proton
signals at δ 3.08 and 3.03 (H-13) and δ 4.33 (H-9) with the
carbon resonance at δ 215.4. Mosher derivatization was
performed on compound 6, which was treated with (R)- and
(S)-MTPA chloride to form (S)-MTPA and (R)-MTPA esters,
The molecular formula of 5 was established as C21H24O5 by
HR-MALDI-TOF-MS (m/z 379.1526 [M + Na]+, calcd for
1
C21H24O5Na, 379.1521) and 13C NMR data. The H NMR
spectrum of 5 displayed signals at δ 7.40 (dd, J = 8.3, 1.9 Hz),
7.39 (dd, J = 8.3, 1.9 Hz), 7.15 (dd, J = 8.3, 1.9 Hz), 7.00 (dd, J
= 8.3, 1.9 Hz), 5.11 (s), 4.03 (s), and 3.71 (s), ascribable to the
two aryl moieties with the same substitution pattern as in 3
(Table 2). Further signals of a disubstituted cis-olefinic function
at δ 6.33 (d, J = 11.6 Hz) and 5.32 (dd, J = 11.6, 8.6 Hz) and of
a proton linked to an oxymethine carbon at δ 3.86 (ddd, J =
D
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1
Table 3. 13C and H NMR Data (J in Hz) of Compound 9
respectively. Analysis of the Δδ(S−R) values of the protons
neighboring the oxygenated methine according to the Mosher
model12 (Figure 1) allowed the assignment of the 9S
configuration of 6. Accordingly, the structure of compound 6,
named giffonin F, was determined as shown.
(600 Mz, δ ppm, in Methanol-d4)
δC
δH (J in Hz)
1
127.4
127.5
152.4
117.6
128.9
137.6
30.4
2
The molecular formula of 7 was established as C21H22O5 by
HR-MALDI-TOF-MS (m/z 377.1367 [M + Na]+, calcd for
C21H22O5Na, 377.1365) and the 13C NMR data. Comparison
of the 1H NMR spectrum of 7 with those of 1−6 suggested that
they share the same aromatic substitution patterns. Further-
3
4
6.83, d (8.0)
5
7.04, dd (8.0, 2.2)
6
1
7
3.17, (2H) m
2.83, (2H) m
more, the H NMR spectrum displayed four signals at δ 6.36
8
40.6
(d, J = 11.4 Hz), 6.01 (dd, J = 11.4, 9.2 Hz), 5.67 (dd, J = 15.4,
9.2 Hz), and 5.64 (dd, J = 15.4, 8.6 Hz), ascribable to Z- and E-
olefinic protons, respectively, and a signal at δ 4.07 (dt, J = 8.6,
3.3 Hz), corresponding to a proton linked to an oxymethine
carbon (Table 2). The COSY experiment showed the
connectivity from H-7 to H-10, allowing a conjugated diene
system (C-7/C-10) flanked by a hydroxy group at C-11 (δ
4.07) to be identified. ROESY experiment showed correlations
of H-15 (δ 7.33) with H-13β (δ 3.04) and of H-19 (δ 7.42)
with H-13α (δ 2.71) and H-11 (δ 4.07). To determine the
absolute configuration of C-11 in 7, (S)- and (R)-MTPA esters
of 7 were synthesized. The Δδ (δS − δR) values observed for
signals of the protons close to C-11 revealed an 11S
configuration for 7. On the basis of the reported data, the
structure of compound 7, named giffonin G, was determined as
depicted.
9
203.0
134.6
150.4
36.2
10
11
12
13
14
15
16
17
18
19
6.62, d (15.6)
7.10, dt (7.6, 15.6)
2.62, (2H) m
33.8
2.93, (2H) br t (5.5)
133.4
129.3
116.4
153.2
136.0
134.2
7.03, dd (8.0, 2.2)
6.76, d (8.0)
6.92, d (2.2)
7.02, d (2.2)
α-Ara (at C-3)
1
2
3
4
5
93.5
5.14, d (3.7)
73.7
3.38, dd (8.0, 3.7)
3.69, dd (8.0, 3.0)
3.81, m
74.5
72.7
The 13C NMR and HR-MALDI-TOF-MS data of 8 (m/z
347.1261 [M + Na]+, calcd for C20H20O4Na, 347.1259)
62.7
3.87, dd (12.5, 3.0); 3.67, dd (12.5, 2.6)
β-Glc (at C-17)
97.9
1
supported a molecular formula of C20H20O4. The H NMR
1
2
3
4
5
6
4.51, d (7.8)
data for the heptadiene moiety were comparable to those of 7,
while differences were observed for signals due to an aryl
76.0
3.15, dd (9.0, 7.8)
3.37, dd (9.0, 9.0)
3.31, dd (9.0, 9.0)
3.31, m
77.7
1
moiety. In particular, the H NMR spectrum showed aromatic
71.6
signals at δ 7.44 (dd, J = 8.3, 1.9 Hz, H-19), 7.36 (dd, J = 8.3,
1.9 Hz, H-15), 7.19 (dd, J = 8.3, 1.9 Hz, H-16), and 7.03 (dd, J
= 8.3, 1.9 Hz, H-18), corresponding to the 1,4-disubstituted
aromatic ring, and at δ 6.30 (d, J = 1.7 Hz, H-4) and 5.51 (d, J
= 1.7 Hz, H-6), corresponding to a 1,2,3,5-tetrasubstituted
aromatic ring (Table 2). Moreover, a signal at δ 4.03 (s), typical
of a methoxy group, was observed. The HMBC correlation
between the proton signal at δ 4.03 and the carbon resonance
at δ 136.9 allowed the methoxy group to be placed at C-2.
Mosher derivatization was performed on compound 8, which
was treated with (R)- and (S)-MTPA chloride to form (S)-
MTPA and (R)-MTPA esters, respectively. Analysis of the
Δδ(S−R) values of the protons neighboring the oxygenated
methine according to the Mosher model12 (Figure 1) allowed
the assignment of an 11S configuration of 8. Therefore, the
structure of compound 8, named giffonin H, was defined as
shown.
77.7
62.5
3.80, dd (12.0, 2.5); 3.70, dd (12.0, 4.5)
9 was established as alnusone, a natural compound first isolated
from Alnus japonica and also available via total synthesis.14,15
The H NMR spectrum displayed in the sugar region signals
1
corresponding to two anomeric protons at δ 5.14 (d, J = 3.7
Hz) and 4.51 (d, J = 7.8 Hz). The NMR data (HSQC, HMBC,
COSY, and 1D-TOCSY) indicated the presence of an α-
arabinopyranosyl unit (δ 5.14) and a β-glucopyranosyl unit (δ
4.51). The configurations of the arabinose and glucose units
were established as L and D, respectively, after hydrolysis of 9
with 1 N HCI, trimethylsilation, and GC analysis.16 The linkage
sites of the sugar units on the diaryl heptanoid moiety were
obtained from the HMBC spectrum, which showed correlations
between H-1ara (δ 5.14) and the 13C NMR resonance of C-3 (δ
152.4) and between H-1glc (δ 4.51) and C-17 (δ 153.2). On the
basis of the reported data, the structure of compound 9, named
giffonin I, was established as shown.
The 13C NMR and HR-MALDI-TOF-MS data of 9 (m/z
611.2107 [M + Na]+, calcd for C30H36O12Na, 611.2104)
supported a molecular formula of C30H36O12. The 13C NMR
spectrum of 9 showed 30 carbon signals, of which 19 were
assigned to a diaryl heptanoid moiety13 and 11 to two sugar
units (Table 3). The 1H NMR spectrum showed signals
ascribable to two 1,2,4-trisubstituted aromatic rings at δ 7.04
(dd, J = 8.0, 2.2 Hz), 7.03 (dd, J = 8.0, 2.2 Hz), 7.02 (d, J = 2.2
Hz), 6.92 (d, J = 2.2 Hz), 6.83 (d, J = 8.0 Hz), and 6.76 (d, J =
8.0 Hz) and signals due to a disubstituted trans-olefinic function
at δ 6.62 (d, J = 15.6 Hz) and 7.10 (dt, J = 7.6, 15.6 Hz). The
structure of the heptanoid moiety was readily deduced from
HSQC, HMBC, and COSY correlations. Thus, the aglycone of
Diarylheptanoid derivatives occur frequently in plants
belonging to the Betulaceae family, but so far only one report
deals with their presence in the leaves of C. avellana.2
Noteworthy, giffonins A−I differ from the compounds reported
by Riethmuller et al.2 in that they represent cyclized
̈
diarylheptanoids, and, in particular, giffonins A−H are diaryl
ether heptanoid derivatives. Diarylheptanoids isolated from
plants belonging to the Betulaceae family have been reported to
possess various biological activities including antioxidant, anti-
inflammatory, anticancer, and antiadipogenic.2,17−19 Therefore,
E
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Journal of Natural Products
Article
Figure 2. Effects of MeOH extract (0.1−100 μg/mL; 30 min), giffonins A−I (1−9) (0.1−100 μM; 30 min), and curcumin (0.1−100 μM; 30 min)
on plasma lipid peroxidation induced by H2O2. The results are representative of three independent experiments and are expressed as means SD.
The effect of five different concentrations of tested compounds (0.1, 1, 10, 50, and 100 μM) and tested extract (0.1, 1, 10, 50, and 100 μg/mL) was
statistically significant according to the ANOVA I test; p < 0.05 (for compounds 1, 5, 7 and curcumin); p < 0.02 (for compounds 4, 8 and extract); p
> 0.05 (for compounds 2, 3, 6, and 9).
Figure 3. Effects of MeOH extract (10 μg/mL; 30 min), giffonins A−I (1−9) (10 μM; 30 min), and curcumin (10 μM; 30 min) on plasma lipid
peroxidation induced by H2O2/Fe2+. The results are representative of three independent experiments and are expressed as means SD. The
statistical significances were confirmed with the paired Student’s t test.
the MeOH extract of C. avellana leaves and giffonins A−I (1−
9) were evaluated for their inhibitory effects on human plasma
lipid peroxidation induced by H2O2 and H2O2/Fe2+, by
measuring the concentration of thiobarbituric acid reactive
substances (TBARS).
Table 4. Inhibitory Effects of the Extract (10 μg/mL; 30
min), Compounds 1−9 (10 μM; 30 min), and Curcumin (10
μM; 30 min) on Plasma Lipid Peroxidation Induced by
a
H2O2 or H2O2/Fe2+
inhibition of lipid
The MeOH extract, compounds 1−9, and curcumin, used as
reference compound, were tested at doses ranging from 0.1 to
100 μg/mL and from 0.1 to 100 μM, respectively. The tested
compounds, curcumin, and the plant extract did not exert any
effect on autoperoxidation of human plasma (data not shown).
The MeOH extract of leaves at 10 μg/mL reduced by about
38% plasma lipid peroxidation stimulated by H2O2 or H2O2/
Fe2+. Compounds 4 and 8 at 10 μM reduced both H2O2- and
H2O2/Fe2+-induced lipid peroxidation by more than 60% and
50%, respectively, being more active than curcumin (Figure 2).
Among the nine compounds, the weakest activity was displayed
by compounds 2, 6, and 9 (Figures 2 and 3 and Table 4), which
exerted a protective action against oxidative stress induced by
H2O2 or H2O2/Fe2+, similar to that shown by curcumin.
This study demonstrates the potential benefits of C. avellana
leaves as a rich source of phenolic compounds. Moreover, it
extends and reinforces the notion of the antioxidant capacity of
peroxidation induced by
H2O2 (%)
inhibition of lipid peroxidation
induced by H2O2/Fe (%)
compound
1
8.1 3.6 (p < 0.05)
2.2 2.4 (p > 0.05)
16.4 3.9 (p < 0.05)
64.3 14.5 (p < 0.002)
20.8 5.1 (p < 0.05)
12.1 4.9 (p < 0.05)
37.6 7.7 (p < 0.02)
55.8 13.9 (p < 0.01)
12.6 4.5 (p < 0.05)
19.2 4.4 (p < 0.05)
38.8 6.1 (p < 0.02)
36.8 8.8 (p < 0.02)
7.3 3.8 (p > 0.05)
28.2 4.9 (p < 0.05)
63.2 10.7 (p < 0.002)
30.8 6.8 (p < 0.02)
12.0 5.2 (p < 0.05)
35.5 7.6 (p < 0.02)
50.6 14.5 (p < 0.01)
18.1 4.8 (p < 0.05)
18.6 5.1 (p < 0.05)
38.3 7.2 (p < 0.02)
2
3
4
5
6
7
8
9
curcumin
extract
a
The results are representative of three independent experiments and
are expressed as means SD. The statistical significances were
confirmed with the paired Student’s t test.
F
dx.doi.org/10.1021/np5004966 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
d4, 600 MHz) data, see Table 1; HR-MALDI-TOF-MS [M + Na]+ m/
z 379.1524 (calcd for C21H24O5Na, 379.1521).
C. avellana leaf extracts, confirming the beneficial value of the
diarylheptanoid derivatives. Furthermore, the discovery of
phytochemicals with health benefits in the PGI byproducts
could be useful to give an interesting and economically feasible
opportunity to use waste materials, such as leaves, as a source of
functional ingredients for nutraceutical, herbal, and cosmetic
formulations.
Giffonin D (4): amorphous, white solid; IR (KBr) νmax 3425, 2930,
1713, 1665 cm−1; 1H and 13C NMR (methanol-d4, 600 MHz) data, see
Table 1; HR-MALDI-TOF-MS [M + Na]+ m/z 377.1367 (calcd for
C21H22O5Na, 377.1365).
Giffonin E (5): amorphous, white solid; [α]25D −28 (c 0.03 MeOH);
1
IR (KBr) νmax 3430, 2935, 1665 cm−1; H and 13C NMR (methanol-
d4, 600 MHz) data, see Table 2; HR-MALDI-TOF-MS [M + Na]+ m/
z 379.1526 (calcd for C21H24O5Na, 379.1521).
EXPERIMENTAL SECTION
Giffonin F (6): amorphous, white solid; [α]25D −24 (c 0.03 MeOH);
IR (KBr) νmax 3432, 2940, 1710, 1665 cm−1; 1H and 13C NMR
(methanol-d4, 600 MHz) data, see Table 2; HR-MALDI-TOF-MS [M
+ Na]+ m/z 393.1319 (calcd for C21H22O6Na, 393.1314).
■
General Experimental Procedures. Optical rotations were
measured on a JASCO DIP 1000 polarimeter. IR measurements
were obtained on a Bruker IFS-48 spectrometer. NMR experiments
were performed on a Bruker DRX-600 spectrometer (Bruker BioSpin
GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI
CryoProbeat 300 K. All 2D NMR spectra were acquired in methanol-
d4 (99.95%, Sigma-Aldrich), and standard pulse sequences and phase
cycling were used for DQF-COSY, HSQC, and HMBC spectra. The
NMR data were processed using UXNMR software. The ROESY
spectra were acquired with tmix = 400 ms. Exact masses were measured
using a MALDI-TOF MicroMX (Micromass) mass spectrometer. A
mixture of analyte solution and α-cyano-4-hydroxycinnamic acid
(Sigma) was applied to the metallic sample plate and dried. Mass
calibration was performed with the ions from the ACTH (fragment
18−39) at 2465.1989 Da, with α-cyano-4-hydroxycinnamic acid at
190.0504 Da as an internal standard. GC analysis was performed on a
Thermo Finnigan Trace GC apparatus with an FID detector and using
a l-Chirasil-Val column (0.32 mm × 25 m). Column chromatography
was performed over Sephadex LH-20 (Pharmacia). HPLC separations
were carried out on a Waters 590 system equipped with a Waters R401
refractive index detector, a Waters XTerra Prep MSC18 column (300
× 7.8 mm i.d.), and a Rheodyne injector.
Giffonin G (7): amorphous, white solid; [α]25D −71 (c 0.1 MeOH);
1
IR (KBr) νmax 3430, 2930, 1620 cm−1; H and 13C NMR (methanol-
d4, 600 MHz) data, see Table 2; HR-MALDI-TOF-MS [M + Na]+ m/
z 377.1365 (calcd for C21H22O5Na, 377.1365).
Giffonin H (8): amorphous, white solid; [α]25 −22 (c 0.03
D
1
MeOH); IR (KBr) νmax 3430, 2930, 1620 cm−1; H and 13C NMR
(methanol-d4, 600 MHz) data, see Table 2; HR-MALDI-TOF-MS [M
+ Na]+ m/z 347.1261 (calcd for C20H20O4Na, 347.1259).
Giffonin I (9): amorphous, white solid; [α]25D −4 (c 0.1 MeOH); IR
1
(KBr) νmax 3425, 2920, 1710 cm−1; H and 13C NMR (methanol-d4,
600 MHz) data, see Table 3; HR-MALDI-TOF-MS [M + Na]+ m/z
611.2107 (calcd for C30H36O12Na, 611.2104).
Preparation of (S)- and (R)-MTPA Esters of 2, 3, and 5−8.
(R)-(−) and (S)-(+)-MTPA-Cl (15 μL) and a catalytic amount of
DMAP were separately added to two different aliquots of 2 (each 1.5
mg) in anhydrous pyridine. The resulting mixtures were maintained at
room temperature under vigorous stirring overnight, and then 1H
NMR spectra were recorded. The related proton signals were assigned
by analyzing COSY spectra. By the same procedure, the (S)- and (R)-
MTPA esters of 3 and 5−8 were prepared, and the related proton
signals were also assigned by analyzing COSY spectra.
Plant Material. The leaves of the “Nocciola di Giffoni” (Corylus
avellana) were collected at Giffoni, Salerno, Italy, in November 2012
and identified by V. De Feo (Department of Pharmacy, University of
Salerno, Italy). A voucher specimen (No. 133) has been deposited in
this Department.
1
(S)-MTPA ester of 2 (2a): selected H NMR values (600 MHz,
methanol-d4) δH 7.80 (dd, J = 8.5, 1.9 Hz, H-19), 7.74 (dd, J = 8.5, 1.9
Hz, H-15), 7.29 (dd, J = 8.5, 1.9 Hz, H-18), 5.79 (dd, J = 5.4, 8.9 Hz,
H-13), 5.25 (ddd, J = 15.5, 10.5, 4.0, H-8), 3.17 (t, J = 5.5 Hz, H-12),
2.65 (dt, J = 16.0, 3.0 Hz, H-10), 2.60 (m, H-9), 2.15 (m, H-9).
Extraction and Isolation. The leaves of “Nocciola di Giffoni” (C.
avellana L.) (910 g) were dried and extracted at room temperature
using solvents of increasing polarity such as n-hexane (2.5 L for 3 days,
three times), CHCl3 (2.5 L for 3 days, three times), and MeOH (2.5 L
for 3 days, three times). After filtration and evaporation of the solvent
to dryness in vacuo, 30 g of crude MeOH extract was obtained. The
dried MeOH extract (3.0 g) was fractionated on a Sephadex LH-20
(Pharmacia) column (100 × 5 cm), using MeOH as mobile phase,
affording 88 fractions (8 mL), monitored by TLC. Fractions 34 and 35
(56.5 mg) were chromatographed by semipreparative HPLC using
MeOH−H2O (3:2) as mobile phase (flow rate 2.5 mL/min) to yield
compounds 2 (1.8 mg, tR = 11.1 min), 6 (1.5 mg, tR = 19.5 min), 3
(2.9 mg, tR = 30.2 min), and 5 (1.6 mg, tR = 52.2 min). Fractions 36−
40 (90.0 mg) were chromatographed by semipreparative HPLC using
MeOH−H2O (13:7) as mobile phase (flow rate 2.5 mL/min) to yield
compounds 8 (2.4 mg, tR = 18.0 min), 1 (2.0 mg, tR = 19.2 min), 7
(3.5 mg, tR = 20.5 min), and 4 (4.1 mg, tR = 21.2 min). Fraction 41
(13.5 mg) was chromatographed by semipreparative HPLC using
MeOH−H2O (9:11) as mobile phase (flow rate 2.5 mL/min) to yield
compound 9 (2.5 mg, tR = 20.0 min). This procedure was repeated
three times to accumulate sufficient amounts of compounds 2, 3, and
5−8 to subject to the modified Mosher’s method.
1
(R)-MTPA ester of 2 (2b): selected H NMR values (600 MHz,
methanol-d4) δH 7.74 (dd, J = 8.5, 1.9 Hz, H-19), 7.72 (dd, J = 8.5, 1.9
Hz, H-15), 7.28 (dd, J = 8.5, 1.9 Hz, H-18), 5.79 (dd, J = 5.4, 8.9 Hz,
H-13), 5.26 (ddd, J = 15.5, 10.5, 4.0, H-8), 3.43 (t, J = 5.5 Hz, H-12),
2.68 (dt, J = 16.0, 3.0 Hz, H-10), 2.61 (m, H-9), 2.17 (m, H-9).
1
(S)-MTPA ester of 3 (3a): selected H NMR values (600 MHz,
methanol-d4) δH 6.49 (d, J = 11.5 Hz, H-7), 5.65 (ddd, J = 11.5, 7.5,
7.5 Hz, H-8), 4.23 (m, H-11), 3.15 (dt, J = 12.5, 3.7 Hz, H-13), 2.85
(td, J = 12.5, 5.2 Hz, H-13), 2.21 (m, H-12), 2.01 (m, H-9), 1.98 (m,
H-12), 1.97 (m, H-9), 1.30 (m, H-10), 0.75 (m, H-10).
1
(R)-MTPA ester of 3 (3b): selected H NMR values (600 MHz,
methanol-d4) δH 6.48 (d, J = 11.4 Hz, H-7), 5.63 (ddd, J = 11.5, 7.5,7.5
Hz, H-8), 4.23 (m, H-11), 3.19 (dt, J = 12.5, 3.7 Hz, H-13), 2.87 (td, J
= 12.5, 5.2 Hz, H-13), 2.31 (m, H-12), 2.00 (m, H-9), 2.03 (m, H-12),
1.95 (m, H-9), 1.24 (m, H-10), 0.68 (m, H-10).
1
(S)-MTPA ester of 5 (5a): selected H NMR values (600 MHz,
methanol-d4) δH 6.43 (d, J = 11.2 Hz, H-7), 5.20 (dd, J = 11.2, 8.6 Hz,
H-8), 5.05 (ddd, J = 12.0, 8.5, 3.4 Hz, H-9), 3.07 (m, H-13), 2.45 (td, J
= 12.5, 5.4 Hz, H-13), 2.16 (m, H-12), 1.98 (q, J = 10.8 Hz, H-11),
1.45 (m, H-10), 1.38 (m, H-12), 1.12 (m, H-11), 0.35 (br t, J = 11.8
Hz, H-10).
Giffonin A (1): amorphous, white solid; IR (KBr) νmax 3440, 2935,
1705, 1660 cm−1; 1H and 13C NMR (methanol-d4, 600 MHz) data, see
Table 1; HR-MALDI-TOF-MS [M + Na]+ m/z 377.1369 (calcd for
C21H22O5Na, 377.1365).
1
(R)-MTPA ester of 5 (5b): selected H NMR values (600 MHz,
methanol-d4) δH 6.40 (d, J = 11.2 Hz, H-7), 5.15 (dd, J = 11.2, 8.6 Hz,
H-8), 5.05 (ddd, J = 12.0, 8.5, 3.4 Hz, H-9), 3.09 (m, H-13), 2.48 (td, J
= 12.5, 5.4 Hz, H-13), 2.21 (m, H-12), 2.05 (q, J = 10.8 Hz, H-11),
1.65 (m, H-10), 1.40 (m, H-12), 1.28 (m, H-11), 0.40 (br t, J = 11.8
Hz, H-10).
Giffonin B (2): amorphous, white solid; [α]25D −14 (c 0.1 MeOH);
IR (KBr) νmax 3430, 2940, 1705, 1660 cm−1; 1H and 13C NMR
(methanol-d4, 600 MHz) data, see Table 1; HR-MALDI-TOF-MS [M
+ Na]+ m/z 393.1318 (calcd for C21H22O6Na, 393.1314).
1
(S)-MTPA ester of 6 (6a): selected H NMR values (600 MHz,
Giffonin C (3): amorphous, white solid; [α]25D −28 (c 0.03 MeOH);
methanol-d4) δH 6.53 (d, J = 11.8 Hz, H-7), 5.25 (dd, J = 11.8, 8.6 Hz,
H-8), 5.15 (t, J = 10.0 Hz, H-9), 3.13 (m, H-13), 3.09 (td, J = 12.5, 5.5
1
IR (KBr) νmax 3450, 2940, 1655 cm−1; H and 13C NMR (methanol-
G
dx.doi.org/10.1021/np5004966 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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Hz, H-13), 2.90 (td, J = 12.5, 5.5 Hz, H-12), 2.59 (m, H-12), 2.75 (m,
supernatant was mixed with 0.2 volume of 0.12 M thiobarbituric acid
in 0.26 M Tris at pH 7.0 and immersed in a boiling water bath for 15
min; then absorbance at 532 nm (UV/vis Helios alpha Unicam
spectrophotometer) was measured.21 The TBARS concentration was
calculated using the molar extinction coefficient (ε = 156 000 M−1
cm−1).
Statistical Analysis. The statistical analysis was done by several
tests. In order to eliminate uncertain data, the Q-Dixon test was
performed. All the values in this study were expressed as mean SD.
The statistical analysis was performed with one-way ANOVA for
repeated measurements. The statistically significant differences were
also assessed by applying the paired Student’s t test.
H-10), 1.90 (br s, H-10).
(R)-MTPA ester of 6 (6b): selected H NMR values (600 MHz,
1
methanol-d4) δH 6.50 (d, J = 11.8 Hz, H-7), 5.20 (dd, J = 11.8, 8.6 Hz,
H-8), 5.15 (t, J = 10.0 Hz, H-9), 3.15 (m, H-13), 3.10 (td, J = 12.5, 5.5
Hz, H-13), 2.92 (td, J = 12.5, 5.5 Hz, H-12), 2.60 (m, H-12), 2.80 (m,
H-10), 2.05 (br s, H-10).
1
(S)-MTPA ester of 7 (7a): selected H NMR values (600 MHz,
methanol-d4) δH 7.52 (dd, J = 8.2, 1.9 Hz, H-19), 7.08 (dd, J = 8.2, 1.9
Hz, H-18), 6.47 (d, J = 11.4 Hz, H-7), 6.05 (dd, J = 11.4, 9.0 Hz, H-8),
5.87 (dd, J = 15.4, 8.4 Hz, H-10), 5.69 (dd, J = 15.4, 9.0 Hz, H-9), 5.57
(dt, J = 8.4, 3.3 Hz, H-11), 3.05 (dt, J = 12.5, 3.0 Hz, H-13), 2.83 (td, J
= 12.5, 3.0 Hz, H-13), 2.27 (dq, J = 14.0, 3.3 Hz, H-12), 1.75 (tt, J =
14.0, 3.3 Hz, H-12).
ASSOCIATED CONTENT
■
1
(R)-MTPA ester of 7 (7b): selected H NMR values (600 MHz,
S
* Supporting Information
methanol-d4) δH 7.53 (dd, J = 8.2, 1.9 Hz, H-19), 7.09 (dd, J = 8.2, 1.9
Hz, H-18), 6.45 (d, J = 11.4 Hz, H-7), 6.02 (dd, J = 11.4, 9.0 Hz, H-8),
5.83 (dd, J = 15.4, 8.4 Hz, H-10), 5.67 (dd, J = 15.4, 9.0 Hz, H-9), 5.57
(dt, J = 8.4, 3.3 Hz, H-11), 3.08 (dt, J = 12.5, 3.0 Hz, H-13), 2.85 (td, J
= 12.5, 3.0 Hz, H-13), 2.41 (dq, J = 14.0, 3.3 Hz, H-12), 1.79 (tt, J =
14.0, 3.3 Hz, H-12).
1H and 13C NMR, HSQC, HMBC, COSY, and ROESY spectra
of compounds 1−9. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +39 089969763. Fax: +39 089969602. E-mail: piacente@
unisa.it.
■
1
(S)-MTPA ester of 8 (8a): selected H NMR values (600 MHz,
methanol-d4) δH 7.41 (dd, J = 8.3, 1.9 Hz, H-19), 7.30 (dd, J = 8.3, 1.9
Hz, H-18), 6.22 (d, J = 11.4 Hz, H-7), 5.98 (t, J = 11.4 Hz, H-8), 5.88
(dd, J = 15.4, 8.6 Hz, H-10), 5.79 (dd, J = 15.4, 11.4 Hz, H-9), 5.57
(dt, J = 8.6, 3.5 Hz, H-11), 3.06 (dt, J = 13.0, 3.3 Hz, H-13), 2.80 (td, J
= 13.0, 3.3 Hz, H-13), 2.24 (dq, J = 14.2, 3.4 Hz, H-12), 1.73 (tt, J =
14.2, 3.4 Hz, H-12).
Notes
The authors declare no competing financial interest.
1
(R)-MTPA ester of 8 (8b): selected H NMR values (600 MHz,
ACKNOWLEDGMENTS
methanol-d4) δH 7.42 (dd, J = 8.3, 2.9 Hz, H-19), 7.31 (dd, J = 8.3, 2.9
Hz, H-18), 6.20 (d, J = 11.2 Hz, H-7), 5.96 (t, J = 11.2 Hz, H-8), 5.84
(dd, J = 15.4, 8.6 Hz, H-10), 5.76 (dd, J = 15.4, 11.2 Hz, H-9), 5.57
(dt, J = 8.6, 3.8 Hz, H-11), 3.08 (dt, J = 12.8, 3.3 Hz, H-13), 2.82 (td, J
= 12.8, 3.3 Hz, H-13), 2.36 (dq, J = 14.2, 3.8 Hz, H-12), 1.75 (tt, J =
14.2, 3.8 Hz, H-12).
Determination of the Sugar Configuration. The configurations
of sugar units of compound 9 were established after hydrolysis of 9
with 1 N HCl, trimethylsilation, and determination of the retention
times by GC operating in the reported experimental conditions.20 The
peaks of the hydrolysate of 9 were detected at 14.74 min (D-glucose)
and at 8.91 and 9.83 min (L-arabinose). Retention times for authentic
samples after being treated in the same manner with 1-(trimethylsilyl)-
imidazole in pyridine were detected at 14.71 min (D-glucose) and 8.92
and 9.80 (L-arabinose).
Biological Assay. DMSO, thiobarbituric acid (TBA), and H2O2
were purchased from Sigma (St. Louis, MO, USA). All other reagents
were of analytical grade and were provided by commercial suppliers.
Stock solutions of tested compounds and tested plant extract were
made in 50% DMSO. The final concentration of DMSO in samples
was lower than 0.05%, and in all experiments its effects were
determined.
■
This work was supported by grant 506/1136 from University of
Lodz.
REFERENCES
■
(1) Shahidi, F.; Alasalvar, C.; Liyana-Pathirana, C. M. J. Agric. Food
Chem. 2007, 55, 1212−1220.
́
́ ́
(2) Riethmuller, E.; Alberti, A.; Toth, G.; Beni, S.; Ortolano, F.;
̈
́
Kerya, A. Phytochem. Anal. 2013, 24, 493−503.
(3) Amaral, J. S.; Ferreres, F.; Nadrade, P. B.; Valentao, P.; Pinheiro,
C.; Santos, A.; Seabra, R. Nat. Prod. Res. 2005, 19, 157−163.
(4) De Salvador, F. R.; Proietti, G.; Lolletti, D.; Tombesi, A.;
Farinelli, D.; Delfini, M.; Di Cocco, M. E. Acta Hortic. 2009, 845, 599−
606.
(5) Stevigny, C.; Rolle, L.; Valentini, N.; Zeppa, G. J. Sci. Food Agric.
2007, 87, 2817−2822.
(6) Kirmizibekmez, H.; Ariburnu, E.; Masullo, M.; Festa, M.;
Capasso, A.; Yesilada, E.; Piacente, S. Fitoterapia 2012, 83, 130−136.
(7) Denizli, N.; Horo, I.; Gulcemal, D.; Masullo, M.; Festa, M.;
Capasso, A.; Koz, O.; Piacente, S.; Alankus-Caliskan, O. Fitoterapia
2014, 92, 211−218.
(8) Pereira do Amaral, F.; Napolitano, A.; Masullo, M.; Campaner
dos Santos, L.; Festa, M.; Vilegas, W.; Pizza, C.; Piacente, S. J. Nat.
Prod. 2012, 75, 547−556.
Fresh human plasma was obtained from medication-free, regular
donors at a blood bank (Lodz, Poland). Samples of human plasma
were incubated with (1) plant extract at final concentrations of 0.1−
100 μg/mL (30 min, at 37 °C); (2) plant extract at final
concentrations of 0.1−100 μg/mL plus 2 mM H2O2 (30 min, at 37
°C); (3) plant extract at a final concentration of 10 μg/mL plus 4.7
mM H2O2/3.8 mM Fe2SO4/2.5 mM EDTA (30 min, at 37 °C); (4)
test compounds (1−9) and curcumin at final concentrations of 0.1−
100 μM (30 min, at 37 °C); (5) test compounds (1−9) and curcumin
at final concentrations of 0.1−100 μM plus 2 mM H2O2 (30 min, at 37
°C); (6) test compounds (1−9) and curcumin at a final concentration
of 10 μM plus 4.7 mM H2O2/3.8 mM Fe2SO4/2.5 mM EDTA (30
min, at 37 °C).
Lipid Peroxidation Measurement. Lipid peroxidation was
quantified by measuring the concentration of TBARS. Incubation of
plasma (control, test compounds, treated plasma) was stopped by
cooling the samples in an ice-bath. Samples of plasma were transferred
to an equal volume of 20% (v/v) cold trichloroacetic acid in 0.6 M
HCl and centrifuged at 1200g for 15 min. One volume of clear
(9) Lee, W. S.; Kim, J. R.; Im, K. R.; Cho, K. H.; Sok, D. E.; Jeong, T.
S. Planta Med. 2005, 71, 295−299.
(10) Jin, W.; Cai, X. F.; Na, M.; Lee, J. J.; Bae, K. Biol. Pharm. Bull.
2007, 30, 810−813.
(11) Martineau, L. C.; Herve, J.; Muhamad, A.; Saleem, A.; Harris, C.
́
S.; Arnason, J. T.; Haddad, P. S. Planta Med. 2010, 76, 1439−1446.
(12) Appendino, G.; Pollastro, F.; Verotta, L.; Ballero, M.; Romano,
A.; Wyrembek, P.; Szczuraszek, K.; Mozrzymas, J. W.; Taglialatela-
Scafati, O. J. Nat. Prod. 2009, 72, 962−965.
(13) Fuchino, H.; Konishi, S.; Satoh, T.; Yagi, A.; Saitsu, K.; Tatsumi,
T.; Tanaka, N. Chem. Pharm. Bull. 1996, 44, 1033−1038.
(14) Nomura, M.; Tokoroyama, T.; Kubota, T. Phytochemistry 1981,
20, 1097−1104.
(15) Semmelhack, M. F.; Helquist, P.; Jones, L. D.; Keller, L.;
Mendelson, L.; Ryono, L. S.; Gorzynski, S. J.; Stauffer, R. D. J. Am.
Chem. Soc. 1975, 97, 3873−3875.
H
dx.doi.org/10.1021/np5004966 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(16) Altunkeyik, H.; Gulcemal, D.; Masullo, M.; Alankus-Caliskan,
O.; Piacente, S.; Karayildirim, T. Phytochemistry 2012, 73, 127−133.
(17) Nagumo, S.; Ishizawa, S.; Nagai, M.; Inoue, T. Chem. Pharm.
Bull. 1996, 44, 1086−1089.
(18) Reddy, V. L. N.; Ravinder, K.; Srinivasulu, M.; Goud, T. V.;
Reddy, S. M.; Srujankumar, D.; Rao, T. P.; Murty, U. S.;
Venkateswarlu, Y. Chem. Pharm. Bull. 2003, 51, 1081−1084.
(19) Tene, M.; Wabo, H. K.; Kamnaing, P.; Tsopmo, A.; Tane, P.;
Ayafor, J. F.; Sterner, O. Phytochemistry 2000, 54, 975−978.
(20) Gulcemal, D.; Masullo, M.; Bedir, E.; Festa, M.; Karayildirim, T.;
Alankus-Caliskan, O.; Piacente, S. Planta Med. 2012, 78, 720−729.
(21) Wachowicz, B. Cell Biochem. Funct. 1984, 2, 167−170.
I
dx.doi.org/10.1021/np5004966 | J. Nat. Prod. XXXX, XXX, XXX−XXX