Lignans from the fruit of Schisandra glaucescens with antioxidant and neuroprotective properties

Article abstract of DOI:10.1021/np4010536

Two rare 7,8-seco-lignans (1, 2), three new lignan glycosides (3, 4a, 4b), and 10 known lignans (5-14) were isolated from the fruit of Schisandra glaucescens Diels. The absolute configurations of 1 and 2 were determined by comparing their experimental and calculated electronic circular dichroism spectra. The molecular structures of the new compounds (3, 4a, and 4b), including their absolute configurations, were determined using various spectroscopic methods and hydrolysis reactions. The antioxidant activities of the isolated compounds were tested using 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays. Compounds 4, 7, 8, 10, 11, and 12 exhibited antioxidant activities of varying potential in both assays. Of these compounds, 7 showed the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, with IC₅₀ values of 15.7 (150 μM DPPH) and 34.6 μM (300 μM DPPH), respectively, and 4, 12, and 7 displayed higher total antioxidant activities than Trolox in the ferric reducing antioxidant power assay. The neuroprotective effects of these compounds against Aβ_25-35-induced cell death in SH-SY5Y cells were also investigated. Compounds 1, 2, 6, 7, 8, 11, and 12 exhibited statistically significant neuroprotective effects against Aβ_25-35-induced SH-SY5Y cell death compared with the group treated only with Aβ_25-35.

Full text of DOI:10.1021/np4010536

Article
pubs.acs.org/jnp
Lignans from the Fruit of Schisandra glaucescens with Antioxidant
and Neuroprotective Properties
Heng-Yi Yu,†,# Zu-Yu Chen,†,‡,# Bin Sun,† Junjun Liu,† Fan-Yu Meng,† Ye Liu,† Tian Tian,† An Jin,†
and Han-Li Ruan*,†
†Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430000, People’s Republic
of China
‡College of Environment Engineering, Wuhan Textile University, Wuhan 430000, People’s Republic of China
S
* Supporting Information
ABSTRACT: Two rare 7,8-seco-lignans (1, 2), three new lignan glycosides (3, 4a, 4b), and 10 known lignans (5−14) were
isolated from the fruit of Schisandra glaucescens Diels. The absolute configurations of 1 and 2 were determined by comparing their
experimental and calculated electronic circular dichroism spectra. The molecular structures of the new compounds (3, 4a, and
4b), including their absolute configurations, were determined using various spectroscopic methods and hydrolysis reactions. The
antioxidant activities of the isolated compounds were tested using 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant
power assays. Compounds 4, 7, 8, 10, 11, and 12 exhibited antioxidant activities of varying potential in both assays. Of these
compounds, 7 showed the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, with IC50 values of 15.7
(150 μM DPPH) and 34.6 μM (300 μM DPPH), respectively, and 4, 12, and 7 displayed higher total antioxidant activities than
Trolox in the ferric reducing antioxidant power assay. The neuroprotective effects of these compounds against Aβ25−35-induced
cell death in SH-SY5Y cells were also investigated. Compounds 1, 2, 6, 7, 8, 11, and 12 exhibited statistically significant
neuroprotective effects against Aβ25−35-induced SH-SY5Y cell death compared with the group treated only with Aβ25−35
.
lants of the genus Schisandra are economically valuable and
widely used in traditional Chinese medicine. Schisandra
basis for the antioxidant and neuroprotective effects of S.
P
glaucescens fruit. As a result, 15 lignans, including two rare 7,8-
seco-lignans and three new lignan glycosides, were isolated,
identified, and tested for their antioxidant and neuroprotective
activities.
glaucescens Diels is a vine primarily found in western Hubei and
southeastern Sichuan Provinces in China. In traditional Chinese
medicine, the stems of this plant have been used for the
treatment of various diseases, including contusions, rheuma-
tism, and arthritis.1 In recent years, the stems of S. glaucescens
have been studied extensively, and a number of triterpenoids
and lignans have been isolated.2−7
The ripe fruit of S. glaucescens is a sweet red berry that is
consumed as a health food by villagers in the mountains around
Shennongjia, China. These berries are believed to be beneficial
for the lungs and kidneys, relieve the symptoms of asthma,
reduce sweating and night sweats, alleviate chronic diarrhea,
and reduce neurasthenia. Preliminary bioactivity screening
revealed that the ethanol extract of S. glaucescens fruit had 2,2-
diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
and provided neuroprotection against Aβ25−35-induced SH-
SY5Y cell death. Phytochemical studies and antioxidant and
neuroprotective assays were used to investigate the chemical
7,8-Seco-lignans (or 1-phenylbutyl benzoates) are a group of
rare lignans that possess various bioactivities, including anti-
HIV,8,9 anti-HSV,10 antiadenovirus,10 and cytotoxic activities.11
These compounds were first isolated from the leaves of Ocotea
foetens (Lauraceae) in 199512 and were then found in Holostylis
reniformis (Aristolochiaceae) and Beilschmiedia tsangii (Laur-
aceae).11,13 In 2009, these compounds were identified for the
first time in a plant in the Schisandraceae family when two 7,8-
seco-lignans were isolated from the fruit of S. sphenanthera.14
Since then, a number of analogues have been isolated from
plants in the Schisandraceae family.8−10,15
Received: December 15, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Structures of compounds 1−14.
Table 1. NMR Spectroscopic Data (400 MHz, CDCl3) for Compounds 1 and 2
1
2
position
δC, type
δH (J in Hz)
7.48, d (1.9)
position
δC, type
δH (J in Hz)
7.40, d (1.6)
1
122.6, C
1
124.2, C
2
112.3, CH
148.9, C
2
109.6, CH
147.9, C
3
3
4
153.4, C
4
151.9, C
6.81, d (8.2)
5
110.5, CH
123.7, CH
165.1, C
6.86, d (8.5)
5
108.2, CH
125.6, CH
164.7, C
7.60, dd (8.2, 1.7)
6
7.62, dd (8.4, 1.9)
6
7
7
8
209.7, C
8
209.9, C
2.24, s
9
28.8, CH3
132.2, C
2.24, s
9
28.8, CH3
130.8, C
1′
2′
3′
4′
5′
6′
7′
8′
9′
1′
2′
3′
4′
5′
6′
7′
8′
9′
6.91, d (1.9)
107.4, CH
148.1, C
6.90, brs
110.5, CH
149.2, C
147.9, C
149.3, C
6.84, d (8.2)
6.99, dd (8.2, 1.9)
5.94, d (10.1)
3.20, dq (10.1, 7.1)
0.96, d (7.1)
3.88, s
108.4, CH
121.5, CH
78.1, CH
52.6, CH
13.7, CH3
56.2, CH3
56.1, CH3
101.3, CH2
6.78, d (8.2)
6.91, overlap
5.92, d (10.1)
3.18, dq (10.1, 7.1)
0.98, d (7.1)
3.90, s
111.3, CH
120.2, CH
78.2, CH
52.5, CH
13.8, CH3
56.1, CH3
56.0, CH3
101.9, CH2
3-OCH3
3′-OCH3
4′-OCH3
3,4-OCH2O−
3.86, s
4-OCH3
3.92, s
6.02, s
3′,4′-OCH2O−
5.94, dd (4.5, 1.2)
Most structural studies of 7,8-seco-lignans have focused on
7,8-seco-holostylone B.13 One- and two-dimensional NMR
experiments have been used to determine the relative
configuration of 7,8-seco-holostylone B, but its absolute
configuration has not been determined.13 Herein, the absolute
configurations of two rare 7,8-seco-lignans (1 and 2, Figure 1)
B
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were defined by comparing their experimental and calculated
electronic circular dichroism (ECD) spectra.
H-2,6/H-9 were observed (Figure 2). However, no correlation
between H-2′,6′/H-9 or between H-2,6/H-9′ was detected in
the NOESY spectrum. The above evidence suggested that 1 has
the same relative configuration as 7,8-seco-holostylone B13 and
schisandlignan B.15
The absolute configuration of 1 was determined by
comparing its experimental and calculated ECD spectra. As
shown in Figure 3, the sequence and signs of the Cotton effects
RESULTS AND DISCUSSION
■
The air-dried fruit of S. glaucescens were extracted with 95%
ethanol at room temperature, and the extract was concentrated
in vacuo. The resulting crude extract was sequentially
partitioned with petroleum ether, EtOAc, and n-BuOH. The
petroleum ether- and EtOAc-soluble fractions were subjected to
silica gel, Sephadex LH-20, and MCI column chromatography
followed by repeated RP C18 HPLC to afford compounds 1−14
(Figure 1).
Compound 1 was obtained as a colorless oil with a negative
specific rotation, [α]2D0 −39 (c 0.9, CHCl3), and its molecular
formula was assigned as C21H22O7 based on the 13C NMR
spectroscopic data and an HRESIMS ion at m/z 409.1258 [M
+ Na]+ (calcd 409.1263). The IR spectrum exhibited
absorption bands from phenyl (1598 cm−1) and carbonyl
(1713 cm−1) moieties.
The 1H NMR data for 1 (Table 1) indicated the presence of
two 1,2,4-trisubstituted benzene systems [δH 7.48 (1H, d, J =
1.9 Hz, H-2), 6.86 (1H, d, J = 8.5 Hz, H-5) and 7.62 (1H, dd, J
= 8.4, 1.9 Hz, H-6); 6.90 (1H, brs, H-2′), 6.78 (1H, d, J = 8.2
Hz, H-5′) and 6.91 (1H, overlapped, H-6′)], one methyl-
enedioxy [δH 5.94 (2H, dd, J = 4.5, 1.2 Hz, 3′,4′-OCH2O−)],
two methoxy groups [δH 3.90 (3H, s, 3-OCH3) and 3.92 (3H,
s, 4-OCH3)], two methyls [δH 2.24 (3H, s, H-9) and 0.98 (3H,
d, J = 7.1 Hz, H-9′)], and two methines [δH 5.92 (1H, d, J =
10.1 Hz, H-7′) and 3.18 (1H, dq, J = 10.1, 7.1 Hz, H-8′)]. The
13C NMR data (Table 1) indicated two carbonyl carbon
resonances at δC 209.7 (C-8) and 165.1 (C-7). The above 1D
NMR characteristics suggested that 1 is a 7,8-seco-lignan.13 In
1
the H−1H COSY spectrum, the presence of a −CH(H-7′)−
CH(H-8′)−CH3(H-9′)− unit was evident. In the HMBC
spectrum (Figure 2), H-9 [δH 2.24 (3H, s)] was correlated to
Figure 3. Experimental ECD spectrum of compound 1 (A) and
calculated ECD spectra for the enantiomers (7′R,8′S)- and (7′S,8′R)-1
(B).
Figure 2. Key HMBC (→) and NOESY (↔) correlations of
compound 1.
in the experimental ECD spectrum were consistent with those
in the calculated ECD curve of (7′R,8′S)-1. Thus, the structure
of 1 was determined to be (7′R,8′S)-3,4-dimethoxy-3′,4′-
methylenedioxy-7,8-seco-7,7′-epoxylignan-7,8-dione.
C-8 (δC 209.7) and C-8′ (δC 52.6), which indicated that a
methyl ketone moiety (δC 209.7 and 28.8) is linked to C-8′.
Additionally, H-7′ [δH 5.91 (1H, d, J = 10.1 Hz)] was
correlated to C-1′ (δC 132.2), C-2′ (δC 107.4), C-6′ (δC 121.5),
and C-7 (δC 165.1) in the HMBC spectrum, which indicated
the linkage of C-7′ to the 3,4-methylenedioxyphenyl unit and
the linkage of C-7′ to the 3,4-dimethoxyphenyl unit via an ester
oxygen. On the basis of the above evidence, the planar structure
of 1 is 3,4-dimethoxy-3′,4′-methylenedioxy-7,8-seco-7,7′-epox-
ylignan-7,8-dione, which is the same as those of the known
compounds 7,8-seco-holostylone B13 and schisandlignan B.15
The coupling constant (J = 10.1 Hz) between H-7′ [δH 5.92
(1H, d, J = 10.1 Hz)] and H-8′ [δH 3.18 (1H, dq, J = 10.1, 7.1
Hz)] indicated that the preferred conformation of these two
protons is anti.13 Key NOESY correlations of H-2′,6′/H-9′ and
Compound 2 was obtained as a colorless oil with a negative
specific rotation, [α]2D0 −73 (c 1.5, CHCl3), and its molecular
formula was assigned as C21H22O7, the same as that of 1, based
on the 13C NMR data and an HRESIMS ion at m/z 409.1256
[M + Na]+ (calcd 409.1263).
1
The H and 13C NMR data for 2 (Table 1) indicated that 2
has all of the functional groups of 1. Analysis of the HSQC and
HMBC spectra of 2 suggested that the positions of the two
aromatic groups are interchanged compared to 1. In the
HMBC spectrum (Figure 4), H-7′ [δH 5.94 (1H, d, J = 10.1
Hz)] was correlated to C-1′ (δC 130.8), C-2′ (δC 110.5), C-6′
(δC 120.2), and C-7 (δC 164.7), which indicated the linkage of
C
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agreed with the experimental data. Therefore, the structure of 2
was established as (7′R,8′S)-3,4-methylenedioxy-3′,4′-dime-
thoxy-7,8-seco-7,7′-epoxylignan-7,8-dione.
Compound 3 was obtained as a white, amorphous powder
with a negative specific rotation, [α]2D0 −44 (c 0.2, CH3OH),
and its molecular formula was assigned as C31H40O15 based on
the 13C NMR data and an HRESIMS ion at m/z 675.2252 [M
+ Na]+ (calcd 675.2265). The IR spectrum exhibited
absorption bands from hydroxy (3392 cm−1) and phenyl
(1610 cm−1) groups.
Figure 4. Key HMBC (→) and NOESY (↔) correlations of
compound 2.
The 1H NMR data for 3 (Table 2) indicated the presence of
two 1,2,4-trisubstituted benzene systems [δH 6.61−6.62 (2H,
overlap, H-2 and H-2′), 6.68 (2H, d, J = 8.0 Hz, H-5 and H-5′),
and 6.59 (2H, overlap, H-6 and H-6′)], two methylenedioxy
groups [δH 5.88 (4H, s, 3,4-OCH2O− and 3′,4′-OCH2O−)],
two methines [δH 2.02 (1H, m, H-8) and 1.95 (1H, m, H-8′)],
and four methylenes (two of which bear oxygen functional
groups). In addition, protons from two monosaccharide
moieties were observed at δH 3.22−5.00 [two anomeric
protons at δH 4.20 (1H, d, J = 7.8 Hz, H-1″) and 5.00 (1H,
brs, H-1‴)]. These 1H NMR characteristics suggested that 3 is
a lignan glycoside.
Because the aglycone of 3 was not stable under acidic
conditions,16 enzymatic hydrolysis of 3 with β-cellulase was
performed, and tetracentronside B (5) and (−)-dihydrocubebin
(aglycone) were obtained (Figure S67, Supporting Informa-
tion). The presence of tetracentronside B was confirmed by
TLC and HPLC analysis compared with 5, and (−)-dihy-
drocubebin was identified from its 1D NMR spectra and
specific rotation, [α]D20 −34 (c 0.2, CHCl3).17 Additionally,
negative Cotton effects at approximately 234 and 276 nm were
observed in the ECD spectra of 3, 5, and (−)-dihydrocubebin
(Figure 6), which confirmed the R configurations of C-8 and C-
8′ in these three compounds.18
C-7′ to the 3,4-dimethoxyphenyl unit and the linkage of C-7′ to
the 3,4-methylenedioxyphenyl unit via an ester oxygen. Thus,
the planar structure of 2 was established as 3,4-methylenedioxy-
3′,4′-dimethoxy-7,8-seco-7,7′-epoxylignan-7,8-dione. The cou-
pling constant (J = 10.1 Hz) between H-7′ [δH 5.94 (1H, d, J =
10.1 Hz)] and H-8′ [δH 3.20 (1H, dq, J = 10.1, 7.1 Hz)] and
the NOESY correlations of H-2′,6′/H-9′ and H-2,6/H-9
suggested that 2 has the relative configuration shown in Figure
4, which is the same as those of rel-(7′R,8′S)-3,4-methyl-
enedioxy-3′,4′-dimethoxy-7,8-seco-7,7′-epoxylignan-7,8-dione14
and schisandlignan A.15
The absolute configuration of 2 was also determined by
comparing its experimental and calculated ECD spectra. As
shown in Figure 5, the calculated ECD spectrum of (7′R,8′S)-2
To identify the monosaccharide moieties in 3, acid hydrolysis
of 3 with 4 mol/L aqueous TFA was performed and yielded D-
glucose and L-arabinose, which were identified through gas
chromatography after treatment with L-cysteine methyl ester
hydrochloride and TMS derivatization (Figure 7). The
coupling constant (J = 7.8 Hz) of the anomeric proton [δH
4.20 (1H, d, J = 7.8 Hz, H-1″)] of D-glucose and the
characteristic carbon resonances [δC 109.8 (C-1‴), 83.1 (C-
2‴), and 85.8 (C-4‴)] of L-arabinose suggest that the two
monosaccharide moieties are β-D-glucopyranosyl and α-L-
arabinofuranosyl, respectively. Moreover, the correlations of
H-1‴ [δH 5.00 (1H, brs)] to C-6″ (δC 68.0) and H-1″ [δH 4.20
(1H, d, J = 7.8 Hz)] to C-9 (δC 70.2) in the HMBC spectrum
revealed the connections of (−)-dihydrocubebin and two
monosaccharide moieties in 3. Finally, the structure of 3 was
determined to be (8R,8′R)-9-O-(6′-O-α-L-arabinofuranosyl)-β-
D-glucopyranosyldihydrocubebin.
Compound 4 was obtained as a white, amorphous powder
with a negative specific rotation, [α]2D0 −18 (c 0.2, CH3OH),
and its molecular formula was assigned as C26H34O11 based on
the 13C NMR data and HRESIMS ions at m/z 545.1984 [M +
Na]+ (calcd 545.1999) and m/z 1067.4086 [2 M + Na]+ (calcd
1067.4100).
1
The H NMR spectrum of 4 was similar to that of 5, except
that one methylenedioxy group in 5 was replaced by a hydroxy
1
and a methoxy group in 4. The H and 13C NMR spectra of 4
Figure 5. Experimental ECD spectrum of compound 2 (A) and
calculated ECD spectra for the enantiomers (7′R,8′S)- and (7′S,8′R)-2
(B).
revealed two sets of resonances, suggesting that 4 was a mixture
of isomers 4a and 4b.
D
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Table 2. NMR Spectroscopic Data (400 MHz, CD3OD) for Compound 3
position
δC, type
δH (J in Hz)
position
δC, type
δH (J in Hz)
4.20, d (7.8)
1
136.1, C
9-O-glucose
2
110.4, CH
148.9, C
6.62, overlap
2″
3″
6.68, d (8.0)
6.59, overlap
2.70, m
2.63, m
2.02, m
3.87, overlap
1‴
1″
2″
3″
4″
104.4, CH
75.1, CH
78.0, CH
72.0, CH
76.7, CH
68.0, CH2
3
3.22, t (8.1)
3.38, m
4
147.0, C
5
108.8, CH
123.1, CH
35.7, CH2
3.29, m
6
5″
3.44, m
7a
7b
8
6″a
6″b
4.03, m
3.63, m
41.8, CH
70.2, CH
3.52, m
9a
9b
1′
2′
3′
4′
5′
6′
7′a
7′b
8′
9′
6″-O-arabinose
1‴
2‴
3‴
4‴
109.8, CH
83.1, CH
78.8, CH
85.8, CH
63.0, CH2
5.00, brs
4.01, m
3.84, m
3.98, m
136.2, C
2‴
110.4, CH
148.9, C
6.61, overlap
4‴
5‴a
147.0, C
5‴a
5‴b
3.73, dd (12.0, 3.2)
3.64, m
108.8, CH
123.2, CH
35.7, CH2
6.68, d (8.0)
6.59, overlap
2.70, m
2.63, m
1.95, m
3.59, overlap
5.88, s
44.3, CH
62.5, CH2
101.9, CH2
101.9, CH2
3,4-OCH2O−
3′,4′-OCH2O−
5.88, s
and 2D NMR spectra, it was not possible to assign either set to
4a or 4b because of the overlap resonances.
The structures of compounds 5−14 were respectively
confirmed as tetracentronside B (5),16 5-[(2S,3R)-4-(3,4-
dimethoxyphenyl)-2,3-dimethylbutyl]-1,3-benzodioxole (6),20
meso-dihydroguaiaretic acid (7),21 (+)-anwulignan (8),22
(−)-galcatin (9),23 (+)-isootobaphenol (10),23 4′,5-O-dideme-
thylcyclogalgravin (11),24 (+)-chicanine (12),25 (+)-zuihonin
C (13),26 and (+)-zuihonin A (14),26 based on comparisons
with the corresponding literature data.
The free radical scavenging activity of each isolated lignan
was tested using a DPPH assay from 6.25 to 100 μM.
Compounds 4, 7, 8, 10, 11, and 12 exhibited free radical
scavenging activities with varying potential, whereas the other
compounds did not exhibit any activity. The IC50 values of
compounds 4, 7, 8, 10, 11, and 12 and the positive controls
(vitamins C and E) are presented in Table 4. The compounds
exhibited DPPH radical scavenging activities in the following
descending order: 7 > 12 > vitamin C > 4 > vitamin E > 8 > 11
> 10. Notably, compounds 7 and 12 showed higher DPPH
radical scavenging capacities than vitamin C, and compounds 7,
12, and 4 showed higher DPPH radical scavenging capacities
than vitamin E.
The ferric reducing antioxidant power (FRAP) assay is a
method for measuring the total antioxidant activity of a sample.
At low pH, the reduction of the TPTZ-Fe(III) complex to
TPTZ-Fe(II), which has an intense blue color, can be
monitored by measuring the change in absorption at 593 nm.
The corresponding FeSO4 values are calculated using standard
curves and regression equations, and a higher FeSO4 value
indicates higher ferric reducing power. In this experiment, all of
the isolated lignans were tested for total antioxidant activity
using the FRAP assay from concentrations of 0.15 to 1.5 mM.
Similarly to the results in the DPPH assay, compounds 4, 7, 8,
10, 11, and 12 showed concentration-dependent reducing
capabilities at the tested concentrations, whereas the other
Figure 6. ECD spectra of compounds 3, 5, and (−)-dihydrocubebin.
Enzymatic hydrolysis of 4 with β-cellulase yielded only one
aglycone, piperphilippinin VI (Figure S68, Supporting
Information), which was identified from its 1D NMR spectra
and specific rotation, [α]2D0 −28 (c 0.1, CHCl3).19 Negative
Cotton effects at approximately 232 and 287 nm were observed
in the ECD spectra of 4 and piperphilippinin VI (Figure 8),
which confirmed the R configurations of C-8 and C-8′ in the
two compounds.18 Acid hydrolysis of 4 with 4 mol/L aqueous
TFA yielded only D-glucose, which was identified by gas
chromatography after treatment with L-cysteine methyl ester
hydrochloride and TMS derivatization (Figure 7). Thus, we
propose that D-glucose is linked to either 9-OH or 9′-OH in
piperphilippinin VI, which explains the two sets of resonances
observed for 4 in the 1H and 13C NMR spectra. Thus,
compound 4 was a mixture of (8R,8′R)-9-O-β-D-glucopyr-
anosylpiperphilippinin VI (4a) and (8R,8′R)-9′-O-β-D-gluco-
pyranosylpiperphilippinin VI (4b). Although the two sets of
NMR resonances (Table 3) are distinguishable in both the 1D
E
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Table 3. NMR Spectroscopic Data (400 MHz, CD3OD) for
Compounds 4a and 4b
a
a
4a
4b
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1
132.5, C
112.0, CH
147.4, C
144.0, C
114.4, CH
121.4, CH
34.1, CH2
2.60, m
132.5, C
2
6.62, overlap 112.2, CH
147.4, C
6.62, overlap
3
4
144.0, C
5
6.68, overlap 114.4, CH
6.59, overlap 121.3, CH
6.68, d (8.0)
6.59, overlap
2.69, m
6
7a
7b
8
2.69, m
2.60, m
2.08, m
34.0, CH2
40.3, CH
68.9, CH
3.53, m
42.9, CH
1.99, m
3.62, m
9a
9b
1′
2′
3′
4′
5′
6′
7′a
7′b
8′
9′a
9′b
3.89, overlap 61.2, CH2
3.56, m
134.8, C
109.0, CH
147.5, C
145.6, C
107.4, CH
121.8, CH
34.0, CH2
2.60, m
134.9, C
6.61, brs
147.5, C
145.6, C
109.0, CH
6.61, s
6.68, overlap 107.4, CH
6.59, overlap 121.7, CH
6.68, d (8.0)
6.59, m
2.69, m
2.60, m
1.99, m
3.62, m
3.53, m
3.79, s
34.2, CH2
2.69, m
42.5, CH
61.3, CH2
3.56, m
40.3, CH
69.0, CH
2.08, m
3.89, overlap
4-OCH3
54.9, CH3
55.0, CH3
3.79, s
3′,4′-OCH2O− 100.6, CH2 5.89, brs
9-O-glucose
100.6, CH2 5.89, brs
9′-O-glucose
1″
2″
3″
4″
5″
6″a
6″b
103.2, CH
73.8, CH
76.7, CH
70.3, CH
76.5, CH
61.4, CH2
4.19, d (7.6) 103.2, CH
4.19, d (7.6)
3.20, m
3.20, m
3.35, m
3.26, m
3.25, m
3.86, m
3.64, m
73.8, CH
76.7, CH
70.3, CH
76.5, CH
61.4, CH2
3.35, m
3.26, m
3.25, m
3.86, m
3.64, m
Figure 7. GC analysis of the aqueous layer of the acid hydrolysis
reaction mixtures of compounds 3 and 4. (A) Separation of
enantiomers of arabinose and glucose after treatment with L-cysteine
methyl ester hydrochloride and TMS derivatization. (B) Gas
chromatogram of component monosaccharides of 3 after treatment
with L-cysteine methyl ester hydrochloride and TMS derivatization.
(C) Gas chromatogram of component monosaccharide of 4 after
treatment with L-cysteine methyl ester hydrochloride and TMS
derivatization.
a
The NMR data of compounds 4a and 4b might be interchangeable.
Table 4. IC50 Values of Compounds 4, 7, 8, 10, 11, and 12,
Vitamin C, and Vitamin E in DPPH Assay
a
DPPH
150 μM
300 μM
compound
IC50 SD (μM)
IC50 SD (μM)
4
26.51 0.98
15.70 1.10
33.17 1.12
181.10 11.10
37.46 0.35
23.33 1.66
24.68 0.04
26.94 0.99
51.41 2.44
34.64 0.46
75.09 1.51
359.70 20.45
83.39 0.86
44.45 0.89
59.33 0.52
62.09 0.29
7
8
10
11
12
vitamin C
vitamin E
a
IC50 values were calculated using the software Graph Pad Prism 5.0.
The data are expressed as the means SD. Three independent
experiments were performed. Vitamins C and E were used as the
positive controls.
compounds did not exhibit such activities. As shown in Figure
9, compounds 4, 7, 8, 10, 11, and 12 and Trolox exhibited total
antioxidant abilities in the following descending order at a
concentration of 1.5 mM: 4 > 12 > 7 > Trolox > 8 > 11 > 10.
Figure 8. ECD spectra of compound 4 and piperphilippinin VI.
F
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Journal of Natural Products
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various spectroscopic methods and hydrolysis reactions. The
antioxidant activities of the isolated compounds were also
investigated. Compounds 4, 7, 8, 10, 11, and 12, all of which
contain phenolic hydroxy groups capable of behaving as
hydrogen atom donors, exhibited antioxidant activities with
varying potential in both the DPPH and FRAP assays, whereas
the other compounds, which do not contain any phenolic
hydroxy groups in their structures, did not show activity in
these assays. Several different types of lignans,6,28−32 especially
dibenzocyclooctenes, obtained from plants belonging to the
Schisandraceae family have been reported to exhibit neuro-
protective effects in various neurocytotoxic models, including
those involving hydrogen peroxide, tert-butyl hydroperoxide,
cobalt chloride, glutamate, homocysteine, and β-amyloid. In
this study, compounds 1, 2, 6, 7, 8, 11, and 12 exhibited
Figure 9. Antioxidant activities of compounds 4, 7, 8, 10, 11, and 12
and Trolox in the FRAP assay.
statistically significant neuroprotective effects against Aβ25−35
-
Compounds 4, 12, and 7 exhibited higher total antioxidant
activities than Trolox in this test.
induced SH-SY5Y cell death compared with the Aβ25−35-treated
group. Although additional studies, including in vivo animal
experiments in particular, are required to provide further
evidence for the use of S. glaucescens fruit as a health food, the
results of the current study further elucidate the bioactivity of S.
glaucescens fruit.
Alzheimer’s disease is a devastating neurodegenerative
disorder that is estimated to affect more than 35 million
people worldwide, and no effective treatment is available.27
Substantial evidence indicates that the amyloid β-protein (Aβ)
plays a key role in the disease and may be a potential
therapeutic target. Herein, all of the isolated lignans were tested
for their neuroprotective effects against Aβ25−35-induced cell
death in SH-SY5Y cells from 0.08 to 10 μM. As shown in Table
5, compounds 1, 2, 6, 7, 8, 11, and 12 exhibited statistically
significant neuroprotective effects against Aβ25−35-induced SH-
SY5Y cell death compared with the Aβ25−35-treated group.
In conclusion, two rare 7,8-seco-lignans, three new lignan
glycosides, and 10 known lignans were isolated from the fruit of
S. glaucescens Diels. The absolute configurations of the two 7,8-
seco-lignans were determined for the first time through
comparison of their experimental and calculated ECD spectra.
Compounds 1 and 2 were determined to have the same
structures as the known compounds schisandlignans B and A,
respectively, based on a comparison of their spectroscopic data
and specific rotations. The molecular structures and absolute
configurations of the new compounds were determined using
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer 341 polarimeter. UV spectra were
measured on a Varian Cary 50 Scan UV/vis spectrophotometer. ECD
spectra were recorded on a Jasco J-810 spectropolarimeter. IR spectra
were recorded on a Bruker VERTEX 70 FT-IR microscopic
spectrometer. NMR spectra were recorded on a Bruker-AM-400
spectrometer. HRESIMS was performed on a Thermo Scientific LTQ-
Orbitrap XL mass spectrometer. MPLC was performed using a Buchi
pump module C-605. Column chromatography was performed on
silica gel (200−300 or 300−400 mesh; Qingdao Marine Chemical Inc.,
Qingdao, China), Sephadex LH-20 gel (GE Healthcare, Uppsala,
Sweden), or MCI gel (CHP20P, 75−150 μm; Mitsubishi Chemical
Industries Ltd., Tokyo, Japan). HPLC was performed on an Agilent
1260 system. A reversed-phase HPLC column (Outstand C18, 250 ×
4.6 mm i.d.; Intramax, Wuhan, China), a normal-phase HPLC column
(SP-120−5-APS, 250 × 4.6 mm i.d.; Global Chromatography Co.,
Table 5. Neuroprotective Effects of Compounds 1−14 against Aβ25‑35-Induced Neuronal Cell Death in Dopaminergic
a
Neuroblastoma SH-SY5Y Cells
concentrations (μM)
compound
0.08
0.4
2
10
Aβ25−35
b
d
c
d
c
d
c
1
58.3 0.9
53.2 2.8
58.0 0.4
60.6 2.0
65.0 1.0
58.1 0.6
62.2 0.7
57.0 1.8
57.4 0.9
57.7 0.2
58.6 0.8
55.4 0.9
58.2 1.0
60.0 1.0
54.1 0.4
65.4 2.4
62.0 2.9
59.2 1.4
62.1 0.6
64.0 1.1
68.9 2.2
63.5 3.0
62.0 0.7
61.5 0.9
63.1 2.1
66.8 2.1
63.4 3.0
58.3 1.3
61.0 1.4
65.2 1.7
49.2 0.4
49.2 0.4
56.9 1.2
56.9 1.2
61.7 0.5
57.1 1.1
61.7 1.2
49.4 1.5
57.1 1.1
57.1 1.1
57.3 0.3
49.4 1.5
57.3 0.3
57.3 0.3
51.3 0.9
2
3
4
5
c
b
c
6
62.2 0.1
61.1 0.7
69.2 1.3
61.9 1.4
c
c
7
63.8 1.6
68.3 0.7
c
c
c
8
61.5 2.0
61.8 3.0
64.4 3.1
9
58.2 1.1
59.9 0.4
60.7 1.6
63.1 2.1
58.1 1.3
59.2 0.4
55.6 0.9
59.8 1.4
60.5 0.5
62.1 0.7
58.7 0.2
60.5 0.2
10
d
11
65.2 1.7
73.3 1.8
c
d
d
12
68.6 1.4
58.6 0.9
60.3 0.4
58.5 1.0
13
60.4 2.2
60.1 1.1
58.0 1.7
14
c
c
vitamin C
a
The data (cell viability, measured by MTT assay) were normalized and are expressed as a percentage of the control group, which is set to 100%.
b
The data are expressed as the means SEM. Three independent experiments were performed. Vitamin C was used as the positive control. p < 0.05.
c
d
p < 0.01. p < 0.001, compared with the Aβ25−35-induced group.
G
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Journal of Natural Products
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Ltd., Suzhou, China), and a chiral HPLC column (Chiralcel OD-H,
150 × 4.6 mm i.d.; Daicel Chemical Industries, Ltd., Tokyo, Japan)
were used for analytical purposes. A YMC-Pack ODS-A HPLC column
(C18, 250 × 10 mm i.d.; YMC, Tokyo, Japan) was used for
semipreparative purposes. Gas chromatography was conducted on an
Agilent 7820A system. An OV-17 capillary column (30 m × 0.32 mm
× 0.5 μm; Lanzhou Zhongke Antai Analysis Technology Co., Ltd.,
Lanzhou, China) was used for the GC analysis. The DPPH, FRAP, and
MTT assays were all performed on a BioTek Synergy 2 multimode
microplate reader. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazo-
lium bromide (MTT), amyloid β-protein fragment 25−35 (Aβ25−35),
2,2-diphenyl-1-picrylhydrazyl, L-ascorbic acid (vitamin C), α-tocopher-
ol (vitamin E), L-cysteine methyl ester hydrochloride, hexamethyl
disilazane, trimethylchlorosilane, D-glucose, L-glucose, D-arabinose, and
L-arabinose were purchased from Aladdin Industry Corporation,
Shanghai, China. β-Cellulase (from Trichoderma viride, activity ≥35
u/mg) was manufactured by Shanghai YuanYe Biotechnology Co.,
Ltd., Shanghai, China. Trifluoroacetic acid was purchased from
Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. A FRAP
assay kit was purchased from the Beyotime Institute of Biotechnology,
Nantong, China.
8.65 × 10−3, CH3OH) λmax (θ) 220 (−10817, tr), 240 (730, pk), 265
(−1415, tr), 281 (−167, pk), 298 (−1183, tr) nm; IR (KBr) νmax 3423,
2963, 2921, 1714, 1595, 1513, 1443, 1366, 1259, 1158, 1106, 1068,
1033, 806, 763, 679 cm−1; 1H and 13C NMR data, see Table 1;
HRESIMS m/z 409.1256 [M + Na]+ (calcd for C21H22O7Na,
409.1263).
(8R,8′R)-9-O-(6′-O-α-L-Arabinofuranosyl)-β-D-glucopyranosyldi-
hydrocubebin (3): white, amorphous powder; [α]2D0 −44 (c 0.2,
CH3OH); UV (CH3OH) λmax (log ε) 236 (3.97), 288 (3.96) nm;
ECD (c 1.63 × 10−3, CH3OH) λmax (θ) 234 (−1852, tr), 260 (−218,
pk), 275 (−380, tr), 296 (344, pk) nm; IR (KBr) νmax 3392, 2928,
2892, 1633, 1610, 1489, 1442, 1360, 1317, 1247, 1190, 1038, 927, 861,
810, 776, 719, 636 cm−1; 1H and 13C NMR data, see Table 2;
HRESIMS m/z 675.2252 [M + Na]+ (calcd for C31H40O15Na
675.2265).
(8R,8′R)-9-O-β-D-Glucopyranosylpiperphilippinin VI (4a) and
(8R,8′R)-9′-O-β-D-glucopyranosylpiperphilippinin VI (4b): white,
amorphous powder; [α]2D0 −18 (c 0.2, CH3OH); UV (CH3OH) λmax
(log ε) 231 (4.10), 284 (3.90) nm; ECD (c 1.578 × 10−3, CH3OH)
λmax (θ) 214 (1729, tr), 218 (2312, pk), 232 (−1290, tr), 243 (−690,
pk), 244 (−693, tr), 258 (−271, pk), 283 (−599, tr), 312 (−101, pk)
nm; IR (KBr) νmax 3417, 2920, 2852, 1644, 1608, 1513, 1490, 1443,
1369, 1247, 1157, 1124, 1076, 1035, 926, 864, 811, 739 cm−1; 1H and
13C NMR data, see Table 3; HRESIMS m/z 545.1984 [M + Na]+
Plant Material. The fruit of S. glaucescens was collected in the
Shennongjia Mountains of Hubei Province, China, in September 2011
and was identified by Mr. Shi-Gui Shi (Shennongjia Institute for Drug
Control). A voucher specimen (ID 20110905) was deposited in the
Herbarium of Materia Medica, Faculty of Pharmacy, Tongji Medical
College of Huazhong University of Science and Technology, Wuhan,
China.
(calcd for C26H34O11Na, 545.1999) and 1067.4086 [2 M + Na]+
(calcd for C52H68O22Na, 1067.4100).
ECD Calculations. The conformational spaces for the 7′R,8′S and
7′S,8′R isomers of compounds 1 and 2 were explored using the
BALLOON program with a genetic algorithm.33 The conformations
generated by BALLOON were subjected to semiempirical PM3
quantum mechanical geometry optimizations using the Gaussian 09
program.34 Duplicate conformations were identified and removed
when the root-mean-square (RMS) distance was less than 0.5 Å for
any two geometry-optimized conformations. The remaining con-
formations were further optimized at the B3LYP/6-31G* level of
theory in MeOH with the IEFPCM solvation model using Gaussian
09,35 and the duplicate conformations emerging after these
calculations were removed according to the same RMS criteria
above. The harmonic vibrational frequencies were calculated to
confirm the stability of the final conformers (Figures S69 and S70,
Supporting Information). The oscillator strengths and rotational
strengths of the 20 weakest electronic excitations of each conformer
were calculated using the TDDFT methodology at the B3LYP/6-
311G** level of theory with MeOH as the solvent with the IEFPCM
solvation model implemented in Gaussian 09. The ECD spectra for
each conformer were simulated using a Gaussian function with a
bandwidth σ of 0.45 eV. The spectra were combined after Boltzmann
weighting according to their population contributions (Tables S1 and
S2, Supporting Information).
Enzymatic Hydrolysis. To obtain the aglycones without the
occurrence of structural changes, compounds 3 and 4 (each 5 mg)
were treated with β-cellulase (from Trichoderma viride, 0.5 mg for both
3 and 4) in aqueous solution (2 mL for both 3 and 4) at 50 °C for 3 h.
After extraction with EtOAc (5 mL × 5), the combined EtOAc layer
was evaporated to dryness and analyzed by TLC and HPLC.
Semipreparative HPLC was used to isolate the aglycones of the two
compounds.
Acid Hydrolysis and GC Analysis. GC was utilized to determine
the absolute configurations of the saccharides of compounds 3 and 4,
according to a slightly modified literature procedure.36 Briefly, 3 and 4
(each 2 mg) were heated at 90 °C with 4 mol/L aqueous TFA (2 mL)
for 3 h. The reaction mixture was freeze-dried overnight and diluted
with H2O (5 mL). After extraction with EtOAc (5 mL × 4), the
aqueous layer was evaporated to dryness, and the residue was dissolved
in pyridine (1 mL) and reacted with 5 mg of L-cysteine methyl ester
hydrochloride at 60 °C for 1 h. Subsequently, 0.9 mL of hexamethyl
disilazane−trimethylchlorosilane (2:1) was added, and the mixture was
heated at 60 °C for 1 h. The mixture was washed with H2O (5 mL ×
2) and extracted with n-hexane, and the n-hexane layer was analyzed
by GC-FID using an OV-17 capillary column (30 m × 0.32 mm × 0.5
Extraction and Isolation. The air-dried fruit of S. glaucescens (15
kg) was extracted with 95% ethanol at room temperature, and the
solution was concentrated in vacuo to yield a crude extract (750 g).
The extract was added to 1.5 L of deionized H2O and sequentially
partitioned with petroleum ether (PE), EtOAc, and n-BuOH. The
petroleum ether-soluble fraction (210 g) was chromatographed on a
silica gel column eluting with PE−EtOAc (from 1:0 to 6:4) to afford
10 fractions. Fraction 6 (18.5 g) was chromatographed on silica gel
(CHCl3−EtOAc, from 1:0 to 3:1) and Sephadex LH-20 (CH2Cl2−
MeOH, 1:1) followed by semipreparative HPLC (MeOH−H2O,
80:20) to yield compounds 1 (11 mg) and 2 (8 mg). The EtOAc-
soluble fraction (150 g) was chromatographed on a silica gel column
eluting with PE−EtOAc (from 99:1 to 1:2) to afford 14 fractions.
Fraction 2 (12.5 g) was chromatographed on Sephadex LH-20
(CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc, from 15:1 to 10:1)
followed by semipreparative HPLC (MeOH−H2O, 85:15) to yield
compounds 6 (16 mg), 8 (560 mg), 9 (18 mg), and 14 (26 mg).
Fraction 3 (4.8 g) was chromatographed on Sephadex LH-20
(CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc, 6:1) columns to
yield compound 13 (70 mg). Fraction 4 (15.3 g) was chromato-
graphed on Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and silica gel
(PE−EtOAc, from 9:1 to 3:1) columns to yield compounds 7 (240
mg) and 12 (110 mg). Fraction 6 (5.3 g) was chromatographed on
Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc,
4:1) followed by semipreparative HPLC (MeOH−H2O, 77.5:22.5) to
yield compounds 10 (15 mg) and 11 (22 mg). Fraction 10 (19.7 g)
was chromatographed on MCI gel (MeOH−H2O, from 40:60 to
100:0) and silica gel (CH2Cl2−MeOH, from 85:15 to 60:40) followed
by semipreparative HPLC (MeOH−H2O, 55:45) to yield compounds
3 (42 mg), 4 (11 mg), and 5 (350 mg).
(7′R,8′S)-3,4-Dimethoxy-3′,4′-methylenedioxy-7,8-seco-7,7′-ep-
oxylignan-7,8-dione (1): colorless oil; [α]2D0 −39 (c 0.9, CHCl3); UV
(CH3OH) λmax (log ε) 228 (4.73), 257 (4.35), 288 (4.03) nm; ECD (c
9.48 × 10−3, CH3OH) λmax (θ) 208 (−5231, tr), 234 (3957, pk), 259
(−1864, tr), 281 (697, pk), 294 (−550, tr) nm; IR (KBr) νmax 3433,
2922, 1713, 1598, 1512, 1446, 1416, 1270, 1248, 1221, 1175, 1137,
1
1100, 1037, 814, 764, 678 cm−1; H and 13C NMR data, see Table 1;
HRESIMS m/z 409.1258 [M + Na]+ (calcd for C21H22O7Na,
409.1263).
(7′R,8′S)-3,4-Methylenedioxy-3′,4′-dimethoxy-7,8-seco-7,7′-ep-
oxylignan-7,8-dione (2): colorless oil; [α]2D0 −73 (c 1.5, CHCl3); UV
(CH3OH) λmax (log ε) 226 (4.56), 259 (4.13), 288 (3.85) nm; ECD (c
H
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Journal of Natural Products
Article
μm) under the following conditions. The injector temperature was 250
°C. The column temperature was initially 200 °C and gradually
increased at a rate of 5 °C/min up to 250 °C and kept at 250 °C for 10
min. For detection, a flame ionization detector was used at 250 °C.
Helium was used as the carrier gas at a constant flow rate of 1 mL/
min. The derivatives of D-glucose, L-glucose, D-arabinose, and L-
arabinose were analyzed simultaneously.
DPPH Radical Scavenging Activity. The abilities of the
compounds to scavenge DPPH radical were evaluated as previously
reported with minor modifications.37 Briefly, reaction mixtures
containing DPPH (final concentrations of 150 or 300 μM) and the
compounds (final concentrations of 6.25, 12.5, 25, 50, 75, and 100
μM) in EtOH (with 5% DMSO) were placed in the wells of 96-well
plates. An EtOH solution (with 5% DMSO) was used as a control.
Vitamins C and E were used as the positive controls. The absorbance
of the reaction mixture at 517 nm was measured at steady state after 30
min of incubation at room temperature in the dark using a microplate
reader. All tests were performed in triplicate, and the results were
averaged. IC50 is defined as the concentration of compound that
scavenges 50% of the DPPH radical and was calculated using the
software program Graph Pad Prism 5.0 through nonlinear regression
(curve fitting) and the equation [log (inhibitor) vs normalized
response] for dose response (variable slope).
Ferric Reducing Antioxidant Power Assay. The FRAP assay
was performed according to the manufacturer’s instruction. Briefly, a
fresh working solution was prepared by mixing TPTZ solution, TPTZ
diluent, and detective buffer in a ratio of 1:10:1 (v/v). The working
solution was warmed to 37 °C before use. Then, 5 μL samples at
concentrations from 0.15 to 1.5 mM were mixed with 180 μL of FRAP
working solution and kept for 5 min at 37 °C. The absorbances of the
reaction mixtures were recorded at 593 nm. A standard curve was
prepared using FeSO4 at concentrations from 0.15 to 5.00 mM, and
the total antioxidant activities of the tested compounds are expressed
in terms of FeSO4 values, which were calculated using the standard
curves.
Neuroprotective Activity. The neuroprotective activities of the
isolated compounds were measured according to a published
procedure.38 Human dopaminergic neuroblastoma SH-SY5Y cells
purchased from the cell bank of the Basic Medical College of
Huazhong University of Science and Technology were maintained in
DMEM culture medium (Solarbio, Beijing, China) with 10% calf
serum (Sijiqing, Hangzhou, China), 100 U/mL penicillin, and 75 U/
mL streptomycin in a 37 °C incubator (Heal Force HF-90, Hong
Kong) in 5% CO2. Cells were trypsinized with 0.25% trypsin, counted,
and seeded in 96-well culture plates (1 × 104 cells/well). After 24 h of
incubation, the cells were pretreated with compounds 1−14 (0.08, 0.4,
2, and 10 μM) for 2 h before incubation in medium containing Aβ25−35
(1 μM). After 24 h of treatment, 15 μL of MTT (5 mg/mL) was
added. For the MTT assays, the supernatant was discarded, and
DMSO (100 μL/well) was added. The 96-well plate was vibrated on a
microvibrator for 10 min, and the optical density at 570 nm was
measured using a microplate reader. All samples were cultured in
triplicate. Statistical analysis was performed using one-way analysis of
variance (ANOVA) followed by post hoc multiple comparisons using
the Newman−Keuls multiple comparison method. The data are
expressed as the means SEM of three assays.
details of isomers 7′R,8′S and 7′S,8′R of compounds 1 and 2.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 86 27 83657870. Fax: 86 27 83692739. E-mail: ruanhl@
mails.tjmu.edu.cn.
■
Author Contributions
#H.-Y. Yu and Z.-Y. Chen contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the staff at the analytical and testing center of
Huazhong University of Science and Technology for collecting
the spectroscopic data. This work was supported by the Natural
Science Foundation of China (Nos. 31270394, 81072547, and
21102050), the Ministry of Science and Technology of the
People’s Republic of China (International Cooperative Project,
Grant No. 2010DFA32430), and the Wuhan Program for
Science and Technology (No. 2013060501010158).
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