Neuroprotective constituents from the fruits of Maclura tricuspidata

Article abstract of DOI:10.1016/j.tet.2017.03.064

Cudraisoflavone L???T (1–11), cudradihydrochalcone A (12), and cudrabibenzyl A (13), together with 41 known constituents, were isolated from the fruits of Maclura tricuspidata (formerly known as Cudrania tricuspidata). Their structures were determined by spectroscopic and chemical methods including electronic circular dichroism. All isolated compounds were evaluated for their neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced cell death in human neuroblastoma SH-SY5Y cells, of these six compounds exhibited activities with EC₅₀ values of 4.5–18.6?μM. Furthermore, the neuroprotective effects against oxygen glucose deprivation (OGD)-induced cell death of all isolated constituents, together with 37 previously reported isoflavones were evaluated. Four compounds showed neuroprotective activities with EC₅₀ values of 3.5–11.9?μM.

Full text of DOI:10.1016/j.tet.2017.03.064

Accepted Manuscript
Neuroprotective constituents from the fruits of Maclura tricuspidata
Nguyen Tuan Hiep, Jaeyoung Kwon, Dong-Woo Kim, Sungeun Hong, Yuanqiang
Guo, Bang Yeon Hwang, Nahyun Kim, Woongchon Mar, Dongho Lee
PII:
S0040-4020(17)30314-9
10.1016/j.tet.2017.03.064
TET 28570
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 28 January 2017
Revised Date: 21 March 2017
Accepted Date: 23 March 2017
Please cite this article as: Hiep NT, Kwon J, Kim D-W, Hong S, Guo Y, Hwang BY, Kim N, Mar W, Lee
D, Neuroprotective constituents from the fruits of Maclura tricuspidata, Tetrahedron (2017), doi: 10.1016/
j.tet.2017.03.064.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Neuroprotective constituents from the fruits
Leave this area blank for abstract info.
of Maclura tricuspidata
a,b
c
d
d
e
Nguyen Tuan Hiep, Jaeyoung Kwon, Dong-Woo Kim, Sungeun Hong, Yuanqiang Guo, Bang Yeon
f
g
d,
a,
Hwang, Nahyun Kim, Woongchon Mar, * Dongho Lee *
a
Department of Biosystems and Biotechnology, College of Life Science and Biotechnology, Korea University,
Seoul 02841, Republic of Korea
b
Department of Extraction Technology, Vietnam National Institute of Medicinal Materials, 3B Quang Trung,
Hoan Kiem, Hanoi, Vietnam
c
Natural Constituents Research Center, Korea Institute of Science and Technology (KIST), Gangneung 25451,
Republic of Korea
d
Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742,
Republic of Korea
e
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin Key Laboratory of
Molecular Drug Research, Nankai University, Tianjin 300350, People’s Republic of China
f
College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
Forest Medicinal Resources Research Center, National Institute of Forest Science, Yeongju 36040, Republic of
g
Korea

ACCEPTED MANUSCRIPT
Neuroprotective constituents from the fruits of
Maclura tricuspidata
a,b
c
d
d
e
Nguyen Tuan Hiep, Jaeyoung Kwon, Dong-Woo Kim, Sungeun Hong, Yuanqiang Guo, Bang Yeon
f
g
d,
a,
Hwang, Nahyun Kim, Woongchon Mar, * Dongho Lee *
a
Department of Biosystems and Biotechnology, College of Life Science and Biotechnology, Korea
University, Seoul 02841, Republic of Korea
b
Department of Extraction Technology, Vietnam National Institute of Medicinal Materials, 3B Quang
Trung, Hoan Kiem, Hanoi, Vietnam
c
Natural Constituents Research Center, Korea Institute of Science and Technology (KIST), Gangneung
2
5451, Republic of Korea
d
Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742,
Republic of Korea
e
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin Key
Laboratory of Molecular Drug Research, Nankai University, Tianjin 300350, People’s Republic of
China
f
College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
g
Forest Medicinal Resources Research Center, National Institute of Forest Science, Yeongju 36040,
Republic of Korea
Corresponding authors:
*
*
Tel: +82-2-3290-3017. Fax: +82-2-953-0737. E-mail: dongholee@korea.ac.kr (D. Lee).
Tel: +82-2-880-2473. Fax: +82-2-888-9122. E-mail: mars@snu.ac.kr (W. Mar).
1

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Abstract
Cudraisoflavone L − T (1−11), cudrahidrochalcone A (12), and cudrabibenzyl A (13), together
with 41 known constituents, were isolated from the fruits of Maclura tricuspidata (formerly known as
Cudrania tricuspidata). Their structures were determined by spectroscopic and chemical methods
including electronic circular dichroism. All isolated compounds were evaluated for their neuroprotective
effects against 6-hydroxydopamine (6-OHDA)-induced cell death in human neuroblastoma SH-SY5Y
cells, of these six compounds exhibited activities with EC50 values of 4.5–18.6 µM. Furthermore, the
neuroprotective effects against oxygen glucose deprivation (OGD)-induced cell death of all isolated
constituents, together with 37 previously reported isoflavones were evaluated. Four compounds showed
neuroprotective activities with EC values of 3.5–11.9 µM.
50
Keywords: Maclura tricuspidata; Cudrania tricuspidata; Snatzke’s method; Circular dichroism;
Neuroprotective activity
2

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1
.
Introduction
Neurodegenerative diseases are caused by the progressive loss of specific neuronal cell
populations from the brain and spinal cord, and are associated with protein aggregates that lead to either
1
,2
functional loss or sensory dysfunction. Of these, Parkinson’s disease (PD) is characterized by the
degeneration of dopamine neurons of the substantia nigra pars compacta, resulting in clinical symptoms
3
of tremor, rigidity, and slowness in movement. The factors that contribute to nigral dopaminergic cell
death include mitochondrial dysfunction, oxidative stress, altered protein handling, inflammation, and
4
,5
proteasomal dysfunction.
Cerebral ischemia, which results from cardiac arrest, cerebral arterial occlusion, or severe
6
vasospasm after subarachnoid ischemia, causes devastating damage to the brain. In general, it is caused
by neuronal cell death by activating a cascade of events, such as excessive release of excitatory amino
acid, intracellular calcium accumulation, oxidative stress, mitochondrial dysfunction, and
7
-9
inflammation.
Maclura tricuspidata Carrière (formerly known as Cudrania tricuspidata) has long been used as
a traditional herb in Korea. The cortex and root bark of this plant has been used for the treatment of
gastrointestinal tumors and for curing gonorrhea, rheumatism, jaundice, boils, scabies, bruising, and
1
0
dysmenorrhea. Besides, its fruits are widely consumed due to their antioxidant, anti-atherosclerotic,
1
1,12
and anti-inflammatory effects.
Previous studies have indicated that this plant contains biologically
1
3,14
active xanthones, flavonoids, and benzenoids.
As a part of an ongoing research program focused on the discovery of neuroprotective
compounds from higher plants, in the previous report, ten new isoflavones, cudraisoflavones B−K,
together with 27 known isoflavones, were isolated from the EtOAc soluble extract of fruits of M.
tricuspidata. Of these, some isoflavones exhibited significant neuroprotective activities against
1
5
SH-SY5Y cells death induced by 6-hydroxydopamine (6-OHDA), with EC values of 0.5–9.2 µM.
50
3

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Accordingly, the further investigation of the EtOAc soluble extract was carried out, leading to the
isolation of 13 new constituents (1−13) together with 41 known ones. Their structures were determined
by spectroscopic and chemical methods, together with calculations of electronic circular dichroism
(
ECD) spectra based on the quantum-mechanical time-dependent density functional theory. Herein
reported are details of the isolation and structural elucidation, as well as neuroprotective activity against
-OHDA-induced SH-SY5Y cells death of the isolated compounds. Furthermore, the neuroprotective
6
effects of all constituents obtained from M. tricuspidata fruits, including 37 reported isoflavones in
preceding paper, were evaluated in the setting of SH-SY5Y cells death as induced by oxygen-glucose
deprivation (OGD).
2
2
.
Result and discussion
.1. Chemical constituents from M. tricuspidata fruit
The MeOH extract of fruits of M. tricuspidata was dissolved in H O and successively
2
partitioned with n-hexane and EtOAc. The EtOAc soluble extract was fractionated by column
chromatography over silica gel, sephadex LH-20, ODS, and preparative HPLC to afford 13 new
16
compounds (1−13), together with 41 known compounds, which were identified as butyrospermol,
1
7
18
lupeol,
β-sitosterol,
4-(methoxymethyl)phenol (14), 5,4′-dihydroxy-8-(3,3-dimethylallyl)-2′′-
1
9
20
methoxyisopropylfurano[4,5:6,7]isoflavone, 3,5,7,3′,5′-pentahydroxydihydroflavonol, kaempferol-7-
2
1
22
O-β-D-glucopyranoside,
4′-O-methylerythrinin
C,
6-prenyl-5-hydroxy-7,3′,4′-
2
3
24
trimethoxyisoflavone,
genistein,
5,3′,4′-trihydroxy-2′′,2′′-dimethylpyrano(5′′,6′′:7,8)isoflavone
2
5
26
27
28
(
15), 6-prenylapigenin, artocarpesin, millewanin G (16), millewanin H (17), aromadendrin,
2
9
30
apigenin,
4-hydroxybenzoic
acid,
orobol
(18),
5,7,4′-trihydroxy-8-p-
31
32
33
hydroxybenzyldihydroflavonol,
gericudranin E, gericudranin D,
6-prenylorobol (19),
3
4
35
36
37
kaempherol, 7-O-methylbiochanin A, genistein-5-O-methyl-8-C-β-D-glucopyranoside, genistine,
3
8
39
40
sphaerobioside, prunin, sophorobioside, genistein-4′-O-β-D-glucopyranoside, apigenin-7-O-β-D-
4
1
glucopyranoside,
kaempferol-3-O-β-D-glucopyranoside,
nicotiflorine,
quercetin-3-O-β-D-
4

ACCEPTED MANUSCRIPT
4
2
43
glucopyranoside,
4-hydroxybenzaldehyde,
aromadendrin-7-O-β-D-glucopyranoside,
4
4
45
dihydroquercetin 7-O-β-D-glucopyranoside, orobol-7-O-β-D-glucopyranoside, gericudranin C, and
4
6
gericudranin A, by comparing their spectroscopic data with reported data.
Chart 1
+
Compound 1 showed a molecular ion peak at m/z 421.1634 [M + H] (HRESIMS),
corresponding to a molecular formula of C H O . The 1D NMR data of (Table 1) were closely
1
2
5
24
6
1
5
similar to those of cudraisoflavone E, except for the absent of a 2-hydroxyl-3-methylbut-3-enyl group
and the presence of a 2,2-dimethylpyran group [δ 6.76 (1H, d, J = 10.0 Hz, H-1′′′), 5.59 (1H, d, J =
H
1
0.0 Hz, H-2′′′), 1.51 (3H, s, Me-4′′′), and 1.47 (3H, s, Me-5′′′)]. Its HMBC showed cross-peaks
H-2/C-9 (δ 152.7), and H-1′′′/C-7 (δ 153.3), C-8 (δ 102.5) and C-9, indicated that the 2,2-
C
C
C
dimethylpyran was linked at the C-7 and C-8 positions. Besides, the HMBC correlations H-1′′/C-5 (δ
C
1
61.3), C-6 (δ 111.7), and C-7, suggested that the 2-(1-hydroxy-1-methylethyl)dihydrofuran group was
C
located at the C-5 and C-6 positions. Due to small amount of material available, the absolute
5

ACCEPTED MANUSCRIPT
configuration of asymmetric carbon center at C-2′′ remains unknown. Therefore, the new compound 1
was formulated as illustrated, and given the trivial name cudraisoflavone L.
6

ACCEPTED MANUSCRIPT
1
13
a
Table 1. H NMR (500 Hz) and C NMR (125 Hz) spectroscopic data of compounds 1–5.
b
c
c
d
d
1
2
3
4
5
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
2
3
4
5
6
7
8
9
1
151.0
125.3
174.7
161.3
111.7
153.3
102.5
152.7
106.6
8.01, s
154.1
121.8
180.8
153.9
104.3
156.5
106.5
155.0
105.0
8.40, s
150.0
124.8
173.6
154.3
104.3
156.1
101.1
153.1
108.0
8.13, s
154.3
123.7
182.0
158.8
110.5
162.1
108.3
154.9
105.9
8.24, s
154.5
123.4
181.9
159.1
110.1
162.1
108.3
154.8
105.8
8.30, s
0
OH-5
13.39, s
13.53, s
13.53, s
1
2
3
4
′
123.6
131.0
116.1
159.0
121.1
130.0
115.0
157.3
122.4
130.2
114.6
156.9
123.3
131.2
115.9
158.3
124.4
131.1
114.4
160.6
′,6′
′,5′
′
7.78, d (8.5)
7.30, d (8.5)
7.38, d (8.5)
6.82, d (8.5)
7.28, d (8.5)
6.79, d (8.5)
7.46, d (8.5)
6.90, d (8.5)
7.56, d (8.5)
7.00, d (8.5)
OH-4′
OCH -4′
9.56, s
9.46, s
3
55.5
29.7
3.84, s
3
3
.57, dd (8.5, 15.5)
.20, dd (10.0, 15.0)
3.15, dd (1.5, 15.0)
2.95, dd (8.0, 15.0)
4.43, d (7.5)
3.14, dd (2.0, 15.0)
2.97, dd (8.0, 15.0)
4.44, d (8.0)
1
′′
26.9
114.8
6.65, d (10.0)
5.79, d (10.0)
70.2
5.28, d (2.5)
29.6
2
3
′′
′′
93.1
70.9
5.05, dd (8.0, 10.0)
128.6
77.6
68.2
77.9
3.76, dd (2.5, 5.0)
77.0
147.8
77.0
147.7
e
e
e
e
e
e
5.02, brs
5.02, brs
4.82, brs
1.84, s
4
5
′′
′′
26.2
1.65, s
27.8
1.44, s
24.3
1.41, s
110.1
18.6
110.5
18.6
4
.82, brs
e
e
e
e
e
e
26.2
1.42, s
27.7
1.43, s
23.3
1.38, s
1.84, s
OH-2′′
5.59, d (5.0)
4.35, (2H), s
1
2
3
4
5
6
7
′′′
′′′
′′′
′′′
′′′
′′′
′′′
115.7
127.0
78.2
6.76, d (10.0)
5.59, d (10.0)
24.6
76.7
71.7
25.5
25.4
2.77, (2H), m
3.45, ddd (4.0, 6.0, 9.0)
73.2
28.0
4.03, (2H), s
28.0
4.03, (2H), s
130.4
129.0
115.1
157.4
115.1
129.0
57.0
132.6
130.1
115.7
156.2
115.7
130.1
132.5
130.1
115.7
156.2
115.7
130.1
7.32, d (8.5)
7.08, d (8.5)
7.13, d (8.5)
6.69, d (8.5)
7.13, d (8.5)
6.68, d (8.5)
e
e
e
e
28.3
1.51, s
1.16, s
1.15, s
e
e
e
e
28.3
1.47, s
7.08, d (8.5)
7.32, d (8.5)
3.26, s
6.69, d (8.5)
7.13, d (8.5)
6.68, d (8.5)
7.13, d (8.5)
3
OCH -1′′′
OH-2′′′
4.13, d (6.0)
1
2
3
4
5
′′′′
′′′′
′′′′
′′′′
′′′′
114.3
127.5
77.7
6.67, d (10.0)
5.70, d (10.0)
e
e
27.1
1.16, s
e
e
27.5
1.25, s
a
b
c
d
e
TMS was used as the internal standard; chemical shifts are shown in the δ scale with J value (Hz) in parentheses.
Pyridine-d
DMSO-d
Acetone-d
5
.
6
.
6
.
Might be interchanged.
7

ACCEPTED MANUSCRIPT
+
Compound
2
has a molecular peak at m/z 439.1759 [M + H] (HRESIMS), corresponding to the
1
13
molecular formula C25
H
26
O
7
. The H and C NMR spectroscopic data (Table 1) were similar to those of
which possesses a 5-hydoxyisoflavone skeleton with a 2,2-dimethylpyran group. The
difference between the two compounds was the replacement of a 2-hydroxyl-3-methylbut-3-enyl group
by a 2,3-dihydroxyl-3-methylbutyl group [ H 2.77 (2H, m, H-1′′′), 3.45 (1H, ddd, = 4.0, 6.0, 9.0 Hz,
H-2′′′), 1.16 (3H, s, Me-4′′′) and 1.15 (3H, s, Me-5′′′)] at C-8 position, as indicated by the HMBC cross
peaks H-1′′′/C-7 ( C 156.5), C-8 ( C 106.5), and C-9 (δC 155.0).
The Snatzke’s method, employing dimolybdenum tetraacetate as an auxiliary chromophore, was
1
5,47
euchrenone b8
,
δ
J
4
6
δ
δ
4
8
applied to determine the absolute configuration of an acyclic 1,2-diols moiety in compound 2. The C-
′′′ asymmetric center was assigned the configuration according to the negative Cotton effect
observed in the ECD spectrum of Mo-complex at 298 nm (ꢀε -2.64) (Figure 1).
quantum chemical ECD calculation method, based on the time dependent density functional theory
TDDFT), was used to give further verification of the absolute configuration of compound . The
predicted ECD of (2′′′R)- was compared with the experimentally measured ECD of , and the result
revealed good agreement (Figure 2). On the basis of the above data, the structure of new compound
cudraisoflavone M) was determined as shown.
2
R
4
9,50
Moreover, the
(
2
2
2
2
(
8

ACCEPTED MANUSCRIPT
Figure 1. (A) ECD spectrum at stationary condition of 2 and 11 in solution of dimolybdenum
4
+
tetraacetate in DMSO. (B) Favored conformation of the Mo2 complex of 2. (C) Favored conformation
4
+
of the Mo
2
complex of 11.
9

ACCEPTED MANUSCRIPT
Figure 2. Comparison between the calculated and experimental ECD of 2, 8–11 in ACN.
10

ACCEPTED MANUSCRIPT
32 8
The molecular formula of compound 3 was determined to be C33H O from HRESIMS (m/z
+
1
5
57.2165 [M + H] ). Its H NMR spectrum showed a characteristic of an isoflavone skeleton [
1H, s, H-2)], a 2,2-dimethylpyran group, a 3,4-dihydroxyl-2,2-dimethyldihydropyran group [ H
1H, d, = 2.5 Hz, H-1′′), 3.76 (1H, dd,
= 5.0 Hz, OH-2′′)], and a 4-(methoxylmethyl)phenolate group [δH 4.35 (2H, s, H-1′′′),
= 8.5 Hz, H-4′′′, 6′′′), 7.32 (2H, d, = 8.5 Hz, H-3′′′, 7′′′), and 3.26 (3H, s, OMe-1′′′)]
Table 1). The 2,2-dimethylpyran was located at the C-7 and C-8 positions, as confirmed by the HMBC
correlations H-2/C-9 ( 153.1) as well as H-1′′′′/C-7 ( 156.1), and H-2′′′′/C-8 ( 101.1). Furthermore,
the position of a 3,4-dihydroxyl-2,2-dimethyldihydropyran group was determined as C-5 and C-6
according to the HMBC correlations from H-1′′ to C-7, C-6 ( C 104.3), and C-5 ( C 154.3). According
to the HMBC correlation H-1′′/C-5′′′ δC 157.4), the 4-(methoxylmethyl)phenolate group was attached
δ
H
8.13
(
(
δ
5.28
J
J = 2.5, 5.0 Hz, H-2′′), 1.41 (3H, s, Me-4′′), 1.38 (3H, s, Me-5′′),
and 5.59 (1H, d,
.08 (2H, d,
J
7
J
J
(
δ
C
δ
C
δ
C
δ
δ
(
to C-1′′ of the 3,4-dihydroxyl-2,2-dimethyldihydropyran group. The relative conformation of 3 was
determined by analyzing the vicinal coupling constant. A gauche orientation between H-1′′ and H-2′′
5
1
was inferred on the basis of small coupling constant JH-1′′/H-2′′ (2.5 Hz). The absolute configuration of
asymmetric carbon centers at C-1′′ and C-2′′ remain unknown due to the limitation of sample amount
available. Thus, the new compound 3 (cudraisoflavone N) was formulated as illustrated.
The HRESIMS spectrum of compound 4 (cudraisoflavone O) showed a molecular ion peak at
–
m/z 459.1425 [M – H] , corresponding to a molecular formula of C H O . Details analysis of its 1D
2
7
24
7
NMR data (Table 1) indicated that 4 possesses the same 5-hydoxyisoflavone skeleton with a p-
1
5
substituted benzyl group at C-8 position as cudraisoflavone G, except the absence of a 2,2-
dimethylpyran group and the presence of a 2-hydroxyl-3-methylbut-3-enyl group [δH 3.15 (1H, dd,
.5, 15.0 Hz, Ha-1′′), 2.95 (1H, dd, = 8.0, 15.0 Hz, Hb-1′′), 4.43 (1H, brd, = 8.0 Hz, H-2′′), 5.02 (1H,
brs, Ha-4′′), 4.82 (1H, brs, Hb-4′′) , and 1.84 (3H, s, Me-5′′)], which was located at the C-6 position on
the basis of HMBC correlations H-1′′/C-5 ( 158.8), C-6 ( 110.5), and C-7 ( 162.1). Accordingly,
J
=
1
J
J
δ
C
δ
C
δ
C
the structure of new compound 4 was established as shown.
11

ACCEPTED MANUSCRIPT
Analysis of the HRESIMS spectrum indicated that compound 5 has molecular formula of
+
1
13
C
28
H
26
O
7
(
m/z 475.1779 [M + H] ). Its H and C NMR spectroscopic data (Table 1) were similar to
those of 4, except for the presence of the methoxyl group at C-4 , as supported by the HMBC correlation,
particularly from the methoxy resonance [δH 3.84 (3H, s)] to C-4 δC 160.6). Therefore, the structure of
new compound 5 was determined as shown, and named 4 -methylcudraisoflavone O (5).
Compound 6 was determined to be C H O from HRESIMS (m/z 443.1501 [M – H] ). Its H
′
′
(
′-O
–
1
2
7
24
7
1
3
and C NMR spectroscopic data were similar to those of 4 (Tables 1 and 2), except for the replacement
of a 2-hydroxyl-3-methylbut-3-enyl group by a prenyl group [ 3.45 (2H, d, = 7.0 Hz, H-1′′), 5.23
1H, t, = 7.5 Hz, H-2′′), 1.78 (3H, s, Me-4′′), and 1.65 (3H, s, Me-5′′)] at C-6 position, as indicated by
the HMBC correlations H-1′′/C-5 (δC 158.5), C-6 ( C 112.2), and C-7 ( C 159.9). Thus, the structure of
new compound 6 (cudraisoflavone P) was established as shown.
The molecular formula of compound 7 was established as C H O by HRESIMS at m/z
δ
H
J
(
J
δ
δ
2
8
26
6
+
1
13
4
59.1807 [M + H] . H and C NMR spectroscopic data were almost the same as those of 6 (Table 2),
except for the presence of a methoxyl group at δH 3.83 (3H, s), which was located at the C-4 position,
according to its HMBC correlation with C-4 160.6). Accordingly, the structure of new compound 7
was established as shown, and named 4 -methylcudraisoflavone P.
′
′ (δ
C
′-O
–
Compound 8, which was obtained as a yellow powder, exhibited an [M – H] ion peak at m/z
1
6
71.1907 in the HRESIMS, corresponding to the molecular formula C H O . Its H NMR spectrum
4
0
32 10
exhibited characteristic signals of isoflavone skeleton at δH 8.19 (1H, s, H-2) and 8.14 (1H, s, H-12′′), as
well as two downfield signals at δH 13.83 (1H, s, OH-5) and 13.58 (1H, s, OH-15′′), suggesting the
5
2
presence of two isoflavone units (Table 3). Besides, the singlet signals at δH 6.58 (1H, s) and 6.30 (1H,
s) were assigned to H-8 and H-18′′, respectively, confirmed by the HMBC correlations H-2/C-9 ( C
58.5), H-8/C-6 ( 112.3), C-7 ( 158.4), C-9, and C-10 ( 106.0) as well as H-12′′/C-19′′ 156.8),
H-18′′/C-16′′ 110.3), C-17′′ 162.2), C-19′′, and C-20 ( 105.7).
δ
1
δ
C
δ
C
δ
C
(δ
C
(
δ
C
(
δ
C
δ
C
12

ACCEPTED MANUSCRIPT
1
13
a
Table 2. H NMR (500 Hz) and C NMR (125 Hz) spectroscopic data of compounds 6, 7, 11–13.
b
b
b
c
c
6
7
11
12
13
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
1
2
3
4
5
6
129.9
129.5
114.9
155.2
114.9
129.5
131.8
128.9
115.0
155.3
115.0
128.9
154.2
123.8
182.0
158.5
112.2
8.23, s
154.5
123.5
182.0
158.8
112.4
8.26, s
150.7
126.1
175.0
152.9
106.8
7.98, s
6.88, d (8.5)
6.55, d (8.5)
7.00, d (8.0)
6.66, d (8.0)
6.55, d (8.5)
6.88, d (8.5)
6.66, d (8.0)
7.00, d (8.0)
3
2
.26, m
.78, dd (7.5, 13.5)
7
159.9
160.1
159.5
38.2
36.5
37.6
2.67, (2H), m
2.97, (2H), m
8
9
1
106.9
154.5
106.3
106.8
154.5
106.3
108.8
157.5
109.5
55.5
205.3
5.36, t, (7.5)
0
OH-4
OH-5
9.10, s
13.37, s
13.35, s
1
2
3
4
5
6
7
′
′
′
′
′
′
′
123.3
131.1
115.9
158.3
115.9
131.1
124.4
131.1
114.5
160.6
114.5
131.1
124.8
131.3
115.6
157.9
115.6
131.3
104.3
160.7
91.8
158.4
126.4
152.9
145.3
109.0
161.9
101.4
160.4
110.1
171.9
7.46, d (8.5)
6.90, d (8.5)
7.55, d (8.5)
6.99, d (8.5)
7.40, d (8.5)
6.85, d (8.5)
6.00, s
6.41, s
6.43, s
6.90, d (8.5)
7.46, d (8.5)
6.99, d (8.5)
7.55, d (8.5)
6.85, d (8.5)
7.40, d (8.5)
OH-2
OH-3
OH-6
′
′
′
12.72, s
11.55, s
10.55, s
3.73, s
OCH
3
-4
′
55.6
3.83, s
55.9
1
2
3
4
5
′′
′′
′′
′′
′′
22.3
122.9
132.6
18.0
25.8
3.45, (2H), d (7.0)
5.23, t (7.5)
22.3
122.9
132.6
18.0
25.8
3.46, (2H), d (7.0)
5.23, t, (7.0)
70.3
75.3
80.5
27.0
19.3
4.87, d (8.5)
3.75, d (8.5)
129.9
129.5
114.7
156.0
114.7
99.7
73.1
77.0
69.6
76.5
4.87, d (7.5)
3.21, m
3.28, m
3.16, m
3.34, m
7.07, d (8.5)
6.62, d (8.5)
d
d
d
d
d
d
1.78, s
1.78, s
1.49, s
d
d
d
d
d
d
1.65, s
1.65, s
1.21, s
6.62, d (8.5)
7.07, d (8.5)
3
3
.69, d (11.5)
.47, dd (5.5, 11.5)
6
′′
129.5
60.6
1
2
3
4
5
′′′
′′′
′′′
′′′
′′′
27.8
4.10, (2H), s
27.8
4.11, s
22.2
3.44, (2H), d (7.5)
5.24, t (7.5)
105.6
73.7
76.0
69.5
77.1
4.39, d (7.5)
3.27, m
3.22, m
3.23, m
3.12, m
131.7
130.0
115.8
156.4
131.7
130.0
115.9
156.5
123.3
131.8
17.9
7.07, d (8.5)
6.70, d (8.5)
7.07, d (8.5)
6.70, d (8.5)
1.81, s
1.65, s
25.8
3
3
.63, dd (2.0, 11.5)
.49, dd (5.0, 11.5)
6
′′′
′′′
115.8
130.0
6.70, d (8.5)
7.07, d (8.5)
115.9
130.0
6.70, d (8.5)
7.07, d (8.5)
60.7
7
a
TMS was used as the internal standard; chemical shifts are shown in the δ scale with J value (Hz) in parentheses.
b
Acetone-d
6.
c
6
DMSO-d .
d
Might be interchanged.
1
3

ACCEPTED MANUSCRIPT
1
13
a
Table 3. H NMR (500 Hz) and C NMR (125 Hz) spectroscopic data of compounds
8
–10
.
b
c
b
8
9
10
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
2
3
4
5
6
7
8
9
1
154.3
124.3
181.9
160.3
112.3
158.4
94.6
8.19, s
154.0
122.1
180.4
159.8
107.1
162.0
93.0
8.32, s
154.1
123.8
181.9
161.6
108.6
161.9
95.3
8.12, s
6.58, s
6.46, s
6.51, s
158.5
106.0
155.6
104.1
157.3
105.8
0
OH-5
13.83, s
13.95, s
13.99, s
1
2
3
4
′
122.9
131.2
115.9
158.3
121.2
130.1
115.0
157.4
123.2
131.2
115.9
158.4
′, 6
′
′
7.47, d (8.5)
6.91, d (8.5)
7.36, d (8.5)
6.81, d (8.5)
7.43, d (8.5)
6.88, d (8.5)
′, 5
′
OH-4
′
9.56, s
2
′′
71.6
40.6
5.72, d (10.5)
78.0
39.3
79.9
40.3
d
e
2
2
.14, dd (6.5, 14.0) e
2.33, dd (7.0, 14.0)
3
′′
2.88, dd (4.0, 10.0)
d
.04, dd (3.0, 14.0)
1.77, dd (10.5, 14.0)
3.85, m
4
5
6
7
′′
′′
′′
′′
31.5
3.96, brs
5.49, brs
27.6
128.0
129.3
3.78, dt (9.5, 4.0)
5.20, d (9.5)
29.9
128.6
130.8
122.5
132.9
23.6
5.09, d (8.5)
f
f
f
f
1.71, s
25.4
1.59, s
25.8
1.71, s
d
2
1
.23, d (17.0)
.57, d (17.0)
f
f
f
f
8
′′
41.1
33.2
30.0
28.9
154.3
123.7
181.8
160.6
110.3
162.2
94.2
e
17.6
1.74, s
18.0
1.78, s
9
1
1
1
1
1
1
1
1
1
1
2
′′
27.6
1.49, s
29.1
1.58, s
f
f
0
1
2
3
4
5
6
7
8
9
0
′′
′′
′′
′′
′′
′′
′′
′′
′′
′′
′′
1.13, s
0.77, s
8.14, s
136.8
116.5
153.8
122.0
180.4
159.8
108.8
159.7
94.3
6.89, d (16.5)
6.72, d (16.5)
8.29, s
135.7
119.3
154.2
124.0
181.9
162.0
110.2
161.7
94.2
6.90, d (16.5)
6.83, d (16.5)
8.12, s
f
f
6.30, s
6.61, s
6.49, s
156.8
105.7
155.2
104.5
156.8
106.1
OH-15′′
13.58, s
13.30, s
13.41, s
1
2
3
4
′′′
123.2
131.1
115.9
158.3
121.0
130.0
115.0
157.3
122.9
131.1
115.9
158.4
′′′, 6′′′
′′′, 5′′′
′′′
7.47, d (8.5)
6.91, d (8.5)
7.36, d (8.5)
6.81, d (8.5)
7.43, d (8.5)
6.88, d (8.5)
OH-4′′′
9.55, s
a
TMS was used as the internal standard; chemical shifts are shown in the
δ
scale with
J
value (Hz) in parentheses.
b
c
d
e
f
Acetone-
6
d .
DMSO-d .
6
β-
face.
α-
face.
Might be interchanged.
14

ACCEPTED MANUSCRIPT
The two isoflavone units were linked by a monoterpene, suggested by the fragment ion peak at
+
m/z 337 [M + H – 336] (Figure 3), obtained by a retro Diels-Alder-type fragmentation in the ESIMS of
5
3
1
8
. Analysis of the H NMR spectrum of 8 indicated that the linkage contained the resonances of one
oxygenated methine proton [ 5.72 (1H, d, = 10.5 Hz, H-2′′)], one allylic methine proton [ 3.96
1H, brs, H-4′′)], one methine proton [ 2.88 (1H, dd, = 4.0, 10.0 Hz, H-3′′)], and one olefinic proton
δH 5.49 (1H, brs, H-5′′)]. Additionally, the resonances for one vinylic methyl [δH 1.71 (3H, s, Me-7′′)],
2.23
= 17.0 Hz, Hb-8′′)] were observed. The gross structure of
δ
H
J
δ
H
(
[
δ
H
J
two methyl [δH 1.13 (3H, s, Me-10′′) and 0.77 (3H, s, Me-11′′)], together with one methylene [ H
δ
(
1H, d, = 17.0 Hz, Ha-8′′) and 1.57 (1H, d,
J
J
the linkage was further established by 2D NMR studies, particularly COSY and HMBC. The spin
system from H-2′′ to H-4′′ was observed in the COSY spectrum. Furthermore, the serial HMBC
correlations H-5′′/C-4′′
C C C
(δ 31.5) and C-7′′ (δ 23.6), H-7′′/C-6′′ and C-8′′ (δ 41.1), Hb-8′′/C-9′′; H-
1
0
′′/C-9′′ and C-3′′ 40.6), H-11′′/C-9′′ and C-3′′ were contributed to elucidation of the skeleton of the
(δ
C
monoterpene unit from C-2′′ to C-11′′. The HMBC correlation between H-2′′ and C-6 indicated that the
first isoflavone was connected through bonding between C-6 and C-2′′. The remaining connection of the
second isoflavone was established from C-4′′ to C-16′′, confirmed by the HMBC correlation from H-5′′
to C-16′′. The aforementioned groups accounted for 24 out of 25 degrees of unsaturation in compound 8,
indicated the presence of an additional ring. Since C-17′′ and C-2′′ were
and methine carbon, respectively, an -bridge was established between C-2′′ and C-17′′ to form the six-
membered ring. Based on the spectral analysis above, the planar structure of 8 was obtained as depicted.
O-bearing quaternary carbon
O
15

ACCEPTED MANUSCRIPT
Figure 3. ESI mass fragments of 8.
The relative configuration of 8 was determined by analyzing vicinal coupling constants with the
aid of NOE correlations obtained from a NOESY experiment. The large coupling constant JH-2′′/ H-3′′
(10.5 Hz) indicated the anti orientation of the H-2′′/H-3′′. In addition, the small coupling constants JH-3′′/
(4.0 Hz) indicated the gauche orientation for H-3′′/H-4′′. Accordingly, the stereochemical
H-4′′
5
1
relationships in the C-2′′−C-4′′ moiety was contributed (Figure 4), which were further confirmed by
the NOESY correlations H-2′′/Ha-8′′ and Me-11′′, as well as H-3′′/H-4′′ and Me-10′′. For the elucidation
of the absolute configuration of compound 8, ECD spectroscopy in combination with quantum-chemical
ECD calculations was the method of choice. As shown in Figure 2, the calculated ECD spectrum for
(2
′′R,3′′S,4′′S)-8 is consistent with the experimental ECD spectrum for 8. Therefore, the structure of 8
was defined as shown, and named cudraisoflavone Q.
16

ACCEPTED MANUSCRIPT
9
8
9
1
0
Figure 4. Key NOESY correlations of compounds 8–10.
17

ACCEPTED MANUSCRIPT
Compound 9 was isolated as a yellow amorphous powder. The molecular formula C H O
32 10
4
0
with 25 degrees of unsaturation, was deduced from its negative HRESIMS, which showed a molecular
–
1
13
31
ion peak [M – H] m/z 671.1932 (calcd 671.1917 for C40H O10). The H and C NMR of 9 were
similar to those of 8 (Table 3), except for the resonances in the bridge linking the two isoflavone units.
1
The H NMR spectrum show that the bridge contained characteristic resonances of a trans double bond
[δH 6.89 (1H, d, J = 16.5 Hz, H-10′′), and 6.72 (1H, d, J = 16.5 Hz, H-11′′)], another olefinic methine
proton [δH 5.20 (1H, d, J = 9.5 Hz, H-5′′)], and one allylic methine proton [δH 3.78 (1H, dt, J = 9.5, 4.0
Hz, H-4′′)] which was bonded to an aromatic ring. Additionally, resonances for two vinylic methyl
groups [δH 1.59 (3H, s, Me-7′′) and 1.74 (3H, s, Me-8′′)], one allylic methyl group [δH 1.49 (3H, s, Me-
9
′′)], and a methylene group [
Hz, Hb-3′′)] were observed. The spin system from H-3′′ to H-5′′ was established from COSY spectrum
data. Furthermore, the serial HMBC correlations H-7′′/ C-5′′ 128.0) and C-6′′ 129.3), H-8′′/C-5′′
and C-6′′, H-3′′/C-2′′ δC 78.0), C-9′′ δC 27.6), and C-10′′ δC 136.8), Me-9′′/C-2′′, H-10′′/C-2′′, H-
′′/C-2′′, confirmed the skeleton of the bridge as depicted. Besides, the HMBC correlations H-11′′/C-5
δC 159.8), C-6 (δC 107.1), and C-7 (δC 162.0) indicated that the first isoflavone unit was connected with
the bridge through bonding between C-6 and C-11′′. The remaining connection of the bridge with the
second isoflavone was established from C-4′′ to C-16′′ δC 108.8), confirmed by the HMBC correlations
between H-5′′, H-4′′, H-3′′ and C-16′′
The relative configuration of the dihydropyran ring was assigned on the basis of vicinal coupling
constants and the NOESY studies. The coupling constants JHa-3′′/H-4′′ (6.5 Hz) and
H
δ 2.14 (1H, dd, J = 6.5, 14.0 Hz, Ha-3′′) and 2.04 (1H, dd, J = 3.0, 14.0
(δ
C
(δ
C
(
(
(
1
1
(
(
.
JHb-3′′/H-4′′ (3.0 Hz)
5
4
support the equatorial position of H-4′′. Besides, the NOESY correlations H-4′′/Me-8′′ and Ha-3′′, H-
5
′′/Me-7′′, H-10′′/Me-9′′, Hb-3′′, and H-5′′, as well as Me-9′′/ Ha-3′′ and Hb-3′′ are all compatible with
pseudo-equatorial position for Me-9′′ group. Therefore, the preferred conformation of the dihydropyran
ring was contributed as depicted (Figure 4). Similar to 8, the experimental ECD spectrum of 9 fits well
with the calculated ECD spectrum of (2′′R,4′′S)-9 (Figure 2). Thus, compound 9 was concluded to be a
18

ACCEPTED MANUSCRIPT
new isoflavone dimer, named cudraisoflavone R.
Compound 10 has the same molecular formula, C H O , as that of 9 based on the HRESIMS
40
32 10
–
1
13
data m/z 671.1893 [M – H] (calcd 671.1917 for C40
H
31
O
10). The H and C NMR of 10 were similar to
those of 9 (Table 3). Besides, the COSY and HMBC spectra of compound 10 showed the same
correlations as those observed in 9, suggesting that both compounds have the same gross structure. The
coupling constants JHa-3′′/H-4′′ (7.0 Hz) and JHb-3′′/H-4′′ (10.5 Hz) support the axial position of H-4′′
[δH 3.85
5
3
(
1H, m)]. Furthermore, the NOESY correlations H-4′′/ Me-8′′ and Ha-3′′, H-10′′/Me-9′′, Ha-3′′, and
H-4′′, H-5′′/Me-7′′ and Hb-3′′, as well as Me-9′′/ Ha-3′′ and Hb-3′′ are compatible only with the pseudo-
equatorial position for Me-9′′ group. Thus, the preferred conformation of the dihydropyran ring was
contributed as shown (Figure 4). Similar to 8 and 9, the calculated ECD spectrum of (2′′R,4′′R)-10
agreed well with the experimental ECD spectrum of 10, suggesting the 2′′R,4′′R configurations for 10
(
Figure 2). Consequently, the structure of 10 was assigned as shown and given the trivial name
cudraisoflavone S.
Compound 11 showed a molecular ion peak at m/z 439.1770 [M + H] (HRESIMS),
+
1
13
corresponding to a molecular formula of C H O . The H and C NMR analysis of compound 11
2
5
26
7
1
5,55
suggested a similar structure to that of eryvarin B,
dimethyldihydropyran group at C-5 and C-6 positions was replaced by a 3,4-dihydroxy-2,2-
dimethyldihydropyran group [ 4.87 (1H, d, = 8.5 Hz, H-1′′), 3.75 (1H, d, = 8.5 Hz, H-2′′), 1.49
3H, s, Me-4′′), and 1.21 (3H, s, Me-5′′)], which was deduced from the HMBC correlations H-1′′/C-5
152.9), C-6 ( 106.8), and C-7 ( 159.5).
Compound 11 possesses asymmetric carbon centers at C-1′′ and C-2′′. The large coupling
excepted that a 3-hydroxy-2,2-
δ
H
J
J
(
(
δ
C
δ
C
δ
C
5
1
constant JH-1′′/H-2′′ (8.5 Hz) indicated the anti orientation for H-1′′/H-2′′. Similar to compound 2, the
Snatzke’s method was used to determine the absolute configuration of the 1,2-diols moiety of
4
8
compound 11 . The negative sign of the Cotton effect observed in the ECD spectrum of Mo-complex
at 298 nm (ꢀε -1.05) indicated that the C-1′′ and C-2′′ asymmetric centers were assigned to have the
R
19

ACCEPTED MANUSCRIPT
5
6
and
S configuration, respectively (Figure 1). The further verification of the absolute configuration of
compound 11 was carried out by comparing its experimentally measured ECD with the predicted ECD
of (1′′R,2′′S)-11. As can be seen in Figure 2, the calculated ECD spectrum is consistent with
experimental result, indicated that the structure of 11 (cudraisoflavone T) was determined as illustrated.
Compound 12 was obtained as a yellowish brown oil. Its molecular formula was established as
–
C H O by HRESIMS (m/z 557.1678 [M – H] ). The IR spectrum of 12 illustrated bands at 3261 and
28
30 12
-1
1
1
676 cm , consistent with the presence of hydroxyl and carbonyl function groups. The H NMR
spectrum showed signals due to two chelated hydroxyl groups [δH 12.72 (1H, s, OH-2 ) and 10.55 (1H, s,
OH-6 )], two 1,4-disubstitued benzene groups [δH 6.88 (2H, d, J = 8.5 Hz, H-2, 6) and 6.56 (2H, d, J =
.5 Hz, H-3, 5), as well as 7.07 (2H, d, J = 8.5 Hz, H-2′′, 6′′) and 6.62 (2H, d, J = 8.5 Hz, H-3′′, 5′′)],
an aromatic proton [ 6.01 (1H, s, H-3 )], an allylic methine proton [ 5.37 (1H, t, = 7.5 Hz, H-8)], a
methoxyl group [ 3.74 (3H, s, OMe-4 )], and a methylene [ 3.26 (1H, m, Ha-7) and 2.79 (1H, dd,
7.5, 13.5 Hz, Hb-7)] (Table 2). In addition, the characteristic resonance for an anomeric proton [ H
.39 (1H, d, J = 7.5 Hz, H-1′′′)] was observed. Based on the large coupling constant of the anomeric
proton, the sugar substituent was suggested to be a -configured glucose unit. The COSY correlation H-
/H-8, together with HMBC correlations H-7/C-1 ( C 129.9), C-2, 6 ( C 129.5), C-8 ( C 55.5), and C-9
δC 205.3), H-8/C-7 (δC 38.2) and C-9, OH-2 /C-1 δC 104.3), C-2 δC 160.7), and C-3 δC 91.8), as
158.4), and C-5 126.4) suggesting that 12 possesses a
was confirmed by the
152.9). The location of the methoxy group was
according to its HMBC correlation with C-4 . The HMBC correlation between
anomeric proton H-1′′′ with C-5 suggested the location of -glucose unit at C-5 . Furthermore, the
′
′
8
δ
H
δ
H
′
δ
H
J
δ
H
′
δ
H
J
=
δ
4
β
7
δ
δ
δ
(
′
′
(
′
(
′ (
well as H-3
′/C-9, C-1
′, C-2
′, C-4
′
(
δ
C
′ (δ
C
dihydrochalcone nucleus. Besides, the presence of a hydroxyl group at C-6
HMBC correlations OH-6 /C-1 , C-5 , and C-6
determined to be at C-4
′
′
′
′
′ (δ
C
′
′
′
β
′
HMBC correlations H-8/C-1′′, C-2′′, and C-6′′, as well as H-2′′, 6′′/C-8 indicated the presence of the 1,4-
disubstitued benzene group at C-8. Additionally, the sugar substituent was confirmed by acid hydrolysis
5
7,58
of 12 to give D-glucose, identified by HPLC analysis.
Accordingly, the structure of compound 12
20

ACCEPTED MANUSCRIPT
was established as shown, and given the trivial name cudradihydrochalcone A.
Analysis of the HRESIMS spectrum indicated that 13 has the molecular formula C H O (m/z
2
1
24 10
–
1
4
8
35.1290 [M – H] ). Its H NMR spectrum showed two sets of aromatic signals at
δ
H
7.00 (2H, d, J =
),
.0 Hz, H-2, 6) and 6.66 (2H, d, J = 8.0 Hz, H-3, 5), as well as 6.41 (1H, s, H-4 ) and 6.43 (1H, s, H-6
′
′
which were assigned as a 1,4-disubstituted benzene ring and one 1,3,5-trisubstituted benzene ring,
respectively (Table 2). Besides, two methylene signals at δH 2.67 (2H, m, H-7) and 2.97 (2H, m, H-8)
5
9
were attributed to a –CH –CH – group. Furthermore, the HMBC correlations H-7/C-1 (δC 131.8), C-2,
2
2
6
1
4
(
δC 128.9), C-8 (δC 37.6), and C-1
09.0), and C-6 δC 110.1) indicated a bibenzyl skeleton for 13. Additionally, the W-couplings from H-
and H-6 to carbonyl group signal at 171.9 (C-7 ) observed in the HMBC spectrum indicated the
of 13. Similar to compound 12, the characteristic doublet at 4.87 (1H, d,
J = 7.5 Hz, H-1′′) was assigned to be an anomeric proton of the -glucose unit, according to its large
coupling constant. Besides, the HMBC correlation between the anomeric proton H-1′′ with C-5 δC
60.4) indicated the attachment of the -glucose unit to the C-5 position. The HPLC analysis of
derivatization products after acid hydrolysis of 13 indicated the D configuration of the -glucose
Thus, the bibenzyl structure of compound 13 was established as shown, and given the trivial
name cudrabibenzyl A.
′ (δC 145.3) together with H-8/C-1, C-7 (δC 36.5), C-1′, C-2′ (δC
′
(
′
′
δ
C
′
presence of carbonyl at C-2
′
δ
H
β
′
(
1
β
′
β
57,58
unit.
2
.2.
Neuroprotective activities of the constituents from M. tricuspidata fruits.
All isolated compounds were assessed against 6-OHDA induced neuronal cell death in SH-
SY5Y cells (Table 4). Of these, orobol (18) (EC 6.4 µM) and 6-prenylorobol (19) (EC 4.5 µM) were
50
50
the most potent constituents obtained (the positive control curcumin gave an EC value of 6.2 µM).
5
0
Besides, 4-(methoxymethyl)phenol (14), 5,3
15), millewanin G (16), and millewanin H (17) possessed moderate neuroprotective potential with the
EC50 values of 11.2–18.6 µM. The other compounds were inactive (EC50 > 20 µM).
′,4′-trihydroxy-2′′,2′′-dimethylpyrano(5′′,6′′:7,8)isoflavone
(
21

ACCEPTED MANUSCRIPT
Table 4. Neuroprotective Activity against 6-OHDA-induced
a
cell death in SH-SY5Y cells
Compound
EC50 (µM)
11.2 ± 0.65
4
5
-(Methoxymethyl)phenol (14)
,3 ,4 -Trihydroxy-2",2"-
′
′
dimethylpyrano(5",6":7,8)
18.6 ± 1.20
isoflavone (15)
Millewanin G (16)
Millewanin H (17)
Orobol (18)
18.5 ± 1.10
15.2 ± 0.93
6.4 ± 0.55
4.5 ± 0.62
6.2 ± 0.41
6
-Prenylorobol (19)
b
Curcumin
a
Data are presented as means ± standard deviation from three
separate experiments.
b
Positive control.
Additionally, all constituents obtained from the EtOAc soluble extract of M. tricuspidata fruits,
1
5
including the 37 reported isoflavones in preceding paper, were evaluated for their neuroprotective
effects against cell death induced by OGD in SH-SY5Y cells.
Of these, cudraisoflavone M (2), 5,7-dihydroxy-6-(2''-hydroxy-3''-methylbut-3''-enyl)-4'-
methoxylisoflavone, cudraisoflavone H, and cudraisoflavone I, exhibited moderate neuroprotective
potential against SH-SY5Y cells death induced by OGD, with their EC50 values of 3.5–11.9 µM (Table
5
). Cudraisoflavone I was the most potent constituent obtained with EC50 value of 3.5 µM, which is
greater than that of carnosine (EC 13.4 µM), a neuroprotective agent that reduces brain damage after
5
0
6
0
cerebral ischemia. The rest of compounds were inactive (EC >20 µM).
5
0
22

ACCEPTED MANUSCRIPT
Table
5.
Neuroprotective
Activity
against
a
OGD-induced cell death in SH-SY5Y cells.
Compound
EC50 (µM)
8.7 ± 0.91
Cudraisoflavone M (2)
5
3
,7-Dihydroxy-6-(2''-hydroxy-
''-methylbut-3''-enyl)-4'-
7.3 ± 0.84
b
methoxylisoflavone
b
Cudraisoflavone H
11.9 ± 1.20
3.5 ± 0.42
13.4 ± 1.50
b
Cudraisoflavone I
c
Carnosine
a
Data are presented as means ± standard deviation
from three separate experiments.
b
15
Reported in previous paper .
c
Positive control.
3
.
Conclusion
In the present study, isolated compounds from the fruits of M. tricuspidata were assessed
against 6-OHDA and OGD-induced cell death in SH-SY5Y cells. Some were found to have promising
neuroprotective effects. Besides, since PD, cerebral ischemia, and other neurodegenerative diseases are
characterized by the progressive loss of neuronal structure and function, strong efforts have been aimed
at exploring dietary to prevent these neuronal damages. In this study, neuroprotection was achieved at
physiologically relevant concentrations of dietary polyphenols, which have been effective in protecting
6
1-64
against both age-related cognitive and motor decline.
Moreover, the fruits of M. tricuspidata have
been widely consumed in Korea as fresh fruits, juices, and jams, for the treatment of various medical
1
2
disorders. Accordingly, these compounds obtained herein show significant potential to be developed
for therapeutic purposes in PD and cerebral ischemia.
23

ACCEPTED MANUSCRIPT
4
.
Experimental
.1. General
Column chromatography was performed using silica gel (Kieselgel 60, 70–230 and 230–400
mesh, Merck), and revered-phase (C ) silica gel (12 m, YMC, Kyoto, Japan). Gel permeation
chromatography was conducted using Sephadex LH-20 (18−111 m, GE Healthcare AB, Stockholm,
Sweden). HPLC was conducted using the Varian Prostar 210 system. For preparative HPLC, a YMC-
Pack ODS-A (5 m, 250 × 20 mm i.d., YMC Co., Ltd., Kyoto, Japan) column was used with an 8
mL/min flow rate. For semipreparative HPLC, an YMC J sphere ODS-H80 (4 m, 250 × 10 mm i.d.,
4
ꢁ
1
8
ꢁ
ꢁ
′
ꢁ
YMC Co., Ltd., Kyoto, Japan) column was used with a 4 mL/min flow rate. Thin layer chromatography
was performed using pre-coated silica gel 60 F254 plates (0.25 mm, Merck). Optical rotations were
measured on a JASCO P-2000 polarimeter, using 10 cm or 1cm cells. UV spectra were obtained using
an OPTIZEN POP spectrophotometer. CD spectra were recorded on a JASCO J-1100 spectrometer. IR
spectra were recorded using a Varian 640-IR spectrometer. NMR spectra were acquired using a Varian
VNMRS 500 MHz system. Low and high-resolution ESI-MS was performed on a Waters Q-TOF
micromass spectrometer.
3
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 6-hydroxydopamine (6-
OHDA), carnosine, molybdenum (II) acetate dimer, and anhydrous dimethyl sulfoxide (DMSO) were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Curcumin was obtained from Enzo Life Science
(
Farmingdale, NY, USA).
.2. Plant material
The fruits of Maclura tricuspidata (syn. Cudrania tricuspidata) were collected from the Korea
Forest Research Institute, Southern Forest Research Center, Jinju, Korea, since September 2008 (GPS
coordinates: 35°12 46.7′′N, 128 10 28.5′′E), and identified by Dr. Hak Ju Lee, Korea Forest Research
4
′
′
′
24

ACCEPTED MANUSCRIPT
Institute, Seoul 130-712, Republic of Korea. The voucher specimen (accession number: KH1-5-090904)
was deposited at the Department of Biosystems and Biotechnology, Korea University, Seoul, Korea.
4
.3. Extraction and isolation
Fresh fruits of M. tricuspidata (10.7 kg) were sliced and extracted with MeOH (3 × 10 L) at
room temperature, for 10 days. After removal of the organic solvent in vacuo, the combined extract
TH1-1-1, 630.9 g) was suspended in H O (2 × 2.5 L) and partitioned successively with -hexane (4 × 2
-hexane (TH1-2-1, 48.4 g) and EtOAc (TH1-2-2, 27.8 g)
(
2
n
L) and EtOAc (5 × 2 L) to afford dried
soluble extracts.
n
Fractionation of the EtOAc soluble extract subjected to silica gel column chromatography
(
CHCl –MeOH, 1:0 to 1:1) to afford six fractions (TH1-4-1–TH1-4-6). Fraction TH1-4-3 (9.68 g) was
3
subjected to silica gel CC and eluted with a gradient of
subfractions (TH1-10-1–TH1-10-7) were obtained. Fraction TH1-10-2 (408.0 mg) was applied to a
silica gel column developed with a gradient of CHCl –MeOH (1:0 to1:1), resulting in seven
subfractions (TH1-12-2–TH1-12-7). Fraction TH1-12-2 (64.5 mg) was eluted by silica gel CC with
-hexane–CHCl –MeOH (1:0:0 to 0:1:1), to afford eight subfractions (TH1-24-1–TH1-24-8). Fraction
n-hexane–EtOAc (1:0 to 0:1), and seven
3
n
3
TH1-24-7 (37.2 mg) was separated using Sephadex LH-20 eluted with CHCl –MeOH (1:1, v/v),
3
producing five subfractions (TH1-32-1–TH1-32-5). Fraction TH1-32-3 (24.2 mg) was subjected to
passage over LH-20 column with CHCl –MeOH (1:1, v/v), to afford butyrospermol (4.8 mg). Fraction
3
TH1-10-3 (161.0 mg) was subjected to a LH-20 column chromatography eluted with CHCl –MeOH
3
(
1:1, v/v), and seven subfractions (TH1-45-1–TH1-45-7) were obtained. Fraction TH1-45-5 (13.6 mg)
was eluted by silica gel CC with -hexane–CHCl –MeOH (1:0:0 to 0:1:1), resulting in six fractions
TH1-50-1–TH1-50-6). Purification of TH1-50-2 (6.0 mg) was carried out by CC with CHCl –MeOH
1:1) leading to isolation of lupeol (1.0 mg). Fraction TH1-10-4 (4.7 g) was eluted by silica gel CC with
-hexane–CHCl –MeOH (1:0:0 to 0:1:1), resulting in sixteen subfractions
n
3
(
(
3
a gradient mixture of
n
3
(TH1-74-1–TH1-74-16). Fraction TH1-74-2 (46.8 mg) was applied to a silica gel column developed
25

ACCEPTED MANUSCRIPT
with a gradient of
n-hexane–CHCl (1:0 to 0:1), to afford β-sitosterol (1.8 mg). Fraction TH1-74-4
3
(
1354.8 mg) was separated using Sephadex LH-20 and eluted with CHCl –MeOH (1:1, v/v) to yield
3
eight subfractions (TH1-84-1–TH1-84-8). Fraction TH1-84-2 (187.3 mg) was applied to a silica gel
column, eluted with a gradient of -hexane–EtOAc (1:0 to 0:1), resulting in preparation of fractions
n
TH1-102-1–TH1-102-3. Fraction TH1-102-2 (168.3 mg) subjected to silica gel CC with gradient
mixtures of CHCl –MeOH (1:0 to 1:1), to afford fractions TH1-108-1–TH1-108-8. Fraction TH1-108-6
3
(
26.7 mg) was purified by preparative HPLC (MeOH–H O, 50–90% MeOH in H O), to afford
2 2
cudraisoflavone L (1, 2.0 mg). Fraction TH1-74-5 (1566.0 mg) was subjected to passage over LH-20
column with CHCl –MeOH (1:1, v/v) to afford five subfractions (TH1-138-1–TH1-138-5). Fraction
3
TH1-138-3 (793.4 mg) was eluted by silica gel CC with a gradient mixture of
n
-hexane–EtOAc gradient
1:0 to 0:1), leading to the isolation of 4-(methoxymethyl)phenol (14, 195.0 mg). Fraction TH1-74-7
301.9 mg) was subjected to silica gel CC with gradient mixtures of -hexane–EtOAc (1:0 to 0:1),
(
(
n
resulting in preparation of fractions TH1-194-1–TH1-194-12. Fraction TH1-194-7 (43.8 mg) was
separated by preparative HPLC (MeOH–H O, 60–93%, MeOH in H O) to afford 4′-O-
2
2
Methylcudraisoflavone O (5, 1.5mg). TH1-74-8 (125.4 mg) was separated on a column of reversed-
phase C silica gel, eluted with MeOH–H O (5:5 to 10:0, MeOH in H O), to give 5 subfractions
1
8
2
2
(
TH3-1-1–TH3-1-5). Fraction TH3-1-4 (63.2 mg) was separated by preparative HPLC (MeOH–H O,
2
6
0–100%, MeOH in H
42.9 mg) was passed over a silica gel column and eluted with a
resulting in the isolation of 5,4
methoxyisopropylfurano[4,5:6,7]isoflavone (2.5 mg). Fraction TH1-74-11 (160.1 mg) was eluted by
silica gel CC with a gradient mixture of -hexane–EtOAc gradient (1:0 to 0:1), resulting in seven
2
O), to afford fractions TH3-45-1–TH3-45-6. Furthermore, fraction TH3-45-4
-hexane–EtOAc gradient (1:0 to 0:1),
-dihydroxy-8-(3,3-dimethylallyl)-2′′
(
n
′
-
n
subfractions (TH3-7-1–TH3-7-7). The purification of fraction TH3-7-6 (39.8 mg) was carried out by
preparative HPLC (MeOH–H O, 60–85%, MeOH in H O), leading to the isolation of cudraisoflavone
2
2
M (2, 3.1 mg) and cudraisoflavone N (3, 2.0 mg). The purification of fraction TH3-69-5 (64.6 mg) was
26

ACCEPTED MANUSCRIPT
carried out by preparative HPLC (MeOH–H O, 65–95%, MeOH in H O), leading to the isolation of 4′-
2
2
O
-methylcudraisoflavone P (7, 2.8 mg). Fraction TH1-10-6 (1672.0 mg) was passed over a column
containing Sephadex LH-20, and eluted CHCl –MeOH (1:1), resulting in eleven subfractions
TH3-101-1–TH3-101-11). Fraction TH3-101-6 (307.0 mg) was subjected to silica gel CC with gradient
3
(
mixtures of
n
-hexane–EtOAc (1:0 to 0:1), producing fractions TH3-113-1–TH3-113-9. Fraction TH3-
1
6
13-4 (48.5 mg) and TH3-113-5 (71.8 mg) were further separated by preparative HPLC (MeOH–H O,
2
0–95%, MeOH in H O), to afford 4
′-
O
-methylerythrinin C (2.3 mg) 6-prenyl-5-hydroxy-7,3
′,4
′-
2
trimethoxyisoflavone (1.2 mg), respectively. Fraction TH1-4-4 (2.30 g) was separated using Sephadex
LH-20 and eluted with CHCl –MeOH (1:1, v/v) to yield eight subfractions (TH4-21-1–TH1-21-8).
3
Fraction TH4-21-7 (757.8 mg) was applied to a silica gel column eluted with gradient mixtures of
CHCl
TH4-77-5 (77.0 mg) was further separated by preparative HPLC (MeOH–H
H O), leading to the isolation of genistein (20.3 mg), 5,3 ,4 -trihydroxy-2",2"-dimethylpyrano(5",6":7,8)
isoflavone (15, 4.1 mg), 6-prenylapigenin (3.5 mg), and millewanin G (16, 12.9 mg). Fraction TH3-77-6
122.1 mg) was subjected to preparative HPLC (MeOH–H O, 50–100%, MeOH in H O), to afford
3
–MeOH (1:0 to 1:1), resulting in the preparation of fractions TH4-77-1–TH3-77-10. Fraction
2
O, 50–95%, MeOH in
′
′
2
(
2
2
aromadendrin (70.1 mg), apigenin (5.1 mg), cudraisoflavone O (4, 4.0 mg) and cudraisoflavone P (6,
.4 mg). The purification of fraction TH4-77-8 (65.1 mg) was carried out by using preparative HPLC
MeOH–H O, 50–100%, MeOH in H O), to give millewanin H (17, 7.0 mg), cudraisoflavone Q (8, 8.8
mg), cudraisoflavone R (9, 7.0 mg), and cudraisoflavone S (10, 8.3 mg). Fraction TH1-4-5 (2.14 g) was
separated using Sephadex LH-20 and eluted with CHCl –MeOH (1:1, v/v) to yield eight subfractions
TH4-89-1–TH1-89-8). Fraction TH4-89-2 (364.0 mg) was eluted on silica gel using an -hexane–
5
(
2
2
3
(
n
CHCl –MeOH (1:0:0 to 0:1:1), to afford five subfractions (TH4-99-1–TH4-99-5). The purification of
3
fraction TH4-99-2 (117.3 mg) was carried out by using preparative HPLC (MeOH–H O, 50–90%,
2
MeOH in H O), to give cudraisoflavone T (11, 9.6 mg). Fraction TH4-89-5 (51.9 mg) was purified by
2
2 2
preparative HPLC (MeOH–H O, 50–90%, MeOH in H O), to afford 4-hydroxybenzoic acid (11.3 mg)
27

ACCEPTED MANUSCRIPT
and artocarpesin (5.0 mg). Fraction TH4-89-6 was further separated by preparative HPLC (MeOH–H O,
2
4
5–70%, MeOH in H O), leading to the isolation of orobol (18, 5.0 mg), 5,7,4′-trihydroxy-8-p-
2
hydroxybenzyldihydroflavonol (9.5 mg), gericudranin E (18.8 mg), gericudranin D (6.7 mg), and 6-
prenylorobol (19, 5.3 mg). The purification of fraction TH4-89-7 (17.8 mg) was carried out by using
2 2
semipreparative HPLC (MeOH–H O, 50–80%, MeOH in H O) to yield kaempherol (5.4 mg). Fraction
TH1-4-6 (5.48 g) was passed over a column containing Sephadex LH-20, and eluted CHCl –MeOH
3
(1:1), resulting in 12 subfractions (TH4-137-1–TH4-137-12). Fraction TH4-137-4 (765.0 mg) was
separated on a column of reversed-phase C silica gel, eluted with MeOH–H O (1:5 to 10:0, MeOH in
1
8
2
H O), to give 9 subfractions (TH4-169-1–TH4-169-9). Fraction TH4-169-5 (111.8 mg) was subjected to
2
2 2
preparative HPLC (MeOH–H O, 47–52%, MeOH in H O), to afford 7-O-methylbiochanin A (3.5 mg).
TH4-137-5 (931.0 mg) was subjected to passage over reversed-phase C18 silica gel using MeOH–H
2
O
(1:5 to 10:0), producing subfractions TH4-171-1–TH4-171-8. Fraction TH4-171-1 (140.1 mg) was
subjected to preparative HPLC (MeOH–H O, 15–55%, MeOH in H O), to give genistein-5-O-Methyl-8-
2
2
C-β-D-glucopyranoside (3.8 mg). Fraction TH4-171-3 (236.7 mg) was passed over a column containing
C silica gel, and eluted MeOH–H O (1:5 to 10:0, MeOH in H O), to afford prunin (2.4 mg),
18
2
2
sophorobioside (2.6 mg), and genistein-4
′-O-
β-D-glucopyranoside (1.1 mg). Further separation of the
impure fraction (TH5-3-2, 126.3 mg) eluted by EtOAc–MeOH gradient (1:0 to 5:1), yielded genistine
5.7 mg) and sphaerobioside (4.0 mg). Fraction TH4-171-4 (133.9 mg) was separated by preparative
HPLC (MeOH–H O, 40–90%, MeOH in H O), to give four subfractions (TH4-197-1–TH4-197-4).
Fraction TH4-197-1 (27.9 mg) was further separated by using semipreparative HPLC (MeOH–H O,
5%, MeOH in H O) to afford apigenin-7- -D-glucopyranoside (7.6 mg). The purification of fraction
TH4-197-2 (83.7 mg) using preparative HPLC (MeOH–H O, 60%, MeOH in H O) led to the isolation
(
2
2
2
5
O-β
2
2
2
of kaempferol-3-O-β-D-glucopyranoside (37.6 mg) and nicotiflorine (16.2 mg). Fraction TH4-171-6
(
74.6 mg) was subjected to preparative HPLC (MeOH–H O, 40–90%, MeOH in H O) to afford
2
2
fractions TH4-199-1–TH4-199-4. The purification of TH4-199-1 (40.3 mg) was carried out by using
28