A chemical investigation of the leaves of morus alba L.

Article abstract of DOI:10.3390/molecules23051018

The leaves of Morus alba L. are an important herbal medicine in Asia. The systematic isolation of the metabolites of the leaves of Morus alba L. was achieved using a combination of liquid chromatography techniques. The structures were elucidated by spectr

Full text of DOI:10.3390/molecules23051018

molecules
Article
A Chemical Investigation of the Leaves of
Morus alba L.
1
2
3
1
4
4
Xiao-yan Chen , Ting Zhang , Xin Wang , Mark T. Hamann , Jie Kang , De-quan Yu
4
,
and Ruo-yun Chen *
1
Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, Medical University of South
Carolina, Charleston, SC 29425, USA; chenxiaoyan8615@gmail.com or chenxi@musc.edu (X.-y.C.);
hamannm@musc.edu (M.T.H.)
2
3
4
Institute of Medical Information & Library, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100020, China; brendatingting@126.com
Beijing Key Laboratory of Bioactive Substances and Function Foods, Beijing Union University,
Beijing 100191, China; shtwangxin@buu.edu.cn
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
jiekang@imm.ac.cn (J.K.); dqyu@imm.ac.cn (D.-q.Y.)
*
Correspondence: rych@imm.ac.cn; Tel.: +86-10-8316-1622
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 31 March 2018; Accepted: 24 April 2018; Published: 26 April 2018
Abstract: The leaves of Morus alba L. are an important herbal medicine in Asia. The systematic
isolation of the metabolites of the leaves of Morus alba L. was achieved using a combination of
liquid chromatography techniques. The structures were elucidated by spectroscopic data analysis
and the absolute configuration was determined based on electronic circular dichroism (ECD)
spectroscopic data and hydrolysis experiments. Their biological activity was evaluated using different
biological assays, such as the assessment of their capacity to inhibit the aldose reductase enzyme;
the determination of their cytotoxic activity and the evaluation of their neuroprotective effects against
the deprivation of serum or against the presence of nicouline. Chemical investigation of the leaves
of Morus alba L. resulted in four new structures
1
–
4
and a known molecule
inhibited aldose reductase with IC50 values of 4.33 M and 6.0 M compared with the potent
M). Pretreatment with compound decreased PC12 cell
5. Compounds 2 and
5
µ
µ
−
3
AR inhibitor epalrestat (IC50 1.88
apoptosis subsequent serum deprivation condition and pretreatment with compound 5 decreased
×
10
µ
3
nicouline-induced PC12 cell apoptosis as compared with control cells (p < 0.001).
Keywords: Morus alba L.; aldose reductase inhibitor; neuroprotective agent; natural products
1
. Introduction
The species Morus alba L., known as white mulberry, belongs to the genus Morus of the family
Moraceae, is native to China and now is cultivated throughout the world [
have been used medicinally in Traditional Chinese Medicine including the leaves, root bark, stem and
fruits [ ]. In East Asia the leaves have been an important herbal medicine for treatment of cold,
fever, headache, cough and rheumatic diseases for thousands of years [ ]. Extracts or constituents
of M. alba L. leaves were reported to possess anti-inflammatory, antioxidant, antiobesity, antidiabetic,
and hypolipidemic properties [ ]. Phytochemical investigations of the leaves of M. alba reported
the presence of flavonoids, lignans, pyrrole alkaloids, polyphenols, fatty acids, and anthocyanin [ ].
1,2]. All parts of this plant
3,4
3,5
5,6
6,7
Previous phytochemical studies and the pharmacological potentials of constituents of the genus Morus
have been reviewed by Yang et al. [8].
Molecules 2018, 23, 1018; doi:10.3390/molecules23051018
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1018
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Aldose reductase is the first and rate-controlling enzyme in the polyol pathway that reduces
glucose into sorbitol and then fructose. Intracellular excess sorbitol is thought to lead to diabetic
complications, including neuropathy, nephropathy, retinopathy, and cataract [9]. Fructose can lead to
the formation of 3-deoxyglucosone, a key intermediate known to accelerate the formation of Advanced
Glycation End products (AGEs) [10]. The presence and accumulation of AGEs contribute to the
development of atherosclerosis and promotes renal damage, diabetic nephropathy, and a series of
cancers [11–14]. Besides reducing glucose, aldose reductase is also involved in the reduction of
oxidative stress-generated lipid aldehydes and their conjugate with GSH, which can alter cellular
signals by mediating transcription factors such as NF-Kb and AP1 [15]. Aldose reductase inhibitors
have been shown to be an effective multi-disease target to prevent diabetic complications, cancers,
cardiovascular diseases, and inflammatory complications [16]. This study investigated the acetone
and chloroform fractions of the ethanol extract of the leaves of M. alba L., which showed effects
against diabetes and human cancer cell lines in our previous study, leading to the identification of
four new structures, namely a sesquiterpenoid glucoside
derivative , a flavan , and a known compound, (9R)-hydroxyl-(10E,12Z,15Z)-octadecatrienoic
acid ( ). In addition, we report the results of aldose reductase inhibitory and neuroprotective
activity evaluations.
1, an aromatic glucoside 2, a farnesylacetone
3
4
5
2
. Results and Discussion
2
.1. Characterization
The crude extract of the leaves of Morus alba. L was divided into four fractions by flash silica gel
column chromatography. The generated acetone and chloroform fractions were further isolated by the
combination of resin column chromatography, silica gel column chromatography, medium pressure
liquid chromatography (MPLC), and high performance liquid chromatography (HPLC), generating
four new compounds and a known one (Figure 1).
2
0
Moralsin (
showed the presence of hydroxy (2957 cm ), alkyl (3402 cm ) and ester carbonyl (1760 cm
functional groups. The molecular formula, C H O , was determined from its sodium adduct
1
) was obtained as a white powder, [α]
−
72 (c 0.19, MeOH). The IR spectrum of
D
−
1
−1
−1
)
1
21
30
8
+
ion in the HRESIMS (433.1826 [M + Na] , calcd. 433.1833), corresponding to seven indices of
hydrogen deficiency. The H-NMR and C-NMR spectra (Tables 1 and 2) revealed the presence
of two trisubstituted double bonds [ 6.82 (t, J = 3.0 Hz, H-4), 5.66 (d, J = 9.3 Hz, H-8); 132.4 (C-4),
1
13
δ
H
δ
C
1
5
30.7 (C-5), 129.8 (C-8), 138.8 (C-9)], one terminal double bond [ H
δ
6.44 (dd, J = 17.5, 10.5 Hz, H-10),
.37 (d, J = 17.5 Hz, H-11a), 5.20 (d, J = 10.5 Hz, H-11b)], one ester carbonyl ( C 168.8), two oxygenated
4.41 (m, H-3), 5.12 (t, J = 9.3 Hz, H-7); 70.4 (C-3), 76.8 (C-7)], three methyls [ H 0.90
), 1.60 (dd, J = 15.0, 6.5 Hz, H-2 )],
2.57 (m, H-6), 5.12 (t, J = 9.3 Hz, H-7)], one tertiary carbon group [ 29.5 (C-1)]
δ
methines [
δ
H
δ
C
δ
(
3H, s), 0.87 (3H, s), 1.88 (3H, s)], one methylene [ H
δ
1.86 (m, H-2
α
β
two methines [
and a glucopyranosyl unit [
analysis of H- H COSY spectrum, the NMR date displayed there were two spin systems C2-C3-C4
and C6-C7-C8 in . In the HMBC spectrum, the correlations of H-12, H-13/C-1, C-2, C-6; H-4/C-5,
δ
H
δ
C
0 0 0
δ 4.35 (d, J = 8.0 Hz, H-1 ), 2.9-3.7 (6H, H-2 -6 )]. In combination with
H
1
1
1
C-14; H-6/C-4; H-11/C-9; and H-15/C-8, C-10 determined the monocyclofarnesane carbon skeleton
0
containing a 14,7-olide ring. In addition, the correlations of H-1 /C-3 indicated the glucopyranosyl
unit was connected to C-3 of the monocyclofarnesane-type sesquiterpenoid aglycone.
3
A
β
-anomeric configuration for the glucosyl unit was assigned via its large J1,2 coupling constant
(
8.0 Hz). The D-configuration of the glucose was determined by GC analysis of the trimethylsilyl
L-cysteine derivatives after acid hydrolysis of . The E-configuration of the 8,9-double bond was
demonstrated by the nuclear Overhauser effect (NOE) effect of H-7/H-15 and H-8/H-10 in the ROESY
D experiment. NOE effect of H-3/H-2 , H-7/H-12, and H-6/H-8, H-2 , and H-13 showed that H-2
H-3, H-6, C-13, and C-8 were cofacial, assigned as the -orientation, and H-7 and C-12 were -oriented.
The absolute configuration of aglycone moiety was assigned by analysis of the electronic circular
1
1
β
β
β,
β
α

Molecules 2018, 23, 1018
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dichroism (ECD) spectroscopy using excitation chirality method [17]. The ECD spectrum showed
positive Cotton effect at 263 nm and negative Cotton effect at 224 nm arising from coupling between
conjugated diene and
was in agreement of a negative chirality of
of 1 was unequivocally assigned as (3S, 6S, 7R) and the structure of moralsin was determined as 1.
α
,β-unsaturated ester chromophores (Supplementary Materials). Such a pattern
1
as depicted in Figure 2. Thus the absolute configuration
1
a
Table 1. H-NMR Spectroscopic Data of Compounds 1–4 .
1
2
3
4
Position
δH (J in Hz)
Position
δH (J in Hz)
Position
δH (J in Hz)
Position
δH (J in Hz)
2
2
3
4
6
7
8
α
β
1.86, (overlapped)
1.60, dd, (15.0, 6.5)
4.41, m
1
3
4
5
7
2.25, s
6.09, d, (16.5)
7.45, dd, (16.5, 11.5)
6.23, d, (11.5)
3.93, t, (6.5)
1.47, m
1.97, t, (7.5)
5.03, t, (7.0)
2.12, dd, (7.5, 7.0)
2.42, t, (7.5)
1.06, s
2
3a
3b
4a
4b
5
5.27, dd, (9.6, 1.8)
2.20, m
3
4
5
2 , 6
3 , 5
6.65, d, (8.4)
7.18, dd, (8.4)
6.55, d, (8.4)
7.47, m
7.36, m
7.36, m
5.33, d, (12.6)
5.22, d, (12.6)
4.84, d, (7.2)
3.21, m
3.49, m
3.09, t, (8.7)
3.25, m
3.86, d, (8.1)
3.44, m
4.83, d, (3.0)
3.75, d, (3.0)
3.89 (1H, d, 9.0)
3.59 (1H. d, 9.0)
3.35 (1H, m)
1.85, m
2.86, m
2.63, m
0
0
0
6.82, t, (3.0)
2.57, m
0
0
5.12, t, (9.3)
5.66, d, (9.3)
6.44, dd, (17.5, 9.0)
5.37, d, (17.5)
5.20, d, (10.5)
0.90, s
8
9
6.79, d, (8.4)
6.30, d, (8.4)
6.40, d, (2.4)
6.42, dd, (8.4, 2.4)
7.25, d, (8.4)
3.73, dd, (7.2, 6.0)
2.92, dd, (17.4, 7.8)
2.54, dd, (17.4, 5.4)
4
0
6
7 a
0
0
1
2
3
4
5
1
0
11
12
13
15
16
17
3
5
6
7 b
0
00
1
1
1a
1b
0
00
0
0
0
00
1
1
1
1
2
3
4
2
3
3
0
00
0.87, 3H, s
1.88, s
1.85 (3H, s)
1.62 (3H, s)
4 a
0
0
00
5
4 b
0
00
b
00
4.35, d, (8.0)
2.91, t, (8.0)
3.15, m
3.03, t, (8.0)
3.15, m
5
6
1.22, s
1.31, s
6 a
0
0
0
0
00
b
00
6 b
000
-OMe
3.75, s
1
2
000
000
5
4
4
a
a
0
0
000
6
6
a
b
3.68, dd, (10.5, 1.0)
3.44, dd, (10.5, 6.0)
000
5
a 1H–NMR data (
δ) were measured at 500 MHz in DMSO-d for 1, 2, 4, and at 600 MHz in methanol-d for 3.
6
4
b
The assignments were based on HSQC and HMBC experiments; Interchangeable.
Figure 1. Structures of Compounds 1–5.
Table 2. 13C-NMR Spectroscopic Data of Compounds 1–4 a.
1
2
3
4
Position
δc, Type
Position
δc, Type
Position
δc, Type
Position
δc, Type
1
2
3
29.5, C
43.2, CH2
70.4, CH
1
2
3
27.1, CH3
198.3, CH
129.9, CH
2
3
4
74.2, CH
29.9, CH2
25.8, CH2
1
2
3
120.0, C
155.3, C
b
105.5 , CH

Molecules 2018, 23, 1018
4 of 11
Table 2. Cont.
1
2
3
4
Position
δc, Type
Position
δc, Type
Position
δc, Type
Position
δc, Type
4
5
6
7
8
9
132.4, CH
130.7, C
52.3, CH
76.8, CH
129.8, CH
138.8, C
140.0, CH
115.6, CH2
21.0, CH3
28.9, CH3
168.8, C
12.3, CH3
102.7, CH
73.5, CH
76.8, CH
70.1, CH
76.9, CH
4
5
6
7
8
139.4, CH
122.3, CH
153.1, C
74.7, CH
33.3, CH2
27.5, CH2
135.3, C
124.0, CH
21.8, CH2
43.1, CH2
208.1, C
29.8, CH3
13.4, CH3
23.2, CH3
5
6
7
8
4a
8a
128.5, CH
109.7, CH
153.0, C
109.3, C
114.4, C
154.5, C
122.4, C
156.1, C
102.1, CH
161.4, C
105.7, CH
128.0, CH
77.4, C
4
5
6
7
1
2 , 6
131.0, CH
109.4 , CH
155.4, C
165.8, C
136.2, C
127.8, CH
128.3, CH
127.8, CH
66.0, CH2
100.4, CH
73.27, CH
75.6, CH
70.0, CH
76.8, CH
67.8, CH2
109.4, CH
75.9, CH
b
0
0
0
0
0
9
0
1
1
1
1
1
1
1
2
3
4
5
0
1
2
3
4
10
11
12
13
14
15
16
17
1
2
3
4
5
6
2
3
3 , 5
0
0
4
7
1
2
3
4
5
6
0
0
0
00
0
00
0
00
5
0
00
00
0
0
0
0
00
00
70.5, CH
27.3, CH2
20.8
00
00
4
5
6
00
000
1
2
00
000
25.8
6
1.2, CH b 4.29,
dd, (11.5, 6)
0
2
000
6
-OMe
55.7
3
78.7, C
0
0
00
00
4
5
73.30, CH2
63.2, CH2
Figure 2. ECD Spectrum of compound 1. The positive Cotton effect of ECD spectrum is in agreement
with the negative chirality of (3S, 6S, 7R) diastereoisomer of compound 1.
Compound
2 was obtained as a yellow oil. The IR spectrum of 2 showed the presence of hydroxy
−
−1
−1
(
3419 cm 1), alkyl (2931 cm ), and carbonyl (1712 cm ) functional groups. The molecular formula,
+
C H O , was determined from its sodium adduct ion in the HRESIMS (301.1785 [M + Na] , calcd.
17
26
3
1
13
3
(
01.1774), corresponding to five indices of hydrogen deficiency. The H-NMR (Table 1), C-NMR
Table 2), and DEPT (Supplementary Information) spectra revealed the presence of three double bonds
at 6.09 (d, J = 16.5 Hz, H-3), 7.45 (dd, J = 16.5, 11.5 Hz, H-4), 6.23 (d, J = 11.5 Hz, H-5), 5.03 (t, J = 7.0 Hz,
δ
H
H-11) and
δ 129.9 (C-3), 139.4 (C-4), 122.3 (C-5), 153.1 (C-6), 135.3 (C-10), 124.0 (C-11), a oxymethine
C
group at
δ 3.93 (t, J = 6.5 Hz, H-7), four methylene groups at δ 1.47 (m, H-8), 1.97 (t, J = 7.5 Hz, H-9),
H H
2
.12 (m, H-12), 2.42 (t, J = 7.5 Hz, H-13), four terminal methyl groups at δ 2.25 (s, H-1), 2.06 (s, H-15),
H
1
.85 (s, H-16), 1.62 (s, H-17), and two carbonyl groups at δ 198.3 (C-2), 208.1 (C-14). The coupling
C
1
1
1
patterns in H-NMR spectrum and the correlations in H- H COSY spectrum showed the presence
of three spin systems of C-3-C-4-C-5, C-7-C-8-C-9, and C-11-C-12-C-13. In the HMBC spectrum the
correlations of H-1, H-3, H-4/C-1, H-4, H-8/C-6, H-5/C-7, H-8, H-12/C-10, H-11/C-9, and H-12,
H-14, H-15/C-14 clarified the connections of the two terminal methyl groups (C-1, 15), the two

Molecules 2018, 23, 1018
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carbonyl groups and the three spin systems, suggesting a linear structure of pentadecatrien-2,14-dione.
The HMBC correlations of H-5, H-7/C-16, H-16/C-5, C-7, H-9, H-11/C-17, and H-17/C-9, C-10, C-11
allowed the attachments of C-16 to C-6 and C-17 to C-10, which also further supported the presence of
a pentadecatrien-2,14-dione moiety.
A 3E geometry was assigned via its large 3J3,4 coupling constant (16.5 Hz). The NOESY
correlations of H-5/H-7 and H-11/H-17 revealed that the geometries of the C-5 and C-10
olefins in
0-dimethyl-pentadecatrien-2,14-dione. The optical rotation of
of enantiomers, which was proved by its separation on HPLC using a chiral chromatography column.
Compound was obtained as a yellow oil. The molecular formula, C H O , was established
2
were 5E and 10Z. Compound
2
was characterized as (3E,5E,10Z)-7-hydroxy-6,
1
2
that is close to 0 suggested is a pair
2
3
2
1
24
5
+
from its proton adduct ion in the HRESIMS (357.16873 [M + H] , calcd. 357.1702), which was also
supported by NMR data. Analysis of the H-NMR spectrum (Table 1) of
a set of ABX system aromatic protons at
and 7.25 (d, J = 8.4 Hz, H-6 ), two ortho-coupled doublet aromatic protons at
1
3
revealed the presence of
0
0
δ
H
6.40 (d, J = 2.4 Hz, H-3 ), 6.42 (dd, J = 8.4, 2.4 Hz, H-5 ),
0
δ 6.79 (d, J = 8.4 Hz,
H
H-5) and 6.30 (d, J = 8.4 Hz, H-6), and a set of aliphatic proton signals at
δ 5.27 (dd, J = 9.6, 1.8 Hz,
H
H-2), 2.20 (m, H-3a), 1.85 (m, H-3b), 2.86 (m, H-4a), 2.63 (m,H-4b), suggesting a flavan skeleton for
1
3
3
, which was consistent with the C-NMR data. The proton signals at
δ
3.73 (dd, J = 7.2, 6.0 Hz,
H
00
0
0
00
H-3 ), 2.92 (dd, J = 17.4, 7.8 Hz, H-4 a), 2.54 (dd, J = 17.4, 5.4 Hz, H-4 b), 1.31 (s, 3H) and 1.22
s, 3H), in combination with 13C-NMR signals at
δ at 77.4 (C-2 ), 70.5 (C-3 ), 27.3 (C-4 ), and 25.8,
000 000 000
(
2
C
00
0.8 (-Me) showed the presence of a 3 -hydroxyprenyl residue forming a furan ring with a hydroxyl
0
0
00
00
group. The correlations of H-3 , H-4 a, H-4 b/C-8 confirmed the isoprenyl substituent was located
at C-8, cyclizing onto 7-hydroxyl group. Accordingly, the structure of
Figure 1.
3 was assigned as shown in
The optical rotation of
proved by the separation on HPLC using a chiral chromatography column.
Compound was obtained as a yellow oil. The IR spectrum of
3 that was close to 0, suggesting 3 is a pair of enantiomers, which was
4
4
showed the presence
−
1
−1
−1
of hydroxy (3347 cm ), carbonyl (1726 cm ) and aromatic (1606 and 1466 cm ) functional
groups. The molecular formula, C H O , was determined from its sodium adduct ion in the
2
5
30 13
+
HRESIMS (561.1583 [M + Na] , calcd. 561.1579) and also supported by the NMR spectroscopic
1
data. The H-NMR (Table 1) spectrum showed signals attributable to a monosubstituted aromatic
0 0 0 0 0
7.47 (2H, m, H-2 , 6 ) and 7.36 (3H, m, H-3 , 4 , 5 ), and an oxymethylene group at δ
H H
ring at
5
δ
0
0
.33 (d, J = 12.6 Hz, H-7 a) and 5.22 (d, J = 12.6 Hz, H-7 b), revealing the presence of a benzyl
0
0
0
group in combination with the correlations of H-7 /C-2 ,6 in HMBC spectrum. An ABC spin
system attributed to anomeric protons at 6.65 (d, J = 8.4 Hz, H-3), 7.18 (dd, J = 8.4 Hz, H-4),
δ
H
and 6.55 (d, J = 8.4 Hz, H-5). The correlations of H-3, H-5/C-7 ( C
showed the presence of a 2,6-bisubstituted benzoyl moiety. Together with the coupling patterns
of oxymethylene and oxymethine protons resonating between H 3.09 and 4.84 indicated the presence
of a glucopyranosyl and a apiofuranosyl units. The correlations of H-7 /C-7, H-1 /C-2, H-1 /C-6
in HMBC spectrum determined a moiety of apiofuranosyl(1 6)-glucopyranose was attached to C-2
δ 165.8) in HMBC spectrum
δ
0
00
000
00
→
of benzoyl moiety and the benzyl group was connected to C-7of the benzoyl moiety. A
β
-anomeric
3
configuration for the glucosyl unit was assigned via its large J
coupling constant (7.2 Hz).
”,2”
1
3
The
the chemical shift of anomeric carbon ( C
a apiofuranosyl units were determined by gas chromatography (GC) analysis of the trimethylsilyl
L-cysteine derivatives after acid hydrolysis of . On the basis of the above data, was characterized as
β
-configuration for apiofuranosyl unit was assigned via its J
coupling constant (3.0 Hz) and
1
”’,2”’
δ
109.4) [6,18]. The D-configurations of glucopyranosyl and
4
4
benzyl 2-O-[β-D-apiofuranosyl(1→6)-β-D-glucopyranosyl]-2,6-dihydroxy-benzoate.
The known compound was identified as (9R)-hydroxyl-(10E,12Z,15Z)-octadecatrienoic acid (
NMR analysis and comparison with literature data [19].
5
) by

Molecules 2018, 23, 1018
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2
.2. Aldose Reductase Inhibitory Effects of 2 and 5 and Neuroprotective Effects of Compounds 1–5
The aldose reductase inhibitory and neuroprotective bioactivities of compounds were assessed.
Compounds and possessed inhibition activities against aldose reductase, with IC50 values of
.33 M and 6.0
M compared with the potent AR inhibitor epalrestat (IC50 1.88 × 10 µM) [20]
Compound exhibited neuroprotective activity against PC12 cell damage induced by serum
deprivation and appeared to protect against PC12 cell damage caused by nicouline (Table 3), an assay
extensively used in screening active agents for Parkinson’s disease [21 24]. Compounds were
1–5
2
5
−
3
4
µ
µ
.
3
5
–
1–5
evaluated for their cytotoxic activities against eight human cancer cell lines (human colon carcinoma
cell line HCT-8, hepatocellular carcinoma cell line Bel-7402, human renal cell carcinoma cell line
KETR3, Human cervical carcinoma cell line HELA, human gastric cancer cell line BGC-823, human
ovarian carcinoma cell line A2780, human breast cancer cell line MCF-7, and human lung carcinoma
cell line A549) by means of the MTT assay [25], using paclitaxel and 5-fluouracil as positive controls.
Nevertheless, all the isolated compounds resulted to be inactive.
−
5
Table 3. Neuroprotective Effects of Compounds 1–5 at concentration of 10 M (means ± SD, n = 6).
Sample
Serum Deprivation (%)
Nicouline 4 µM (%)
100.0 ± 1.4
control
model
100.0 ± 3.7
#
##
###
41.4 ± 3.8
74.3 ± 1.4
1
2
3
4
5
50.2 ± 12.7
66.2 ± 12.6
59.8 ± 2.7***
50.9 ± 7.8
78.6 ± 2.9
78.4 ± 2.0 *
75.7 ± 3.0
76.9 ± 3.6
70.2 ± 16.1
86.2 ± 7.6 ***
#
##
p < 0.001 vs. control,* p < 0.05,** p < 0.001, *** p < 0.0001 vs. model.
2
.3. Discussion
We found it was helpful to subject the chloroform fraction of the ethanol extract to flash silica gel
column chromatography repeatedly before isolation to remove pigments. The 80% MeOH-H O mobile
2
phase of Sephadex LH-20 column chromatography worked well for all kinds of structures in our
study. We obtained two pairs of enantiomers, (2a
phenomenon whereby the R and S configurations of C-2 of a similar flavan were interconvertible,
which (3a 3b) may be subject to [ ]. In addition, considering the wide range of examples that the
,2b) and (3a,3b). Yang et al. reported an interesting
,
5
isomers of enantiomers showed differences in pharmacological processes, further separation and
research of the two pairs of enantiomers is needed.
Polyphenols from Morus plants have indicated extensive antioxidative activities, especially the
kind of Diels-Alder type adducts [8,26]. Oxidative stress played a key role in neurodegenerative
diseases, which implied the potential of polyphenols from Morus plants against neurodegenerative
disorders [27]. Unfortunately, most of the previous studies on Morus polyphenols had been focused
on their anti-oxidant properties. More efforts are suggested to explore the neuroprotective action of
constituents of Morus plants.
3
. Materials and Methods
3
.1. Plant Material
The leaves of Morus alba L. were collected in the Anding Mulberry Garden (Beijing, China), in
July 2011, and identified by Prof. Lin Ma (Institute of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College, Beijing, China). A voucher specimen (No. ID-S-2543)
has been deposited at the Herbarium of Institude of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College.

Molecules 2018, 23, 1018
7 of 11
3
.2. General Experimental Procedures
Optical rotations were measured with a P-2000 polarimeter (Jasco, Tokyo, Japan) and UV
spectra with a Jasco V-650 spectrophotometer. ECD spectra were measured on a Jasco J-815
spectrometer. IR spectra were recorded on a model 5700 spectrometer (Nicolet, Madison, SD, USA)
by an FT-IR microscope transmission method. NMR measurements were performed using VNS-600
(
Varian Medical Systems, Inc., Palo Alto, CA, USA), Mercury-300 (Varian Medical Systems, Inc.,
Palo Alto, CA, USA), Bruker-AV-III-500 (Bruker Corporation, Karlsruhe, Germany), and Inova-500
Varian Medical Systems, Inc., Palo Alto, CA, USA) spectrometers. ESIMS was performed on
(
Agilent 1100 Series LC/MSD Trap SL mass spectrometer and HRESIMS data were obtained using
an Agilent 6520 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Ltd., Santa Clara, CA, USA).
Gas chromatography (GC) was operated on Agilent 7890A system. HPLC was performed on a Lumtech
instrument (Lumiere Tech Ltd. Beijing, China) equipped with a 500 ELSD detector (Alltech, Deerfield,
IL, USA) and a YMC-Pack ODS-A column (250
×
20 mm, 5
300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (GE), and ODS
m, YMC) were used for column chromatography. TLC was carried out with GF254 plates (Qingdao
µm, YMC, Tokyo, Japan). Silica gel
(200
50
−
(
µ
Marine Chemical Factory). Spots were visualized by spraying with 10% H SO in EtOH followed
2
4
by heating.
3
.3. Cell Lines, Chemicals and Biochemicals
PC12 cells (adrenal gland; pheochromocytoma) were purchased from the American Type Culture
Collection (Manassas, VA, USA). Dimethyl sulphoxide (DMSO), nicouline, more commonly known as
rotenone, and 3-(3,4-dimehylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were obtained from
Sigma (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS),
and horse serum were purchased from Gibco BRL (New York, NY, USA). Epelrestat was purchased
from Dayin Marine Bio-Pharmaceutical Co., Ltd. (Rongcheng, Shandong, China). NADPH-Na4,
paclitaxel and 5-fluorouracil were purchased from Sigma-Aldrich (Beijing, China). All other chemicals
were of analytical grade and were commercially available.
3
.4. Extraction and Isolation
Air-dried leaves of Morus alba L. (30 kg) were exhaustively extracted with 95% aqueous EtOH
(
3 × 100 L, 2 h) at reflux. The combined extracts were concentrated under reduced pressure to
dryness. The residue (2.9 kg) was subjected to column chromatography on silica gel and eluted
with petroleum ether, chloroform, acetone and methanol. The acetone residue (423 g) was subjected
to D101 macroporous resins column chromatography by a gradient elution with EtOH/H O (0:100,
2
30:70, 60:40, 95:5) to yield four fractions (fractions A–D). The separation of fraction B (120 g) was
carried out on silica gel column chromatography eluted with CHCl /MeOH (10:1–3:1) to provide
3
three subfractions B1–B3. Subfraction B2 (80 g) was further purified by MPLC (ODS, 50
µm, YMC)
and eluted with 15, 35, 55, 75 and 100% MeOH H O, to afford 40 subfractions. Fraction B2-18
−
2
(
42 mg) was purified by preparative HPLC using 20% MeCN H O (8 mL/min) as the mobile phase
−
2
to yield compound
2
1
(4 mg) and fraction B2-20 (20 mg) was purified by preparative HPLC using
(18 mg). The chloroform residue (355 g) was
subjected to silica gel column chromatography by a gradient elution with petroleum ether/acetone
100:0, 95:5, 90:10, 80:20, 70:30, 60:40) to yield six fractions (fractions E–M). The separation of fraction
M (38 g) was carried out by MPLC (ODS, 50 m, YMC) and eluted with 5, 15, 35, 55, 75, and 100%
MeOH H O, to afford 25 subfractions. Fraction M-7 (100 mg) was purified by preparative HPLC
using 20% MeOH
of fraction N (20 g) was carried out by MPLC (ODS, 50
00% MeOH H O, to afford 28 subfractions. Fraction N-6 (50 mg) was purified by preparative HPLC
using 30% MeOH
5% MeCN H O (8 mL/min) to yield compound 4
−
2
(
µ
−
2
−
H O (8 mL/min) as the mobile phase to yield compound
5
(45 mg). The separation
2
µ
m, YMC) and eluted with 10, 30, 55, 75, and
1
−
2
−
H O (8 mL/min) as the mobile phase to yield compound 2 (10 mg). The separation
2

Molecules 2018, 23, 1018
8 of 11
of fraction g (139 g) was carried out by MPLC (ODS, 50
µm, YMC) and eluted with 15, 35, 55, 75, 85,
and 100% MeOH-H2O, to afford 38 subfractions. Fraction G-10 (385 mg) was subjected to fractionation
using Sephadex LH-20 column chromatography (80% MeOH-H O) to provide 40 subfractions. Fraction
2
G-10-18 (42 mg) was purified by the preparative HPLC using 35% MeCN H O (8 mL/min) as the
−
2
mobile phase to yield compound 3 (15 mg).
3
.5. Characterization
): white powder; [α]2
0
1
−
72 (c 0.19, MeOH); UV(MeOH): λmax (log ε) 228 (2.10) nm; IR νmax
Moralsin (
1
D
−
1
13
3402, 2957, 1760, 1606, 1080 cm ; H-NMR (DMSO-d , 500 MHz) and C-NMR (DMSO-d , 125 MHz)
6
6
+
see Table 2; positive-ion HRESIMS m/z 433.1826 [M + Na] (calcd. 433.1833).
(
2
5
3E,5E,10Z)-7-Hydroxy-6,10-dimethyl-pentadecatrien-2,14-dione ( ): yellow oil; UV(MeOH): λmax (log ε)
2
−
1 1
07 (4.31) nm, 247 (0.11) nm; IR νmax 3347, 2926, 1726, 1606, 1466, 1051, 1025 cm ; H-NMR (DMSO-d6,
1
3
00 MHz) and C-NMR (DMSO-d , 125 MHz) see Tables 1 and 2; positive-ion HRESIMS m/z 561.1583
6
+
[
M + Na] (calcd. 561.1579).
0
0 000 000 000 000 000
-Hydroxy-4 -methoxyl-2H-(2 , 2 -dimethyl-3 -hydroxy)-pyran-(5 ,6 :8,7)-flavane (3): yellow oil;
2
−
1
1
UV(MeOH): λmax (log
ε
) 206 (3.90) nm, 280 (2.80) nm; IR νmax 3372, 1718, 1602 cm ; H-NMR
13
(
MeOH-d , 600 MHz) Table 1; C-NMR (MeOH-d , 150 MHz) Table 2; positive-ion HRESIMS m/z
4
4
+
3
57.16873 [M + H] (calcd. for C H O , 357.1702).
21 25 5
Benzyl 2-O-[
β
-D-apiofuranosyl(1
→
6)-β-D-glucopyranosyl]-2,6-dihydroxybenzoate (4): yellow oil; UV(MeOH):
−1 1
λmax (log ε) 207 (4.32) nm, 247 (0.1) nm; IR νmax 3347, 2926, 1726, 1606, 1466, 1051, 1025 cm ; H-NMR
13
(
DMSO-d , 300 MHz) and C-NMR (DMSO-d , 125 MHz) see Tables 1 and 2; positive-ion HRESIMS m/z
6
6
+
561.1583 [M + Na] (calcd. 561.1579).
◦
3.04 (0.58 CHCl ); ESIMS
3
(
9R)-Hydroxy-(10E,12Z,15Z)-octadecatrienoic acid (
5
): Yellow powder, [α]20
−
D
+
1
m/z 317.2 [M + Na] , H-NMR (DMSO-d , 500 MHz)
δ
: 2.18 (2H, t, 9.6), 1.47 (2H, m, H-3), 1.38 (2H, m,
6
H-8), 2.04 (2H, m, H-17), 0.92 (3H, t, J = 9.6 Hz, H-18), 3.97 (1H, m, H-9), 5.66 (1H, dd, J = 15.5, 6.0 Hz,
H-10), 6.44 (1H, dd, J = 15.5, 11.5 Hz, H-11), 5.26-5.40 (3H, m, H-12, 15, 16), 5.96 (1H, t, J = 11.0 Hz
,
:
H-13), 2.88 (2H, dd, J = 11.0, 7.5, Hz, H-14), 1.24 (6H, m, H-4-6). 13C-NMR (DMSO-d , 125MHz)
δ
6
174.5 (C-1), 138.6 (C-10), 131.7 (C-16), 128.7 (C-13), 128.3 (C-12), 126.7 (C-15), 123. 5 (C-11), 70.44 (C-9),
3
7.2 (C-8), 33.7 (C-2), 28.5, 28.8, 28.9 (C-4-6), 25.5 (C-14), 24.9 (C-7), 24.5 (C- 3), 20.0 (C-17), 14.1 (C-18).
3
.6. Acid Hydrolysis of the Saponins and Determination of the Absolute Configuration of the Monosaccharides
◦
Compound
1
(2 mg) was hydrolyzed in 2 M HCl/H O at 80 C for 2 h. The residue was
2
reacted sequentially with L-cysteine methyl ester hydrochloride and N-trimethylsilylimidazole.
The resulting monosaccharide N-trimethylsilylimidazole derivatives were analyzed by GC. D-Glucose
was confirmed by comparison of the retention time of the derivatives with those of authentic sugars
derivatized in a similar way, which showed retention times of 27.93 min. The constituent sugars of
compounds
4 were identified by the same method as 1. Retention times of authentic samples were
detected at 17.87 min (D-apiofuranose) and at 27.93 (D-glucose). The reaction and GC conditions were
as described in the literature [28].
3
.7. Aldose Reductase Assay
The assay was operated in 96 well culture plate. A 100 µL mixture that contained 10 mM
DL-glyceraldehyde, 0.16 mM NADPH-Na4 and aldose reductase in 0.1 M sodium phosphate buffer
◦
(
pH 6.2), with or without test compounds was prepared at 0 C. Appropriate blank were employed
◦
for corrections. The assay mixture was incubated at 25 C. After 10 min of incubation, the plate was
20 C for 5 min to stop the reaction. The change in the absorbance at 340 nm
due to NADPH oxidation was measured in a plate reader [29].
◦
immediately cooled at
−

Molecules 2018, 23, 1018
9 of 11
3
.8. Neuroprotection Bioassays
The PC12 cells were cultured in DMEM medium supplemented with 5% horse serum and 5%
4
fetal bovine serum. Then, 100
µ
L of cells with an initial density of 5
×
10 cells/mL was seeded
in each well of a poly-L-lysine-coated, 96-well culture plate and precultured for 24 h. The medium
was then replaced by different fresh medium including the control (complete medium), the model
(
complete medium with 4
with different drug concentrations, 10, 1, and 0.1
medium), and the cells were cultured for 48 h. Then, 10
After incubation for 4 h, the medium was removed, and 150
formazan crystals. The optical density (OD) of the PC12 cells was measured on a microplate reader at
µ
M rotenone or serum free medium), and the sample (the test compounds
M, were added to the aforementioned model
L of MTT (5 mg/mL) was added to each well.
L of DMSO was added to dissolve the
µ
µ
µ
5
70 nm [30].
4
. Conclusions
The plants of genus Morus have been extensively investigated for their medicinal constituents and
a series of unique structures were characterized [31 37]. This studied focused on the fractions that had
been rarely researched before and generated four new structures with aldose reductase inhibitory or
neuroprotective activities. The known molecule was isolated for the first time from the genus Morus
–
5
and its neuroprotective activity reported for the first time. The compounds reported here provide new
potential aldose reductase inhibitory or neuroprotective agents for further research.
Supplementary Materials: The following are available online.
Author Contributions: D.-q.Y. and R.-y.C. conceived and designed the experiments; X.-y.C. performed the
experiments and wrote the paper; T.Z. and X.W. contributed data analysis, M.T.H. and J.K. contributed writing.
Acknowledgments: The author is grateful to Li Li who helped measure the CD spectra, Zhu-Fang Shen for the
aldose reductase assay and Nai-Hong Chen for the neuroprotection bioassay. This work was supported by CAMS
Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-010).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Sample of the compound 5 is available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).