Neuroprotective effects of triterpenoid saponins from Medicago sativa L. against H2O2-induced oxidative stress in SH-SY5Y cells

Article abstract of DOI:10.1016/j.bioorg.2018.11.008

Medicago sativa L. is a forage legume plant widely distributed in all continents. Six new triterpenoid saponins, Medicagosides A-F (1–6) and five known ones (7–11) were isolated from M. sativa. Their structures were determined via HRESIMS, 1D and 2D NMR analysis. Biologically, all the isolates displayed neuroprotective activities against H₂O₂-induced damage in SH-SY5Y cells. Among them, compounds 1, 3–5 and 10 exhibited striking neuroprotective activities at 100 μM, restoring cell viability range from 79.66% to 89.03%, relative to 79.46% (100 μM) of Trolox used as the positive control.

Full text of DOI:10.1016/j.bioorg.2018.11.008

BioorganicChemistry83(2019)468–476
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Neuroprotective effects of triterpenoid saponins from Medicago sativa L.
against H2O2-induced oxidative stress in SH-SY5Y cells
⁎
⁎
Xue-Gui Liua,b, Yu-Qiu Suna, Jun Biana, Ting Hana, Dan-Dan Yuea, Dan-Qi Lia,b, , Pin-Yi Gaoa,b,
a College of Pharmaceutical and Biological Engineering, Shenyang University of Chemical Technology, Shenyang, Liaoning 110142, People’s Republic of China
b Institute of Functional Molecules, Shenyang University of Chemical Technology, Shenyang, Liaoning 110142, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Medicago sativa L. is a forage legume plant widely distributed in all continents. Six new triterpenoid saponins,
Medicagosides A-F (1–6) and five known ones (7–11) were isolated from M. sativa. Their structures were de-
termined via HRESIMS, 1D and 2D NMR analysis. Biologically, all the isolates displayed neuroprotective ac-
tivities against H2O2-induced damage in SH-SY5Y cells. Among them, compounds 1, 3–5 and 10 exhibited
striking neuroprotective activities at 100 μM, restoring cell viability range from 79.66% to 89.03%, relative to
79.46% (100 μM) of Trolox used as the positive control.
Medicago sativa L.
Triterpenoid saponins
Neuroprotective effects
SH-SY5Y cells
1. Introduction
neuroprotective effects of various compounds in neurodegenerative
diseases.
The incidence of neurodegenerative diseases has been increasing
due to the rising aging population. These diseases, primarily
Alzheimer’s and Parkinson’s diseases, are characterized by progressive
and irreversible degeneration neuronal cells [1]. With the advance-
ments in research on neurodegenerative diseases, more factors related
to the occurrence and development of neurodegenerative diseases are
beginning to be revealed. The most striking risk factor of neurodegen-
erative diseases is aging, as it is a major cause of poor memory, com-
munication difficulties, bad learning ability and changes in personality,
which seriously affects human health [2,3]. Synthetic neuroprotective
agents used to treat neurodegenerative diseases have some side effects
including dry mouth, fatigue, somnolence, dysphoria and difficulty
walking [4]. Thus, more attention has been paid to natural products as
potential neuroprotective agents because they possess fewer side effects
and leave no drug residues in the human body [4–6]. Previous studies
indicate that neurodegenerative diseases are caused by mitochondrial
DNA mutation, oxidative stress damage, excitatory toxin and immune-
related inflammation, of which oxidative stress plays an important role
[7]. Oxidative stress occurs when reactive oxygen species containing
H2O2 are excessively generated [8]. Oxidative stress can lead to the
neuronal cell injury because these cells possess low oxidative resistance,
high metabolic capacity and non-replicative nature [9]. Therefore,
H2O2-induced damage in the neuron-like cells, such as the human
neuroblastoma SH-SY5Y cells [10], is used as a model to investigate the
Medicago sativa L. (Leguminosae) was initially discovered in Iran
and is now extensively grown in Europe, USA, Canada, Australia, China
and India [11]. M. sativa is a perennial plant of 30–100 cm height. Its
roots are thick and strong, and grow deep into the soil layers. Its leafy
stems are erect, clumped and even supine. M. sativa is used as a forage
crop and its leaves are used to prepare proteins as nutritional supple-
ment, hence M. sativa. is an important plant in agricultural sectors and
food industries [12]. M. sativa, is also used as a medicinal plant to boost
memory, treat cough, kidney stones, gastric ulcers, arthritis and central
nervous system diseases [13,14]. Previous phytochemical investiga-
tions on M. sativa have reported the presence of several chemical
compounds such as saponins [15], flavonoids [12], alkaloids [16],
polysaccharides [17], coumarins [18] and amino acids [19], of which
saponins are among the major chemical components.
Saponins are a class of glycosides in which aglycones are triterpenes
or steroid. These compounds have been extensively isolated from nat-
ural plants, of which triterpenoid-type compounds are the main con-
stituents. Saponins possess various biological activities including anti-
tumor, antifungal, anti-inflammatory, antiviral and immunomodulation
activities [20,21]. Saponins isolated from M. sativa are pentacyclic tri-
terpenoid glycosides, of which hederagenin-type and medicagenic acid-
type saponins are present in high quantities [14,22]. Based on the
previous findings that saponins confer neuroprotection against neuro-
degenerative disorders [23,24], we performed neuroprotective assays
⁎ Corresponding authors at: College of Pharmaceutical and Biological Engineering, Shenyang University of Chemical Technology, Shenyang, Liaoning 110142,
People’s Republic of China.
E-mail addresses: lidanqi@yeah.net (D.-Q. Li), gpy1981@126.com (P.-Y. Gao).
https://doi.org/10.1016/j.bioorg.2018.11.008
Received 4 October 2018; Received in revised form 7 November 2018; Accepted 9 November 2018
Availableonline13November2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

X.-G. Liu et al.
BioorganicChemistry83(2019)468–476
Table 1
1H NMR (600 MHz) and 13C NMR (150 MHz) for aglycone moieties of compounds 1–6 in Pyridine‑d5.
NO.
1
2
3
4
5
6
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
1.59 m; 1.10 m
38.8
26.2
81.7
43.5
47.5
18.2
32.9
39.9
48.1
36.9
23.9
1.56 m; 1.04 m
38.7
26.0
82.7
43.5
48.1
18.3
32.8
39.9
48.1
36.9
23.8
1.44 m; 0.93 m
38.6
25.8
83.4
43.3
48.5
18.3
32.8
39.9
48.1
36.8
23.8
1.52 m; 0.96 m
38.7
25.8
82.2
43.4
47.8
18.2
32.5
39.9
48.1
36.9
23.8
2.31 m; 1.31 m
4.87 m
44.0
70.2
85.5
52.6
52.5
20.8
33.0
40.1
48.7
36.8
23.9
2.24 m; 1.19 m
43.4
69.0
81.9
54.3
47.8
20.2
32.5
40.2
48.4
36.2
23.3
122.7
144.2
42.3
28.1
23.9
46.9
41.7
2
2.21 m; 2.00 m
2.18 m; 2.01 m
2.26 m; 1.94 m
2.10 m; 1.91 m
4.65 m
3
4.30 m
4.15 m
4.10 m
4.13 m
4.75 d (3.2)
–
4.10 d (3.5)
4
–
–
–
–
–
5
1.74 m
1.60 m
1.50 m
1.56 m
1.99 m
1.44 m
6
1.79 m; 1.44 m
1.71 m; 1.36 m
1.69 m; 1.35 m
1.65 m; 1.33 m
1.83 m; 1.66 m
1.60 m; 1.29 m
–
1.55 m; 1.02 m
7
1.47 m; 1.70 m
1.58 m; 1.34 m
1.58 m; 1.30 m
1.82 m; 1.75 m
1.84 m; 1.77 m
8
–
–
–
–
–
9
1.78 m
1.76 m
1.70 m
1.71 m
1.77 m
1.67 m
–
10
11
12
13
14
15
16
17
18
–
–
–
–
–
1.93 m; 1.81 m
1.74 m; 1.01 m
2.03 m; 1.90 m
1.92 m; 1.94 m
2.12 m; 2.03 m
1.97 m
5.46 t
122.9 5.45 t
122.9 5.42 t
122.8 5.43 t
122.9 5.50 t
122.4 5.44 t
–
144.1
42.1
28.3
23.1
47.3
–
144.1
42.1
28.2
23.4
47.0
–
144.1
42.1
28.2
23.3
46.9
–
144.1
42.1
28.2
23.3
46.9
–
144.8
42.2
28.2
23.6
46.6
41.9
–
–
–
–
–
–
–
2.10 m; 1.26 m
2.08 m; 2.00 m
–
2.35 m; 1.13 m
2.06 m; 1.96 m
–
2.32 m; 1.08 m
2.03 m; 1.92 m
–
2.33 m; 1.09 m
2.03 m; 1.93 m
–
2.14 m; 1.11 m
2.09 m; 1.93 m
–
2.33 m; 1.15 m
2.08 m
–
3.29 dd (13.7, 3.9) 41.6
3.21 dd (13.9, 3.9) 41.7
3.18 dd (13.2, 3.4) 41.7
3.19 dd (13.7, 4.1) 41.7
3.31 dd (13.3,
2.94)
3.21 dd (13.4,
3.7)
19
20
21
22
23
1.76 m; 1.26 m
–
46.1
30.9
34.1
32.7
64.0
1.74 m; 1.26 m
–
46.1
30.7
33.9
32.5
65.2
1.73 m; 1.24 m
–
46.1
30.7
33.9
32.5
65.9
1.73 m; 1.24 m
–
46.1
30.7
33.9
32.7
65.0
1.79 m; 1.31 m
–
46.4
30.9
34.1
33.1
1.79 m; 1.27 m
–
46.1
30.8
33.9
1.37 m; 1.16 m
2.02 m; 1.74 m
4.35 m; 3.77 d
(10.5)
1.58 m; 1.34 m
1.82 m; 1.76 m
4.30 m; 3.78 d
(10.5)
1.32 m; 1.06 m
1.80 m; 1.74 m
4.32 m; 3.76 d
(10.6)
1.33 m; 1.07 m
1.55 m; 1.31 m
4.08 m; 3.73 d
(10.6)
1.44 m; 1.20 m
2.05 m; 1.82 m
–
1.36 m; 1.09 m
1.43 m; 1.22 mm 32.5
180.8 9.81 s
208.2
24
25
26
27
28
29
30
0.97 s
13.7
16.1
17.5
26.0
176.3
33.1
23.7
1.11 s
13.4
16.1
17.5
26.0
176.4
33.1
23.6
1.11 s
13.3
16.1
17.5
26.0
176.4
33.1
23.6
0.95 s
13.4
16.1
17.5
26.0
176.4
33.1
23.6
2.06 s
1.54 s
1.05 s
1.26 s
–
14.1
16.8
17.3
26.2
180.1
33.2
23.7
1.76 s
1.47 s
1.12 s
1.24 s
–
11.6
16.7
17.5
26.1
176.4
33.1
23.6
0.95 s
0.97 s
0.91 s
0.93 s
1.12 s
1.15 s
1.11 s
1.13 s
1.22 s
1.19 s
1.19 s
1.18 s
–
–
–
–
0.92 s
0.91 s
0.88 s
0.89 s
0.94 s
1.01 s
0.93 s
0.91 s
1.00 s
0.90 s
0.87 s
0.88 s
on the isolated triterpenoid saponins from M. sativa. Specifically, neu-
roprotective properties of the saponins from M. sativa were studied for
the first time. Here, we report that compounds 1–11 isolated and
characterized from M. sativa have neuroprotective effects against H2O2-
induced injury in SH-SY5Y cells.
(Shenyang Pharmaceutical University). The sample (No. 2016-MSL)
was kept in our research group laboratory (Shenyang University of
Chemical Technology).
2.3. Extraction and isolation
2. Materials and methods
M. sativa (7.0 kg) was extracted with EtOH-H2O (70:30, v/v) by
refluxing three times for two hours and concentrated to obtain the re-
sidue, which was successively partitioned three times with CH2Cl2. The
aqueous phase (1.5 kg) was analyzed by a macroporous resin D101
column (EtOH/H2O, 0, 30%, 50%, 70% and 95%, v/v) to obtain Fr.
1–5. The combined fractions 4–5 (600.0 g) based on TLC profiles were
subjected to vacuum-liquid chromatography over silica gel with
CH2Cl2/MeOH (50:1–0:1, v/v) as an eluent, which yielded eight frac-
tions (A-H). Fr. C (43.4 g) was analyzed by a silica gel column (CH2Cl2/
MeOH, 50:1–0:1, v/v) to form Fr. C1-C4. Subsequently, Fr. C3 (14.7 g)
was repeatedly applied to the silica gel column and eluted using
CH2Cl2/MeOH (30:1–0:1, v/v) to produce Fr. C3-1-C3-4. Fr. C3-3
(4.7 g) was separated through a column chromatography with ODS
using MeOH/H2O (40%, 60%, 80% and 100%, v/v) to form Fr. C3-3a-
C3-3e. Fr. C3-3c was chromatographed over HPLC (70% MeOH/H2O,
v/v) to obtain compounds 7 (392.9 mg), 2 (4.9 mg), 8 (12.9 mg) and 4
(6.1 mg). Compounds 10 (93.8 mg), 3 (33.0 mg) and 6 (4.5 mg) from Fr.
C3-3b were separated under identical conditions as for Fr. C3-3c.
Separation of Fr. C3-3d via HPLC (72% MeOH/H2O, v/v) produced
compounds 5 (5.5 mg), 1 (4.3 mg) and 9 (25.0 mg).
2.1. General experimental procedures
1H NMR, 13C NMR, HMBC, HSQC, 1H-1H COSY, TOCSY and NOESY
spectra were accomplished by Bruker AVIII 600 MHz spectrometers
(Bruker Co., Billerica, MA, USA) in pyridine‑d5. HRESIMS analysis was
conducted using an Agilent 6540 Q-TOF MS (Agilent Technologies,
USA). LC-20AR HPLC fitted with an RID-20A detector (Shimadzu
Corporation, Japan) was employed for analysis and preparation of
Promosil C18 column and YMC-pack ODS-A column, respectively. HP-
Amino column (Sepax Technologies, USA) was used for acid hydrolysis
experiment. The isolation and purification of compounds were per-
formed via D101 macroporous resin (Tianjin Ruijin Special Chemicals
Co., China), silica gel (Qingdao Haiyang Chemical Co., China), octa-
decylsilanized (ODS) (ODS-A-HG, YMC Co. Ltd). Methanol, acetonitrile,
30% H2O2 and all other solvents were purchased from Shenyang-Lab
Science and Trade Co., Ltd. (Shenyang, China). Trolox was obtained
from Biotopped Science and Technology Co., Ltd. (Beijing, China). The
microplate reader made by Bio-Rad Laboratories (Hercules, California,
USA) was used.
In addition, Fr. D (34.2 g) was separated on a silica gel column
(CH2Cl2/MeOH, 10:1–0:1, v/v) to obtain Fr. D1-D5. Fr. D2 (3.1 g) was
then purified via an ODS column (MeOH/H2O, 40%, 60%, 80% and
100%, v/v), yielding three fractions (Fr. D2-1-D2-3). Finally, compound
11 (9.0 mg) was obtained from Fr. D2-3 via HPLC with MeOH/H2O
(80%, v/v).
2.2. Plant material
M. sativa used in this study was obtained from Inner Mongolia
Prataculture (Chifeng, China) and authenticated by Professor Jin-Cai Lu
469

X.-G. Liu et al.
BioorganicChemistry83(2019)468–476
Table 2
1H NMR (600 MHz) and 13C NMR (150 MHz) for sugar moieties of compounds 1–6 in Pyridine‑d5.
NO.
1
2
3
4
5
6
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
3-O-
1
Glc
Xyl
Glc
Ara
Glc
Glc
5.02 d (7.3)
4.07 m
106.4
76.0
88.3
71.0
78.3
62.5
5.12 d (6.9)
4.17 m
104.3
82.3
77.8
70.4
66.5
5.53 d (7.4)
4.10 m
103.4
83.5
78.7
70.7
77.7
62.1
5.37 d (4.1)
4.59 m
103.6
80.0
72.7
67.8
63.6
5.21 d (7.7)
3.87 m
102.7
84.5
77.4
70.1
78.5
61.6
4.97 d (7.7)
3.93 m
101.1
83.3
77.8
70.5
78.4
62.5
2
3
4.01 m
4.27 m
4.30 m
4.66 m
4.24 m
4.16 m
4
4.20 m
4.12 m
4.12 m
4.56 m
4.11 m
4.08 m
5
4.32 m
4.32 m; 3.63 m
4.20
4.35 m; 3.88 m
3.94 m
3.95 m
6
4.58 m; 4.33 m
4.46 m; 4.42 m
4.49 m; 4.45 m
4.54 m; 4.26 m
Xyl
Glc
Glc
Glc
Glc
Glc
1
2
3
4
5
6
5.30 d (7.8)
4.06 m
105.8
74.2
78.6
69.5
66.4
5.59 d (7.7)
4.22 m
103.4
84.2
77.9
71.2
77.9
62.2
5.11 d (7.6)
4.21 m
103.3
84.6
77.8
70.9
78.2
62.3
5.33 d (7.9)
4.16 m
103.5
85.4
77.7
71.3
78.0
62.3
5.30 d (7.7)
3.90 m
104.5
85.6
78.0
69.9
77.6
62.5
5.37 d (7.7)
4.21 m
103.6
84.7
77.9
70.8
78.0
62.4
4.11 m
4.28 m
4.27 m
4.28 m
4.45 m
4.20 m
4.10 m
4.24 m
4.37 m
4.21 m
4.25 m
4.32 m
4.31 m; 3.58 m
3.86 m
3.84 m
3.79 m
3.73 m
3.91 m
4.51 m; 4.44 m
4.48 m; 4.40
4.44 m; 4.28 m
4.49 m; 4.26 m
4.53 m; 4.45 m
Ara
Xyl
Glc
Glc
Xyl
1
2
3
4
5
6
5.33 d (6.9)
4.61 m
106.3
73.6
74.2
69.1
67.3
5.35 d (7.0)
4.08 m
106.5
76.0
77.7
70.6
67.5
5.34 d (8.0)
4.11 m
106.7
76.9
77.8
71.0
79.1
62.1
5.09 d (7.6)
4.04 m
106.6
76.9
77.3
71.0
79.5
62.5
5.40 d (7.1)
4.13 m
106.6
76.1
78.3
70.7
67.5
4.20 m
4.11 m
4.22 m
4.15 m
4.32 m
4.28 m
4.12 m
4.14 m
4.10 m
4.15 m
4.46 m; 3.83 m
4.33 m; 3.71 m
4.04 m
3.93 m
4.33 m; 3.72 m
4.48 m; 4.44 m
4.58 m; 4.30 m
28-O-
Ara
Glc
Glc
Glc
Glc
1
2
3
4
5
6
6.51 d (2.3)
4.62 m
93.5
75.2
70.0
66.2
63.1
6.37 d (8.2)
4.26 m
95.7
74.2
78.9
71.0
79.4
62.5
6.32 d (8.0)
4.23 m
95.7
74.1
78.9
71.0
79.3
62.7
6.35 d (8.2)
4.22 m
95.7
74.1
78.9
70.9
79.3
62.6
6.36 d (8.1)
4.23 m
95.7
74.1
78.9
71.0
79.4
62.2
4.58 m
4.34 m
4.35 m
4.31 m
4.41 m
4.44 m
4.41 m
4.29 m
4.37 m
4.39 m
4.53 m; 3.98 m
4.06 m
4.04 m
4.04 m
4.08 m
4.49 m; 4.38 m
4.50 m; 4.35 m
4.64 m; 4.39 m
4.49 m; 4.46 m
Rha
1
2
3
4
5
6
5.85 brs
4.64 m
4.65 m
4.41 m
4.45 m
1.84 d (5.8)
101.1
72.7
71.9
84.4
68.6
18.4
Xyl
1
2
3
4
5
5.17 d (7.1)
4.12 m
107.2
75.6
78.7
71.6
67.5
4.09 m
4.24 m
4.26 m; 3.54 m
2.3.1. Medicagoside A (1)
data in C5D5N, as shown in Tables 1 and 2; HRESIMS, m/z 1011.4778
White amorphous powder; 1H (600 MHz) and 13C NMR (150 MHz)
data in C5D5N, as shown in Tables 1 and 2; HRESIMS, m/z 1211.5417
[M+Cl]− (calcd for C57H92O25Cl, 1211.5611).
[M+Na]+ (calcd for C48H76O21Na, 1011.4771).
2.3.6. Medicagoside F (6)
White amorphous powder; 1H (600 MHz) and 13C NMR (150 MHz)
data in C5D5N, as shown in Tables 1 and 2; HRESIMS, m/z 1103.5303
[M−H]− (calcd for C53H83O24, 1103.5269).
2.3.2. Medicagoside B (2)
White amorphous powder; 1H (600 MHz) and 13C NMR (150 MHz)
data in C5D5N, as shown in Tables 1 and 2; HRESIMS, m/z 1059.5387
[M−H]− (calcd for C52H83O22, 1059.5371).
2.4. Acid hydrolysis of triterpenoid saponins
2.3.3. Medicagoside C (3)
New compounds (1–6) were hydrolyzed with 2 M CF3COOH for 6 h at
95 °C. The hydrolysates were concentrated with CH3OH repeatedly to
obtain sugar residues. The residues were analyzed by HPLC on an HP-
Amino column, using acetonitrile-water (84:16) as an eluent [25]. The
saccharide composition of compounds were confirmed through com-
parisons with standard sugars [tR (L-rhamnose) = 18.6 min; tR (D-xy-
lose) = 22.2 min; tR (L-arabinose) = 24.8 min; tR (D-glucose) = 34.9 min;
tR (D-galactose) = 37.2 min].
White amorphous powder; 1H (600 MHz) and 13C NMR (150 MHz)
data in C5D5N, as shown in Tables 1 and 2; HRESIMS, m/z 1089.5474
[M−H]− (calcd for C53H85O23, 1089.5476).
2.3.4. Medicagoside D (4)
White amorphous powder; 1H (600 MHz) and 13C NMR (150 MHz)
data in C5D5N, as shown in Tables 1 and 2; HRESIMS, m/z 1089.5476
[M−H]− (calcd for C53H85O23, 1089.5476).
2.5. CCK-8 cell viability assay
2.3.5. Medicagoside E (5)
White amorphous powder; 1H (600 MHz) and 13C NMR (150 MHz)
Neuroprotective properties of compounds 1–11 against H2O2-induced
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BioorganicChemistry83(2019)468–476
Fig. 1. Structures of triterpenoid saponins (1–11) from M. sativa.
damage in SH-SY5Y cells were measured by the Cell Counting Kit-8 (CCK-
8, Dojindo Laboratories, Japan) method as reported previously [5,26].
SH-SY5Y cells, initially introduced in 2016, and 1:1 mixture of MEM and
F12 medium were provided by Stem Cell Bank, Chinese Academy of
Sciences. SH-SY5Y cells were added into 96-well microplates (1 × 104/
well) and placed in the incubator for 24 h at 37 °C. Different concentra-
tions of compounds (25, 50 and 100 μM) freshly prepared with medium
were added to plates for 1 h before they were damaged with H2O2
(600 μM) for 4 h. Thereafter, CCK-8 (10 μL) was added into the 96-well
microplates. The absorbance readings were measured at 450 nm by the
microplate reader. Trolox, which was used as the reference group, was
handled with the same methods as the drug group. Finally, cell viability
was compared with that of the control group of 100%.
silica gel and ODS) and HPLC method which yielded six new com-
pounds (1–6) and five known ones (7–11) (Fig. 1). Compounds (7–11)
were identified as 3-O-[α-L-arabinopyranosyl(1 → 2)-β-D-glucopyr-
anosyl(1 → 2)-α-L-arabinopyranosyl]-28-O-[β-D-glucopyranoside] he-
deragenin (7) [27,28], 3-O-[β-D-glucopyranosyl(1 → 2)-α-L-arabinopyr-
anosyl] hederagenin 28-O-β-D-glucopyranosyl ester (8) [29], 3-O-[α-L-
rhamnopyranosyl(1 → 2)-β-D-glucopyranosyl(1 → 2)-β-D-glucopyrano-
side]medicagenic acid (9) [22], medicagenic acid 3,28-di(O-glucupyr-
anoside) (10) [27] and soyasaponin I methyl ester (11) [30].
Compound 1 formed a white amorphous powder. Its molecular
formula was confirmed as C57H92O25 by the negative-ion HRESIMS at
m/z 1211.5417 [M+Cl]− (calcd for C57H92O25Cl, 1211.5611). The 1H
NMR spectrum (Table 1) showed six single methyl signals at δH 0.92 (H-
29), 0.95 (H-25), 0.97 (H-24), 1.00 (H-30), 1.12 (H-26) and 1.22 (H-
27), one primary hydroxymethyl group at δH 4.35 and 3.77 (H-23) and
one olefinic proton signal at δH 5.46 (H-12). The 13C NMR spectrum
(Tables 1 and 2) together with the HSQC spectrum showed 30 carbon
signals of the sapogenin portion and 27 of the sugar component. From
3. Results and discussion
The 70% ethanol extract of Medicago sativa L. was isolated and
purified through column chromatography (D101 macroporous resin,
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BioorganicChemistry83(2019)468–476
the 13C NMR spectrum, six tertiary methyl carbons at δc 13.7 (C-24),
16.1 (C-25), 17.5 (C-26), 23.7 (C-30), 26.0 (C-27), 33.1 (C-29), and two
olefinic carbons at δC 122.9 (C-12) and 144.1 (C-13) showed a △12
oleanane skeleton. The C-3 and C-28 positions were glycosylated ac-
cording to the downshift of δc 81.7 and upshift of δc 176.3, respec-
tively, indicating that compound 1 was a 3,28-bidesmosidic saponin.
Five anomeric protons were found at δH 5.02, 5.17, 5.30, 5.85 and 6.51
corresponding to five anomeric carbons at δC 106.4, 107.2, 105.8,
101.1 and 93.5 in the HSQC spectrum, respectively. Furthermore, acid
hydrolysis experiment and NMR analysis confirmed the presence of D-
glucose, D-xylose, L-arabinose and L-rhamnose in compound 1. Coupling
xylopyranosyl]hederagenin 28-O-β-D-glucopyranosyl ester and given
the trivial name Medicagoside B.
Compound 3 was a white amorphous powder. Its molecular formula
was determined as C53H86O23 by the negative-ion HRESIMS (m/z
1089.5474 [M−H]−, calcd for C53H85O23, 1089.5476). Compared with
the NMR data (Tables 1 and 2) of compound 2, compound 3 was
identified as a bidesmosidic hederagenin saponin. The only difference
between compounds 2 and 3 was the types of saccharide units assigned
to C-3 of compound 3. From acid hydrolysis experiment, D-glucose and
D-xylose were found in compound 3. Moreover, the saccharide portions
were defined with β orientation by coupling constants [31]. The sac-
charide portion at C-3 of the aglycone was verified as β-D-xylopyranosyl
(1 → 2)-β-D-glucopyranosyl(1 → 2)-β-D-glucopyranosyl moiety based on
HMBC cross-peak (Fig. 2) of δH 5.35 (Xyl-H-1) with δC 84.6 (Glc-C-2′),
constants (3JH1
, H2 = 7.1–7.8 Hz) revealed a β-configuration for the
glucose and xylose [31]. The α-anomeric configuration of the arabinose
was identified by the 3JH-1, H-2 value of 2.3 Hz on the basis of a study by
Ishii et al. [32] and the NOESY correlations from δH 6.51 (Ara-H-1) to
δH 4.58 (Ara-H-3) and δH 4.44 (Ara-H-4) [33]. The correlations of δH
5.85 (Rha-H-1) with δH 4.41 (Rha-H-4) and δH 1.84 (Rha-H-6) in the
NOESY spectrum (Fig. 3) represented the α-anomeric configuration of
the rhamnose unit [34]. All proton signals were assigned by the COSY,
TOCSY, HMBC and HSQC spectra. The HMBC correlations (Fig. 2) from
δ
H 5.11 (Glc-H-1′) with δC 83.5 (Glc-C-2), and δH 5.53 (Glc-H-1) with δC
83.4 (C-3). The existence of β-D-glucopyranosyl ester at C-28 of com-
pound 3 was confirmed on the basis of HMBC correlations from δH 6.32
(Glc-H-1″) to δC 176.4 (C-28). Thus, compound 3 was identified as 3-O-
[β-D-xylopyranosyl(1 → 2)-β-D-glucopyranosyl(1 → 2)-β-D-glucopyr-
anosyl]hederagenin 28-O-β-D-glucopyranosyl ester and named as Med-
icagoside C.
δ
H 5.30 (Xyl-H-1) to δC 88.3 (Glc-C-3), and δH 5.02 (Glc-H-1) to δC 81.7
(C-3) indicated that the sequence of the disaccharide at the C-3 position
was β-D-xylopyranosyl(1 → 3)-β-D-glucopyranosyl moiety. The sequence
of the saccharide portion at C-28 was β-D-xylopyranosyl(1 → 4)-α-L-
rhamnopyranosyl(1 → 2)-α-L-arabinopyranosyl moiety as supported by
the HMBC spectrum, showing correlations from δH 5.17 (Xyl-H-1′) to δC
84.4 (Rha-C-4), δH 5.85 (Rha-H-1) to δC 75.2 (Ara-C-2), and δH 6.51
(Ara-H-1) to δC 176.3 (C-28). From the NOESY spectrum, the α-con-
figuration of H-3 of the aglycone was identified by the observation of
the correlations from δH 4.30 (H-3) to δH 1.74 (H-5), and δH 1.74 (H-5)
to δH 1.22 (H-27) [35]. Based on these data sets, compound 1 resembled
the known compound 7 [27,28] except for the sugar types assigned to
C-3 and C-28. Accordingly, compound 1 was identified as 3-O-[β-D-
xylopyranosyl(1 → 3)-β-D-glucopyranosyl]-28-O-[β-D-xylopyranosyl
(1 → 4)-α-L-rhamnopyranosyl(1 → 2)-α-L-arabinopyranoside]heder-
agenin and named as Medicagoside A.
Compound 4 was isolated as a white amorphous powder and had
the same molecular formula (C53H86O23) as compound 3 in accordance
with the negative-ion HRESIMS (m/z 1089.5476 [M−H]−, calcd for
C
53H85O23, 1089.5476). Compound 4 was almost identical to com-
pound 3 except for the presence of L-arabinose unit instead of the D-
xylose unit as revealed by the NMR (Tables 1 and 2) analyses and acid
hydrolysis experiment. Coupling constants (3JH1
, H2 = 4.1 Hz) and
NOESY (Fig. 3) analysis showed the α-configuration for arabinose unit
[32,33]. β-D-glucopyranosyl(1 → 2)-β-D-glucopyranosyl(1 → 2)-α-L-ara-
binopyranosyl portion and β-D-glucopyranosyl portion were bound to C-
3 and C-28 of compound 4, respectively, which were confirmed by the
HMBC cross-peaks (Fig. 2) from δH 5.34 (Glc-H-1′) to δC 85.4 (Glc-C-2),
δ
H 5.33 (Glc-H-1) to δC 80.0 (Ara-C-2), δH 5.37 (Ara-H-1) to δC 82.2 (C-
3), and δH 6.35 (Glc-H-1″) to δC 176.4 (C-28). Thus, compound 4 was
found to be 3-O-[β-D-glucopyranosyl(1 → 2)-β-D-glucopyranosyl(1 → 2)-
α-L-arabinopyranosyl]hederagenin 28-O-β-D-glucopyranosyl ester and
given the trivial name Medicagoside D.
Compound 2, a white amorphous powder, was determined as
C
52H84O22 by the negative-ion HRESIMS (m/z 1059.5387 [M−H]−
,
calcd for C52H83O22, 1059.5371). Its 1H NMR spectrum (Tables 1 and 2)
exhibited signals for six angular methyl groups at δH 0.90 (H-30), 0.91
(H-29), 0.97 (H-25), 1.11 (H-24), 1.15 (H-26) and 1.19 (H-27), one
primary oxygenated methylene at δH 4.30 and 3.78 (H-23) and one
olefinic proton at δH 5.45 (H-12). The NMR data were similar to those
of 1, showing △12 and 3,28-bidesmosidic triterpenoid saponin of 2.
Moreover, according to the HSQC spectrum, four anomeric protons at
Compound 5 was obtained as a white amorphous powder with the
molecular formula C48H76O21 by the positive-ion HRESIMS at m/z
1011.4778 [M + Na]+ (calcd for C48H76O21Na, 1011.4771). In the 1H
NMR spectrum (Tables 1 and 2) and HSQC spectra, six tertiary methyl
singlets [δH 0.94 (H-29), 1.01 (H-30), 1.05 (H-26), 1.26 (H-27), 1.54
(H-25) and 2.06 (H-24)], one olefinic proton [δH 5.50 (H-12)], as well
as three anomeric protons (δH 5.09, 5.21 and 5.30) were found. The 13
C
δ
H 5.12, 5.33, 5.59 and 6.37 were assigned to four anomeric carbons at
NMR spectrum showed representative olefinic carbon signals (δC 122.4
and 144.8) attributable to olean-12-ene moiety, two carboxyl groups
(δC 180.1 and 180.8) and three anomeric carbons (δC 102.7, 104.5 and
106.6). It was evident from the NMR data that only C-3 position of
aglycone was glycosylated and C-28 position contained one free car-
boxyl group. The presence of D-glucose in compound 5 was verified via
acid hydrolysis experiment, NMR analysis along with the reported lit-
erature [36]. Their β-anomeric configurations were confirmed by the
δC 104.3, 106.3, 103.4 and 95.7, respectively, showing the existence of
four sugar molecules in compound 2. The sugar constituents of 2 were
defined as
D-xylose and
experiment.DT-ghleucβo-asen,omeric configurLa-tairoanbifnoorsxeylaofsteeranadcigdluhcyodsreomlyosiis-
3
eties were identified by the JH1
,
values of 6.9–8.2 Hz [31]. The
arabinose unit was deduced by the αH-a2nomeric configuration depending
3
on the JH1
,
value and NOESY data (Fig. 3) [32,33]. The saccharide
H2
sequences were determined from HMBC, HSQC, COSY and TOCSY ex-
periments. Based on the HMBC cross-peaks (Fig. 2) from δH 5.33 (Ara-
H-1) to δC 84.2 (Glc-C-2), δH 5.59 (Glc-H-1) to δC 82.3 (Xyl-C-2), and δH
5.12 (Xyl-H-1) to δC 82.7 (C-3), the α-L-arabinopyranosyl(1 → 2)-β-D-
glucopyranosyl(1 → 2)-β-D-xylopyranosyl portion was assigned to C-3.
Additionally, the β-D-glucopyranosyl unit was labeled at C-28 of com-
pound 2 through the HMBC cross-peak from δH 6.37 (Glc-H-1′) to δC
176.4 (C-28). The α-configuration of H-3 was identified via NOESY
cross-peaks of δH 4.15 (H-3) with δH 1.60 (H-5), and δH 1.60 (H-5) with
δH 1.19 (H-27) [35]. The NMR data resembled those of the isolated
compound 7 [27,28] except for the α-L-arabinopyranosyl portion at-
tached to C-3. Therefore, compound 2 was determined as 3-O-[α-L-
arabinopyranosyl(1 → 2)-β-D-glucopyranosyl(1 → 2)-β-D-
3JH1
, H2 coupling constants [37]. All sugar signals of compound 5 were
revealed by a combination of HMBC, HSQC, COSY and TOCSY spectra.
The sequence of three sugar molecules localized at C-3 of compound 5
was inferred via the HMBC correlations (Fig. 2) between δH 5.09 (Glc-
H-1″) and δC 85.6 (Glc-C-2′), δH 5.30 (Glc-H-1′) and δC 84.5 (Glc-C-2),
δH 5.21 (Glc-H-1) and δC 85.5 (C-3), showing that the linkage was β-D-
glucopyranosyl(1 → 2)-β-D-glucopyranosyl(1 → 2)-β-D-glucopyranosyl
portion. From the NOESY spectrum (Fig. 3), the stereochemistry of H-2
and H-3 of the aglycone was determined by long-range correlations of
δ
δ
H 4.87 (H-2) with δH 1.99 (H-5) and δH 1.77 (H-9), δH 4.75 (H-3) with
H 1.99 (H-5) and δH 1.77 (H-9), and δH 1.99 (H-5) with δH 1.26 (H-27)
[38], suggesting the presence of the 2β,3β-dihydroxyoleanane skeleton.
From the NMR analysis, the aglycone moiety of 5 was confirmed as
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BioorganicChemistry83(2019)468–476
Fig. 2. Key HMBC correlations of compounds 1–6.
2β,3β-dihydroxy medicagenic acid similar to that of the isolated com-
pound 9 [22]. Consequently, compound 5 was determined as 3-O-[β-D-
glucopyranosyl(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucopyrano-
side]medicagenic acid and given the trivial name Medicagoside E.
Compound 6 was isolated as a white amorphous powder. Its mole-
cular formula, C53H84O24, was identified via the HRESIMS data (m/z
1103.5303 [M−H]−, calcd for 1103.5269). The 1H NMR (Table 1) and
HSQC data revealed the presence of six methyl singlets [δH 0.91 (H-30),
0.93 (H-29), 1.12 (H-26), 1.24 (H-27), 1.47 (H-25) and 1.76 (H-24)],
one aldehydic proton [δH 9.81 (H-23)] with a corresponding carbon (δC
208.2), and one olefinic proton [δH 5.44 (H-12)] with two typical ole-
finic carbon signals (δC 122.7 and 144.2), which were consistent with
one △12 pentacyclic triterpene derivative. The 13C NMR results (Tables
1 and 2) showed 30 carbon signals of the aglycone and 23 of the sac-
charide unit. One hydroxyl group was bound to C-2 of the aglycon as
revealed by the 1D NMR data and the HMBC correlation (Fig. 2) from
δH 2.24 (H-1) to δC 69.0 (C-2). The α-configuration for H-2 and H-3
were identified by analysis of the NOESY correlations (Fig. 3) from δH
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BioorganicChemistry83(2019)468–476
Fig. 3. Key NOESY correlations of compounds 1–6.
4.65 (H-2) to δH 4.10 (H-3), δH 4.10 (H-3) to δH 1.44 (H-5), δH 1.44 (H-
5) to δH 1.67 (H-9), and δH 1.44 (H-5) to δH 1.24 (H-27) [38]. One
aldehyde group was assigned to C-4 of the aglycon by the HMBC cross-
peak of δH 1.76 (H-24) with δC 208.2 (C-23). Therefore, the aglycone
portion of compound 6 was designated as 2β,3β-dihydroxyolean-12-en-
23-ale-28-oic acid [39]. Four sugar units were further determined using
acid hydrolysis experiment, which confirmed the existence of the D-
glucose and D-xylose. The β-anomeric configurations for these sugar
84.7 (Glc-C-2′), δH 5.37 (Glc-H-1′) to δC 83.3 (Glc-C-2), and δH 4.97
(Glc-H-1) to δC 81.9 (C-3), suggesting that β-D-xylopyranosyl(1 → 2)-β-
D-glucopyranosyl(1 → 2)-β-D-glucopyranosyl portion was bound to C-3.
Likewise, the β-D-glucopyranosyl portion was bound to C-28. Therefore,
compound 6 was identified as 3-O-[β-D-xylopyranosyl(1 → 2)-β-D-glu-
copyranosyl(1 → 2)-β-D-glucopyranosyl]-28-O-β-D-glucopyranoside-2β,
3β-dihydroxyolean-12-en-23-ale-28-oic acid and named as Medicago-
side F.
units were also identified by the 3JH1
,
H2 values of 7.1–8.1 Hz [31]. The
The neuroprotective effects of compounds 1–11 isolated from M.
sativa were examined in vitro by the typical model of H2O2-induced
HMBC correlation peaks were observed from δH 5.40 (Xyl-H-1) to δC
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BioorganicChemistry83(2019)468–476
Fig. 4. Neuroprotective effects of compounds 1–11 against H2O2-induced injury in SH-SY5Y cells using CCK-8 assay. Detailed data were showed in the
Supplementary Information Table S1. ###P < 0.001 vs. the control group, *P < 0.05, **P < 0.01 and ***P < 0.001 vs. the H2O2-treated cells group.
damage in SH-SY5Y cells. Cell viability was remarkably reduced to
around 61% in H2O2-treated cells compared to the cells of the control
group in which cell viability was 100%. As shown in Fig. 4, cell viability
of H2O2-treated cells was improved following treatment with the iso-
lated compounds compared with the H2O2-treated group. Compounds 2
and 6 restored the cell viability to 78.23% (100 μM) and 78.71%
(100 μM), respectively, which was nearer to that of Trolox (79.46%,
100 μM). Notably, compounds 1, 3–5 and 10 exhibited striking pro-
tective effects as the concentration was increased. Accordingly, cell
viability at 100 μM reached 81.94%, 84.84%, 81.94%, 89.03%, and
79.66%, respectively, compared to Trolox. Among the compounds, the
neuroprotective activity of compound 5 was the highest, while com-
pound 9 (62.13%, 100 μM) showed a weak protective effect. Interest-
ingly, the structures of compounds 5 and 9 only differed by the types of
saccharide molecules bound to C-3 of the aglycon, suggesting that these
types of saccharide molecules had an important influence on the neu-
roprotective effects of the compounds against the H2O2-induced da-
mage in SH-SY5Y cells. In recent years, some researchers investigated
the neuroprotective effects of oleanane-type triterpenoid saponins
[40–41]. Zhang et al. [5] showed that congmusaponin I acted as a
protective agent in neurodegenerative disease by inhibiting H2O2-in-
duced apoptosis and fighting against oxidative stress. Wang et al. [24]
indicated that autophagy was conducive to the protective effect of
Eclalbasaponin I. Although mechanisms of neuroprotection by tri-
terpenoid saponins remain obscure, those findings about neuroprotec-
tive activities of saponins (1–11) from M. sativa may provide some ideas
for the development of agents to treat neurodegenerative diseases.
from the Project of Education Department of Liaoning province of P. R.
China, Foundation(No. 20170023) from the Project of Science and
Technology Department of Liaoning province of P. R. China and
Foundation (No. 20170520250) from Department of Science and
Technology of Liaoning province of P. R. China.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2018.11.008.
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This study was supported by the National Natural science
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