Alkaloids and lignans with acetylcholinesterase inhibitory activity from the flower buds of: Magnolia biondii Pamp

Article abstract of DOI:10.1039/d0nj01537g

Two previously undescribed alkaloids, 4,4′-dihydroxy-3-methoxy-paucine-4′-O-β-d-glucopyranoside and (S)-2-(1,3-propanediol-2-yl)-isococlaurine, and three new lignans, (7R,8S)-9,9′-dihydroxy-3,4-dimethoxy-7,8-dihydrobenzofuran lignan, (7R,8S)-4,9,9′-trihydroxy-3,3′-dimethoxy-9′-acetoxy-7,8-dihydrobenzofuran lignan-4-O-β-d-glucopyranoside, and (7S,8R)-4,9,9′-trihydroxy-3,3′,5-trimethoxy-9′-acetoxy-7,8-dihydro-benzofuran lignan-4-O-β-d-glucopyranoside, together with eleven known compounds, were isolated from the flower buds of Magnolia biondii Pamp. Their structures were determined by extensive spectroscopic analysis, and their absolute configurations were deduced by analysis of optical rotation and electronic circular dichroic (ECD) spectra. All the isolated compounds were evaluated in vitro for their acetylcholinesterase (AChE) inhibitory activity. As a result, it was found that (+)-isococlaurine exhibited the strongest AChE inhibitory effect. Molecular docking experiments were carried out to identify the probable binding mode of this compound in the binding sites of AChE and molecular dynamics simulations were performed to evaluate the stability over time of the main interactions observed during docking calculations.

Full text of DOI:10.1039/d0nj01537g

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Alkaloids and lignans with acetylcholinesterase inhibitory
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activity from the flower buds of Magnolia biondii Pamp
Yangang Caoa, b, Hongwei Lia, b, Yanli Zhanga, b, Jianchao Wanga, Yingjie Rena, b,
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Yanling Liua, b, Mengna Wanga, b, Chen Hea, b, Xu Chena, b, Xiaoke Zhenga, b*
Weisheng Fenga, b*
,
a School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, P.R. China.
b Co-Construction Collaborative Innovation Center for Chinese Medicine and Respiratory
Diseases by Henan & Education Ministry of P.R. China, Zhengzhou 450046, P.R. China.
Abstract
Two previously undescribed alkaloids, 4,4'-dihydroxy-3-methoxy-paucine-4'-O-β-D-
glucopyranoside, (S)-2-(1,3-propanediol-2-yl)-isococlaurine, and three new lignans,
(7R,8S)-9,9'-dihydroxy-3,4-dimethoxy-7,8-dihydrobenzofuran lignan, (7R,8S)-4,9,9'-
trihydroxy-3,3'-dimethoxy-9'-acetoxy-7,8-dihydrobenzofuran
lignan-4-O-β-D-
glucopyranoside, (7S,8R)-4,9,9'-trihydroxy-3,3',5-trimethoxy-9'-acetoxy-7,8-dihydro-
benzofuran lignan-4-O-β-D-glucopyranoside, together with eleven known compounds,
were isolated from the flower buds of Magnolia biondii Pamp. Their structures were
determined by extensive spectroscopic analysis, and their absolute configurations were
deduced by analysis of optical rotation and electronic circular dichroic (ECD) spectra.
All the isolated compounds were evaluated in vitro for their acetylcholinesterase (AChE)
inhibitory activity. As a result, (+)-isococlaurine exhibited strongest AChE inhibitory
effect. Molecular docking experiments were carried out to identify the probable binding
mode of this compound in the binding sites of AChE and molecular dynamics
simulations were performed to evaluate the stability over time of the main interactions
observed in docking calculations.
Key words: Magnolia biondii Pamp; Magnoliaceae; alkaloids; lignans;
acetylcholinesterase inhibitory activity; molecular docking; molecular dynamic
*
Corresponding author.
E-mail address: zhengxk.2006@163.com (X.-K Zheng), fwsh@hactcm.edu.cn (W.-S Feng)

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simulations
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Introduction
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The plant of Magnolia biondii Pamp (family Magnoliaceae) is a perennial herb
mainly distributed in Henan, China.1 Its flower buds, commonly known as Xin-yi in
China, have been used in traditional medicine to treat allergic rhinitis, sinusitis, and
headache.2 Previous phytochemical studies on this herb resulted in the identification of
variety of constituents including esstenial oils, lignans, neolignans, monoterpends,
sesquiterpends, and alkaloids.3–7
Alkaloids have been reported as important chemical constituents in M. biondii,
which possess significant activity, especially antitumor, antibacterial, anti-
inflammatory, antioxidative, and anti-neurodegenerative activity.8,9 In our efforts to
discover more bioactive constituents from M. biondii, sixteen compounds, including
two new alkaloids, 4,4'-dihydroxy-3-methoxy-paucine-4'-O-β-D-glucopyranoside (1),
(S)-2-(1,3-propanediol-2-yl)-isococlaurine (2), and three new lignans, (7R,8S)-9,9'-
dihydroxy-3,4-dimethoxy-7,8-dihydrobenzofuran
lignan
(3),
(7R,8S)-4,9,9'-
lignan-4-O-β-D-
trihydroxy-3,3'-dimethoxy-9'-acetoxy-7,8-dihydrobenzofuran
glucopyranoside (4), (7S,8R)-4,9,9'-trihydroxy-3,3',5-trimethoxy-9'-acetoxy-7,8-
dihydrobenzofuran lignan-4-O-β-D-glucopyranoside (5), together with eleven known
compounds (6–16), were isolated (Fig. 1). Herein, the isolation, structure elucidation,
acetylcholinesterase inhibitory activity of all the compounds, and molecular docking
experiment, molecular dynamic simulations of 8 with AChE would be described.

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Fig. 1 Structures of compounds 1–16.
Results and Discussion
Compound 1 was isolated as a yellow crystal with a molecular formula of C20H29NO9
established by the HRESIMS and NMR data. The 1H NMR data of 1 (Table 1) revealed
signals for an 1,3,4-trisubstituted aromatic ring [δH 7.11 (1H, d, J = 1.6 Hz), 7.02 (1H,
dd, J = 8.2, 1.6 Hz), and 6.78 (1H, d, J = 8.2 Hz)], a conjugated olefinic group [δH 7.43
(1H, d, J = 15.6 Hz) and 6.42 (1H, d, J = 15.6 Hz)], an oxygenated methylene [δH 3.94
(1H, m) and 3.59 (1H, m)], a methoxyl group [δH 3.88 (s)], and a nitrogenated
methylene [δH 3.32 (2H, m)]. Furthermore, an anomeric proton resonance was observed
at δH 4.25 (1H, d, J = 7.8 Hz), while additional sugar signals appeared at δH 3.15–3.85.

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13
Its C NMR data (Table 1) indicated the presence of a carbonyl carbon (δC 169.2), three
sp2 quaternary carbons (δC 150.0, 149.3, and 128.2), five sp2 methine carbons (δC 142.0,
123.2, 118.7, 116.5, and 111.5), a sp3 oxymethlene carbon (δC 70.3), a methoxyl group
(δC 56.4), as well as six monosaccharide carbons (δC 104.4, 78.1, 78.0, 75.2, 71.7, and
62.8). The above NMR data resembled those of paucine 3'-β-D-glucopyranoside,10 but
for the replacement of the nitrogenated methylene group by the oxygenated methylene
group and the presence of the methoxyl group. Acid hydrolysis of 1 yielded D-glucose,
which was detected in the hydrolysate by chiral-phase HPLC analysis. β-configuration
of the D-glucose was determined based on coupling constant (J = 7.8 Hz) of the
anomeric proton. The D-glucose was attached to C-4' due to the HMBC correlation of
H-1'' (δH 4.25)/C-4' (δC 70.3). The methoxyl group was located at C-3 based on the
HMBC correlation from protons of the methoxyl group (δH 3.88) to C-3 (δC 149.3) (Fig.
2). Therefore, the structure of 1 (Fig. 1) was elucidated as 4,4'-dihydroxy-3-methoxy-
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paucine-4'-O-β-D-glucopyranoside.
Table 1 1H and 13C NMR Spectroscopic Data for 1 in CD3OD (500 MHz for 1H, 125 MHz for 13C).
Position δH (J in Hz)
δC
Position
3'
δH (J in Hz)
1.67, m
δC
1
128.2
111.5
149.3
150.0
116.5
27.2
70.3
2
3
4
5
6
7
8
9
1'
2'
7.11, d (1.6)
4'α
3.94, m
4'β
3.59, m
1''
4.25, d (7.8)
3.17, dd (8.8, 8.0)
3.26a
104.4
75.2
78.0
71.7
78.1
62.8
6.78, d (8.2)
2''
7.02, dd (8.2, 1.6) 123.2
3''
7.43, d (15.6)
6.42, d (15.6)
142.0
118.7
169.2
40.2
4''
3.27a
5''
3.34, m
3.85a
6''α
6''β
3-OCH3
3.32, m
1.68, m
3.66, dd (11.8, 5.2)
3.88, s
28.1
56.4
a overlapped signals.

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Fig. 2 Key HMBC and 1H-1H COSY correlations of 1–4.
Compound 2 was isolated as a yellow crystal. Its molecular formula was determined
to be C20H25NO5 by the HRESIMS and NMR data. The 1H NMR spectra of 2 (Table 2)
displayed resonances for an 1,4-disubstituted aromatic ring [δH 7.12, (2H, d, J = 8.0 Hz)
and 6.80 (2H, d, J = 8.0 Hz)], an 1,2,4,5,-tetrasubsituted aromatic ring [δH 6.77 (1H, s)
and 6.64 (1H, s)], two nitrogenated methines [δH 4.60 (1H, dd, J = 8.6, 5.6 Hz) and 3.65
(1H, m)], two oxygenated methylenes [δH 3.58 (2H, dd, J = 11.1, 4.7 Hz) and 3.51 (2H,
dd, J = 11.1, 5.8 Hz)], a nitrogenated methylene [δH 3.48 (1H, m) and 3.25 (1H, m)],
and a methoxyl group [δH 3.84 (3H, s)]. Twenty carbon signals in its 13C NMR (Table
2) and DEPT135 spectra were attributed to six sp2 quaternary carbons (δC 158.3, 149.3,
146.9, 126.9, 125.1, and 123.6), six sp2 methine carbons [δC 131.7 (2 × C), 117.0 (2 ×
C), 114.2, and 112.6], two sp3 nitrogenated methine carbons (δC 73.8 and 57.9), two sp3
oxymethylene carbons (δC 64.4), a sp3 nitrogenated methylene carbon (δC 40.8), and a
methoxyl group (δC 56.4). The 1D NMR data of 2 were similar to those of
isococlaurine,11 with the main difference being the presence of an 1,3-propanediol-2-yl
moiety attached to the N-2, which was determined by the HMBC correlation of H-1''
(δH 3.65) with C-1 (δC 57.9) (Fig. 2).
Table 2 1H and 13C NMR Spectroscopic Data for 2 in CD3OD (500 MHz for 1H, 125 MHz for 13C).
Position δH (J in Hz)
δC
Position
3'
δH (J in Hz)
δC
1
4.60, dd, (8.6, 5.6) 57.9
6.80, d (8.0)
117.0

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3α
3β
4
3.48, m
3.25, m
3.03, m
40.8
4'
158.3
5'
6.80, d (8.0)
7.12, d (8.0)
3.39a
117.0
25.9
6'
131.7
40.4
7'α
7'β
1''
4a
5
123.6
114.2
146.9
149.3
112.6
125.1
126.9
131.7
2.98a
6.64, s
3.65, m
73.8
64.4
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6
2''α
2''β
3''α
3''β
7-OCH3
3.58, dd (11.1, 4.7)
3.51, dd (11.1, 5.8)
3.58, dd (11.1, 4.7)
3.51, dd (11.1, 5.8)
3.84, s
7
8
6.77, s
64.4
56.4
8a
1'
2'
7.12, d (8.0)
a overlapped signals.
The (S) absolute configuration of 2 was determined by comparing its optical rotation
[α] 2D0 –15.7 (c 0.08; MeOH) with that of the similar published compound (+)-
isococlaurine.12 Thus, the structure of compound 2 (Fig. 1) was defined as (S)-2-(1,3-
propanediol-2-yl)-isococlaurine.
Compound 3 was isolated as a pale yellow solid with a molecular formula of
C20H24O5 determined by the HRESIMS and NMR data. In its 1H NMR spectrum (Table
3), the signals indicated the presence of two 1,3,4-trisubstituted benzene rings [δH 7.08
(1H, s), 7.00 (1H, d, J = 8.2 Hz), 6.95 (1H, s), 6.92 (1H, d, J = 8.5 Hz), 6.90 (1H, d, J
= 8.5 Hz), and 6.72 (1H, d, J = 8.2 Hz)], an oxymethine [δH 5.48 (1H, d, J = 5.9 Hz)],
two oxymethylenes [δH 3.83 (1H, dd, J = 13.4, 8.0 Hz), 3.76 (1H, dd, J = 13.4, 4.4 Hz),
and 3.55 (2H, t, J = 6.5 Hz)], and two methoxyl groups [δH 3.80 (3H, s) and 3.78 (3H,
s)]. In the 13C NMR spectrum of 3 (Table 3), 20 carbon signals, including six sp2
quaternary carbons (δC 159.4, 150.6, 150.2, 136.5, 135.7, and 128.7), six sp2 methine
carbons (δC 129.7, 125.9, 119.4, 112.9, 110.6, and 109.8), a sp3 oxygenated methine
carbon (δC 88.2), two sp3 oxygenated methylene carbons (δC 65.2 and 62.2), and two
methoxyl groups (δC 56.5 and 56.4) were observed. These NMR data mentioned above
was
similar
to
those
of
4-[3-hydroxymethyl-5-(3-hydroxypropyl)-2,3-
dihydrobenzofuran-2-yl]-2-methoxyphenol,13 except for an extra methoxyl group
attached to C-3, which was confirmed by the HMBC correlation of methoxyl group
protons (δH 3.80) with C-3 (δC 150.2) (Fig. 2).

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Table 3 1H and 13C NMR Spectroscopic Data for 3–5 in CD3OD (500 MHz for 1H, 125DMOI:H10z.1f0o3r9/D0NJ01537G
13C).
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8
9
3
4
5
Position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
135.7
136.3
135.9
104.4
154.4
136.4
154.4
104.4
88.5
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2
6.95, s
110.6 7.02, s
150.2
111.2 6.73, s
151.0
3
4
150.6
147.6
5
6.92, d (8.5)
6.90, d (8.5)
5.48, d (5.9)
3.44, m
112.9 7.13, d (8.4)
119.4 6.92, d (8.4)
118.1
6
119.4 6.73, s
7
88.2
55.2
5.55, d (5.6)
3.45, m
3.85a
88.5
55.7
65.0
5.56, d (5.6)
3.46, m
8
55.7
9α
9β
1'
3.83, dd (13.4, 8.0) 65.2
3.76, dd (13.4, 4.4)
136.5
3.85, dd (12.1, 6.1) 65.0
3.77, dd (12.1, 4.8)
136.4
3.74a
137.1
2'
7.00, d (8.2)
6.72, d (8.2)
129.7 6.71, s
114.2 6.73, s
138.3
114.3
3'
109.8
159.4
128.7
140.3
4'
145.3
145.4
5'
129.6
129.6
118.0
6'
7.08, s
125.9 6.70, s
118.0 6.70, s
7'
2.61, t (7.6)
1.79, m
32.6
35.9
62.2
2.63, dd (14.6, 7.0) 32.9
2.63, dd (14.8, 7.1) 32.9
8'
1.92, m
31.7
65.1
173.1
20.8
1.91, m
31.7
65.1
173.1
20.8
105.3
75.7
77.8
71.3
78.3
62.6
9'
3.55, t (6.5)
4.05, t (6.3)
4.07, t (6.5)
1''
2''
1'''
2'''
3'''
4'''
5'''
6'''α
6'''β
2.02, s
2.01, s
4.89, d (7.8)
3.48a
3.46a
102.8 4.88, d (7.8)
74.9
77.9
71.4
78.2
62.5
3.86a
3.84a
3.85a
3.37a
3.38, m
3.19, m
3.67, br d (10.4)
3.57, t (6.4)
3.82, s
3.65, dd (9.8, 4.6)
3.45, d (9.8)
3.81, s
3-OCH3 3.80, s
4-OCH3 3.78, s
5-OCH3
56.5
56.4
56.8
56.7
57.0
3.81, s
3.87, s
57.0
56.8
3'-OCH3
3.86, s
a overlapped signals.
The relative-trans configuration of 3 was established on the basis of the coupling
constant J7,8 (5.9 Hz) in accordance with literature reports.9 The (7R,8S) absolute
configuration was determined by the negative Cotton effect at 291 nm (Δε – 1.7)
observed in its electronic circular dichroic (ECD) spectrum (Fig. 3).14 Thus, the

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structure of 3 was elucidated as (7R,8S)-9,9'-dihydroxy-3,4-dimethoxy-7,8-
5
6
dihydrobenzofuran lignan.
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Fig. 3 Experimental ECD spectra of 3–5.
Compound 4 was isolated as a pale yellow solid with a molecular formula of
C28H36O12 established by the HRESIMS and NMR data. The 1D NMR data of 4 (Table
3) revealed similarities with those of 3, with the main difference being the presence of
an acetyl group and β-glucopyranosyl moiety. The acetyl group was linked to C-9' based
on the correlation from H-9' (δH 4.05) to C-1'' (δC 173.1) in the HMBC spectrum. The
methoxyl groups was located at C-3 and C-3', respectively, based on the HMBC
correlations from the protons of 3-OCH3 (δH 3.82) to C-3 (δC 151.0) and from the
protons of 3'-OCH3 (δH 3.86) to C-3' (δC 138.3). The hexose moiety was identified as
D-glucose by chiral-phase HPLC analysis of the acid hydrolysate of 4, which was
attached to C-4 due to the HMBC correlation of H-1''' (δH 4.89)/C-4 (δC 147.6). The
relative-trans configuration of 4 was also established on the basis of the coupling
constant J7,8 (5.6 Hz) in accordance with 3 and literature report.9 The negative Cotton
effect at 294 nm (Δε – 1.7) observed in its ECD spectrum (Fig. 3), indicating the (7R,8S)
absolute configuration of 4.14 Therefore, the structure of 4 was defined as (7R,8S)-
4,9,9'-trihydroxy-3,3'-dimethoxy-9'-acetoxy-7,8-dihydrobenzofuran lignan-4-O-β-D-

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glucopyranoside.
5
6
7
8
Compound 5 was isolated as a pale yellow solid with a molecular formula of
C29H38O13 deduced from the HRESIMS and NMR data. The 1D NMR data of 5 (Table
3) resembled to those of 4 and differed only in the methoxyl group. The connection
locations of methoxyl groups and β-glucopyranosyl moiety were determined based on
the HMBC correlations. The coupling constant J7,8 (5.7 Hz) suggested the relative-trans
configuration of 5. The (7S,8R) absolute configurations of 5 were determined due to the
positive Cotton effect at 294 nm (Δε + 1.6) observed in its ECD spectrum (Fig. 3).15–17
Therefore, the structure of 5 was defined as (7S,8R)-4,9,9'-trihydroxy-3,3',5-
trimethoxy-9'-acetoxy-7,8-dihydrobenzofuran lignan-4-O-β-D-glucopyranoside.
By comparison of the NMR data with those reported in the literatures, eleven known
compounds were identified as (+)-reticuline N-oxide (6),18 4'-hydroxybenzyl-6-
methoxy-7-hydroxyisoquinoline (7),19 (+)-isococlaurine (8),11,12 (–)-isococlaurine
(9),11,12 α-magnoflorine (10),20 β-magnoflorine (11),20 3',4-O-dimethylcedrusin (12),21
(+)-fargesin (13),22 4,4'-dimethoxy-3'-hydroxy-7,9':7',9-diepoxy lignan-3-O-β-D-
glucopyranoside (14),23 fargesol (15),24 scaphopetalone (16).25
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All the isolated compounds (1–16) were evaluated in vitro for their AChE inhibitory
activity. As a result, compounds 1, 6, and 8 exhibited moderate AChE inhibitory effect
with IC50 values of 12.5 ± 2.4, 10.4 ± 2.5, and 8.2 ± 1.8 μM (Table 4).
Table 4 Acetylcholinesterase inhibitory effects of compound 1–16.
Compounds
IC50 (μM)
12.5 ± 2.5
26.1 ± 2.8
30.4 ± 3.2
>50
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4
>50
5
10.4 ± 2.4
29.9 ± 3.7
8.2 ± 1.8
25.7 ± 2.9
>50
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7
8
9
10
11
12
13
14
>50
45.2 ± 3.6
>50
>50

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34.7 ± 3.2
15
16
42.6 ± 2.7
7.7 ± 2.3 × 10–3
a. Positive control compound.
tacrinea
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The docking results (Fig. 4.) indicated that two conventional hydrogen bonds
between 6-OH and residue SER203 and between 7-OCH3 and residue GLY122, a
carbon hydrogen bond between H-3 and residue TYR337, four π-π stacked interactions
between 4-hydroxy-benzyl moiety and residue TRP286 and between benzene ring of
the isoquinoline moiety and residue TRP86, and a π-π T-shaped interaction between
benzene ring of the isoquinoline moiety and residue TYR337 were observed in the
complexes of compound 8 and AChE proposed by docking studies.
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Fig. 4 The receptor-ligand interaction of 8 with AChE.
In order to evaluate the stability over time of the main interactions observed in
docking calculations, molecular dynamics simulations (1000 ps, 300 K) of the docked
complexes (8 with AChE) were performed. The root mean square deviation (RMSD)
data suggested that the binding of 8 stabilized AChE and lead to less structural
deviations from its first frame (Fig. 5). Comparison between initial and final pose of
each accommodated ligand confirmed the essential role of the conserved triad TRP86-
SER203-TRP286 for carbohydrate binding (Fig. 6).

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Fig. 5 RMSD plot of AChE complexed with 8 during MD simulations (1000 ps).
Fig. 6 The initial (A) and final (B) pose of 8 docked in the AChE during the MD
simulations.
Conclusion
In this study, two new alkaloids, 4,4'-dihydroxy-3-methoxy-paucine-4''-O-β-D-
glucopyranoside, (S)-2-(1,3-propanediol-2-yl)-isococlaurine, and three new lignans,
(7R,8S)-9,9'-dihydroxy-3,4-dimethoxy-7,8-dihydrobenzofuran lignan, (7R,8S)-4,9,9'-
trihydroxy-3,3'-dimethoxy-9'-acetoxy-7,8-dihydrobenzofuran
lignan-4-O-β-D-
glucopyranoside, (7S,8R)-4,9,9'-trihydroxy-3,3',5-trimethoxy-9'-acetoxy-7,8-dihydro-
benzofuran lignan-4-O-β-D-glucopyranoside, as well as eleven known compounds,

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were isolated from flower buds of M. biondii. Biologically, all the isolated compounds
were evaluated for their inhibitory effects against AChE. Among them, compound 8
exhibited a moderate inhibitory AChE activity with an IC50 value of 8.2 ± 1.8 μM. The
results of molecular docking and molecular dynamics simulations experiments
suggested that compound 8 bound to AChE stably.
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Experimental
General experimental procedures
NMR spectra were measured by a Bruker Avance III 500 spectrometer (Bruker,
Germany). MS spectra were acquired using a Bruker maXis HD mass spectrometer
(Bruker, Germany). UV spectra were measured by a Thermo EVO 300 spectrometer
(Thermo, Waltham, MA, USA). IR spectra were recorded on a Thermo Nicolet IS 10
spectrometer (Thermo, Waltham, MA, USA). Optical rotations were record by a
Rudolph AP-Ⅳ polarimeter (Rudolph, Hackettstown, NJ, USA). ECD spectra were
obtained using an Applied Photophysics Chirascan qCD spectropolarimeter
(AppliedPhotophysics, Leatherhead, Surrey, UK). Semisemipreparative HPLC
separations were conducted on a Saipuruisi LC 50 HPLC system, equipped with an
UV/VIS 50 detector (Saipuruisi, Beijing, China). Monosaccharide elucidation was
performed on a Waters 2695 separation module equiped with an evaporative light
scattering detector (ELSD) (Waters, Milford, MA, USA) using a CHIRALPAK AD-H
column (250 × 4.6 mm) (Daicel Chiral Technologies Co., Ltd., China). Column
chromatographies were performed using MCI gel CHP-20 (TOSOH Corp., Tokyo,
Japan), ODS gel (50 μm) (YMC Group, Kyoto, Japan), Sephadex LH-20 (40–70 μm)
(Amersham Pharmacia Biotech AB, Uppsala, Sweden), Toyopearl HW-40C (TOSOH
Corp., Tokyo, Japan), and silica gel (160–200 mesh and 200–300 mesh) (Marine
Chemical Industry, Qingdao, China).
Plant material
The dry flower buds of M. biondii collected in Nanzhao, Henan province, China,
was identified by Professor Chengming Dong, School of Pharmacy, Henan University

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of Chinese Medicine, Zhengzhou, China. A voucher specimen (20140609) was
deposited at the Department of Pharmaceutical Chemistry, Henan University of Chinese
Medicine.
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Extraction and isolation
The powder flower buds (5.0 kg) were extracted with 50% aqueous acetone three
times (20 L, smashed tissue extraction) at room temperature. The combined solutions
were evaporated under vacuum to give a crude extract (463 g). The crude extract was
suspended in H2O (2 L) and then successively extracted five times with petroleum ether,
EtOAc and n-BuOH, respectively. The n-BuOH fraction (60.0 g) was subjected to
Diaion HP-20 column chromatography (CC) eluted with an EtOH-H2O (0:100, 20:80,
40:60, 80:20, 95:5) gradient to give five subfractions (B1–B5). B2 (14.0 g) was then
applied to ODS CC eluted with a MeOH-H2O (0:100, 5:95, 10:90, 15:85, 20:80, 25:75,
35:65,45:55, 100:0) gradient to give nine subfractions (B2-1–B2-9). B2-3 (1.9 g) was
subjected to MCI gel CHP-20 CC eluted with a MeOH-H2O (10:100, 30:70, 50:50,
70:30, 100:0) gradient to yield 5 subfractions (B2-3-1–B2-3-5). B2-3-4 (241.3 mg) was
purified by semipreparative HPLC eluted with MeOH-H2O (50:50) to give 7 (2.7 mg,
tR = 20.0 min). B2-4 (3.2 g) was subjected to silica gel CC eluted with a CH2Cl2-MeOH
(100:0, 80:20, 60:40, 40:60, 20:80, 0:100) gradient to yield 5 subfractions (B2-4-1–B2-
4-5). B2-4-4 (102.4 mg) was chromatographed by Toyopearl HW-40C CC eluted with
MeOH-H2O (30:70) to give 3 subfractions (B2-4-4-1–B2-4-4-3). B2-4-4-1 (24.3 mg)
was purified by semipreparative HPLC eluted by MeOH-H2O (20:80) to give 2 (4.1 mg,
tR = 48.1 min). B2-4-4-3 (50.8 mg) was purified by semipreparative HPLC eluted with
MeOH-H2O (30:70) to give 8 (35.2 mg, tR = 17.6 min), 9 (7.8 mg, tR = 23.8 min), and
10 (16.1 mg, tR = 32.4 min). B2-4-6 (982.4 mg) was applied to Sephadex LH-20 CC
eluted by MeOH to give 4 subfractions (B2-4-6-1–B2-4-6-4). B2-4-6-3 (46.1 mg) was
purified by semipreparative HPLC eluted with MeOH-H2O (35:65) to give 6 (3.3 mg,
tR = 28.5 min) and 1 (6.6 mg, tR = 30.3 min). B3 (14.5 g) was subjected to silica gel CC
eluted with a CH2Cl2-MeOH (100:0, 80:20, 60:40, 40:60, 20:80, 0:100) gradient to
yield 9 subfractions (B3-1–B3-9). B3-6 (1.4 g) was applied to Sephadex LH-20 CC

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eluted with MeOH to give 3 subfractions (B3-6-1–B3-6-3). B3-6-2 (122.4 mg) was
purified by semipreparative HPLC eluted by MeOH-H2O (50:50) to give 5 (66.6 mg,
tR = 18.1 min) and 4 (19.3 mg, tR = 40.2 min). B4 (1.1 g) was subjected to silica gel CC
eluted with a CH2Cl2-MeOH (100:0, 80:20, 60:40, 40:60 0:100) gradient to yield 8
subfractions (B4-1–B4-8). B4-2 (114.4 mg) was applied to Toyopearl HW-40C CC
eluted with MeOH-H2O (70:30) to give 2 subfractions (B4-2-1–B4-2-2). B4-2-2 (35.2
mg) was chromatographed by semipreparative HPLC eluted by MeOH-H2O (45:55) to
give 14 (4.7 mg, tR = 25.2 min). B4-3 (278.1 mg) was applied to Sephadex LH-20 CC
eluted with MeOH-H2O (70:30) to give 4 subfractions (B4-3-1–B4-3-4). B4-3-2 (56.3
mg) was chromatographed by semipreparative HPLC eluted by MeOH-H2O (40:60) to
give 12 (16.5 mg, tR = 19.3 min) and 16 (6.3 mg, tR = 23.7 min). B4-3-4 (47.8 mg) was
chromatographed by semipreparative HPLC eluted by MeOH-H2O (40:60) to give 3
(7.4 mg, tR = 19.3 min), 15 (27.2 mg, tR = 23.7 min), and 13 (3.6 mg, tR = 40.7 min).
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Compound 1: yellow crystal; [α]20 –20.9 (c 0.13, MeOH); UV (MeOH) λmax 219,
D
234, 294, 319 nm; IR (iTR) νmax: 3365, 2940, 2881, 2844, 1653, 1594, 1517, 1456,
1429, 1274, 1073, 1032 cm–1; HRESIMS m/z 450.1737 [M + Na]+ (calcd for 450.1734,
C20H29NO9Na); 1H NMR (500MHz, CD3OD) and C NMR (125MHz, CD3OD) data
13
see Table 1.
Compound 2: yellow crystal; [α]20 –15.7 (c 0.08, MeOH); UV (MeOH) λmax 206,
D
281 nm; IR (iTR) νmax: 3244, 2950, 2839, 1675, 1518, 1455, 1203, 1136, 1067 cm–1;
HRESIMS m/z 360.1803 [M + H]+ (calcd for 360.1805, C20H26NO5); H NMR
1
(500MHz, CD3OD) and 13C NMR (125MHz, CD3OD) data see Table 2.
Compound 3: pale yellow solid; [α]20 –13.6 (c 0.09, MeOH); UV (MeOH) λmax 203,
D
226, 284 nm; IR (iTR) νmax: 3352, 2936, 1652, 1595, 1515, 1488, 1453, 1262, 1234,
1022 cm–1; HRESIMS m/z 367.1537 [M + Na]+ (calcd for 367.1515, C20H24O5Na); 1H
NMR (500MHz, CD3OD) and 13C NMR (125MHz, CD3OD) data see Table 3.
Compound 4: pale yellow solid; [α]20 –9.4 (c 0.07, MeOH); UV (MeOH) λmax 205,
D
281 nm; IR (iTR) νmax: 3368, 1683, 1207, 1140, 1073, 1036 cm–1; HRESIMS m/z
599.1874 [M + Cl]– (calcd for 599.1889, C28H36O12Cl); 1H NMR (500MHz, CD3OD)
and 13C NMR (125MHz, CD3OD) data see Table 3.

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Compound 5: pale yellow solid; [α]D –55.5 (c 0.08, MeOH); UV (MeOH) λmax 207,
5
6
280, 318 nm; IR (iTR) νmax: 3369, 2942, 1678, 1455, 1205, 1137, 1070, 1038 cm–1;
7
8
1
HRESIMS m/z 629.1981 [M + Cl]– (calcd for 629.1995, C29H38O13Cl); H NMR
9
(500MHz, CD3OD) and 13C NMR (125MHz, CD3OD) data see Table 3.
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Acid hydrolysis of compounds 1, 4, and 5
Compounds 1 (1.1 mg), 4 (0.9 mg) and 5 (1.0 mg) were hydrolyzed with 2 M HCl
(2.5 mL) (80 °C, 3 h). For each compound, the mixture was dried and the residue was
dissolved in H2O (2 mL) and then extracted with EtOAc (3 × 2 mL).26 The dry aqueous
was subjected to chiral-phase HPLC equipped with a Chiralpack AD-H column (250 ×
4.6 mm) and an evaporative light scattering detection (ELSD), using
hexane:alcohol:trifluoroacetic acid mixture (750:250:0.25) as the mobile phase (0.5
mL·min–1).27 The retention time of the sugar from hydrolysate was similar to that of D-
glucose (tR = 16.9 min), so the sugar was identified as D-glucose.
AChE inhibition assays
AChE inhibition was determined using a modified Ellman’s method.28 5,5'-dithiobis
(2-nitrobenzoic) acid (DTNB, Ellman’s reagent), Electrophorus electricus
acetylcholinesterase (EeAChE), and acetylcholine iodide were purchased from Sigma
Chemical. The assay solution, containing 100 μL of phosphate buffer PH 8.0, 20 μL of
DTNB (0.625 mM), 40 μL of EeAChE (0.02 U·mL–1), and 20 μL of test compound in
DMSO, was taken in a 96-well microtiter plate and pre-incubated at 37 oC for 20 min.
Then, 20 μL of acetylcholine iodide (0.625 mM) was added into each well. The
hydrolysis of acetylthiocholine was monitored at 412 nm after 30 min using a
microplate reader. Tacrine was used as positive control. All the reactions were
performed in triplicate. The percentage inhibition was calculated as follows: %
inhibition = (E–S)/E × 100 (E is the activity of the enzyme without test compound and
S is the activity of enzyme with test compound).
Molecular modeling and molecular dynamic simulations

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Molecular docking studies were conducted using Discovery Studio 2018 program
(Acclrys Inc., San Diego, USA). The X-ray crystal structure of the recombinant human
AChE in complex with donepezil was obtained from the RCSB protein data bank with
PDB ID: 4EY7.29 All the water molecules and ligands in the PDB file were removed
and hydrogen atoms along with Gasteiger-Marsili charges were added. The energy was
minimized with CHARMm force field. The dock ligands in the set parameters are as
follows: top hit 10, pose cluster radius parameter value of 0.1. Evaluation of the
molecular docking was performed according to the scores in order to obtain the best
pose of compound 8.30
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Molecular dynamic simulations for 1000 ps were performed on AChE with 8 at 300
K using the CHARMm force field in Discovery Studio 2018 program (Acclrys Inc.,
San Diego, USA). The system was minimized using 2000 steps of steepest descent and
2000 steps of conjugate gradient for energy minimization. The system was slowly
driven from an initial temperature of 50 K to the target temperature of 300 K for 20 ps
and equilibration simulations were run for 20 ps. The molecular dynamic simulations
(production) were performed for 1000 ps with NPT system at a constant temperature of
300 K.31
Funding
This work was supported by the Central Leading Local Science and Technology
Development Special Foundation ([2016]149), Science and Technology Key Project of
Henan Province (182102410080), and Henan Province High-level Personnel Special
Support (ZYQR201810080).
Conflicts of interest
The authors have declared that there is no conflict of interest.
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New Journal of Chemistry
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View Article Online
DOI: 10.1039/D0NJ01537G
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Sixteen compounds, including two new alkaloids and three new lignans, were isolated
and their AChE inhibitory activities were evaluated.