Two new glycoalkaloids from Stephania succifera

Article abstract of DOI:10.1016/j.phytol.2019.10.001

Two new glycoalkaloids, manshurienine C (1) and 8-oxotetrahy-corydalmine-1-O-β-D-glucopyranoside (2), along with eleven known alkaloids (3–13), were isolated from the tubers of Stephania succifera. Their structures were elucidated by spectroscopic methods, including NMR and MS spectra. Compounds 5, 6, 8, and 10 showed in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).

Full text of DOI:10.1016/j.phytol.2019.10.001

PhytochemistryLetters34(2019)99–102
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Phytochemistry Letters
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Two new glycoalkaloids from Stephania succifera
Shi-Ting Wanga, Wen-Qi Qiana, Peng Heb, Mei-Qing Fengb, Yun Kanga, Ya-Qin Wanga,
Jian-Ming Huanga,⁎
a Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, 201203, China
b Department of Microbiological and Biochemical Pharmacy, School of Pharmacy, Fudan University, Shanghai, 201203, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two new glycoalkaloids, manshurienine C (1) and 8-oxotetrahy-corydalmine-1-O-β-D-glucopyranoside (2),
along with eleven known alkaloids (3–13), were isolated from the tubers of Stephania succifera. Their structures
were elucidated by spectroscopic methods, including NMR and MS spectra. Compounds 5, 6, 8, and 10 showed
in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).
Stephania succifera
Alkaloid
Antibacterial activity
1. Introduction
the presence of 22 carbons, including 14 aromatic carbons (δC
105.3 − 148.2), a carbonyl (δC 167.2), a methylenedioxy (δC 103.2),
Stephania succifera H. S. Lo et Y. Tsoong (Menispermaceae) is mainly
distributed in southern China. The tubers are used in Chinese folk
medicine for acesodyne, sedation, and detoxication (The Chinese
Academy of Science Flora of China Edition Board, 1996). Several
classes of alkaloids have been isolated from S. succifera, including
aporphine, proaporphine, and protoberberine alkaloids (Chen et al.,
1989; He et al., 2017; Semwal et al., 2010). Most of these alkaloids
exhibited significant pharmacological effects such as cytotoxic, anti-
bacterial, and analgaesic activities (Yang et al., 2010; Zeng et al., 2017).
The continuous search for novel and bioactive compounds from S.
succifera has led to the isolation of two new glycoalkaloids (1 and 2)
(Fig. 1) and 11 known alkaloids (3–13) (Supplementary Fig. S1).
Compound 3 is found in nature for the first time, and compounds 4–5,
7, 8, 11, and 13 are isolated from this plant for the first time. The
isolates were assayed for their antibacterial activities against methi-
cillin-resistant Staphylococcus aureus (MRSA). Compounds 5, 6, 8, and
10 showed antibacterial activities.
and a glucopyranosyl group (δC 60.9, 70.0, 73.7, 76.2, 76.9, and 104.4).
The 1H NMR data (Table 1) presented a methylenedioxy group at δH
6.49 (1H, d, J =1.0 Hz) and 6.50 (1H, d, J =1.0 Hz); an aromatic
AA'BB' coupling system at δH 7.66 (1H, dd, J = 7.5, 2.0 Hz, H-6), 7.67
(1H, dd, J = 7.5, 2.0 Hz, H-7), 8.59 (1H, dd, J = 7.5, 2.0 Hz, H-5), and
8.62 (1H, dd, J = 7.5, 2.0 Hz, H-8); a typical amide proton at δH 10.10
(s, 1 H); and a singlet aromatic proton at δH 7.68 (1H, s, H-2). Further
examination of the 1H-NMR spectrum of 1 revealed the presence of the
glucopyranosyl moiety, which should be in a β-configuration based on
the coupling constant (8.0 Hz) of the anomeric proton (H-1′). The ab-
solute configuration was assigned as D-form by comparing the TLC and
optical rotation value between the enzymatic hydrolysis product of 1
and the authentic standards (D-glucose and L-glucose). Therefore, it
was concluded that this compound was an aristolactam derivative with
a β-D-glucopyranose. When comparing the 1H- and 13C-NMR data of 1
with those of manshurienine A and manshurienine B (Zhang and Jiang,
2006), the only difference was that the glucose of compound 1 was
attached at C-9 (δC 132.1), which was confirmed by the HMBC corre-
lation (Fig. 2) from H-1′ (δH 4.82) to the aromatic carbon C-9. A
combination of the HMBC and HSQC experiments permitted the as-
signment of all protonated carbons. Thus, compound 1 was elucidated
as aristolactam Ⅱ-9-O-β-D-glucopyranoside and given the trivial name
manshurienine C.
2. Results and discussion
Compound 1 was obtained as a yellow amorphous powder. Its
molecular formula was identified as C22H19NO9 by HRESIMS with an
[M+H]+ ion at m/z 442.1114 (calcd 442.1133). The IR spectrum ex-
hibited absorption bands for lactam (1668 cm−1) and aromatic groups
(1557 and 1417 cm−1). The UV absorptions at 381, 323, 289, 277, 267,
and 231 nm resembled those of aristolactam derivatives (Yang et al.,
2010; Zhang and Jiang, 2006). The 13C NMR data of 1 (Table 1) showed
Compound 2 was obtained as a yellow amorphous solid. The mo-
lecular formula was assigned as
C26H31NO11 by HRESIMS (m/z
534.1950 [M+H]+, calcd 534.1970). The IR spectrum exhibited ab-
sorption bands for lactam (1630 cm−1) and aromatic groups (1618 and
⁎ Corresponding author.
E-mail address: jmhuang@shmu.edu.cn (J.-M. Huang).
https://doi.org/10.1016/j.phytol.2019.10.001
Received 20 July 2019; Received in revised form 14 September 2019; Accepted 3 October 2019
1874-3900/©2019PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

S.-T. Wang, et al.
PhytochemistryLetters34(2019)99–102
Fig. 1. Structures of compounds 1 and 2 from Stephania succifera.
Table 1
1H and 13C NMR spectroscopic data of 1 and 2 (δ in ppm).
No.
1
No.
2
a
a
b
b
δC
δH (J in Hz)
δC
δH (J in Hz)
1
118.6
105.3
148.2
146.8
108.8
125.1
126.1
126.4
127.5
123.7
130.1
132.1
122.1
126.0
167.2
103.2
1
148.6
141.8
153.9
107.8
133.2
124.0
25.1
2
7.68, s
2
3
3
4
4
6.79, s
4a
4a
4b
5
4b
5
8.59, dd (7.5, 2.0)
7.66, dd (7.5, 2.0)
7.67, dd (7.5, 2.0)
8.62, dd (7.5, 2.0)
α 2.57, ddd (16.1, 12.5, 4.5)
β 3.36, overlapped
6
7
6
39.4
α 2.83, td (12.5, 2.9)
8
β 4.90, ddd (12.5, 4.5, 2.9)
8a
8
164.9
123.0
149.5
151.5
120.9
124.0
9
8a
9
10
10a
10
11
12
11
7.00, d (8.1)
6.95, d (8.1)
-OCH2O-
6.50, d (1.0)
6.49, d (1.0)
10.10, s
N-H
1′
12a
13
131.8
39.7
104.4
73.7
76.2
70.0
76.9
60.9
4.82, d (8.0)
3.46, m
α 3.16, dd (15.5, 3.0)
β 2.74, t (15.5)
4.79, dd (13.3, 3.0)
4.98, d (7.5)
3.47, overlapped
3.44, overlapped
3.39, m
2′
3′
3.36, m
13a
1′
56.8
105.2
75.7
78.0
71.6
78.2
62.6
4′
3.23, dd (8.0, 5.1)
3.29, m
5′
2′
6′
3.82, dt (11.3, 4.2)
3.51, dt (11.3, 6.0)
5.79, d (5.5)
5.22, d (4.9)
5.13, d (5.4)
5.16, dd (6.0, 4.2)
3′
4′
Glc-OH
Glc-OH
Glc-OH
6′-OH
5′
3.19, m
6′
3.77, dd (11.9, 5.3)
3.65, dd (11.9, 2.3)
3.85, s
2-OCH3
3-OCH3
9-OCH3
61.7
56.7
62.0
3.86, s
3.84, s
a
b
Measured at 600 MHz (1H) and 150 MHz (13C) in DMSO-d6.
Measured at 600 MHz (1H) and 150 MHz (13C) in methanol-d4.
1497 cm−1). The 13C NMR data of 2 (Table 1) showed the presence of
26 carbons, including 12 aromatic carbons (δC 107.8 − 153.9), a car-
bonyl (δC 164.9), three methoxy groups (δC 56.7, 61.7, and 62.0), three
methylenes (δC 39.7, 39.4, and 25.1), a methine (δC 56.8), and a glu-
copyranosyl group (δC 62.6, 71.6, 75.7, 78.0, 78.2, and 105.2). The 1H
NMR spectrum revealed the presence of two ortho-coupled aromatic
protons at δH 6.95 (1H, d, J =8.1 Hz, H-12) and 7.00 (1H, d, J =8.1 Hz,
H-11); one singlet aromatic proton at δH 6.79 (1H, s, H-4); three
methoxy groups at δH 3.86, 3.85, and 3.84 (each 3H, s). The glucosyl
unit with its anomeric proton appearing at δH 4.98 (1H, d, J =7.5 Hz,
comparing the TLC and optical rotation data with those of the authentic
standards (D-glucose and L-glucose). The absolute configuration at C-
13a was confirmed as S (α-orientation) by comparing the negative op-
tical rotation ([α]25D –71.1°) of the aglycone moiety with the literature
value for this aglycone (Lee et al., 2009). A typical 1H NMR signal at δH
4.79 (1H, dd, J = 13.3, 3.0 Hz, H-13a) also supported the S config-
uration at C-13a (Lee et al., 2009). Thus, compound 2 was deduced as
(-)-8-oxo-10-hydroxy-2,3,9-trimethoxyberberine-1-O-β-D-glucopyrano-
side, which could also be named as (-)-8-oxotetrahy-corydalmine-1-O-β-
D-glucopyranoside.
H-1′) was speculated as
a
β-configuration. Therefore, an ox-
The known compounds (3–13) (Supplementary Fig. S1)
were identified as secocrebanine (3) (Wang et al., 2016),
norcrebanine (4) (Rayanil et al., 2016), 8-hydroxy-9-methoxy-1,2-me-
thylenedioxyaporphine (5) (Thuy et al., 2012), crebanine (6)
(Makarasen et al., 2011), N-formyl-asimilobine-2-O-β-D-glucopyrano-
side (7) (Choudhary et al., 2010), asimilobine-2-O-β-D-glucopyranoside
(8) (Likhitwitayawuid et al., 1993), N-methylasimilobine-2-O-β-D-glu-
copyranoside (9) (Kashiwaba et al., 2000), asimilobine (10) (Costa
oprotoberberine with a glucosyl moiety was proposed. A comparison of
the 1H- and 13C-NMR data with those of (-)-8-oxo-10-hydroxy-2,3,9-
trimethoxyberberine revealed that compound 2 had an additional glu-
cosyl moiety attached at C-1. The location of the β-glucosyl moiety was
supported by the HMBC correlation (Fig. 3) of H-1′ (δH 4.98) with C-1
(δC 148.6). Enzymatic hydrolysis of compound 2 afforded its sugar and
aglycone moieties. The sugar moiety was assigned as D-glucose by
100

S.-T. Wang, et al.
PhytochemistryLetters34(2019)99–102
3. Material and methods
3.1. General experimental procedures
Optical rotations were recorded using a Rudolph Autopol IV–T au-
tometic polarimeter (Rudolph Research Analytical, USA). UV spectra
were measured on a U–2900 spectrophotometer (Hitachi, Japan). IR
spectra were obtained on a Nicolet i5 spectrometer (Thermo Fisher,
USA) with KBr pellets. HRESIMS spectra were recorded on a TripleTOF
5600+ mass spectrometer (Applied Biosystems Sciex, USA). NMR
spectra were recorded on a Bruker Ascend™ 600 MHz spectrometer with
TMS as internal standard. Column chromatography (CC) was performed
using silica gel (200–300 and 300–400 mesh, Qingdao Haiyang
Chemical Co., Ltd., China), HZ818 macroporous resin (HuaZhen
Technology, China), C18 (40–60 μm, Agela Technologies, China), and
Sephadex LH–20 (Amersham Biosciences, Sweden). Fractions were
monitored by TLC using precoated GF254 silica gel plates (Qingdao
Haiyang Chemical Co., Ltd., China) and RP–18 F254S silica gel plates
(Merck KGaA, Germany). Semipreparative HPLC was carried out on an
Fig. 2. Selected 1H–1H COSY
pound 1.
and HMBC (H→C) correlations of com-
LC–15C (Shimadzu, Japan) with
a
Gemini C18 column
(10 mm × 150 mm, 5 μm, Phenomenex, Germany).
3.2. Plant material
The tubers of S. succifera were collected at Wuzhishan, Hainan
Province, China, in 2014, and authenticated by Dr. Yun Kang, Fudan
University. A voucher specimen (2014057 G) has been deposited in the
Department of Pharmacognosy, Fudan University, China.
Fig. 3. Selected 1H–1H COSY
relations of compound 2.
, HMBC (H→C), and ROESY
cor-
3.3. Extraction and isolation
The dried and powdered tubers (5.5 kg) of S. succifera were ex-
tracted with 95% EtOH (50 L) at room temperature. After concentration
under reduced pressure, the crude extract (798 g) were dissolved in
water (2 L) to get a homogeneous dispersion and then partitioned
successively with petroleum ether (PE) and dichloromethane (CH2Cl2)
to afford a PE extract (85 g) and a CH2Cl2 extract (196 g). The re-
maining aqueous solution was applied to an HZ818 macroporous resin
column eluted with EtOH–H2O (95:5, v/v). The EtOH–H2O (95:5) el-
uate (99 g) was chromatographed on a silica gel column (200–300
mesh) with a gradient CH2Cl2–MeOH (15:1, 10:1, 5:1, 2:1, 0:1, v/v) as
eluent to give nine fractions (Fr. A–I). Fr.G was loaded on another
HZ818 macroporous resin column eluted with a gradient MeOH–H2O
(5:5, 6:4, 7:3, 1:0, v/v) to yield subfractions G1–G5. Fr.G3 was sub-
jected to C18 CC eluted with MeOH–H2O (3:7, 4:6, 5:5, 6:4, 1:0, v/v) to
give Fr.G3.1 to Fr.G3.5. Fr.G3.2 was further separated on a Sephadex
LH-20 CC (MeOH) followed by semipreparative HPLC (MeOH–H2O,
45:55, v/v, flow rate 3 mL/min) to yield 2 (5.4 mg) and 7 (10.0 mg).
Fr.G3.3 was purified by Sephadex LH-20 CC (MeOH) and semi-
preparative HPLC (MeOH–H2O, 35:65, v/v, flow rate 3 mL/min) to
yield 13 (2.7 mg). Fr.G4 was subjected to silica gel CC (300–400 mesh)
eluted with a gradient CH2Cl2–MeOH (15:1, 10:1, 8:1. 5:1, v/v) and
further purified by semipreparative HPLC (MeOH–0.05% Et2NH aqu-
eous solution, 40:60, v/v, flow rate 3 mL/min) to give 10 (12.9 mg).
Fr.G5 was purified by Sephadex LH-20 CC (MeOH) and semipreparative
HPLC (MeOH–H2O, 55:45, v/v, flow rate 3 mL/min) to yield 1 (2.4 mg).
Fr.H was subjected to semipreparative HPLC (MeOH–0.02% Et2NH
aqueous solution, 30:70, v/v, flow rate 3 mL/min) to yield 8 (6.8 mg).
Compound 9 (5.1 mg) was obtained from Fr.I using the same procedure
as compound 8. The CH2Cl2 extract was fractionated on a silica gel CC
(200–300 mesh) using a gradient CH2Cl2–MeOH (1:0, 50:1, 20:1, 10:1,
1:1, 0:1, v/v) as eluent to give eight fractions (Fr.J–Q). Fr.L was loaded
on another silica gel CC (300–400 mesh) eluted with a gradient
CH2Cl2–MeOH (30:1, 20:1, 10:1, v/v) to give subfractions L1–L6. Fr.L3
was purified by Sephadex LH-20 CC (MeOH) followed by semi-
preparative HPLC (MeOH–0.02% Et2NH aqueous solution, 88:12, v/v,
et al., 2015), liriodenine (11) (Fujita et al., 2010), 4-hydroxycrebanine
(12) (Kunitomo et al., 1985), and trans-feruloyl-(3-O-methyl)-dopa-
mine-4-O-β-D-glucopyranoside (13) (Yim et al., 2012) by comparison of
experimental and reported spectroscopic data. Compound 3 is found in
nature for the first time, and compounds 4, 5, 7, 8, 11, and 13 are
isolated from S. succifera for the first time.
All isolated compounds were tested for their in vitro antibacterial
activity against MRSA by the broth microdilution method. Vancomycin
was used as a positive control. As shown in Table 2, compounds 5, 6, 8,
and 10 showed antimicrobial activity against MRSA. The lowest
minimum inhibitory concentration (MIC) was observed for compound 6
(128 μg/mL) followed by compounds 5, 8, and 10 (256 μg/mL).
Table 2
Anti-MRSA activities of the compounds isolated from S. succifera.
Compounds
MIC(μg/mL)
manshurienine C (1)
> 256
> 256
> 256
> 256
256
8-oxotetrahy-corydalmine-1-O-β-D-glucopyranoside (2)
secocrebanine (3)
norcrebanine (4)
8-hydroxy-9-methoxy-1,2-methylenedioxyaporphine (5)
crebanine (6)
128
N-formyl-asimilobine-2-O-β-D-glucopyranoside (7)
asimilobine-2-O-β-D-glucopyranoside (8)
N-methylasimilobine-2-O-β-D-glucopyranoside (9)
asimilobine (10)
> 256
256
> 256
256
liriodenine (11)
> 256
> 256
> 256
0.5
4-hydroxycrebanine (12)
trans-feruloyl-(3-O-methyl)-dopamine- 4-O-β-D-glucopyranoside (13)
vancomycina
a
Vancomycin was used as a positive control.
101

S.-T. Wang, et al.
PhytochemistryLetters34(2019)99–102
flow rate 3 mL/min) to yield 6 (18.7 mg). Fr.L4 was subjected to C18 CC
eluted with MeOH–H2O (1:4, 1:1, 7:3, 9:1, 1:0, v/v) to give Fr.L4.1 to
Fr.L4.5. Fr.L4.3 was separated by semipreparative HPLC (flow rate
3 mL/min) eluted with MeOH–0.02% Et2NH aqueous solution (90:10,
v/v), MeOH–0.02% Et2NH aqueous solution (85:15, v/v), and
CH3CN–0.02% Et2NH aqueous solution (6:4, v/v) to afford 4 (8 mg), 5
(4.9 mg), and 12 (33 mg), respectively. Fr.L4.4 was purified by
Sephadex LH-20 CC (MeOH) followed by semipreparative HPLC
(MeOH–0.02% Et2NH aqueous solution, 60:40 and 95:5 for the pur-
ification of 3 and 11, respectively, v/v, flow rate 3 mL/min) to yield 3
(2.1 mg) and 11 (1.8 mg).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
There are no conflicts of interest to declare.
Acknowledgment
The present study was financially supported by the grant from the
National Natural Science Foundation of China (31100238).
3.4. Spectroscopic data
Appendix A. Supplementary data
3.4.1. manshurienine C (1)
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.phytol.2019.10.001.
Yellow amorphous powder; [α]25D –155.0° (c 0.10, MeOH); UV
(MeOH) λmax (log ε): 381 (3.59), 323 (3.75), 289 (4.17), 277 (4.22),
267 (4.21), 231 (4.26) nm; IR (KBr) νmax 3734, 3703, 3627, 3595, 1668,
References
1557, 1417, 1288, 1071 cm−1
;
1H and 13C NMR data see Table 1;
HRESIMS m/z 442.1114 [M+H]+ (calcd for C22H19NO9, 442.1133).
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3.4.2. 8-oxotetrahy-corydalmine-1-O-β-D-glucopyranoside (2)
Yellow amorphous solid; [α]25D –179.0° (c 0.10, MeOH); UV
(MeOH) λmax (log ε): 278 (4.07) nm; IR (KBr) νmax 3643, 2938, 2334,
1630, 1618, 1497, 1483, 1331, 1026, 666 cm−1
;
1H and 13C NMR data
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separately dissolved in H2O (500 μL) and mixed with β-cellulase (2 mg).
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EtOAc. The EtOAc layer of compound 2 was concentrated to dryness
under reduced pressure and redissolved with methanol (1 mL) for op-
tical rotation measurement of the aglycone moiety. The aqueous phases
were used for optical rotation measurement and TLC analysis of the
sugar moieties. The liberated sugars of 1 and 2 were confirmed as
glucose because of the same Rf value (0.35) in TLC (EtOAc: MeOH:
H2O: HOAc = 13:3:3:4) as the authentic standards (D-glucose and L-
glucose), and were identified as D-form based on their nearly identical
optical rotation values to the D-glucose standard ([α]25D +52.6°).
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3.6. Antibacterial assay
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The isolated compounds were tested for in vitro antibacterial ac-
tivities against MRSA strains ATCC 43,300 (American Type Culture
Collection (ATCC), USA) by the broth microdilution method (Luo et al.,
2014). The compounds and vancomycin (positive control) were dis-
solved in methanol, respectively, diluted in cation-adjusted Mueller
Hinton broth (CAMHB) to achieve nine twofold serially diluted con-
centrations, and then added to the wells of microtiter plates. For in-
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