Phytochemistry 181 (2021) 112567

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Diterpenoid alkaloids from Aconitum barbatum var. puberulum Ledeb

Nurfida Ablajan a, c, Bo Zhao a, Jiang-Yu Zhao a, Bian-Lin Wang a, Sh.Sh. Sagdullaev b, H.
A. Aisa a, *
a
State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization and Key Laboratory of Plants Resources and Chemistry of Arid Zone, Xinjiang
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing South Road 40-1, Urumqi, 830011, Xinjiang, PR China
b
S.Yu.Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, Uzbekistan
c
University of Chinese Academy of Sciences, Beijing, 100039, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Seven previously undescribed diterpenoid alkaloids, including five C20-diterpenoid alkaloids, barpuberudine,
Aconitum barbatum var. puberulum Ledeb. barpubesines A-D, and two C18-diterpenoid alkaloids, barpubenines A-B, along with 11 known diterpenoid al­
Ranunculaceae kaloids were isolated from the whole plant of Aconitum barbatum var. puberulum Ledeb. (Ranunculaceae). Bar­
Diterpenoid alkaloids
puberudine is an unprecedented carbon skeleton of C20-diterpenoid alkaloid, while barpubenines A-B are the first
Barpuberudine
Barpubesines
example of rearranged types in C18-diterpenoid alkaloids. Their structures were elucidated based on a
Barpubenines comprehensive spectroscopic data analysis. The probable pathway of biogenesis of barpuberudine and barpu­
Biological activities benines A-B were discussed. Additionally, the antiarrhythmic, cytotoxic and antimicrobial activities of isolates
were also evaluated.

1. Introduction
Aconitum barbatum var. puberulum Ledeb., a member of genus Aco­
nitum belonging to Ranunculaceae family, is a perennial herb mainly
distributed in the northern part of China, Siberia region of Mongolia and
Russia (Wang et al., 1979). Its roots have been widely used as a Chinese
folk medicine to treatment of stomach-ache, tracheitis, rheumatic
arthritis, lymphatic tuberculosis and some other inflammations for a
long period of time (State Administration of Traditional Chinese Medi­
cine, 1999). Previous phytochemical investigations on this plant have
reported the occurrence of diterpenoid alkaloids, benzamide de­
rivatives, flavonoids, steroids and monoterpene glycosides, respectively,
in which diterpenoid alkaloids are considered as the main characteristic
constituents (Mu et al., 2012; Sun et al., 2009; Yu et al., 1983). Diter­
penoid alkaloids, structurally classified into three categories as C18-, C19-
and C20-subtypes, are a group of structurally complex natural products
and display many interesting biological activities, such as
anti-inflammatory, analgesic, anti-arrhythmia, anti-tumor, as well as
insecticidal and antifeedant activities (Dzhakhangirov et al., 1997; Liu
et al., 2011; Reina and González-Coloma 2007; Tang et al., 2012; Ulu­
belen et al., 2001; Wang et al., 2010; Wangchuk et al., 2015).
There are also a large number of A. barbatum var. puberulum plant
resources in Central Asia (Commissione Redactorum Florae
Xinjiangensis, 1994), but their chemical constituents have not been re­
ported. A phytochemical investigation of the whole plants of
A. barbatum var. puberulum from Central Asia was thus performed as part
of our continuous work on the discovery of compounds with unique
structures and strong bioactivities from Central Asia plant resources.
This led to isolation of seven previously undescribed diterpenoid alka­
loids, barpuberudine (1), barpubesines A-D (2-5), barpubenines A-B
(6–7), and 11 known diterpenoid alkaloids (8–18) (Fig. 1). Compounds
1–15, and 18 were isolated from A. barbatum var. puberulum for the first
time, among which barpuberudine is an unprecedented carbon skeleton
of C20-diterpenoid alkaloid, while barpubenines A-B (6–7) are the first
example of rearranged types in C18-diterpenoid alkaloids, which pro­
vided important scientific support for phytochemical taxonomy. Herein,
the isolation, structure elucidation, biogenesis, cytotoxicity, antimicro­
bial activity and antiarrhythmic activity of these compounds are
discussed.
2. Results and discussion
2.1. Phytochemical investigation
Air-dried whole plants of A. barbatum var. puberulum (5.8 kg) were
crushed in a blender, soaked with 5% Na2CO3 for 3 h, and extracted with

* Corresponding author.
E-mail address: haji@ms.xjb.ac.cn (H.A. Aisa).

https://doi.org/10.1016/j.phytochem.2020.112567
Received 19 April 2020; Received in revised form 28 September 2020; Accepted 21 October 2020
Available online 13 November 2020
0031-9422/© 2020 Elsevier Ltd. All rights reserved.
N. Ablajan et al.
CHCl3–MeOH (4:1) at room temperature. The extract was concentrated
under reduced pressure to leave a residue, which was suspended in 3%
aq. H2SO4 and then successively extracted with EtOAc. The aqueous
layer was alkalized with Na2CO3 to pH 10 and then extracted with
EtOAc to give the crude alkaloids (30 g). The crude alkaloids were then
subjected to column chromatographic purifcation procedures using a
silica gel, octadecyl silica (ODS), Sephadex LH-20, pHPLC, and pTLC,
respectively, to afford seven previously undescribed diterpenoid alka­
loids (1–7), and 11 known diterpenoid alkaloids, 15-ketotetrahydroati­
sine (8) (Pelletier et al., 1980), leucostomine A (9) (Xu et al., 2016),
N-acetylsepaconitine (10) (Tel’nov et al., 1988), N-deacetyllappaconi­
tine (11) (Ross et al., 2010), sepaconitine (12) (Usmanova et al., 1987),
acosepticine (13) (Sayed et al., 1992), lappaconidine (14) (Ross et al.,
1992), trabzonine (15) (Mericli et al., 2000), lappaconitine (16) (Yu
et al., 1983) and puberuline C (17) (Sun et al., 2009), leucostine (18)
(Wei et al., 1996). The structures of the known compounds were eluci­
dated by ESI-MS, 1H and 13C NMR techniques, and their spectroscopic
data agreed well with the literature values. The 13C NMR data of
15-ketotetrahydroatisine (8) was reported for the first time.
Barpuberudine (1) was obtained as a white amorphous powder, and
gave a positive reaction with Dragendorff’s reagent. Its molecular for­
mula was deduced as C23H37NO3 based on (+)-HRESIMS (m/z 376.2834
[M+H]+, calcd for 376.2846) analysis. Its 1H and 13C NMR data (Ta­
bles 1 and 2) indicated the presence of two tertiary methyl groups [(δH
0.81 3H, s; 1.68, 3H, br. s); δC 26.8, 19.9], a trisubstituted double bond
(δH 5.38, br. s; δC 123.7, 136.5), a methoxy group (δH 3.35, 3H, s; δC
55.8), and a typical N-ethoxyl group (δH 2.38, 2H, t, J = 6.0 Hz, 3.62, 2H,
t, J = 6.0 Hz; δC 61.9, 60.2). Its 13C NMR and DEPT spectra showed
compound 1 contains twenty-three carbon signals, including three
methyls groups (δC 19.9, 26.8 and 55.8), eleven methylenes (δC 18.6,
23.0, 23.7, 31.4, 36.1, 39.6, 42.9, 60.0, 60.2, 61.0 and 61.9), five
Fig. 1. Structures of compounds 1–19.
2
Phytochemistry 181 (2021) 112567
methines (δC 47.1, 51.5, 88.5, 109.2 and 123.7), and four quaternary
carbons (δC 34.8, 37.0, 44.3 and 136.5). These characteristic spectro­
scopic data suggested that 1 was a C20-diterpenoid alkaloid. Comparison
of the NMR data of 1 with those of atisine-type alkaloids spiramines H
and I (Hao et al., 1994) showed that the rings A and B have the same
structure. However, the structure of rings C and D were quite different.
Further inspection of its 2D NMR spectra indicated that 1 was an
abnormal C20-diterpenoid alkaloid.
Comparison of the NMR data of 1 with those of known compound
(19) (Wang et al., 1992) showed that the ring C and ring D have the same
skeleton. The methoxy group was located at C-11 based on the HMBC
correlations (Fig. 2) between 11-OCH3 (δH 3.35, 3H, s) and C-11 (δC
109.2). The chemical shifts of H-11 and C-11 (δH 4.97, d, J = 6.0 Hz; δC
109.2) were shifted downfield, inferring that an another oxygenated
group was linked to C-11. Analysis of the chemical shifts of H-15 and
C-15 (δH 3.79, s; δC 88.5), combined with the HMBC correlations from
H-11 to C-15, from H-15 to C-8 (δC 44.3) and C-11, suggested that C-15
was connected with C-11 through oxygen atoms. The unique carbon
skeleton of 1 was assigned on the basis of the fragments established
above.
The relative configuration of 1 was deduced according to analysis of
NOESY spectroscopic data. As a reference point, β stereochemistry of H-
5 was used, which is characteristic for diterpenoid alkaloids. The NOESY
correlations (Fig. 3) of H-5 with H-7β, H-11 and H3-18 indicated that H-
11 and C-18 were β-oriented. The relative configuration of C-8 was
inferred by the correlations of H2-13 with H-20α and H2-21. However, it
was impossible to infer the relative configuration of H-9 and H-15 by
NOESY spectrum because of overlapping or missing NOESY signals.
A plausible biosynthetic pathway for the formation of 1 was pro­
posed in Fig. 4. Compound 1 is obtained by initial breakage of C (11)–C
(12) bond accompanied epimerization at C-9 from compound 4,
N. Ablajan et al. Phytochemistry 181 (2021) 112567

Table 1
1
H NMR spectroscopic data for compounds 1–7 (δ in ppm, J in Hz).
position 1a 2a 3d 4b 5c 6b 7c

1α 1.57, dd (13.6, 6.4) 1.58, m 2.19, m 2.26, dd (13.8, 5.4)

1β 1.38, m 2.05, m 1.43, m 1.38, m 3.74, dd (10.8, 4.2) 3.37, overlapped 3.07, m
2α 2.90, m 1.14, m 1.64, m 2.44, br.s 2.20, m 1.41, m 1.99, m
2β 1.44, m 1.34, m 1.47, m 1.63, m 1.94, m 2.02, m 1.78, m
3α 1.64, m 1.48, dd (14.0, 4.4) 1.69, m 1.71, dd (13.2, 6.0) 1.59, m 1.93, m 1.74, m
3β 1.42, m 1.72, m 1.39, m 1.51, m 1.27, m 1.80, m 1.54, m
4 – – – - - 2.76, br.s 2.11, m
5 1.27, dd (13.6, 6.4) 1.35, m 1.18, m 1.09, d (11.4) 1.27, m 2.63, t (2.4) 2.36, t (3.0)
6α 1.65, m 1.72, m 1.78, m 1.59, m 1.82, m - -
6β 1.52, m 1.28, m 1.58, m 1.43, dd (14.4, 4.8)
7α 1.43, m 1.84, dd (13.6,4.0) 1.73, m 1.19, dt (13.2, 3.0) 2.23, d (5.4) 4.02, s 4.31, s
7β 1.70, m 1.13, m 1.44, m 1.79, td (13.2, 5.4)
8 – – – – – – –
9 1.64, m 1.65, m 1.73, m 1.47, m 0.88, m 2.13, m 2.01, m
10 – – – – – 2.23, m 1.91, m
11 α 2.06, m 1.76, m 3.70, t (4.2) 2.11, t (12.0) – –
11 β 4.97, d (6.0) 1.34, m 2.06, m 1.65, m
12 α 5.38, br.s 2.05, m 2.08, br.s 2.30, br.s 2.39, t (4,8) 2.28, m 2.22, d (8.4)
12 β 2.16, m 1.97, m
13 α 1.98, m 1.94, m 1.54, m 1.63, m 1.76, m 2.25, t (6.0)
13 β 1.98, m 1.56, m 1.62, m 1.42, m 1.56, m 2.34, t (7.8) 3.42, t (3.6)
14 α 2.16, dt (14.0, 3.6) 1.59, m 1.72, m 1.95, t (12.6) 1.86, m
14 β 1.42, m 1.09, m 1.27, m 0.84, m 1.73, m 3.54, t (5.4)
15 α 3.79, s 3.13, s – 3.53, s 4.25, s 2.44, dd (12.0, 7.2) 2.06, dd (12.0, 7.8)
15 β 2.13, m 2.17, m
16 – – 2.20, m – – 3.75, t (8.0) 3.61, t (8.4)
17 a 1.68, br.s 1.28, s 1.12, d (7.2) 5.28, br.s 4.99, s 4.28, d (2.0) 3.64, d (1.2)
17 b 5.15, br.s 5.01, s
18 0.81, s 1.06, s 1.04, s 0.87, s 0.79, s
19 a 2.48, d (10.8) 3.81, d (17.2) 3.67, d (16.4) 2.79, overlapped 2.92, d (12.6) 7.30, d (4.8) 2.89, m
19 b 2.27, d (10.8) 3.69, d (17.2) 3.54, d (16.4) 2.57, br.s 2.33, d (12.6) 2.78, dd (11.4, 7.2)
20 α 2.40 m 8.71, s 9.71, s 3.12, br.s 4.10, s
20 β 2.66 d (10.8) 2.87, m
21 a 2.38, t (6.0) 4.17, m 4.59, m 2.78, br.s 2.80, m 3.09, m
21 b 2.38, t (6.0) 4.12, m 2.89, m
22 3.62, t (6.0) 4.04, m 4.03, m 3.76, t (5.4) 1.26, br.s 1.13, t (7.2)
3.62, t (6.0) 3.95, m 3.98, m
1-OCH3 3.32, s 3.28, s
11-OCH3 3.35, s
14-OCH3 3.33, s 3.31, s
16-OCH3 3.38, s 3.34, s
a
Data were recorded in CD3OD at 400 MHz.
b
Data were recorded in CD3OD at 600 MHz.
c
Data were recorded in CDCl3 at 600 MHz.
d
Data were recorded in CDCl3 at 400 MHz.

followed by acetalization and O-methylation to give 1 (Wang et al.,
1997, 2002). It can be inferred from the plausible biosynthetic pathway
that the H-9 and H-15 were all α-oriented. Considering the dilute sulfuric
acid was used in the extraction of the total alkaloids, the LC-MS analyses
of CH3Cl/MeOH extract of Aconitum barbatum var. puberulum was car­
ried out in order to verify whether compound 1 was a natural product or
a synthetic product during extraction and isolation. The result (Fig. S2)
showed that compound 1 was a natural product from Aconitum barbatum
var. puberulum, which is an unprecedented carbon skeleton of
C20-diterpenoid alkaloid.
Barpubesine A (2) was isolated as a white needle crystal, with the
molecular formula C22H36NO+ 3 determined based on the (+)-HRESIMS
(m/z 362.2676 [M]+, calcd for 362.2690) analysis. The 1H NMR data
(Table 1) indicated the presence of two tertiary methyl groups (δH 1.06,
1.28, each 3H, s). Its 13C NMR, HSQC and (+)-HRESIMS spectra showed
22 carbon signals including two methyls (δC 23.9 and 24.9), eleven
methylenes (δC 20.1, 20.4, 21.6, 25.4, 26.6, 32.0, 35.8, 41.8, 58.5, 60.4
and 64.9), five methines (δC 39.5, 39.5, 46.1, 82.9 and 184.1) and four
quaternary carbons (δC 34.4, 38.4, 47.2 and 76.5). The aforementioned
spectral features along with the biogenetic considerations suggested that
compound 2 could be a C20-diterpenoid alkaloid (Mody et al., 1978;
Wang et al., 2010; Yunusov et al., 1993). The resonances at C-4 (δC
34.4), C-10 (δC 47.2), C-20 (δH 8.71, 1H, s; δC 184.1), C-21 (δH 4.12, 4.17,
each 1H, m; δC 64.9) and C-22 (δH 3.95, 4.04, each 1H, m; δC 58.5)
strongly indicated the existence of a quaternary-N center in the mole­
cule. Comparison of the NMR data of 2 with those of the known
atisine-type alkaloid leucostomine A (9) (Xu et al., 2016) indicated that
there was a methyl group and a hydroxyl group at C-16 in 2 instead of a
typical exocyclic double bond in 9, which was also confirmed by the
HMBC correlations from H-17 (δH 1.28 3H, s) to C-12 (δC 39.5), C-15 (δC
82.9) and C-16 (δC 76.5). In addition, 2 lacked a hydroxyl group at C-11
when compared with 9. The complete planar structure of 2 was further
verified by analysis of the HMBC and 1H–1H COSY spectra (Fig. 2). The
NOESY correlations (Fig. 3) between H-1β/H-9/H3-17,
H3-18/H-5/H-9/H-11β, H-11α/H-20/H-14α, and H-14β/H-15/H-7α
indicated that H-5, and H-9 were in β-oriented, and H-15 and 16-OH was
in α-orientation. Because formic acid (FA) was used in the pHPLC pu­
rification of 2 and the alkaloid iminium must be positively charged,
compound 1 was considered being obtained as a formate (Zhang et al.,
2013; Meng et al., 2016). Thus, compound 2 was elucidated as shown in
Fig. 1, and named barpuberudine B.
The (+)-HRESIMS spectrum of barpubesine B (3) displayed a pro­
tonated molecule at m/z 344.2569 [M]+ (calcd for 344.2584), which
corresponded to a molecular formula of C22H34NO+ 2 . As compound 2,
compound 3 was also obtained as a formate. The NMR data of com­
pounds 3 and 2 were demonstrated similarities. The difference included

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N. Ablajan et al. Phytochemistry 181 (2021) 112567

Table 2
13
C NMR spectroscopic data for compounds 1–9 (δ in ppm).
position 1a 2a 3d 4b 5c 6b 7c 8d 9d

1 39.6, CH2 35.8, CH2 35.3, CH2 41.0, CH2 69.4, CH 81.5, CH 82.7, CH 40.3, CH2 37.3, CH2
2 23.7, CH2 20.4, CH2 18.9, CH2 23.4, CH2 30.2, CH2 25.8, CH2 24.8, CH2 21.2, CH2 21.4, CH2
3 42.9, CH2 41.8, CH2 41.2, CH2 41.6, CH2 38.1, CH2 26.4, CH2 30.4, CH2 41.5, CH2 42.8, CH2
4 34.8, C 34.4, C 33.6, C 34.6, C 34.5, C 39.0, CH 33.3, CH 33.5, C 35.6, C
5 47.1, CH 46.1, CH 45.0, CH 50.4, CH 51.1, CH 55.7, CH 57.1, CH 49.7, CH 46.7, CH
6 18.6, CH2 20.1, CH2 19.6, CH2 18.2, CH2 23.4, CH2 211.0, C 214.5, C 17.2, CH2 21.6, CH2
7 36.1, CH2 32.0, CH2 28.2, CH2 32.0, CH2 40.3, CH 79.9, CH 79.5, CH 29.5, CH2 32.1, CH2
8 44.3, C 38.4, C 44.6, C 39.5, C 51.7, C 48.3, C 54.1, C 43.4, C 40.3, C
9 51.5, CH 39.5, CH 44.5, CH 52.1, CH 48.9, CH 47.0, CH 45.7, CH 39.6, CH 52.5, CH
10 37.0, C 47.2, C 46.7, C 38.9, C 43.4, C 47.3, CH 46.7, CH 49.7, C 48.4, C
11 109.2, CH 21.6, CH2 21.8, CH2 71.0, CH 27.2, CH2 50.4, C 49.1, C 23.5, CH2 71.8, CH
12 123.7, CH 39.5, CH 32.7, CH 46.3, CH 37.2, CH 31.6, CH2 31.2, CH2 33.9, CH 45.9, CH
13 23.0, CH2 25.4, CH2 25.6, CH2 24.8, CH2 27.1, CH2 40.8, CH 39.8, CH 28.2, CH2 25.2, CH2
14 31.4, CH2 26.6, CH2 23.6, CH2 27.4, CH2 20.1, CH2 83.5, CH 83.1, CH 25.3, CH2 26.1, CH2
15 88.5, CH 82.9, CH 218.3, C 77.7, CH 72.2, CH 28.7, CH2 28.7, CH2 219.3, C 77.0, CH
16 136.5, C 76.5, C 46.5, CH 151.3, C 156.5, C 84.4, CH 83.8, CH 45.7, CH 151.2, C
17 19.9, CH3 23.9, CH3 14.7, CH3 115.5, CH2 111.9, CH2 74.0, CH 65.0, CH 15.1, CH3 117.4, CH2
18 26.8, CH3 24.9, CH3 25.1, CH3 26.6, CH3 26.1, CH3 26.7, CH3 26.5, CH3
19 61.0, CH2 60.4, CH2 59.4, CH2 60.3, CH2 56.8, CH2 143.5, CH 51.5, CH2 60.4, CH2 61.8, CH2
20 60.0, CH2 184.1, CH 183.3, CH 55.9, CH2 66.1, CH 53.9, CH2 185.4, CH
21 61.9, CH2 64.9, CH2 63.2, CH2 61.8, CH2 53.0, CH2 49.8, CH2 61.0, CH2 66.5, CH2
22 60.2, CH2 58.5, CH2 58.2, CH2 58.6, CH2 11.5, CH3 13.9, CH2 58.2, CH2 59.9, CH2
1-OCH3 57.2, CH3 57.3, CH3
11-OCH3 55.8, CH3
14-OCH3 57.4, CH3 57.5, CH3
16-OCH3 56.9, CH3 56.9, CH3
a
Data were recorded in CD3OD at 100 MHz.
b
Data were recorded in CD3OD at 150 MHz.
c
Data were recorded in CDCl3 at 150 MHz.
d
Data were recorded in CDCl3 at 100 MHz.

Fig. 2. Key 1H–1H COSY and HMBC correlations of 1–7.

3 lack of a hydroxyl group at C-16, and 3 bearing a carbonyl group at C-
15 instead of hydroxyl group in 2. It was also confirmed by the HMBC
cross-peaks (Fig. 2) from H-17 (δH 1.12, 3H, d, J = 7.2 Hz) to C-12 (δC
32.7) and C-15 (δC 218.3), and from H-16 (δH 2.20, m) to C-13 (δC 25.6)
and C-15 (δC 218.3). The relative configuration of 3 (Fig. S1) was the
same as that of 2 by interpretation of the NOESY data, and the
α-orientation of H-16 was defined via the NOESY correlations of H-13β/
H-16/H3-17 and H-12/H-16.
The molecular formula C22H35NO3 of barpubesine C (4) was deduced
by (+)-HRESIMS at m/z 362.2688 [M+H]+ (calcd for 362.2690). The 1H
and 13C NMR spectra of 4 (Tables 1 and 2) showed characteristic pattern
of an atisine-type C20-diterpenoid alkaloid, with an angular methyl
group (δH 0.87, 3H, s; δC 26.6), a typical N-ethoxyl group [(δH 2.78, 2H,
br. s; 3.76, 2H, t, J = 5.4 Hz); δC 61.8, 58.6] and a typical exocyclic
double bond (δH 5.15, 5.28, each 1H, br. s; δC 115.5, 151.3). Comparison
of the NMR data of 4 with those of leucostomine A (9) (Xu et al., 2016)
indicated that the only different was bearing a typical ternary nitrogen
in 4 instead of the quaternary-N center in 9. The NOESY (Fig. S1) cor­
relations of H-18/H-5/H-9/H-7β, H-7α/H-15/H-14β,
H-20α/H-11/H-13α, and H-13β/H-12/H-17b suggested that H-5 and
H-9 were in β-configuration, and H-11 and H-15 were α-oriented.
The 1H (Table 1) and 13C (Table 2) NMR data of barpubesine D (5)
which had a molecular formula of C22H33NO2 determined by
(+)-HRESIMS at m/z 344.2573 [M+H]+ (calcd for 344.2584), showed
many similarities to those of denudatine-type C20-diterpenoid alkaloids
lepenine and 11α-hydroxy lepenine (Miao et al., 1999), except that the

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N. Ablajan et al. Phytochemistry 181 (2021) 112567

Fig. 3. Key NOESY correlations of 1, 2, 5 and 6.

Fig. 4. Plausible biosynthetic pathway of barpuberudine (1).

chemical shifts of C-9, C-11 and C-12 signals, which caused by lacked of
a hydroxyl group at C-11 in 5. It was also determined by (+)-HRESIMS
analysis and the 1H–1H COSY correlations (Fig. 2) of H-9 (δH 0.88
m)/H-11 (δH 2.11, t, J = 12.0 Hz; 1.65 m)/H-12 (2.39, t, J = 4.8 Hz). The
correlations of H-1/H-5/H-9 and H-1/H-9/H-15 in NOESY spectrum
(Fig. 3) indicated that H-1, H-5, H-9 and H-15 were all in
β-configuration.
Barpubenine A (6) was obtained as a white, amorphous powder. It’s
(+)-HRESIMS spectrum displayed a protonated molecule at m/z
392.2081 [M+H]+ (calcd for C21H30NO6, 392.2068), which corre­
sponded to a molecular formula of C21H29NO6. The 1H NMR spectrum
(Table 1) indicated the presence of three methoxyl groups (δH 3.32, 3.33

5
N. Ablajan et al.
and 3.38, each 3H, s). The 13C NMR (Table 2) and HSQC spectra showed
that 6 contains 21 carbons including three methyl groups (δC 56.9, 57.2
and 57.4), four methylenes (δC 25.8, 26.4, 28.7 and 31.6), eleven
methines (δC 39.0, 40.8, 47.0, 47.3, 55.7, 74.0, 79.9, 81.5, 83.5, 84.4
and 143.5), two quaternary carbons (δC 48.3 and 50.4), and one
carbonyl carbon (δC 211.0). The aforementioned data suggested that 6
might be a C18-diterpenoid alkaloid. The three methoxyl groups were
attributed to C-1, C-14 and C-16 based on the HMBC correlations (Fig. 2)
from 1-OCH3 (δH 3.32, 3H, s) to C-1 (δC 81.5), 14-OCH3 (δH 3.33, 3H, s)
and C-14 (δC 83.5), and from 16-OCH3 (δH 3.38, 3H, s) to C-16 (δC 84.4).
The HMBC cross-peaks of H-17 (δH 4.28, d, J = 2.0 Hz) to C-5 (δC 55.7),
C-7 (δC 79.9), C-15 (δC 28.7) and C-19 (δC 143.5) indicated that C-17 was
connected with C-8. It was the first example of rearranged-type skeleton
in C18-diterpenoid alkaloids. A carbonyl group was placed at C-6 and a
hydroxyl group was located at C-7 based on the HMBC cross-peaks from
H-5 (δH 2.63, t, J = 2.4 Hz) to C-6 (δC 211.0) and C-7 (δC 79.9), and from
H-7 (δH 4.02, s) to C-8 (δC 48.3), C-9 (δC 47.0), C-15 (δC 28.7) and C-17
(δC 74.0). A nitrone group was placed between C-17 and C-19 which was
confirmed by the deshielding shifts of H-17 (δH 4.28), H-19 (δH 7.30), C-
17 (δC 74.0) and C-19 (δC 143.5), as well as the (+)-HRESIMS analysis
(Chen et al., 2010).
The NOESY correlations (Fig. 3) of H-2β/H-1/H-5/H-9/H-14, H-1/H-
10/H-14, H-4/H-5/H-10, H-15α/H-7/H-19, and H-16/H-20 indicated
that ring “A” was in boat conformation and H-1, H-4, H-5, H-9, H-10 and
H-14 were β-oriented, and H-7 and H-16 was α-oriented. Therefore,
compound 6 was identified as shown in Fig. 1, and named barpuber­
udine F.
The (+)-HRESIMS data of barpubenine B (7) indicated a molecular
formula as C23H35NO5 at m/z 406.2579 [M+H]+ (calcd for 406.2588).
Its 1H-NMR data (Table 1) displayed signals for a typical N-ethyl group
(δH 1.13, 3H, t, J = 7.2 Hz; 3.09, 2.89, each 1H, m). The NMR data for 7
were similar to those for 6 except that the nitrone group in 6 was
replaced by an N-ethyl group in 7, which was supported by the chemical
shifts of C-4 (ΔδC − 5.7 ppm), C-8 (ΔδC +5.8 ppm), C-17 (ΔδC − 9.0 ppm)
and C-19 (ΔδC − 92.0 ppm). The NOESY correlations (Fig. S1) of 7
suggested it to possess the same relative configuration as 6.
A plausible biosynthetic pathway for the formation of 6 and 7 was
proposed in Fig. 5. As rearranged type C19-diterpenoid alkaloids, com­
pound 7 has been afforded from alkaloid with a 7,8-diol system (18) via
pinacol rearrangement and reduction reaction (Wang et al., 1999a;
Wang et al., 1999b). Compound 7 is further subjected to N-deethylation
and oxidation to obtain 6. Therefore, the absolute configuration of 7
should be the same as that of 6.
2.2. Biological activity assay
All compounds were evaluated for their cytotoxicity against A549
and HeLa human cancer cell lines using the MTT method (Xue et al.,
2019). Unfortunately, all the compounds showed no cytotoxicity (IC50 ˃
50 μM) (Table S1). The antibacterial activities of the isolates were also
determined by the gar well diffusion method (Begmatov et al., 2018)
with ampicillin and amphotericin B as positive control. The results
showed that compounds 4 presented weak inhibitory activity against the
Candida albicans; compounds 1, 4, 7 and 13 presented weak inhibitory
Fig. 5. Plausible biosynthetic pathway of barpubenines A-B (6–7).
6
Phytochemistry 181 (2021) 112567
capacities against the Staphylococcus aureus (Table S2).
The antiarrhythmic activity of alkaloids 7, 9–12, 15 and 16 was
preliminary tested at a dose of 8 mg/kg in a mouse model of aconitine
induced arrhythmia (Table 3). As a control group, lappaconitine (16)
had significant antiarrhythmic effect at this dose. N-acetylsepaconitine
(10) could significantly prolong the ventricular premature (VP) action,
which the effect was better than that of lappaconitine. Its inhibition of
ventricular tachycardia (VT) and ventricular flutter (VFL) were similar
to that of lappaconitine, and the inhibition of ventricular fibrillation
(VFib) and death was slightly weaker than lappaconitine (Heubach
et al., 1998). The antiarrhythmic effect of N-deacetyllappaconitine (11)
was stronger than that of lappaconitine except inhibition of VFib
(Tomshich et al., 1997). Although sepaconitine (12) had a certain
antiarrhythmic activity, it was significantly weaker than 11, showing
that acetyl and hydroxyl groups had a significant impact on the activity.
Due to diterpenoid alkaloids are inhibitors of multi-ion channels (Chen
et al., 2011). It is not easy to define the direct action of these functional
groups on the activity. Compounds 7, 9 and 15 had no obvious effect on
VP, although they could inhibit VT, VFL and VFib to some extent, they
couldn’t be regarded as potential antiarrhythmic compounds.
3. Conclusions
In summary, seven undescribed and ten known diterpenoid alkaloids
were isolated and identified from the whole plant of A. barbatum var.
puberulum. Barpuberudine (1) was an unprecedented carbon skeleton of
C20-diterpenoid alkaloid, while barpubenines A-B were the first example
of rearranged types in C18-diterpenoid alkaloids. All the compounds
showed no cytotoxicity, while some of them showed weak inhibitory
capacities against the C. albicans and S. aureus. N-acetylsepaconitine
(10) and N-deacetyllappaconitine (11) showed significant antiar­
rhythmic activity at a dose of 8 mg/kg in a mouse model of aconitine
induced arrhythmia. Unfortunately, the weight of specialised alkaloids
1–6 is not enough to test antiarrhythmic activity, which needs us to
continue in-depth study. This research provided important scientific
support for the sustainable utilization and development of characteristic
plant resources in Central Asia.
4. Experimental
4.1. General experimental procedures
The optical rotations were recorded using a Rudolph RS Autopol VI
automatic polarimeter. A Chirascan spectropolarimeter (Applied Pho­
tophysics, UK) was used to obtain ECD spectra. The NMR spectra were
recorded using a Varian VNMRS-600 spectrometer (Varian, Palo Alto,
CA, USA) (600 MHz for 1H and 150 MHz for 13C NMR) and INOVA–400
spectrometer (Varian, Palo Alto, CA, USA) (400 MHz for 1H and 100
MHz for 13C NMR) in CDOD3 or CDCl3 using TMS as an internal stan­
dard. The (+)-HRESIMS spectra were measured using a QStar Elite mass
spectrometer (AB SCIEX, Framingham, MA, USA). Semi-preparative
HPLC was conducted on a Shimadzu LC-10 A instrument (Shimadzu
Corporation, Kyoto, Japan), with UV detection, using a XCharge C18
(10*250 mm, 5 μm, Waters Co., Milford, MA, USA) column. The
N. Ablajan et al. Phytochemistry 181 (2021) 112567

Table 3
Antiarrhythmic activity of compounds 7, 9–12, 15 and 16.
Sample a Aconitine dosage in ECG change (μg/kg) Aconitine dosage for death (μg/kg)

VP VT VFL VFib

Model group 47.10 ± 9.86 90.55 ± 12.25 120.83 ± 28.58 227.34 ± 44.52 287.82 ± 46.77
16 Lappaconitine 68.89 ± 11.35 b 152.70 ± 21.39 239.98 ± 39.69 346.11 ± 53.51 395.82 ± 44.96
** ** ** ** **
7 44.79 ± 9.66 134.10 ± 6.74 204.79 ± 12.21 261.89 ± 25.42 325.91 ± 23.56
** **
9 48.15 ± 4.20 140.27 ± 20.59 228.04 ± 38.83 305.99 ± 46.80 328.12 ± 23.33
** ** *
10 93.57 ± 16.71 146.63 ± 25.27 242.86 ± 60.24 318.66 ± 26.04 371.29 ± 42.11
** ** ** ** *
11 89.59 ± 11.40 183.47 ± 26.57 272.49 ± 49.97 331.26 ± 35.92 403.08 ± 46.63
** ** ** ** **
12 58.74 ± 18.21 93.21 ± 17.43 208.43 ± 26.31 295.93 ± 65.47 339.84 ± 54.54
**
15 45.73 ± 12.17 132.65 ± 23.36 222.97 ± 27.71 315.05 ± 41.32 364.22 ± 40.93
* ** *
b
**P < 0.01, *P < 0.05, the sample groups were compared with the model group.
a
The dosage of the compounds was 8 mg/kg, the method of administration was intraperitoneal administration at 0.4 ml/20 g.

detection wavelengths were set to 200 nm and 230 nm. All of the sol­
vents used were analytical grade and purchased from Tianjin chemical
factory (Tianjin, China). Column chromatography separations were
performed on silica gel GF254 (Qingdao Ocean Chemical Factory,
China), LiChroprep RP-18 gel (20–45 μm, Merck, Germany) and
Sephadex LH-20 (GE Healthcare, Sweden). Fractions were monitored by
TLC on silica gel plates (silica gel G, Qingdao Ocean Chemical Factory,
China) and were visualized by spraying with modified Dragendorff’s
reagent.
4.2. Plant material
The whole plant was collected from the Guozigou of Yili Region,
Xinjiang of China (east longitude 81◦ 4′ , north latitude 44◦ 27′ , eleva­
tion1728 m), in July of 2017, and authenticated by Prof. Ying Feng
(Xinjiang Institute of Ecology and Geography, CAS) as Aconitum barba­
tum var. puberulum Ledeb. (Ranunculaceae). A voucher specimen
(XJIPC150727) was deposited in Xinjiang Technical Institute of Physics
and Chemistry, Chinese Academy of Sciences, PR China.
4.3. Extraction and isolation
The air-dried, whole plants of Aconitum barbatum var. puberulum (5.8
kg) were crushed in a blender, soaked with 5% Na2CO3 for 3 h at the
room temperature, and extracted with CHCl3:CH3OH (4:1) under cold
extraction method. After evaporation, the crude extract was extracted
exhaustively with 3% aq. H2SO4. The filtrate was then alkalized with
Na2CO3 to pH 10 and extracted with EtOAc to give the crude alkaloids
(30.0 g) after the solvent had been evaporated. The crude alkaloids were
separated using silica gel column chromatography and eluted using a
CHCl3–CH3OH (100:1, 60:1, 40:1, 20:1, 10:1, 5:1, 3:1 and 1:1) to obtain
nine fractions (A→I). Compound 16 (800.0 mg) was obtained from Fr. A
after recrystallized by methanol several times. The chromatographic
separation of Fr. A (4.60 g) on a silica gel column eluted with petroleum
ether-acetone-diethylamine (20:1:1 → 1:1:1) to give subfractions (Fr. A-
1→A-5). Compounds 10 (156.3 mg), 11 (55.6 mg) and 12 (36.6 mg)
were isolated from Fr. A-2 by semipreparative HPLC (XCharge C18 5 μm
10*250 mm, (CH3CN–H2O with 0.1%FA, 13:87). The chromatographic
separation of fraction B (1.71 g) on a silicagel column eluted with pe­
troleum ether-acetone-diethylamine (20:1:1 → 1:1:1), and petroleum
ether-acetone-methanol (1:1:1), to give subfractions (Fr. B-1→B-6).
Compounds 6 (2.2 mg) and 7 (20.8 mg) were isolated from Fr. B-4 by
semipreparative HPLC (CH3CN–H2O with 0.1%FA, 13:87). Compound
18 (10.5 mg) was obtained by semipreparative HPLC (CH3CN–H2O with
0.1%FA, 10:90 to 35:65 for 18 min, v/v) from fraction B-5. Fr. H (4.2 g)
was subjected to flash chromatography on a LiChroprep RP-18 gel col­
umn (H2O–MeOH, 95:5 to 20:80, v/v) to afford subfractions (Fr. H-
1→H-5). Fr. H-1 was chromatographed on Sephadex LH-20 column,
eluted with CHCl3–CH3OH (1:1) and further purified by preparative TLC
(silica gel H), eluted with CHCl3–CH3OH–H2O (82:16:2) to yield 13
(40.3 mg). The chromatographic separation of Fr. C (2.10 g) on a silica
gel column eluted with petroleum ether-acetone-diethylamine (10:1:1
→ 1:1:1) to give subfractions (Fr. C-1→C-7). Fr. C-2 was separately
chromatographed over a semipreparative HPLC (CH3CN–H2O with 0.1%
FA, 11:89) to afford 5 (2.1 mg). Compound 15 (19.3 mg) was isolated
from Fr. C-3 by semipreparative HPLC (CH3CN–H2O with 0.1%FA,
15:85). The chromatographic separation of Fr. D (3.60 g) on a silica gel
column eluted with petroleum ether-acetone-diethylamine (10:1:1 →
1:1:1) to give subfractions (Fr. D-1→D-5). Compound 17 (4.2 mg) was
obtained from Fr. D-2 by semipreparative HPLC (CH3CN–H2O with 0.1%
FA, 15:85–30:70 for 20 min, v/v). Fr. I (3.04 g) was subjected to flash
chromatography on a LiChroprep RP-18 gel column (H2O–MeOH, 95:5
to 20:80, v/v) to afford six subfractions (I-1→I-6). I-1 and I-3 were
chromatographed on Sephadex LH-20 column, eluted with
CHCl3–CH3OH (1:1) and further purified by semipreparative HPLC
(CH3CN–H2O with 0.1%FA, 3:97, v/v) and (CH3CN–H2O with 0.1%FA,
7:93, v/v) respectively to afford 1 (7.7 mg), 2 (6.4 mg), 3 (10.5 mg), 4
(5.5 mg) and 8 (11.8 mg). Fr. I-5 was chromatographed on Sephadex LH-
20 column, eluted with CHCl3–CH3OH (1:1) to give subfractions (Fr. I-5-
1→I-5-3). I-5-1 was separately chromatographed over a semipreparative
HPLC (CH3CN–H2O with 0.1%FA, 4:96) and further purified by pre­
parative TLC (silica gel H), eluted with CHCl3–CH3OH–H2O (82:16:2) to
yield 9 (24.9 mg). Fr. F was further separated using silica gel column
chromatography and eluted with Petroleum ether-acetone-diethylamine
(10:1:1 → 1:1:1) and further purified by semipreparative HPLC
(CH3CN–H2O with 0.1%FA,11:89, v/v) to afford 14 (44.6 mg).
4.3.1. Barpuberudine
White, amorphous powder; -66 (c 0.2, MeOH); ECD (MeOH) 201 (Δε
− 47.0), 214 (Δε +7.0) nm; 1H and 13C NMR data, see Tables 1 and 2;
(+)-HR-ESI-MS m/z 376.2834 [M+H]+ (calcd for C23H37NO3,
376.2846).
4.3.2. Barpubesine A
White, needle crystal; +15 (c 0.2, MeOH); ECD (MeOH) 200 (Δε
− 4.6), 221 (Δε +12.0) nm; IR (KBr) vmax 3361, 2923, 2857, 1465, 1053,
938, 883 cm− 1; 1H and 13C NMR data, see Tables 1 and 2; (+)-HR-ESI-
MS m/z 362.2676 [M]+ (calcd for C22H36NO+ 3 , 362.2690).

7
N. Ablajan et al.
4.3.3. Barpubesine B
White, amorphous powder; +14 (c 0.4, MeOH); IR (KBr) vmax 3143,
2936, 2871, 1708, 1670, 1452, 1117, 987 cm− 1; 1H and 13C NMR data,
see Tables 1 and 2; (+)-HR-ESI-MS m/z 344.2569 [M]+ (calcd for
C22H34NO+ 2 , 344.2584).
4.3.4. Barpubesine C
White, amorphous powder; -3 (c 0.1, MeOH); ECD (MeOH) 217 (Δε
− 4.8), 203 (Δε +9.6) nm; 1H and 13C NMR data, see Tables 1 and 2;
(+)-HR-ESI-MS m/z 362.2688 [M+H]+ (calcd for C22H35NO3,
362.2690).
4.3.5. Barpubesine D
White, amorphous powder; -60 (c 0.02, MeOH); ECD (MeOH) 233
(Δε +8.0), 205 (Δε +30.6) nm; 1H and 13C NMR data, see Tables 1 and 2;
(+)-HR-ESI-MS m/z 344.2563 [M+H]+ (calcd for C22H33NO2,
344.2584).
4.3.6. Barpubenine A
White, amorphous powder; -7 (c 0.1, MeOH); ECD (MeOH) 267 (Δε
− 7.7), 233 (Δε +1.8) nm; 1H and 13C NMR data, see Tables 1 and 2;
(+)-HR-ESI-MS m/z 392.2081 [M+H]+ (calcd for C21H30NO6,
392.2068).
4.3.7. Barpubenine B
White, amorphous powder; +5 (c 0.6, MeOH); ECD (MeOH) 296 (Δε
− 20.3), 201 (Δε +19.1) nm; IR (KBr) vmax 3447, 2925, 2853, 1717,
1670, 1457, 1387, 1124, 1089, 973 cm− 1; 1H and 13C NMR data, see
Tables 1 and 2; (+)-HR-ESI-MS m/z 406.2579 [M+H]+ (calcd for
C23H35NO5, 406.2588).
4.4. Cell culture and cytotoxicity assay
The cytotoxic activities of the compounds against human tumor cell
lines A549 and HeLa cells were evaluated in this study using the MTT
method in 96-well microplates (Xue et al., 2019). The cells were plated
in a 96-well plate 24 h before treatment and then exposed to different
concentrations of compounds or control for 72 h. Three replicates were
prepared for each treatment. DMSO (0.1% v/v) was used as negative
control and Doxorubicin was used as the positive control. Finally, the
OD value was read on a plate reader at a wavelength of 570 nm. The IC50
values were calculated for the inhibition rate. Inhibition rate = (OD
value of control group – OD value of experiment group)/(OD value of
control group − OD value of blank group). The results were expressed as
the percentage of inhibition that produced a reduction in the absorbance
by the treatment of the compounds compared to the controls.
4.5. Antibacterial activity assay
The antibacterial activities of isolates were measured using the agar
well diffusion method. Bacterial and fungal pathogens such as C. albicans
(ATCC 10231), E. coli (ATCC 11229) and S. aureus (ATCC 6538) were
used as indicator strains for this analysis. These microorganisms were
aseptically inoculated into appropriate liquid media and incubated at
37 ◦ C. After 16 h, the cells were centrifuged at 6000 rpm for 10 min and
then suspended in sterile water. The different cells (1 ml) were added to
appropriate agar media (100 ml) prior to plating, and the wells were
made using an agar well borer. To these wells, different concentrations
of the tested compounds were added and subsequently incubated at
37 ◦ C for 24 h. Zone of inhibitions were estimated by measuring the
diameter of the microbial growth inhibition zone. Ampicillin and
amphotericin B was used as the positive control. Values were averaged
from three independent experiments.
8
Phytochemistry 181 (2021) 112567
4.6. Antiarrhythmic activity test
A model of aconitine-induced arrhythmia in rats was used for anti­
arrhythmic activity test (Bryzgalov et al., 2013). The ICR mice of SPF
grade, male, weighing 18–22 g, certificate No. 201933363, were pur­
chased from Shanghai jiesijie experimental animal Co., Ltd. The dosage
of the compounds was 8 mg/kg, the method of administration was
intraperitoneal administration at 0.4 ml/20 g, 6 mice in each dose
group, and another model control group was intraperitoneally injected
with the same amount of saline solution. The mice were randomly
divided into groups. 30 min after intraperitoneal injection of the cor­
responding concentration of the tested drug, the mice were anesthetized
with 2% chloral hydrate (0.2 ml/10 g), connected with the electrocar­
diograph electrode of BL-420 biological function experimental system
instrument, and detected the second lead of electrocardiograph. At the
same time, 10 μg/ml aconitine solution (0.1 ml/min) was injected into
the femoral vein. The dosage of aconitine for VP, VT, VFL, VFib and
death of mice were observed and recorded.
Calculation formula of aconitine dosage (μg/kg) = 10 μg/ml × in­
jection volume (ml)/(mouse weight (kg)). All the data are expressed by
mean ± standard deviation. The one-way ANOVA method in IBM SPSS
statistic 20 program was used to analyze the data between groups. If the
variance between the two groups was the same, Bonferroni method
would use for analyze; if the variance was not the same, Dunnett’s T3
method would use for analyze. The results of statistical analysis (P <
0.05) showed significant difference.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was supported by the Xinjiang International Science
&Technology Cooperation Program under Grant No. 2019E01017; In­
ternational Partnership Program of Chinese Academy of Sciences under
Grant No. 153631KYSB20160004; and the Central Asian Drug Discovery
& Development Center of Chinese Academy of Sciences.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2020.112567.
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