Two new sesquiterpenoid glycosides from Nicotiana tabacum

Article abstract of DOI:10.1080/10286020.2014.922553

Two new sesquiterpenoid glycosides, nicotabalactonecoside (1) and nicotabadiolcoside (2), along with four known terpenoids (3-6) were isolated from the leaves of Nicotiana tabacum. The structures of compounds 1 and 2 were determined as dihydrodeacetylphytuberin-2-one 11-O-β-D-glucopyranoside and 1,2-dehydro-4- epieremophil-9-ene-11,12-diol 12-O-β-D-glucopyranoside by extensive spectroscopic analyses (HR-ESI-MS, UV, IR, 1D, and 2D NMR) and chemical method. Compound 1 is an unusual phytuberin-type sesquiterpenoid with a 6/5/5 tricyclic system.

Full text of DOI:10.1080/10286020.2014.922553

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Two new sesquiterpenoid glycosides
from Nicotiana tabacum
Cai-Yan Yangab, Chang-An Gengb, Yun-Bao Mab, Xiao-Yan Huangb,
Xue-Mei Zhangb, Jun Zhoub & Ji-Jun Chenb
a School of Life Sciences of Yunnan University, Kunming 650091,
China
b State Key Laboratory of Phytochemistry and Plant Resources
in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, China
Published online: 09 Jun 2014.
To cite this article: Cai-Yan Yang, Chang-An Geng, Yun-Bao Ma, Xiao-Yan Huang, Xue-Mei Zhang,
Jun Zhou & Ji-Jun Chen (2014) Two new sesquiterpenoid glycosides from Nicotiana tabacum,
Journal of Asian Natural Products Research, 16:6, 611-616, DOI: 10.1080/10286020.2014.922553
To link to this article: http://dx.doi.org/10.1080/10286020.2014.922553
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 6, 611–616, http://dx.doi.org/10.1080/10286020.2014.922553
Two new sesquiterpenoid glycosides from Nicotiana tabacum
Cai-Yan Yangab, Chang-An Gengb, Yun-Bao Mab, Xiao-Yan Huangb, Xue-Mei Zhangb,
Jun Zhoub and Ji-Jun Chenb*
b
aSchool of Life Sciences of Yunnan University, Kunming 650091, China; State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy
of Sciences, Kunming 650201, China
(Received 28 January 2014; final version received 5 May 2014)
Two new sesquiterpenoid glycosides, nicotabalactonecoside (1) and nicotabadiolco-
side (2), along with four known terpenoids (3–6) were isolated from the leaves of
Nicotiana tabacum. The structures of compounds 1 and 2 were determined as
dihydrodeacetylphytuberin-2-one 11-O-b-D-glucopyranoside and 1,2-dehydro-4-
epieremophil-9-ene-11,12-diol 12-O-b-D-glucopyranoside by extensive spectroscopic
analyses (HR-ESI-MS, UV, IR, 1D, and 2D NMR) and chemical method. Compound 1
is an unusual phytuberin-type sesquiterpenoid with a 6/5/5 tricyclic system.
Keywords: nicotabalactonecoside; nicotabadiolcoside; sesquiterpenoids; Nicotiana
tabacum
1. Introduction
(4) [10], lupeol palmitate (5) [11], and
(24R)-cycloartane-3b,24,25-triol 3-O-pal-
mitate (6) [12] (Figure 1). Their structures
were determined based on extensive
spectroscopic analyses and chemical
method. Compound 1 is an important
precursor for the understanding of the
biogenesis of phytuberin-type sesquiter-
penoids, and compounds 4–6 were iso-
lated from N. tabacum for the first time.
Phytuberin represents an unusual tricyclic
sesquiterpenoid with a 6/5/5 fused system.
So far, only three additional cases of this
type of sesquiterpenoid, named phytu-
berol, 2b-methoxydihydrophytuberol, and
2a-methoxydihydrophytuberol, have been
reported from Nicotiana tabacum since its
initial isolation from microbially infected
potato [1–5]. The leaves of N. tabacum are
well-known raw materials in tobacco
production, and its aerial parts are also
used for sedative, anesthetic, diaphoretic,
and emetic purposes in Chinese folk
medicines [6]. Nicotiana plants are rich
in sesquiterpenoids including eudesmane,
vatispirane, monocyclofarnesane, and ere-
mophilane types [7,8]. Our chemical
investigation on N. tabacum afforded two
new sesquiterpenoid glycosides, nicotaba-
lactonecoside (1) and nicotabadiolcoside
(2), along with four known terpenoids, 9-
O-b-D-glucoside of 3-hydroxy-7,8-dide-
hydro-b-ionol (3) [9], b-amyrin palmitate
2. Results and discussion
Nicotabalactonecoside (1) had a molecular
formula of C21H34O9 with five degrees of
unsaturation by means of positive HR-
ESI-MS which exhibited a quasi-molecu-
lar ion peak at m/z 453.2047 [M þ Na]þ.
Its IR spectrum exhibited hydroxyl
(3417 cm21), ester (1737 cm21), and
CZOZC (1149, 1118, and 1035 cm21
)
groups. Acid hydrolysis of compound 1
yielded a D-glucose moiety which was
evidenced from its [a] value of þ51.0
D
(c, 0.06, MeOH). Four methyls [dH 1.52,
*Corresponding author. Email: chenjj@mail.kib.ac.cn
q 2014 Taylor & Francis

612
C.-Y. Yang et al.
14
10
9
6
9
1
1
10
2
8
O
12
5
5
OGlc
7
4
11
4
3
7
OGlc
O
15
11 12
OH
15
13
3
13
2
14
O
1
2
Figure 1. The structures of compounds 1 and 2.
1.36, 1.35, and 1.00 (each 3H, s)] and one
anomeric proton (4.99, d, J ¼ 7.5 Hz)
those of deacetylphytuberin except that
one double bond [dH 6.41 and 4.64 (each
1H, d, J ¼ 2.8 Hz); dC 146.2 and 104.4] in
deacetylphytuberin was replaced by one
carbonyl group (174.4) and one methylene
[dH 2.90 and 2.79 (each 1H, d,
J ¼ 18.5 Hz); dC 44.0], and the chemical
shift of C-11 was down-fielded from 72.2
in deacetylphytuberin to 78.8 in compound
1
were observed in its H NMR spectrum.
Besides a set of signals due to a b-D-
glucosyl group, its 13C NMR (DEPT) data
(Table 1) exhibited four methyls, five
methylenes, one methine, and five qua-
ternary carbons. The NMR data of its
aglycone part (Table 1) were similar to
Table 1. 1H and 13C NMR spectral data of compounds 1 and 2 (d in ppm, J in Hz).
dH
dC
No.
1
2
1
2
1a
1b
2
3a
3b
4
3.53, d, 8.4
3.59, d, 8.4
5.90, dd, 9.8, 2.2
76.9, t
129.3, d
5.33–5.37, m
1.67, dd, 18.4, 5.0
2.47, d, 18.4
174.4, s
44.0, t
123.6, d
33.0, t
2.90, d, 18.5
2.79, d, 18.5
1.48–1.52, m
88.1, s
97.2, s
29.6, t
40.6, d
38.0, s
39.1, t
5
6a
6b
7
8a
8b
9a
9b
10
11
12
2.26–2.30, m
1.86–1.89, m
1.91–1.94, m
1.39–1.43, m
1.61–1.65, m
1.29–1.33, m
1.78, d, 12.5
1.76–1.79, m
1.28, t, 13.1
1.72–1.75, m
1.67–1.69, m 2.10–2.14, m
45.5, d
21.5, t
42.7, d
27.8, t
5.66, d, 7.2
34.3, t
127.1, d
45.0, s
78.8, s
23.6, q
141.0, s
74.9, s
76.9, t
1.36, s
3.89, d, 10.4
3.30, d, 10.4
1.04, s
0.82, d, 6.8
1.02, s
4.19, d, 7.8
3.22, dd, 9.1, 7.8
3.24–3.28, m
3.20–3.23, m
3.29–3.33, m
3.83, dd, 11.8, 2.2
3.62, dd, 11.8, 5.0
13
14
15
10
1.35, s
1.00, s
1.52, s
4.99, d, 7.5
3.94–3.96, m
3.94–3.96, m
4.17–4.21, m
4.23–4.27, m
4.35, dd, 12.0, 2.0
4.32, dd, 12.0, 5.1
24.8, q
16.6, q
25.0, q
98.7, d
75.4, d
78.6, d
71.6, d
78.2, d
62.8, t
20.8, q
18.0, q
31.3, q
104.9, d
75.2, d
78.0, d
71.6, d
78.0, d
62.7, t
20
30
40
50
60

Journal of Asian Natural Products Research
613
OH
O
OH
OH
OH
OH
OH
O
O
O
HO
O
OH
O
HO
13
O
2
1
COSY
HMBC
Figure 2. The key 1H–1H COSY and HMBC correlations of compounds 1 and 2.
1 [2]. The above analyses were confirmed
by the HMBC correlations of H-15 (dH
1.52)/C-3 (dC 44.0), C-4 (88.1), and C-5
(97.2), and H-3 (2.90 and 2.79)/C-2
(174.4), C-4 (88.1), and C-5 (97.2). In
addition, the HMBC correlation from H-10
(dH 4.99) to C-11 (dC 78.8) indicated the
b-D-glucosyl group attached at C-11
(Figure 2). The ROESY correlation of H-
6b (dH 1.86–1.89)/H-15 (1.52) and the
undetected ROESY correlations of H-6a
(dH 2.26–2.30)/H-14 and H-6b (1.86–
1.89)/H-14 (1.00) indicated that C-6 and
C-15 were situated to the same face of the
tetrahydrofuran ring, while C-14 was
directed to the opposite face (Figure 3)
suggesting the same stereochemistry with
deacetylphytuberin (Figure 1). Thus, the
structure of compound 1 was characterized
as dihydrodeacetylphytuberin-2-one 11-O-
b-D-glucopyranoside, and named as
nicotabalactonecoside.
ESI-MS which provided a quasi-molecular
ion peak at m/z 421.2190 [M þ Na]þ. Its
UV spectrum showed the presence of a
conjugated double bond [lmax (log 1): 234
(4.38) nm], and IR spectrum exhibited
hydroxyl (3425 cm21), double bond
(1630 cm21), and CZOZC (1076 and
1032 cm21) groups. Acid hydrolysis of
compound 2 provided a D-glucose moiety
which was approved by its [a]D value of
þ50.6 (c ¼ 0.09, MeOH). Three methyls
(dH 1.04, s; 1.02, s; and 0.82, d,
J ¼ 6.8 Hz), three olefinic protons (5.90,
dd, J ¼ 9.8 and 2.2 Hz; 5.66, d,
J ¼ 7.2 Hz; and 5.33–5.37, m) and
anomeric proton (4.19, d, J ¼ 7.8 Hz)
were revealed by its 1H NMR data
(Table 1). Besides one b-D-glucosyl
group, three methyls, four methylenes,
five methines, and three quaternary car-
bons were displayed in its 13C NMR
1
(DEPT) spectrum. The H and 13C NMR
Nicotabadiolcoside (2) showed a mol-
ecular formula of C21H34O7 with five
degrees of unsaturation by its positive HR-
data of its aglycone part were similar with
those of 4-epieremophil-9-ene-11,12-diol
(2a) except that two methylenes (dC 31.9,
2
1
ROESY
Figure 3. The key ROESY correlations of compounds 1 and 2.

614
C.-Y. Yang et al.
C-1 and 22.4, C-2) in 2a were changed to
be a pair of olefinic carbons (129.3 and
123.6) in 2, together with the obvious
down-fielded shift of C-12 from 68.5 in 2a
to 76.9 in 2 [13]. The above analyses
suggested a double bond between C-1 and
C-2 in 2 and the glucosyl group attached to
C-12, which was confirmed by COSY
correlations of H-1 (dH 5.90)/H-2 (5.33–
5.37)/H-3a (1.67) and HMBC correlations
of H-10 (dH 4.19)/C-12 (dC 76.9), H-9
(5.66)/C-1 (129.3), and H-1 (5.90)/C-3
(33.0), C-5 (38.0), and C-10 (141.0). The
ROESY correlations of H-4 (dH 1.48–
1.52)/H-15 (1.02)/H-6b (1.28), H-15
(1.02)/H-12 (3.89 and 3.30), and H-14
(0.82)/H-6a (1.76–1.79) indicated that the
configuration of 2 was identical with 4-
epieremophil-9-ene-11,12-diol. Therefore,
the structure of compound 2 was eluci-
dated as 1,2-dehydro-4-epieremophil-9-
ene-11,12-diol 12-O-b-D-glucopyrano-
side, and named as nicotabadiolcoside.
The analog of compound 1, phytuberin
lactone, was an important precursor for the
synthesis of phytuberin [2,5]; however,
phytuberin lactone and its analogs have
not been reported from natural resources.
Thus, the first isolation of nicotabalacto-
necoside (1) as natural product was
significant for understanding the biogen-
esis of phytuberin-type sesquiterpenoids.
Compounds 1–6 were assayed on
HEK293 cell line in vitro for the agitating
activities on 5-HT2C receptor using ser-
otonin hydrochloride (5-HT) as the posi-
tive control [14] and did not display
agitating activities at the concentration of
about 0.75 mM.
FTS-135 spectrometer (Bio-Rad, Her-
cules, CA, USA). NMR spectra were
obtained on an Advance III 600 NMR or
Bruker DRX-500 spectrometer (Bruker,
Bremerhaven, Germany) using TMS as the
internal standard. HR-ESI-MS data were
obtained using a Shimadzu LC-MS-IT-
TOF mass spectrometer (Shimadzu,
Kyoto, Japan). Semi-preparative HPLC
was conducted on a Waters Alliance 2695
with a reversed-phase (RP) C18 column
(9.4 mm £ 250 mm, 5 mm; Agilent, Santa
Clara, CA, USA). Silica gel (200–300
mesh; Qingdao Makall group Co., Ltd,
Qingdao, China), Sephadex LH-20 (Amer-
sham Bioscience, Uppsala, Sweden), MCI
CHP 20P (Mitsubishi Chemical Institute,
Tokyo, Japan), and C18 (Merck, Darm-
stadt, Germany) were applied to column
chromatography (CC).
3.2 Plant material
The leaves of N. tabacum were collected
from Luliang County, Yunnan Province,
China, and identified as N. tabacum Linn.
by Prof. Dr Li-Gong Lei, Kunming
Institute of Botany, Chinese Academy of
Sciences. The specimen (No. 2011-09-16)
was stored in the Laboratory of Antivirus
and Natural Medicinal Chemistry, Kunm-
ing Institute of Botany, Chinese Academy
of Sciences.
3.3 Extraction and isolation
The dried leaves (3.5 kg) were extracted
using 90% EtOH (72 h each, 20 l £ 3
times) at room temperature and parti-
tioned between EtOAc and H2O. The
EtOAc fraction (615 g) was chromato-
graphed on a silica gel column (3.0 kg,
18.0 cm £ 70 cm) using H2O–MeOH–
CHCl3 (0:0:100, 0:5:95, 0:10:90,
2:20:80, v/v/v) as the eluent to give Frs
1–8. Fr 5 (29.5 g) was fractionated by a
MCI CHP 20P gel column (310 g, 4.0 cm
£ 40 cm) eluted with MeOH–H2O
(20:80, 40:60, 60:40, 80:20, 100:0) to
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a
Jasco model 1020 polarimeter (Horiba,
Tokyo, Japan). UV spectrum was recorded
using a Shimadzu UV-2401A spectropho-
tometer (Shimadzu, Kyoto, Japan). IR
spectra were carried out on a Bio-Rad

Journal of Asian Natural Products Research
615
produce Frs 5.1–5.5. Fr 5.3 (3.2 g) was
separated on a silica gel CC (100 g,
4.0 cm £ 50 cm) eluted with H2O–
MeOH–CHCl3 (0:10:90, 1.0:15:85) to
obtain Frs 5.3.1–5.3.6. Fr 5.3.6 (40 mg)
was applied to HPLC on an RP C18
column eluted with MeCN–H2O (20:80,
n ¼ 4 ml/min) to generate compounds 1
(3 mg, tR: 11.3 min) and 3 (2 mg, tR:
9.6 min). Fr 5.3.3 (500 mg) was applied to
HPLC on an RP C18 column eluted with
MeOH–H2O (70:30, n ¼ 3 ml/min) to
give 2 (5 mg, tR: 24 min). Fr 8 (15 g)
was fractionated by a MCI CHP 20P gel
column (100 g, 2.54 cm £ 40 cm) eluted
with MeOH–H2O (20:80, 40:60, 60:40,
80:20, 100:0) to yield Frs 8.1–8.5. Fr 8.1
(5.5 g) was subjected to a silica gel CC
(20 g, 1.5 cm £ 40 cm) using CHCl3 –
petroleum ether (PE, 10:90) as the eluent
to generate 4 (9 mg) and 5 (44 mg). Fr 3
(40 g) was chromatographed on a MCI
CHP 20P gel column (310 g, 4.0 cm
£ 40 cm) eluted with MeOH–H2O
(60:40, 80:20, 100:0) to provide Frs
3.1–3.4. Fr 3.3 (300 mg) was submitted
to a silica gel CC (20 g, 1.5 cm £ 40 cm)
eluted with EtOAc–PE (10:90) to obtain
6 (22 g).
m/z 421.2190 [M þ Na]þ (calcd for
C21H34O7Na, 421.2197).
3.3.3 Acid hydrolysis and sugar
identification
Compound 1 (1.0 mg) was treated with 1.0
M HCl (MeOH–H2O, 1:1, 1 ml) on a
boiling water bath for 2 h, and neutralized
by NaHCO3 and partitioned between
CH2Cl2 and H2O. The H2O part was
purified on a silica gel CC (8 g, 1.0 cm
£ 40 cm) eluted with H2O–MeOH–
CHCl3 (3:30:70) to give glucose
(0.2 mg). Acid hydrolysis of compound 2
(1 mg) by the same method of 1 gave
glucose (0.3 mg). The glucoses from
compounds 1 and 2 were determined to
be D-glucoses by their optical rotations
21
½aꢀD þ 51.0 (c ¼ 0.06, MeOH) and
þ50.6 (c ¼ 0.09, MeOH), respectively.
Acknowledgments
This work was financially supported by the
National Natural Science Foundation of China
for Distinguished Young Scholars (No.
81025023) and the Youth Innovation Pro-
motion Association, CAS.
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16
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