Noreudesmane sesquiterpenoids from the leaves of Nicotiana tabacum

Article abstract of DOI:10.1016/j.fitote.2014.04.010

Six new 14-noreudesmane sesquiterpenoids, nicotabacosides A-F (1-6), along with five known sesquiterpenoids (7-11), were isolated from the leaves of Nicotiana tabacum. The structures of compounds 1-6 were elucidated as isorishitin 3-O-β-d-glucopyranoside (1), rishitin 3-O-β-d- glucopyranoside (2), rishitin 2-O-β-d-glucopyranoside (3), 1, 6-dehydro-rishitin 3-O-β-d-glucopyranoside (4), 2-hydroxyl-ligudentatol 3-O-β-d-glucopyranoside (5) and oxyglutinosone 3-O-β-d-glucopyranoside (6) based on extensive spectroscopic analyses (HRESIMS, UV, IR, 1D and 2D NMR). Their absolute configurations were determined by X-ray single-crystal diffraction and comparison of their electronic circular dichroism (ECD) spectra.

Full text of DOI:10.1016/j.fitote.2014.04.010

Fitoterapia 96 (2014) 81–87
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Noreudesmane sesquiterpenoids from the leaves
of Nicotiana tabacum
Cai-Yan Yang a,b, Chang-An Geng a, Xiao-Yan Huang a, Hao Wang a,b, Hong-Bo Xu a,b
,
Wen-Juan Liang a,b, Yun-Bao Ma a, Xue-Mei Zhang a, Jun Zhou a, Ji-Jun Chen a,
⁎
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, PR China
University of Chinese Academy of Sciences, Beijing 100049, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new 14-noreudesmane sesquiterpenoids, nicotabacosides A–F (1–6), along with five known
sesquiterpenoids (7–11), were isolated from the leaves of Nicotiana tabacum. The structures of
compounds 1–6 were elucidated as isorishitin 3-O-β-D-glucopyranoside (1), rishitin 3-O-β-D-
glucopyranoside (2), rishitin 2-O-β-D-glucopyranoside (3), 1, 6-dehydro-rishitin 3-O-β-D-
glucopyranoside (4), 2-hydroxyl-ligudentatol 3-O-β-D-glucopyranoside (5) and oxyglutinosone
3-O-β-D-glucopyranoside (6) based on extensive spectroscopic analyses (HRESIMS, UV, IR, 1D
and 2D NMR). Their absolute configurations were determined by X-ray single-crystal diffraction
and comparison of their electronic circular dichroism (ECD) spectra.
Received 18 February 2014
Accepted in revised form 10 April 2014
Available online 24 April 2014
Keywords:
Nicotabacosides A–F
14-Noreudesmane sesquiterpenoids
Nicotiana tabacum
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
from natural sources, our investigation on N. tabacum
afforded six new 14-noreudesmane sesquiterpenoids,
Nicotiana tabacum belonging to Nicotiana genus of the
Solanaceace family, is an important economic crop whose
leaves are well-known in tobacco production. In addition, its
aerial part is also used for sedative, diaphoretic, anesthetic and
emetic purposes [1]. Phytochemical investigation revealed that
Nicotiana plants were rich in terpenoids, alkaloids and
flavonoids [2–7]. Most of the sesquiterpenoids in Nicotiana
plants are structurally categorized as monocyclofarnesane,
vatispirane and eudesmane-types including 3 cases of unusual
noreudesmane-type. Currently, about 20 noreudesmane-type
sesquiterpenoids were obtained from natural sources and
classified into 13-noreudesmane, 14-noreudesmane and 11,
12, 13-tri-noreudesmane sesquiterpenoids according to the
positions of carbon decreasing [8–13]. Pharmacological studies
showed that nicotine, the most important alkaloid in Nicotiana
plants, was closely related to smoking addiction, and possessed
neuroprotective effect against the toxicity of amyloid-β (Aβ)
oligomers [14]. As a continuous search for active compounds
nicotabacosides A–F (1–6), as well as five known
sesquiterpenoids, actinidioionoside (7), byzantionoside B
(8), (Z)-4-[3′-(β-D-glucopyranosyloxy) butylidene]-3, 5, 5-
trimethyl-2-cyclohexen-l-one (9), (6R, 9R)-3-oxo-α-ionol
β-D-glucopyranoside (10) and (6R, 9S)-3-oxo-α-ionol
β-D-glucopyranoside (11) based on extensive spectroscopic
analyses (HRESIMS, UV, IR, 1D and 2D NMR). The absolute
configurations of compounds 1 and 4 were determined by
X-ray single-crystal diffraction, and compounds 2, 3, 5 and 6
were determined by comparing their electronic circular
dichroism (ECD) spectra. Herein, we described their isola-
tion and structural elucidation.
2. Experimental
2.1. General apparatus and chemicals
Melting points (mp) were measured by a SGW®X-4B
melting point apparatus (Shanghai Precision & Scientific
Instrument Co., Ltd. Shanghai, China). Optical rotations were
obtained on a Jasco model 1020 polarimeter (Horiba, Tokyo,
Japan). HRESIMS data were recorded on a LCMS–IT–TOF mass
⁎
Corresponding author. Tel.: +86 871 65223265; fax: +86 871
65227197.
E-mail address: chenjj@mail.kib.ac.cn (J.-J. Chen).
http://dx.doi.org/10.1016/j.fitote.2014.04.010
0367-326X/© 2014 Elsevier B.V. All rights reserved.

82
C.-Y. Yang et al. / Fitoterapia 96 (2014) 81–87
spectrometer (Shimadzu, Kyoto, Japan). UV spectra were
conducted on a UV-2401A spectrophotometer (Shimadzu,
Kyoto, Japan). Electronic circular dichroism (ECD) spectra
were performed on an Applied Photophysics Chirascan instru-
ment (Agilent, America). IR spectra were collected on a Bio-Rad
FTS-135 spectrometer (Bio-Rad, Hercules, CA). 1D and 2D NMR
spectra were acquired using AM-400, DRX-500 or Advance
III-600 NMR spectrometers (Bruker, Bremerhaven, Germany)
with TMS as internal standard. Semi-preparative HPLC was
performed on a Waters Alliance 2695 (pump: Waters 600,
detector: Waters 2996) with a reversed-phase (RP) C18 column
(9.4 × 250 mm, 5 μm, Agilent). Silica gel (200–300 mesh,
Qingdao Makall group Co., Ltd; Qingdao, China), C18 (Merck,
Darmstadt, Germany) and Sephadex LH-20 (Amersham Bio-
science, Sweden) were used for column chromatography.
was purified through HPLC on a RP C18 column eluted with
MeOH–H2O (55:45) to generate 2 (12 mg) and 3 (30 mg).
Fr.5.4 (3.7 g) was chromatographed on a silica gel column
(100 g, 3.5 × 50 cm) using HCOOH–MeOH–CHCl3 (1:10:90) as
the eluent to give Frs.5.4.1–5.4.4. Fr.5.4.4 (2.2 g) was subjected to
a silica gel CC (40 g, 2.0 × 50 cm) eluted with H2O–MeOH–
EtOAc (0.5:5:95) to generate Frs.5.4.4.1–5.4.4.3. Fr.5.4.4.1
(130 mg) was separated on a sephadex LH-20 column (50 g,
1.4 × 150 cm) to produce compound 5 (5 mg). Fr.8 (15.0 g) was
fractionated by a MCI CHP 20P gel column (100 g, 2.54 × 40 cm)
eluted with MeOH–H2O (20:80, 40:60, 60:40, 80:20, 100:0) to
produce Frs.8.1–8.5. Fr.8.1 (5.5 g) was chromatographed on a
silica gel column (100 g, 4.0 × 50 cm) eluted with H2O–MeOH–
CHCl3 (2:20:80) to generate 7 (20 mg).
Nicotabacoside A (1): Colorless acicular crystal [MeOH–
H2O (1:1, v/v)], mp 107.2–108.2 °C; [α]1D6: +37.1 (c 0.20,
2.2. Plant material
MeOH); ECD (MeOH) Δε
+ 15.66, Δε
+ 6.29,
205
195
Δε + 7.56, Δε − 0.01; IR (KBr) vmax 3423, 1642,
212
231
The leaves of N. tabacum Linn. were collected from Luliang
County of Yunnan Province of China, on September 16, 2011
and identified by Prof. Dr. Li-Gong Lei, Kunming Institute of
Botany, Chinese Academy of Sciences. A voucher specimen (No.
2011-09-16) was deposited in the Laboratory of Antivirus and
Natural Medicinal Chemistry, Kunming Institute of Botany,
Chinese Academy of Sciences.
1439, 1374, 1167, 1077, 1031 cm−1 1H (400 MHz) and 13C
;
NMR (100 MHz) data (see Tables 1 and 3); HRESIMS m/z
407.2040 [M + Na]+ (calcd for C20H32O7Na, 407.2040,
0 mDa).
Crystal data of compound 1: C20H32O7·2H2O, M =
420.49, monoclinic, a = 21.4086 (7) Å, b = 7.3541 (3) Å,
c = 28.0134 (9) Å, α = 90.00°, β = 95.0100 (10)°, γ =
90.00°, V = 4393.6(3) Å3, T = 100 (2) K, space group C2,
Z = 8, μ (CuKα) = 0.830 mm−1, 15,414 reflections mea-
sured, 5780 independent reflections (Rint = 0.0565). The
final R1 values were 0.1023 (I N 2σ (I)) and 0.1054 (all data).
The final wR (F2) values were 0.2790 (I N 2σ (I)) and 0.2875
(all data). The goodness of fit on F2 was 1.368, flack
parameter was 0.2 (3), the Hooft parameter was 0.24 (13)
for 1820 Bijvoet pairs.
2.3. Extraction and isolation
The dried leaves of N. tabacum (3.5 kg) were extracted
with 90% EtOH at room temperature for 3 times, each for
72 h. All the extract was combined and condensed under
reduced pressure (b60 °C), which was partitioned between
EtOAc and H2O.
The EtOAc part (615 g) was subjected to silica gel column
chromatography (CC) (3.0 kg, 18.0 × 70 cm) using H2O–
MeOH–CHCl3 (0:0:100, 0:5:95, 0:10:90, 2:20:80, v/v) as the
eluent to afford Frs.1–8. Fr.4 (35.5 g) was fractionated by a MCI
CHP 20P gel column (310 g, 4.0 × 40 cm) eluted with MeOH–
H2O (20:80, 40:60, 60:40, 80:20, 100:0) to get Frs.4.1–4.5.
Nicotabacoside B (2): White powder; [α]1D7: −49.2 (c 0.09,
MeOH); ECD (MeOH) Δε
− 11.91, Δε
− 15.42,
195
200
Δε 218 + 0.98, Δε 228 − 0.33; IR (KBr) vmax 3473, 3406, 1643,
1443, 1373, 1163, 1079, 1031 cm−1 1H (400 MHz) and 13C
;
(100 MHz) NMR data (see Tables 1 and 3); HRESIMS m/z
407.2053 [M + Na]+ (calcd for C20H32O7Na, 407.2040, +
1.3 mDa).
Fr.4.3 (1.8 g) was subjected to
a silica gel CC (40 g,
2.0 × 50 cm) eluted with MeOH–CHCl3 (10:90) to provide
Frs.4.3.1–4.3.2. Fr.4.3.2 (538 mg) was separated on a sephadex
LH-20 column (50 g, 1.4 × 150 cm) to yield Frs.4.3.2.1–4.3.2.2.
Fr.4.3.2.1 (427 mg) was purified by HPLC on a RP C18 column
using MeOH–H2O (48:52) as the eluent to obtain compounds 8
(23 mg), 9 (8 mg), 10 (46 mg) and 11 (36 mg). Fr.5 (29.5 g)
was fractionated by a MCI CHP 20P gel column (310 g,
4.0 × 40 cm) eluted with MeOH–H2O (20:80, 40:60, 60:40,
80:20, 100:0) to produce Frs.5.1–5.5. Fr.5.2 (1.6 g) was
chromatographed on a RP C18 column (124 g, 2.54 × 40 cm)
eluted with MeOH–H2O (10:90, 30:70, 50:50, 70:30, 100:0) to
obtain Frs.5.2.1–5.2.7. Fr.5.2.7 (100 mg) was purified through
HPLC on a RP C18 column with MeCN–H2O (25:75) as mobile
phase to afford compound 6 (4 mg). Fr.5.3 (3.2 g) was
submitted to a silica gel CC (100 g, 4.0 × 50 cm) eluted with
H2O–MeOH–CHCl3 (0:10:90, 1.0:15:85) to give Frs.5.3.1–5.3.6.
Fr.5.3.3 (500 mg) was separated by HPLC on a RP C18 column
eluted with MeOH–H2O (72:28) to provide Fr.5.3.3.1 and
Fr.5.3.3.2. Fr.5.3.3.1 (60 mg) was purified by HPLC on a RP C18
column using MeCN–H2O (35:65) as the eluent to yield
compounds 1 (10 mg) and 4 (10 mg). Fr.5.3.3.2 (200 mg)
Nicotabacoside C (3): White powder; [α]1D6: −42.5 (c 0.60,
MeOH); ECD (MeOH) Δε 195 − 11.91, Δε 196 − 8.95, Δε 200
−
13.30, Δε 216 + 3.72, Δε 239 − 0.55; IR (KBr) vmax 3408, 1644,
1602, 1450, 1375, 1284, 1164, 1076, 1042, 1023 cm−1 1H
;
(400 MHz) and 13C (100 MHz) NMR data (see Tables 1 and 3);
HRESIMS m/z 407.2038 [M + Na]+ (calcd for C20H32O7Na,
407.2040, −0.2 mDa).
Nicotabacoside D (4): Colorless acicular crystals [MeOH–H2O
(6:4)], mp 195.0–196.0 ºC; [α]1D6: +5.8 (c 0.08, MeOH); UV
(MeOH) λmax (log ε) 241 (4.45) nm; ECD (MeOH) Δε 195 + 8.40,
Δε
− 4.44, Δε
+ 14.88, Δε
− 0.49; IR (KBr) vmax
210
239
259
3419, 1643, 1452, 1372, 1165, 1079, 1030 cm−1; 1H (400 MHz)
and 13C (100 MHz) NMR data are seen in Tables 2 and 3;
HRESIMS m/z 405.1879 [M + Na]+ (calcd for C20H30O7Na,
405.1884, −0.5 mDa).
Crystal data of compound 4: C20H30O7·H2O, M = 400.46,
monoclinic, a = 8.0041 (6) Å, b = 63.282 (5) Å, c = 8.9989
(7) Å, α = 90.00°, β = 116.076 (4)°, γ = 90.00°, V =
4094.1 (5) Å3, T = 100 (2) K, space group P21, Z = 8,
μ
(CuKα) = 0.831 mm−1
,
27,133 reflections measured,
12,552 independent reflections (Rint = 0.1413). The final

C.-Y. Yang et al. / Fitoterapia 96 (2014) 81–87
83
Table 1
Table 3
1H NMR data for compounds 1–3 (δ in ppm, J in Hz).
13C NMR data for compounds 1–6.
No.
1a
2a
2b
3a
No. 1a
2a
38.7, t
2b
37.7, t
3a
4a
5a
6a
1a
1b
2
2.10, m
2.26, m
2.18, m
3.65, m
3.33, m
2.04, m
1.91, m
3.54, m
3.08, m
2.26, m
2.13, m
3.69, m
3.32, m
1
2
3
4
5
6
7
8
9
36.4, t
66.8, d 70.6, d 68.2, d 80.3, d 83.3, d 148.2, s 201.0, s
85.8, d 91.6, d 89.8, d 77.9, d 76.8, d 143.3, s
40.9, d 41.9, d 39.1, d 43.2, d 39.8, d 131.5, s
129.9, s 130.2, s 128.4, s 130.1, s 138.1, s 127.8, s
36.6, t 122.4, d 114.7, d 123.7, d
3.88, m
3.73, dd,
11.7, 5.5
2.34, m
2.13, m
1.73, m
2.10, m
1.73, m
1.43, m
1.94, m
2.08, m
4.71, s
80.2, d
47.0, d
72.9, s
44.1, t
3
4
2.06, m
2.26, m
1.65, m
2.06, m
1.77, m
1.60, m
1.88, m
1.98, m
4.74, s
4.64, s
1.73, s
1.21, d, 7.2
4.39, d, 7.8
3.24, m
3.35, m
3.26, m
2.12, m
2.14, m
1.65, m
2.12, m
1.62, m
1.47, m
1.76, m
1.93, m
4.69, s
2.07, m
2.26, m
1.77, m
2.26, m
1.69, m
1.60, m
1.88, m
1.98, m
4.73, s
33.8, t
32.5, t
30.7, t
32.3, t 127.7, d 33.4, t
6a
6b
7
43.2, d 42.4, d 40.0, d 41.7, d 44.3, d 43.6, d 40.6, d
28.8, t
31.5, t
27.8, t
30.4, t
26.3, t
29.1, t
27.6, t
30.3, t
28.1, t
29.0, t
29.1, t
30.9, t
32.8, t
33.3, t
8a
8b
9a
9b
12a
12b
13
15
1′
2′
3′
4′
5′
10 126.0, s 125.8, s 124.2, s 125.7, s 136.9, s 134.7, s 166.2, s
11 151.1, s 150.1, s 148.5, s 149.8, s 149.3, s 151.1, s 150.4, s
12 109.2, t 109.7, t 109.2, t 109.6, t 111.9, t 109.5, t 110.0, t
13
15
20.9, q 21.5, q 20.9, q 21.4, q 21.6, q 20.9, q 21.2, q
18.5, q 17.1, q 16.4, q 17.2, q 14.0, q 12.8, q 10.6, q
4.61, s
1.69, s
4.62, s
1.73, s
1′ 104.4, d 105.7, d 103.9, d 102.8, d 103.6, d 107.6, d 104.9, d
1.73, s
2′
3′
4′
5′
6′
74.7, d 75.5, d 73.7, d 74.9, d 74.9, d 75.6, d 74.7, d
78.0, d 78.2, d 76.8, d 77.9, d 78.1, d 78.4, d 78.3, d
71.6, d 71.5, d 69.9, d 71.5, d 71.6, d 71.2, d 71.1, d
78.0, d 78.1, d 76.7, d 77.7, d 78.0, d 78.1, d 77.8, d
1.08, d, 7.2
4.32, d, 8.0
3.21, m
3.28, m
3.27, m
1.13, d, 6.8
4.24, d, 7.8
3.07, m
3.12, m
2.99, m
3.12, m
3.62, dd,
11.1, 1.9
3.41, dd,
11.1, 5.1
1.16, d, 6.8
4.36, d, 8.0
3.21, m
3.28, m
3.21, m
3.35, m
3.85, dd,
11.5, 2.0
3.66, dd,
11.5, 5.4
62.6, t
62.6, t
60.9, t
62.6, t
62.6, t
62.5, t
62.5, t
a
Data were reported in CD3OD.
Data were recorded in DMSO-d6.
3.34, m
3.39, m
3.86, dd, 11.5, 1.8
b
6′a
3.87, dd,
11.7, 2.2
3.63, dd,
11.7, 5.5
6′b
3.68, dd, 11.5, 5.1
Data Centre. Copies of these data can be obtained free of
charge via www.ccdc.cam.ac.uk/conts/retrieving.htm (or
from the Cambridge Crystallographic Data Centre, 12, Union
Road, Cambridge CB21EZ, U.K.; fax: (+44) 1223-336-033; or
deposit@ccdc.cam.ac.uk).
a
Data were reported in CD3OD.
Data were recorded in DMSO-d6.
b
R1 values were 0.2022 (I N 2σ (I)) and 0.2407 (all data). The
final wR (F2) values were 0.4110 (I N 2σ (I)) and 0.4483 (all
data). The goodness of fit on F2 was 1.606, flack parameter
was 0.5 (5), the Hooft parameter was 0.2 (2) for 5300 Bijvoet
pairs.
Crystallographic data for the structures of 1 (deposition
no.: CCDC 978782) and 4 (deposition no.: CCDC 978783) (Fig.
1) have been deposited in the Cambridge Crystallographic
Nicotabacoside E (5): White powder, [α]2D0: +12.4 (c 0.20,
MeOH); UV (MeOH) λmax (log ε) 280 (3.49) nm; ECD (MeOH)
Δε
+ 5.12, Δε
+ 6.58, Δε
− 3.05, Δε
+ 0.31,
195
196
208
219
Δε
+ 0.72; IR (KBr) vmax 3422, 1642, 1483, 1451, 1294,
230
1070 cm−1
;
1H (600 MHz) and 13C (150 MHz) NMR data
(see Tables 2 and 3); HRESIMS m/z 403.1750 [M + Na]+ (calcd
for C20H28O7Na, 403.1727, +2.3 mDa).
Nicotabacoside F (6): White powder, [α]2D0: +54.8 (c 0.20,
MeOH); UV (MeOH) λmax (log ε) 230 (4.00) nm; ECD
(MeOH) Δε
+ 1.53, Δε
− 1.62, Δε
+ 0.93,
195
198
215
Table 2
Δε
+ 0.64, Δε
+ 0.91; IR (KBr) vmax 3425, 1680,
221
218
1H NMR data for compounds 4–6 in CD3OD (δ in ppm, J in Hz).
1641, 1381, 1165, 1078, 1027 cm−1 1H (500 MHz) and 13C
;
(125 MHz) NMR data (see Tables 2 and 3); HRESIMS m/z
421.1786 [M + Na]+ (calcd for C20H30O8Na, 421.1833,
−4.7 mDa).
No.
4
5
6
1
2
3
4
6a
6b
7
8a
8b
9a
9b
12a
5.47, br.s
6.44, s
5.79, s
4.20, br.d, 6.7
3.19, dd, 9.4, 6.7
2.31, m
4.45, d, 12.4
2.07, dq, 12.4, 6.7
2.25, dd, 9.6, 2.6
1.28, dd, 9.6, 3.2
2.55, m
1.91, m
1.37, m
2.44, ddd, 14.2, 3.1, 3.1
2.70, ddd, 14.2, 5.1, 3.3
4.74, s
4.67, s
1.75, s
1.23, d, 6.7
4.37, d, 7.8
3.28, m
3.23, m
3.38, m
3.37, m
5.60, br.s
2.67, m
2.34, m
2.30, m
1.93, m
1.53, m
2.71, m
1.65, m
4.76, s
2.4. Acid hydrolysis and sugar identification
2.86, br.s
1.73, m
1.61, m
2.34, m
2.20, m
4.81, s
Compounds 2, 3, 5 and 6 (each 1 mg) were hydrolyzed
with 1.0 M HCl (MeOH–H2O, 1:1, 1 ml) under reflux for 2 h.
The reaction mixture was neutralized by NaHCO3, and
partitioned between CH2Cl2 and H2O. The H2O part was
separated on a silica gel CC (8 g, 1.0 × 40 cm) using H2O–
MeOH–CHCl3 (3:30:70) as the eluent to give glucose. The
glucoses from compounds 2, 3, 5 and 6 were determined to
be D-glucoses by PC comparison with the authentic sample
(H2O–AcOEt–BuOH 5:1:4, upper layer, Rf 0.45; H2O–PhOH
1:4, Rf 0.50) that was visualized by spraying with phthalic
acid–aniline reagent (1.66 g phthalic acid and 0.93 g aniline
dissolved in 100 ml H2O–sat. BuOH), followed by heating and
their [α]2D1 values [+50.4 (c 0.09, MeOH), 49.8 (c 0.07,
12b 4.65, s
13
15
1′
2′
3′
4′
5′
6′a
6′b
1.76, s
1.80, s
2.22, s
1.22, d, 6.5
4.41, d, 7.8
3.22, m
3.28, m
3.27, m
4.46, d, 7.8
3.52, m
3.26, m
3.45, m
3.42, m
3.32, m
3.88, dd, 11.7, 2.2 3.85, dd, 11.9, 2.2 3.80, dd, 11.9, 2.1
3.65, dd, 11.7, 5.7 3.75, dd, 11.9, 4.9 3.67, dd, 11.9, 4.9

84
C.-Y. Yang et al. / Fitoterapia 96 (2014) 81–87
Fig. 1. The structures of compounds 1–6.
MeOH), +51.0 (c 0.08, MeOH) and 49.2 (c 0.06, MeOH)],
respectively [15].
(1079, 1031 cm−1) groups were deduced by its IR spectrum.
Acid hydrolysis of compound 2 provided a D-glucose moiety
which was deduced by its [α]D value [+50.4 (c 0.09, MeOH)]. In
addition to a β-D-glucosyl group, the NMR data of its aglycone
part were similar to those of rishitin except for the down-fielded
shift of C-3 from 79.2 in rishitin to 89.8 in 2 [16–18], revealing
that the glucosyl group was connected to C-3. This deduction
was affirmed by the HMBC correlation from H-1′ (δH 4.24) to
C-3 (δC 89.8). ROESY correlations (Fig. 3) of H-3 (δH 3.08)/H-15
(1.13), H-4 (2.12)/H-6b (1.65)/H-12a (4.69) indicated that its
relative configuration of C-3, C-4, and C-7 was identical with
those of compound 1. Different from compound 1, the ROESY
correlations of H-2/Me-4 in compound 2 were not detected, and
the correlations of H-2/H-1a and H-2/H-1b were observed,
which suggested that OH-2 was α-oriented. In the ECD
spectrum of 2, the first cotton effect (CE) is positive and the
second CE is negative, which is identical to those of compound 1.
Therefore, its absolute configuration was determined to be 2R,
3R, 4S and 7R (see Fig. S1 in Supporting Information).
Nicotabacoside C (3) had a molecular formula of C20H32O7
by the quasi-molecular ion peak at m/z 407.2038 [M + Na]+
in its positive HRESIMS. Its IR spectrum showed hydroxyl
(3408 cm−1), double-bond (1644 cm−1) and ether-bond
(1076, 1042, 1023 cm−1) groups. The D-glucose from acid
hydrolysis of compound 3 was revealed by its [α]D value
[49.8 (c 0.07, MeOH)]. The 1H and 13C NMR spectral data of 3
(Tables 1 and 3) were similar to those of 2 except for the
obvious down-fielded shift of C-2 from 70.6 in 2 to 80.3 in 3
and up-fielded shift of C-3 from 91.6 in 2 to 77.9 in 3,
suggesting the glucosyl group at C-2 which was confirmed by
3. Results and discussion
Nicotabacoside A (1) had a quasi-molecular ion peak at m/z
407.2040 [M + Na]+ in the positive HRESIMS, indicating
the molecular formula of C20H32O7 with five degrees of
unsaturation. Its IR spectrum exhibited hydroxyl (3423 cm−1),
double-bond (1642 cm−1) and ether-bond (1077, 1031 cm−1
)
groups. Two methyls (δH 1.73, s and 1.08, d, J = 7.2 Hz), two
protons of exocyclic double-bond (4.71, s) as well as one
anomeric proton (4.32, d, J = 8.0 Hz) were observed by its 1H
NMR spectrum. Besides a set of signals due to β-D-glucosyl
group, its 13C NMR (DEPT) spectrum showed 2 methyls, 5
methylenes, 4 methines and 3 quaternary carbons. The NMR
data of its aglycone part (Tables 1 and 3) were similar to those of
rishitin except for the obvious down-fielded shift of C-3 from
79.2 in rishitin to 85.8 in 1, indicating that the glucosyl group
was linked to C-3 [16–18]. This deduction was verified by the
HMBC correlation from H-1′ (δH 4.32) to C-3 (δC 85.8). ROESY
correlations of H-2 (3.88)/H-15 (1.08), H-3 (3.73)/H-15 (1.08)
and H-4 (2.34)/H-6b (1.73)/H-12a (4.71) suggested that H-2,
C-15 and H-7 were situated at the same face (Fig. 3). Finally, its
absolute configuration was determined by an X-ray single-
crystal diffraction to be 2S, 3R, 4S and 7R (Fig. 4).
Nicotabacoside B (2) showed a quasi-molecular ion peak
at m/z 407.2053 [M + Na]+ in the positive HRESIMS,
suggesting the molecular formula of C20H32O7. Hydroxyl
(3473, 3406 cm−1), double-bond (1643 cm−1) and ether-bond
Fig. 2. The key 1H–1H COSY and HMBC correlations of compounds 1–6.

C.-Y. Yang et al. / Fitoterapia 96 (2014) 81–87
85
Fig. 3. The key ROESY correlations of compounds 1–3 and 6.
HMBC correlation from H-1′ (δH 4.36) to C-2 (80.3). ROESY
correlations of H-3 (δH 3.32)/H-15 (1.16)/H-6a (2.26) and
H-6b (1.77)/H-12a (4.73) suggested that the C-2, C-3, C-4
and C-7 of 3 had the same relative configuration with those of
2. Thus, the stereochemistry of 3 was determined to be 2R, 3R,
4S and 7R by comparing the ECD spectrum with 2 (see Fig. S1 in
Supporting Information).
produced the D-glucose by its [α]D value [+51.0 (c 0.08,
MeOH)]. Its 1H NMR spectrum indicated two methyls (δH 1.80,
s and 2.22, s), a penta-substituted phenyl ring (6.44, s) and one
anomeric proton (4.46, d, J = 7.8 Hz). The 13C NMR data
showed the presence of one set of β-D-glucosyl signals
Nicotabacoside D (4) had a quasi-molecular ion at m/z
405.1879 [M + Na]+ in the positive HRESIMS, indicating
the molecular formula of C20H30O7 with six degrees of
unsaturation. The presence of conjugated double-bonds was
speculated by its UV spectrum [λmax (log ε) 241 (4.45) nm]
while hydroxyl (3419 cm−1), double-bond (1643 cm−1) as
well as ether-bond (1079, 1030 cm−1) groups were revealed
by its IR spectrum. The 1H and 13C NMR data of 4 were similar
with 3 except that two methylenes (δC 36.6, C-1; 32.3, C-6) in 3
were replaced by two olefinic carbons (δC 122.4, C-1; 127.7,
C-6) in 4, suggesting that Δ5, 10 double-bond in 3 was changed
10
6
to be Δ1,
and Δ5, double-bonds in 4. This speculation
was reinforced by the key 1H–1H COSY correlations of H-1
(δH 5.47)/H-2 (4.20) and H-6 (5.60)/H-7 (2.86) together with
the key HMBC correlations of H-1 (5.47)/C-3 (δC 76.8), C-5
(138.1) and C-9 (29.0); H-6 (5.60)/C-4 (39.8), C-8 (28.1) and
C-10 (136.9) (Fig. 2). In addition, the glucosyl group attaching
to C-2 was confirmed by the HMBC correlation from H-1′
(4.41) to C-2 (83.3). The absolute configuration of compound 4
was elucidated as 2R, 3R, 4S and 7R by an X-ray single-crystal
diffraction (Fig. 4).
Nicotabacoside E (5) had a molecular formula of C20H28O7
with seven degrees of unsaturation by its positive HRESIMS
which gave a quasi-molecular ion at m/z 403.1750 [M + Na]+.
The presence of hydroxyl (3422 cm−1), aromatic ring
(1642 cm−1) and ether-bond (1294, 1070 cm−1) groups was
revealed by its IR spectrum. Acid hydrolysis of compound 5
Fig. 4. X-ray crystal structures of compounds 1 and 4.

86
C.-Y. Yang et al. / Fitoterapia 96 (2014) 81–87
(δC 107.6, 75.6, 78.4, 71.2, 78.1, 62.5). The 1H and 13C NMR data
of its aglycone part were similar to those of ligudentatol [19]
except that one methine (C-2) in ligudentatol was changed to
be quaternary carbon in 5, as well as the up-fielded shift of C-3
from 151.3 in ligudentatol to 143.3 in 5. The above analyses
suggested that one glucosyl group was connected to C-3 and
one additional hydroxyl was located at C-2 in 5, which was also
confirmed by the HMBC correlations H-1′ (δH 4.46)/C-3 (143.3)
and H-1 (6.44)/C-2 (δC 148.2), C-3 (143.3) and C-5 (127.8).
Therefore, its absolute configuration was determined to be 7R
by comparing its ECD spectrum with that of compound 4 (see
Fig. S1 in Supporting Information).
(No. 81025023) and the Youth Innovation Promotion Asso-
ciation, CAS.
Appendix A. Supplementary data
HRESIMS, UV, IR, 1H, 13C (DEPT), 2D NMR (HSQC, HMBC,
1H–1H COSY and ROESY) and ECD spectra for nicotabacosides
A–F, X-ray crystal structures of nicotabacosides A and D,
bioassay of compounds 3 and 7–11 on MT1 and MT2 as well
as the eluent for each fraction are available as Supporting
Information. Supplementary data to this article can be found
online at http://dx.doi.org/10.1016/j.fitote.2014.04.010.
Nicotabacoside F (6) had a molecular formula of C20H30O8
with six degrees of unsaturation by means of positive HRESIMS
which exhibited the quasi-molecular ion peak at m/z 421.1786
[M + Na]+. Its IR spectrum exhibited hydroxyl (3425 cm−1),
References
[1] The Editorial Committee of the Administration Bureau of Flora of China.
Flora of China, 67. Beijing: Beijing Science & Technology Press; 2005
152.
[2] Stedman RL. Chemical composition of tobacco and tobacco smoke.
Chem Rev 1968;68:153–207.
[3] Fuchs A, Slobbe W, Mol PC, Posthumus MA. GC/MS analysis of
fungitoxic terpenoids from tobacco. Phytochemistry 1983;22:1197–9.
[4] Wahlberg I, Arndt R, Nishida T, Enzell CR. Tobacco chemistry. 63
syntheses and stereostructures of six tobacco seco-cembranoids. Acta
Chem Scand B 1986;40:123–34.
carbonyl (1680 cm−1) and ether-bond (1078, 1027 cm−1
)
groups. Acid hydrolysis of compound 6 yielded the D-glucose
which was revealed by its [α]D value [+49.2 (c 0.06, MeOH)].
Its 1H NMR spectrum showed signals of two methyls (δH 1.75, s
and 1.23, d, J-6.7 Hz), one double-bond (5.79, s) and one
anomeric proton (4.37, d, J = 7.8 Hz). Besides
a set of
β-D-glucosyl signals, its 13C NMR (DEPT) data were similar
with those of (+)-oxyglutinosone [20,21] except for the
down-fielded shift of C-3 from 75.1 in (+)-oxyglutinosone to
80.2 in 6, which suggested that one additional glucosyl group
was assigned at C-3. The above inference was verified by HMBC
correlation from H-1′ (δH 4.37) to C-3 (δC 80.2). The ROESY
correlations of H-3 (δH 4.45)/H-15 (1.23)/H-6a (2.25) and H-4
(2.07)/H-6b (1.28)/H-12a (4.74) indicated that H-3, H-15,
OH-5 and H-7 were situated on the same side, consistent with
the relative configuration of (+)-oxyglutinosone (Fig. 3).
Consequently, its absolute configuration was determined to
be 3R, 4R, 5R and 7R by comparing its ECD spectrum with that
of compound 4 (see Fig. S1 in Supporting Information).
The known compounds 7–11 were identified to be
actinidioionoside (7) [22,23], byzantionoside B (8) [24], (Z)-4-
[3′-(β-D-glucopyranosyloxyl) butylidene]-3, 5, 5-trimethyl-
2-cyclohexen-l-one (9) [25], (6R, 9R)-3-oxo-α-ionol β-D-
glucopyranoside (10) and (6R, 9S)-3-oxo-α-ionol β-D-
glucopyranoside (11) [26] by comparing their spectral data
with literatures.
[5] Uegaki R, Kubo S, Fujimori T. Stress compounds in the leaves of
Nicotiana undulata induced by TMV inoculation. Phytochemistry
1988;27:365–8.
[6] Enzell CR, Nasiri A, Forsblom I, Johansson E, Lundberg C, Arnarp J, et al.
Tobacco chemistry. 81. Biotransformations of (Z)-abienol using plant
cell cultures of Nicotiana silvestris. Acta Chem Scand 1995;49:375–9.
[7] Xu YJ, Bai CM, Zhong KJ, Huang JG, Tang WY, Lu X, et al. Analysis of
alkaloids in tobacco by gas chromatography–mass spectrometry. Fenxi
Huaxue 2006;34:382–4.
[8] Yang JS. 13C NMR spectroscopic databook of natural products. Beijing:
Chemical Industry Press; 2011 536–715.
[9] Zan K, Chen XQ, Chai XY, Wu Q, Fu Q, Zhou SX, et al. Two new cytotoxic
eudesmane sesquiterpenoids from Artemisia anomala. Phytochem Lett
2012;5:313–5.
[10] Xie WD, Wang XR, Ma LS, Li X, Row KH. Sesquiterpenoids from
Carpesium divaricatum and their cytotoxic activity. Fitoterapia
2012;83:1351–5.
[11] Li GS, Yao F, Zhang L, Yue XD, Dai SJ. Two new cytotoxic sesquiterpenoids
from Solanum lyratum. Chin Chem Lett 2013;24:1030–2.
[12] Liu HY, Ran XH, Gong NB, Ni W, Qin XJ, Hou YY, et al. Sesquiterpenoids
from Chloranthus multistachys. Phytochemistry 2013;88:112–8.
[13] Luo J, Liu XY, Li EW, Guo LD, Che YS. Arundinols A–C and arundinones A
and B from the plant endophytic fungus Microsphaeropsis arundinis. J
Nat Prod 2013;76:107–12.
[14] Inestrosa NC, Godoy JA, Vargas JY, Arrazola MS, Rios JA, Carvajal FJ, et al.
Nicotine prevents synaptic impairment induced by amyloid-β oligomers
through α 7-nicotinic acetylcholine receptor activation. Neuromolecular
Med 2013;15:549–69.
[15] Escalante-Sanchez E, Rosas-Ramirez D, Linares E, Bye R, Pereda-Miranda
R. Batatinosides II–VI, acylated lipooligosaccharides from the resin
glycosides of sweet potato. J Agric Food Chem 2008;56:9423–8.
[16] Masamune T, Murai A, Takasugi M, Matsunaga A, Katsui N, Sato N, et al. The
isolation and structure elucidation. Bull Chem Soc Jpn 1977;50:1201–5.
[17] Feng X, Wang JS, Luo J, Kong LY. Two new sesquiterpene glucosides from
the leaves of Nicotiana tabacum. J Asian Nat Prod Res 2009;11:675–80.
[18] Kodama H, Fujimori T, Kato K. A nor-sesquiterpene glycoside, rishitin-
β-sophoroside, from tobacco. Phytochemistry 1984;23:690–2.
[19] Naya K, Okayama T, Fujiwara M, Nakata M, Ohtsuka T, Kurio S.
Sesquiterpenes from the rhizomes of Ligularia dentata Hara. Bull Chem
Soc Jpn 1990;63:2239–45.
Compounds 1–6 are six new 14-noreudesmane
sesquiterpenoid glycosides, which enrich the constituents of
nor-sesquiterpenoids in N. tabacum. Compounds 3 and 7–11
were evaluated for the agonistic effects on human melatonin
receptor 1 (MT1) on HEK/293/MT1 cell lines and human
melatonin receptor 2 (MT2) on HEK/293/MT2 cell lines in vitro.
However no obviously agonistic activity was observed at the
tested concentrations (see Table S1 in Supporting Information).
Conflict of interest statement
[20] Katsui N, Yagihashi F, Murai A, Masamune T. Structure of oxyglutinosone
and epioxylubimin, stress metabolites from diseased potato tubers. Chem
Lett 1978:1205–6.
There are no conflicts of interests among all authors in
this manuscript.
[21] Murai A, Taketsuru H, Yagihashi F, Katsui N, Masamune T. The structure
and configuration of (+)-glutinosone and (+)-oxyglutinosone. Bull
Chem Soc Jpn 1980;53:1045–8.
[22] Murai F, Tagawa M. Relationship between ionone glycosides and
terpenoids in Actinidia polygama. Abstract Papers of the 33rd Sympo-
sium on the Chemistry of Terpenes, Essential Oils, and Aromatics
(TEAC), Sendai; 1989. p. 68–70.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China for Distinguished Young Scholars

C.-Y. Yang et al. / Fitoterapia 96 (2014) 81–87
87
[23] Otsuka H, Hirata E, Shinzato T, Takeda Y. Stereochemistry of
megastigmane glucosides from Glochidion zeylanicum and Alangium
premnifolium. Phytochemistry 2003;62:763–8.
[25] Khan SH, Mosihuzzaman M, Nahar N, Rashid MA, Rokeya B, Ali L, et al. Three
megastigmane glycosides from the leaves of Pterospermum semisagittatum.
Pharm Biol 2003;41:512–5.
[24] Matsunami K, Otsuka H, Takeda Y. Structural revisions of blumenol C
glucoside and byzantionoside B. Chem Pharm Bull 2010;58:438–41.
[26] Pabst A, Barron D, Sémon E, Schreier P. Two diastereomeric 3-oxo-α-ionol
β-D-glucosides from raspberry fruit. Phytochemistry 1992;31:1649–52.