Absolute structure assignment of an iridoid-monoterpenoid indole alkaloid hybrid from Dipsacus asper

Article abstract of DOI:10.1016/j.fitote.2019.04.015

Iridoid-monoterpenoid indole alkaloid hybrids (IMIAHs)represent a rare class of natural products reported from only several plants of Rubiaceae and Dipsacaceae families, while their structural assignments remain a very challenging work due to complexity and flexibility. In the current study, a new IMIAH (1)was isolated from the roots of Dipsacus asper and its structure with absolute configuration was unambiguously established by a combination of spectroscopic analyses, chemical degradation and ECD calculation. A new oleanane-type triterpenoid saponin (2)and 15 known co-metabolites were also obtained and structurally characterized. Our biological evaluations showed that compound 2 exhibited moderate inhibition against acetylcholine esterase (AChE)with an IC₅₀ value of 15.8 ± 0.56 μM, and compound 15 displayed potent cytotoxicity selectively against human A549 and H157 lung cancer cells with IC₅₀ values of 6.94 ± 0.24 and 9.06 ± 0.12 μM, respectively.

Full text of DOI:10.1016/j.fitote.2019.04.015

Fitoterapia135(2019)99–106
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Absolute structure assignment of an iridoid-monoterpenoid indole alkaloid
hybrid from Dipsacus asper
Zhi-Pu Yua,b, Yin-Yin Wangb, Shu-Juan Yua,b, Jie Baob, Jin-Hai Yub,⁎, Hua Zhangb,⁎
a School of Chemistry and Chemical Engineering, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan 250022, China
b School of Biological Science and Technology, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan 250022, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Iridoid-monoterpenoid indole alkaloid hybrids (IMIAHs) represent a rare class of natural products reported from
only several plants of Rubiaceae and Dipsacaceae families, while their structural assignments remain a very
challenging work due to complexity and flexibility. In the current study, a new IMIAH (1) was isolated from the
roots of Dipsacus asper and its structure with absolute configuration was unambiguously established by a com-
bination of spectroscopic analyses, chemical degradation and ECD calculation. A new oleanane-type triterpenoid
saponin (2) and 15 known co-metabolites were also obtained and structurally characterized. Our biological
evaluations showed that compound 2 exhibited moderate inhibition against acetylcholine esterase (AChE) with
Dipsacus asper
Glucoindole alkaloid
Iridoid glucoside
Cytotoxicity
Calculated ECD
AChE inhibition
an IC50 value of 15.8
A549 and H157 lung cancer cells with IC50 values of 6.94
0.56 μM, and compound 15 displayed potent cytotoxicity selectively against human
0.24 and 9.06 0.12 μM, respectively.
1. Introduction
computations. To the best of our knowledge, it is only the second report
of IMIAH with a C-22 linkage. In addition, a new oleanane-type tri-
terpenoid saponin (2) and 15 known compounds were also isolated and
identified.
The in vitro cytotoxicity of all the isolates were evaluated against
four human cancer cell lines, A549 (lung), H157 (lung), HepG2 (liver)
and MCF-7 (breast), and compound 15 exhibited selective inhibitory
activity against the two lung cancer cells. Moreover, the acetylcholine
esterase (AChE) inhibitory assay established that compound 2 was a
moderate AChE inhibitor. Herein, the isolation, structural elucidation
and biological evaluations of these natural products are described
below.
Indole alkaloids with a non-rearranged iridoid unit, e.g. strictosi-
dine, are key biosynthetic intermediates of the commonly known
monoterpenoid (rearranged) indole alkaloids (MIAs) [1] which have
afforded several famous anticancer drugs such as vinblastine and vin-
cristine. As the number of non-rearranged MIAs increases in the lit-
erature, a few hybrids of them with an extra iridoid moiety have also
been reported [2–5]. Biogenetically, these iridoid-monoterpenoid in-
dole alkaloid hybrids (IMIAHs) originate from the condensation of a
trypotamine/tryptophan with two iridoid glucosides, and so far they
have only been obtained from several plants of Rubiaceae and Dipsa-
caceae families [2–5]. The core structure of a typical IMIAH is as shown
in 5S-5-carboxyvincoside and 5S-5-carboxystrictosidine (Fig. 1), and C-
3, N-4, C-6 and C-22 are the general connecting sites of the second
iridoid unit. These molecules present tremendous flexibilities which
have brought great challenge to their structural elucidation especially
for the assignment of absolute configuration.
2. Experimental section
2.1. General experimental procedures
Optical rotations were measured on a Rudolph VI polarimeter
(Rudolph Research Analytical, Hackettstown, USA) with a 10 cm length
Following our earlier investigation into the EtOAc partition of the
ethanol extract from D. asper [6], the current study on the polar n-BuOH
partition afforded an new IMIAH (1) incorporating a 22-O-iridoid glu-
coside unit via an ester bond. The planar structure of 1 was first es-
tablished via spectroscopic analyses, and the absolute configuration was
further determined by a series of chemical methods and theoretical
cell. UV and CD spectra were obtained on
a
Chirascan
Spectrophotometer (Applied Photophysics Ltd., Leatherhead, UK) with
a 0.1 cm pathway cell. IR spectra were recorded on a VERTEX70
spectrometer (Bruker Optics Inc., Billerica, USA) with KBr disks. NMR
experiments were performed on a Bruker Avance DRX600 spectrometer
⁎ Corresponding authors.
E-mail addresses: bio_yujh@ujn.edu.cn (J.-H. Yu), bio_zhangh@ujn.edu.cn (H. Zhang).
https://doi.org/10.1016/j.fitote.2019.04.015
Received 2 April 2019; Received in revised form 26 April 2019; Accepted 27 April 2019
Availableonline30April2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

Z.-P. Yu, et al.
Fitoterapia135(2019)99–106
OH
NH
OH
NH
O
O
O
H
O
11'
O
O
O
O
O
H
O
H
6'
OH
O
3'
5'
9'
O
O
H
7'
4
5
O
H
22
16
H
H
8' H
NH14
3
1'
NH
NH
H
H
H
H
17
O
10'
7
15
O
O
9
2
20
H
O
1'''
21
O
O
O
19
5'''
6'''
NH
2''' OH
H
H
OH
OH
13
1
18
HO4'''
O
3'''
HO
HO
HO
HO
11
HO
O
1'' HO
2'' OH
HO
HO
5''
6''
4''
HO
HO
3''
5S-5-carboxyvincoside
5S-5-carboxystrictosidine
HO
29
21
30
1
H19
OH
OH
18
OH
HO
OH
OH
22O
11
9
O
HO
O
17
25
13
26
O
1
28
II
Glc-
14
III
Glc-
O
10
5
OH
HO
8
15
H
27
7
O
O
3
O
H
24
23
Ara
OH
HO
O
O
HO
O
Rha
OH
HO
HO
OH
I
Glc-
2
Fig. 1. Structures of compounds 1 and 2.
(Bruker BioSpin AG, Fällanden, Switzerland) and referenced to solvent
peaks (δC 49.00 and δH 3.31 ppm in CD3OD). ESIMS analyses were
carried out on an Agilent 1260–6460 Triple Quad LC-MS instrument
(Agilent Technologies Inc., Waldbronn, Germany). HRESIMS data were
acquired on an Agilent 6520 QTOF mass spectrometer (Agilent
Technologies Inc., Waldbronn, Germany). All HPLC analyses and se-
parations were performed on Agilent 1260 series LC instruments with
1260 MWD detector (Agilent Technologies Inc., Waldbronn, Germany).
All solvents used for column chromatography were of analytical grade
(Tianjin Fuyu Fine Chemical Co. Ltd., Tianjin, China) and solvents used
for HPLC were of HPLC grade (Oceanpak Alexative Chemical Ltd.,
Goteborg, Sweden). Agilent SB-C18 (9.4 × 250 mm) column (Agilent
Technologies Inc., Santa Clara, USA) was used for HPLC separations.
chromatographed on a silica gel CC, eluted with CHCl3-MeOH-EtOAc-
H2O (15:10:40:5, v/v), to afford five fractions (B3a–B3e), which were
purified in turn by RP-18 CC (10% to 60% MeOH-H2O, v/v) and semi-
preparative HPLC using 45% MeCN-H2O (v/v, 3.00 mL/min) to obtain
7 (11.6 mg, tR = 7.0) and 9 (9.1 mg, tR = 16.2 min), and using 55%
MeCN-H2O (v/v, 3.00 mL/min) to afford compounds 10 (22.3 mg,
tR = 12.5 min), 11 (42.6 mg, tR = 14.0 min) and 12 (39.3 mg,
tR = 15.2 min). Fraction B4 (4.1 g) was chromatographed on a RP-18
column, eluted with MeOH-H2O (10% to 60% v/v), to obtain two major
components, which were further purified by semi-preparative HPLC
using 45% MeCN-H2O (v/v, 3.00 mL/min) as mobile phase to yield
compounds 2 (3.0 mg, tR = 7.5 min) and 14 (12.0 mg, tR = 8.6 min).
Fraction C (52.4 g) was separated by silica gel CC, eluted with CHCl3-
MeOH-H2O (65:15:10, v/v), to obtain five fractions (C1–C5). Fraction
C2 (10.2 g) was subjected to RP-18 CC, eluted with MeOH-H2O (20% to
40%, v/v), to give three major fractions (C2a–C2c), each of which was
purified by semi-preparative HPLC, using 25% MeCN-H2O (v/v,
2.2. Plant material
The roots of Dipsacus asper Wall. ex C. B. Clarke (Caprifoliaceae)
were bought in Kunming ‘Juhuayuan’ herbal market (collected in
September 2016 from An'ning county of Yunnan Province, China) and
were authenticated by Prof. Jie Zhou from University of Jinan. A
voucher specimen has been deposited at School of Biological Science
and Technology, University of Jinan (Accession number: npmc-013).
3.00 mL/min) as mobile phase, to afford compounds
4 (6.0 mg,
tR = 9.5 min), 16 (9.5 mg, tR = 11.2 min) and 17 (7.1 mg,
tR = 10.5 min), respectively. Using similar procedures, fraction C4
(15.2 g) was purified by semi-preparative HPLC (3.00 mL/min, 35%
MeCN-H2O, v/v) to yield 3 (144.1 mg, tR = 13.2 min). Fraction C5
(10.5 g) was separated by RP-18 CC, eluted with MeOH-H2O (20% to
40%, v/v), to obtain three fractions (C5a–C5c), which were subse-
quently purified by semi-preparative HPLC (3 mL/min, 40% MeCN-
2.3. Extraction and isolation
H2O, v/v) to obtain
1 (19.3 mg, tR = 12.0 min), 13 (36.4 mg,
The air-dried roots of Dipsacus asper (10 kg) were smashed into
powder and then extracted with 95% EtOH at room temperature for
four times (one week per time). The crude extract (1.0 kg) was parti-
tioned successively with EtOAc/H2O and n-BuOH/H2O to get EtOAc
and n-BuOH layers. After evaporation of the solvent of n-BuOH parti-
tion under reduced pressure, the extract (500 g) was subjected to
column chromatography (CC) over D101 macroporous absorption resin,
eluted with EtOH-H2O (0%, 20%, 50%, and 100%, v/v) to get four
fractions (A, B, C and D). Fraction B (35 g) was subjected to passage
over a silica gel column, eluted with CHCl3-MeOH-H2O (65:15:10, v/v),
to give four subfractions (B1–B4). Fraction B3 (8.8 g) was then
tR = 9.7 min) and 5 (27.3 mg, tR = 8.2 min). Fraction D (45.5 g) was
subjected to silica gel CC, eluted with CHCl3-MeOH-H2O (65:35:10, v/
v), to afford four fractions (D1–D4). Fraction D3 (28.2 g) was separated
firstly using repeated RP-18 CC and further via semi-preparative HPLC
(3 mL/min, 45% MeCN-H2O, v/v) to afford compounds 6 (42.8 mg,
tR = 8.8 min),
tR = 15.3 min).
8
(78.2 mg, tR = 9.6 min) and 15 (14.2 mg,
2.3.1. Dipsaperine (1)
White amorphous powder; [α]D25–137.2 (c 1.1, MeOH); UV (MeOH)
100

Z.-P. Yu, et al.
Fitoterapia135(2019)99–106
Table 1
Table 2
1H (600 MHz) and 13C (150 MHz) NMR data for 1 in methonal-d4.
1H (600 MHz) and 13C (150 MHz) NMR data for 2 in methonal-d4.
Position
δH (J in Hz)
δC
Position δH (J in Hz)
δC
Position δH (J in Hz)
δC
2
3
5
6
130.3
53.4
59.8
24.2
1
2
α 0.94, m
40.1
26.8
2
3
3.64, m
3.64, m
76.8
74.1
4.54, ddd (12.1, 3.0, 2.5)
3.90, dd (12.1, 4.9)
α 3.46, dd (16.2, 4.9)
β 3.04, ddd (16.2, 12.1, 2.5)
β 1.74, m
α 1.89, m
β 1.77, td (13.3, 3.5)
3.61, dd (12.0, 4.5)
3
4
82.4
44.1
4
5
3.76, m
3.84, dd (12.7,3.7)
3.51, dd (12.7, 1.8)
69.8
65.8
7
8
9
10
11
12
13
14
108.6
127.6
119.4
120.8
123.6
112.4
138.7
34.3
7.48, d (7.9)
5
6
1.21, m
48.6
18.7
Rha
1
7.05, dd (7.9, 7.0)
7.14, dd (8.2, 7.0)
7.32, d (8.2)
α 1.45, m
β 1.36, m
1.45, m (2H)
5.18, d (2.0)
101.7
7
8
9
10
11
35.2
41.9
52.6
37.9
22.2
2
3
4
5
6
4.24, dd (3.0, 2.0)
3.88, dd (9.5, 3.0)
3.56, dd (9.5, 9.5)
3.92, dq (9.5, 6.1)
1.25, d (6.1)
71.1
82.9
72.6
70.1
18.1
a 2.46, ddd (15.1, 12.5, 3.0)
b 2.26, ddd (15.1, 12.1, 3.7)
3.11, m
1.35, m
15
16
17
18
32.8
α 1.59, m
109.0
157.4
120.0
β 1.33, m
7.88, br s
12
α 1.24, m
27.2
Glc-I
a 5.40, br d (17.8)
b 5.28, br d (10.6)
5.86, ddd (17.8, 10.6, 7.4)
2.82, m
β 1.58, m
13
14
15
2.28, br d (11.8)
42.3
43.8
30.5
1
2
3
4.50, d (7.8)
3.30, dd (9.2, 7.8)
3.37, dd (9.2, 8.7)
105.9
75.4
77.8
19
20
21
22
23
1′
3′
4′
5′
135.4
45.5
97.7
171.3
173.7
97.8
152.8
113.2
33.0
α 1.18, m
5.93, d (9.2)
β 1.71, m
16
17
18
α 1.44, m
34.6
49.4
138.4
4
5
6
3.33, dd (9.7, 8.7)
71.3
77.8
62.4
β 2.22, dt (13.7, 3.3)
5.27, d (5.2)
7.43, br s
3.30, ddd (9.7, 5.1,
1.7)
3.87, dd (12.0, 1.7)
3.67, dd (12.0, 5.1)
3.12, m
6′
α 1.77, ddd (14.6, 8.2, 5.0)
β 2.36, ddd (14.6, 6.6, 2.4)
5.30, td (5.0, 2.5)
2.17, m
2.09, td (8.7, 5.2)
1.10, d (6.8)
40.7
19
20
21
5.14, s
134.0 Glc-II
33.0
34.2
1
2
5.48, d (8.2)
3.31, dd (9.3, 8.2)
95.8
74.0
7′
8′
9′
10′
11′
OCH3
1′′
2′′
3′′
80.0
41.4
47.2
14.2
169.4
51.9
100.7
74.8
78.1
71.9
79.0
63.2
α 1.60, m
β 2.02, ddd (13.6,
5.8, 3.2)
22
α 1.35, m
34.5
3
3.41, dd (9.3, 8.8)
78.4
β 1.45, m
3.69, s
4.84, d (7.9)
23
24
0.69, s
13.6
64.5
4
5
3.48, dd (9.7, 8.8)
3.52, ddd (9.7, 4.5,
1.9)
70.8
78.0
3.56, d (11.4)
3.31, d (11.4)
0.93, s
3.25, dd (9.3, 7.9)
3.26, dd (9.3, 9.2)
3.43, dd (9.2, 8.9)
3.41, ddd (8.9, 7.1, 2.0)
4.02, dd (11.8, 2.0)
3.70, dd (11.8, 7.1)
4.65, d (7.9)
3.18, dd (9.4, 7.9)
3.25, dd (9.4, 9.3)
3.37, dd (9.3, 8.9)
3.31, ddd (8.9, 6.2, 2.1)
3.89, dd (11.9, 2.1)
3.63, dd (11.9, 6.2)
25
17.9
6
4.13, dd (11.7, 1.9)
3.78, dd (11.7, 4.5)
69.6
4′′
5′′
6′′
26
27
28
29
30
Ara
1.02, s
0.82, s
16.8
15.6
177.0
29.4
30.9
Glc-III
1
2
3
4
5
4.32, d (7.8)
104.9
75.2
77.8
71.6
78.0
3.21, dd (9.2, 7.8)
3.35, dd (9.2, 8.5)
3.28, dd (9.7, 8.5)
3.24, ddd (9.7, 5.6,
2.6)
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
100.4
74.9
78.1
71.8
78.5
62.9
0.99, s
0.97, s
1
4.49, m
105.1
6
3.86, dd (11.8, 2.6)
3.66, dd (11.8, 5.6)
62.8
searches using mixed torsional/Low-mode sampling method with
OPLS3 force field in an energy window of 3.01 kcal/mol were carried
out by the conformational search module in the Maestro 10.2 software,
returning 48 conformers for 1a and 47 conformers for 1b. After elim-
inating the conformers that did not match the observed coupling con-
stants and NOESY data, 6 and 12 conformers were left for 1a and 1b,
respectively. The re-optimization and the following TDDFT calculations
of the re-optimized conformers were all performed with Gaussian 09
software [7] at the B3LYP/6-311G(d,p) level in vacuo. Frequency ana-
lysis was performed as well to confirm that the re-optimized conformers
were at the energy minima. Finally, the SpecDis 1.64 software [8] was
used to obtain the Boltzmann-averaged ECD spectra of 1a and 1b.
λ
max (log ε) 222 (4.02) nm; ECD (c 0.08, MeOH) λ (Δε) 221 (3.10), 236
(−7.07) nm; IR (KBr) νmax 3416, 2924, 2854, 1690, 1630, 1438, 1400,
1076, 744 cm−1; 1H and 13C NMR spectroscopic data (methanol‑d4) see
Table 1; (−)-ESIMS: m/z 931.7 [M - H]−; (+)-HRESIMS: m/z 933.3497
[M + H]+ (calcd for C44H57N2O20, 933.3499).
2.3.2. 3-O-β-D-Glucopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-
arabinopyranosyl-23-hydroxyolean-18-en-28-oic
acid
28-O-β-D-
glucopyranosyl-(1 → 6)-β-D-glucopyranosyl ester (2)
White amorphous powder; [α]D25–30.2 (c 0.2, MeOH); IR (KBr) νmax
3402, 2934, 1736, 1639, 1454, 1382, 1070, 630 cm−1; 1H and 13C NMR
spectroscopic data (methanol‑d4) see Table 2; (−)-ESIMS: m/z 1235.7
[M
- , ; (+)-HRESIMS: m/z 1259.6040
H]− 1271.9 [M + Cl]−
2.5. Determination of D-glucoses of 1
[M + Na]+ (calcd for C59H96O27Na, 1259.6031).
A solution (1.5 mg) of 1 in 2.0 mol/L HCl (3.0 mL) was stirred at
90 °C for 4 h. After removal of excess HCl under reduced pressure, the
residual aqueous mixture was filtered to remove the aglycone part and
then evaporated under reduced pressure to afford the free mono-
saccharides. The obtained monosaccharides were dissolved in ethanol
2.4. ECD calculation of compounds 1a and 1b
The initial conformations of 1a and 1b were established via the
MM2 force field in the ChemDraw_Pro_14.1 software. Conformational
101

Z.-P. Yu, et al.
Fitoterapia135(2019)99–106
and then excess (S)-(−)-1-phenylethylamine and NaBH3CN were
added. With a catalytic amount of acetic acid added, the mixture was
stirred at 40 °C for 3 h. After evaporation of the ethanol, the residue was
stirred with acetic anhydride in pyridine at room temperature for 5 h to
obtain the amino derivatives. Using the same method, the amino deri-
vatives of authentic L- and D-glucoses were obtained. These derivatives
were subjected to HPLC analyses using the following conditions [HPLC
column: Agilent ZORBAX SB-C18, 5 μm, 4.6 × 250 mm; Mobile phase:
MeCN-H2O (60%, v/v); Flow rate: 1.00 mL/min]. As shown in Fig. S34
[Supplementary material (SM)], the retention times of standard D- and
L-glucose derivatives were observed at tR 5.254 and 4.987 min, re-
spectively, while that of the sugar derivative of 1 was observed at tR
5.285 min, thus assigning D-configuration for the glucoses in 1.
m/z 375.1 [M - H]−
.
2.8. Cytotoxic assay
The cytotoxic activities of all the compounds were tested using SRB
method as we described previously [6]. Adriamycin was used as a po-
sitive control.
2.9. AChE inhibitory assay
The AChE inhibitory activities of all the compounds were tested
using modified Ellman's method as we described previously [9]. Tacrine
was used as a positive control.
2.6. Determination of D-glucoses, L-arabinose and L-rhamnose of 2
3. Results and discussion
Using the same procedure as 1, the amino derivatives of the hy-
drolytic sugars from 2 and their corresponding standard ones (D- and L-
glucoses, D- and L-arabinoses, and L-rhamnose) were prepared. These
derivatives were then subjected to HPLC analyses using the following
conditions [HPLC column: Agilent ZORBAX SB-C18, 5 μm,
4.6 × 250 mm; Mobile phase: MeCN-H2O (50%, v/v); Flow rate:
1.00 mL/min]. As shown in Fig. S35 (SM), the retention times for
standard D- and L-glucoses, D- and L-arabinoses, and L-rhamnose were at
tR 7.597, 7.219, 6.361, 6.257 and 8.652 min, respectively. The retention
times of hydrolytic samples were observed at tR 7.595, 6.247 and
8.632 min, and thus the D-glucoses, L-arabinose and L-rhamnose were
determined for 2.
Compound 1 was obtained as a white amorphous powder. Its mo-
lecular formula of C44H56N2O20 with 18 indices of hydrogen deficiency
was deduced from the protonated molecule ion peak at m/z 933.3497
[M + H]+ (calcd 933.3499) in (+)-HRESIMS analysis and 13C NMR
data. The IR spectrum showed strong absorption bands at 3416, 1690
and 1630 cm−1 attributable to hydroxyl, ester carbonyl and olefinic
groups, respectively. Two anomeric proton signals at δH 4.84 and 4.65
(both d, J = 7.9 Hz), along with the corresponding anomeric carbon
signals observed at δC 100.7 and 100.4, suggested the presence of two
monosaccharide units. Analysis of 1D TOCSY experiments by exciting
the aforementioned anomeric protons (Figs. S13 and S14, SM) returned
two separate coupling networks, which together with the 1He1H COSY
and HSQC data enabled the unambiguous assignments of 1H and 13C
NMR data for the sugar moieties (Table 1). Such information revealed
that both monosaccharide units were glucopyranoses [3,10], with β-
anomeric configurations being determined by the coupling constants of
anomeric protons (both J = 7.9 Hz) [11]. Further HPLC analyses of the
(S)-(−)-1-phenylethylamine derivatives from the hydrolyzed sugars
and authentic D- and L-glucoses (Fig. S34, SM) permitted the assignment
of D-configurations for both monosaccharide units. Besides those as-
signable to the sugar part, the 1H and 13C NMR data (Table 1) also
displayed signals for a carboxyl (δC 173.7), two ester carbonyls (δC
171.3, 169.4), an ortho disubstituted phenyl (δH 7.48, d, J = 7.9 Hz;
7.32, d, J = 8.2 Hz; 7.14, dd, J = 8.2, 7.0 Hz; 7.05, dd, J = 7.9, 7.0 Hz),
four double bonds including a monosubstituted terminal one (δH 5.40,
br d, J = 17.8 Hz; 5.28, br d, J = 10.6 Hz; 5.86, ddd, J = 17.8, 10.6,
7.4 Hz), two trisubstituted ones (δH 7.88, 7.43, each br s) and a tetra-
substituted one, two acetal protons (δH 5.93, d, J = 9.2 Hz; 5.27, d,
J = 5.2 Hz), three oxygenated or ammoniated methines (δH 5.30, 4.54,
3.90), and a secondary methyl (δH 1.10, d, J = 6.8 Hz). These func-
tionalities and the two glucopyranoses accounted for 13 out of 18
2.7. Alkaline hydrolysis
Pure compound 1 (5.0 mg) was digested with 5% NaOH solution
(3.0 mL) at 80 °C for 3 h. The reaction mixture was neutralized with 5%
HCl solution and then concentrated to dryness. The residue was sus-
pended with ethanol and then filtered to remove NaCl. Compounds 1a
(2.0 mg, tR = 12.3 min) and 1b (1.0 mg, tR = 8.1 min) were finally ob-
tained by HPLC purification using 30% MeCN-H2O (v/v) as mobile
phase.
2.7.1. 1a
White amorphous powder; UV (MeOH) λmax (log ε) 221 (3.96) nm;
ECD (c 0.05, MeOH) λ (Δε) 218 (1.09), 233 (−6.11) nm; (−)-ESIMS:
m/z 559.3 [M - H]−
.
2.7.2. 1b
White amorphous powder; UV (MeOH) λmax (log ε) 232 (3.91) nm;
ECD (c 0.04, MeOH) λ (Δε) 224 (−2.34), 253 (0.21) nm; (−)-ESIMS:
O
5'
9'
O
1H-1H
22
Unit A
Unit B
COSY
HMBC
23
6
11'
16
17
21
6'
15
14
OH
20
5
23
O
6
NOESY
3'
7'
19
3
O
O
B
C
5
O
4
b
d
22
NH
2
8'
1'
15
14
O
10'
17
8
c
3
1
A
2
9
O
O
1'''
OH
9
13
3'
19
18
5'''
6'''
4'''
NH
1
21
2'''
O
HO
a
3'''
12
1'
11
HO
5'
O
6'
HO
1''
OH
9'
8'
Unit A
Unit B
5''
6''
4''
2''
3''
HO
7'
HO
HO
Fig. 2. (A) 1He1H COSY and Key HMBC correlations for 1; (B) Key NOESY correlations for 1.
102

Z.-P. Yu, et al.
Fitoterapia135(2019)99–106
O
H
O
OH
NH
O
HO
H
O
OH
O
OH
NH
O
H
O
O
O
O
H
O
H
HO
O
H
O
H
O
H
O
H
O
NaOH
NH
H
H
H
+
O
NH
OH
H
H
HO
H
2O
O
O
OH
OH
HO
O
HO
OH
HO
HO
HO
HO
HO
HO
HO
HO
HO
1
1a
Scheme 1. Alkaline hydrolysis of 1 into 1a and 1b.
1b
indices of hydrogen deficiency, thus requiring another five rings in the
aglycone part of 1. Further comprehensive analyses of 2D NMR data
(Fig. 2) enabled the assembly of the aforementioned functionalities. In
detail, examination of the 1He1H COSY data for the aglycone part of 1
(Fig. 2A) furnished four spin-spin coupling systems (a–d). The frag-
ments (a) and (b), together with the HMBC correlations from H-9 to C-7
(δC 108.6), C-8 (δC 127.6) and C-13 (δC 138.7), H2–6 to C-2 (δC 130.3),
C-7, C-8 and C-23 (δC 173.7), H-3 to C-2, C-5 (δC 59.8) and C-7, and H-
12 to C-8 and C-13, allowed the construction of a tetrahydro-β-carbo-
line-5-carboxylic acid moiety. Afterwards, the fragment (c) along with
the HMBC correlations from H-17 to C-15 (δC 32.8), C-16 (δC 109.0), C-
21 (δC 97.7) and C-22 (171.3), as well as H-21 to C-1′′ (δC 100.7), as-
sembled another structural motif of a seco-iridoid glucoside and also set
up the unit A (Fig. 2A) in 1 as shown. Accordingly, the unit B of an
iridoid glucoside residue was established by fragment (d) together with
the HMBC correlations from H-3′ to C-1′ (δC 97.8), C-4′ (δC 113.2), C-5′
(δC 33.0) and C-11′ (δC 169.4), H-1′ to C-1′′′ (δC 100.4), and OCH3 to C-
11′. Finally, the key HMBC cross-peak from H-7′ to C-22 indicated that
units A and B were connected with each other via an ester bond. The
whole planar structure of 1 was thus elucidated as shown and was the
same as that of pterocephaline obtained from Pterocephalus pinardii [3].
Comparison of their NMR data revealed that compound 1 differed from
pterocephaline mainly in the tetrahydro-β-carboline-5-carboxylic acid
moiety. Two downfield proton resonances at δH 4.54 (ddd, J = 12.1,
3.0, 2.5 Hz, H-3) and 3.90 (dd, J = 12.1, 4.9 Hz, H-5) in 1 were ob-
served, in place of those of δH 3.71 (m, H-3) and 3.38 (dd, 13.2, 5.8 Hz,
H-5) in pterocephaline, suggesting that the configuration at C-3 in 1
was inverted. However, the structural elucidation of pterocephaline
was assigned only by NMR spectroscopic analyses and data comparison
without additional evidences [3]. Moreover, compared with those of
5S-5-carboxyvincoside (Fig. 1), the H-3 and H-5 resonances in pter-
ocephaline displayed quite obvious differences with each ΔδH of 0.46
and 0.94 ppm [12], which seemed too large to be convincing. Such
inconsistency of NMR data together with the increasing reports of en-
antiomorphism in nature reminded us that it was not rigorous to per-
form the structural elucidation of 1 simply via NMR data comparison,
and the structure of 1 required to be systematically investigated using
more strict and credible methods.
14a,b (12.1, 3.0 Hz), assigned the α-direction for H-3 and H-5. For unit B,
the diagnostic NOESY correlation of H-9′/H-5′ revealed that rings B and
C were cis-fused, and the two protons were arbitrarily assigned to be β-
oriented. The relative configurations of other chiral centers were fur-
ther established as shown on the basis of the strong NOESY correlations
of H-5′ with H-6'β, H-6'α with H-7′ and H-1′, and H-8′ with H-1'and H-
7′, which was consistent with loganin (5) [13] as also supported by
excellent NMR comparisons.
The absolute configuration of 1 was eventually established by se-
parate assignments of its degraded products 1a and 1b via comparing
their respective experimental and calculated ECD data, the latter being
acquired by using the time-dependent density functional theory elec-
tronic circular dichroism (TDDFT-ECD) method [14]. It should be noted
that taking the whole molecule to perform ECD calculation would oc-
cupy inestimable computing time due to the reasons listed below: (i) it
would generate a great mass of conformations in the conformational
search procedure due to the flexibility of the whole molecule; (ii) it
would geometrically increase the computing time for each of the con-
formations in the conformational optimization and ECD calculation
procedures due to the large number of atoms; (iii) it would use double
computing time due to the unassigned relative configuration of the
whole molecule. Fortunately, compound 1 was formed by an indole
alkaloid glucoside and an iridoid glucoside via an ester bond, so we
could selectively cleave the ester bond through alkaline hydrolysis. As
described in Scheme 1, compound 1 was hydrolyzed into 1a and 1b,
and all their ECD spectra were presented in Fig. 3. It was found that the
ECD curve of 1a was much closer than 1b to that of 1, demonstrating
that unit A had a dominant contribution to the final ECD data of 1.
The OPLS3 force field with an energy window of 3.01 kcal/mol was
used
to
perform
the
conformational
search
of
(3S,5S,15S,16R,21S,1′S,2′R,3′S,4′S,5′R)-1a to obtain 49 conformers.
The set of NOEs described in Fig. 2B was employed as a postprocessor
Due to the rotational nature of the ester bond linking units A and B,
the whole relative configuration of 1 was unable to be directly estab-
lished, but those of units A and B could be assigned separately by
analyzing the NOESY data (Fig. 2B). For unit A, the key strong NOESY
correlation between H-21 and H-14a revealed that the CH2–14 group
and H-21 were quasi-axially bonded and located at the same side of the
half-chair-like A-ring and were randomly assigned an α-orientation,
thus leaving H-15 β-directed. Subsequently, the cross-peaks of H-14a/
H-19, H-14b/H-18b and H-18a/H-20 indicated that the terminal double
bond was α-oriented and located as shown in Fig. 2B. Then the strong
correlations of H-14a/H-3 and H-3/H-5, along with the values of J3/
Fig. 3. ECD curves of compounds 1, 1a and 1b.
103

Z.-P. Yu, et al.
Fitoterapia135(2019)99–106
(Fig. 4) furnished a negative Cotton effect at 234 nm and a positive
Cotton effect at 219 nm, well matching the experimental ECD spectrum
of 1a (Fig. 4). The absolute configuration of 1a was then assigned as
shown. Using the same force field and energy window as those of 1a,
the conformational search of (1S,5S,7S,8R,9S,1′S,2′R,3′S,4′S,5′R)-1b
gave 47 conformers. After removal of the conformers that did not sa-
tisfy the NOEs (Fig. 2B), the remaining 12 conformers were successively
re-optimized and ECD calculated using the same methods as those for
1a. The Boltzmann-averaged ECD spectrum of 1b was highly consistent
with the experimental one (Fig. 5), thus unambiguously assigning the
absolute configuration of 1b as shown. The structure of 1 was thus
completely characterized, and this compound was named dipsaperine.
Compound 2 had a molecular formula of C59H96O27 as confirmed by
the (+)-HRESIMS ion peak at m/z 1259.6040 ([M + Na]+, calcd
1259.6031) and the 13C NMR data. The 1H and 13C NMR spectra
showed diagnostic signals for five anomeric protons (δH 4.50, 4.49,
4.32, 5.18 and 5.48) and five corresponding carbons (δC 105.9, 105.1,
104.9, 101.7 and 95.8), revealing that the structure of 2 contained five
monosaccharide units. Further analyses of the 1D-TOCSY (Figs. S29-
S33, SM), 2D-HSQC and TOCSY-HSQC (Figs. S22 and S23, SM), and 2D-
COSY and HMBC data (Fig. 6A) enabled the assignments of 1H and 13C
NMR data for the aforementioned sugar parts (Table 2). Based on these
observations and the HPLC analysis of the sugar derivatives (Fig. S35,
SM), the monosaccharides in 2 were finally identified as one α-L-ara-
binopyranose (Ara), one α-L-rhamnopyranose (Rha) and three β-D-glu-
copyranoses (Glc) [11,15]. Besides the sugar units, the aglycone moiety
featuring a 23-hydroxy-oleanane type triterpenoid skeleton was also
uncovered by the NMR data (Table 2), where the diagnostic signals for
an ester carbonyl (δC 177.0), a trisubstituted double bond (δH 5.14, s; δC
138.4, 134.0), an oxymethine (δH 3.61; δC 82.4), an oxygenated me-
thylene (δH 3.56, 3.31; δC 64.5) and six tertiary methyls (δH 1.02, 0.99,
0.97, 0.93, 0.82 and 0.69, each 3H, s), were clearly resolved. The planar
structure of 2 was further confirmed by inspection of the HMBC data
(Fig. 6A). Particularly, the correlations from H-19 to C-13 (δC 42.3), C-
17 (δC 49.4) and C-18 (δC 138.4), and H3–29/30 to C-19 (δC 134.0),
located the double bond at Δ18. Furthermore, the HMBC data revealed
long-rang correlations between C-3 (δC 82.4) of the aglycone and H-1
(δH 4.49) of the Ara, as well as between C-2 (δC 76.8) of the Ara and H-1
(δH 5.18) of the Rha, suggesting the connections of the three structural
fragments as shown. In addition, C-4 (δC 72.6) of the Rha showed a
HMBC correlation with H-1 (δH 4.50) of the Glc-I, and thus the con-
struction of a trisaccharide side chain at C-3 of the aglycone moiety was
completed. Similarly, a disaccharide side chain was linked to C-28
based on the HMBC correlations from H-1 (δH 4.32) of the Glc-III to C-6
(δC 69.6) of the Glc-II and H-1 (δH 5.48) of the Glc-II to C-28 (δC 177.0)
of the aglycone.
Fig. 4. Experimental ECD spectrum of 1a (black solid) compared with the
calculated ECD spectra of 1a (red dashed) and its enantiomer (blue dashed).
Calculated spectra were plotted as sums of Gaussians with a 0.20 eV ex-
ponential half-width. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
Fig. 5. Experimental ECD spectrum of 1b (black solid) compared with the
calculated ECD spectra of 1b (red dashed) and its enantiomer (blue dashed).
Calculated spectra were plotted as sums of Gaussians with a 0.35 eV ex-
ponential half-width. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
filter to eliminate conformers that did not satisfy the observed NOEs.
The filtered conformational conformers were then re-optimized with
density functional theory (DFT) at the B3LYP/6-311G(d,p) level. Using
these DFT-optimized conformers, the quantum-mechanical ECD calcu-
lations were carried out with the method of TDDFT at the B3LYP/6-
311G(d,p) level. Finally, the Boltzmann-averaged ECD spectrum of 1a
30
29
COSY
21
OH
OH
OH
18
17
HO
HMBC
OH
OH
11
O
HO
O
28
O
25
26
13
O
NOESY
OH
1
9
O
10
5
15
3
27
7
O
O
O
HO
30
20
23
24
OH
12
11
25
10
HO
19
18
21
13
26
1
O
O
HO
HO
O
17
2
9
24
HO
8
OH
5
14
27
29
OH
15
7
3
4
6
23
A
B
Fig. 6. (A) 1He1H COSY and Key HMBC correlations for 2; (B) Key NOESY correlations for 2.
104

Z.-P. Yu, et al.
Fitoterapia135(2019)99–106
The relative configuration of the aglycone of 2 was established
Acknowledgements
mainly by examination of ROESY data (Fig. 6B). The coupling constants
of H-3 with H2–2 (J2β/2α,3 = 12.0, 4.5 Hz) revealed that H-3 and H-2β
were α- and β-axially directed in the chair-like A-ring, respectively
[16,17]. It was followed by the assignment of α-orientation for H-5, H-
1α, H-9, H3-27, H-16α and H3-29 as deduced from the ROESY correla-
tions of H-3/H-5, H-5/H-1α and H-9, H-9/H3-27, H3-27/H-16α and H-
16α/H3-29. In turn, the ROESY cross-peaks of H-2β/H3-25 and H3-24,
H3-24/H-6β, H3-25/H-11β, H-11β/H3-26, and H3-26/H-13 and H-15β,
allowed these protons to be β-oriented. The structure of 2 was thus
characterized as 3-O-β-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-
(1 → 2)-α-L-arabinopyranosyl-23-hydroxyolean-18-en-28-oic acid 28-O-
β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl ester.
This work was funded by Natural Science Foundation of Shandong
Province [No. JQ201721 & ZR2018BB023], the Young Taishan Scholars
Program [No. tsqn20161037], National Natural Science Foundation of
China [No. 21672082], Innovation Team Project of Jinan Science &
Technology Bureau [No. 2018GXRC003] and Shandong Talents Team
Cultivation Plan of University Preponderant Discipline [No. 10027].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2019.04.015.
Fifteen known compounds (Fig. S1, SM) were identified to be coc-
culoside (3) [18], swerosid (4) [19], loganin (5) [13], hederagenin 28-
O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (6) [20], 3-O-α-L-
arabinopyranosylhederagenin 28-O-β-D-glucopyranoside (7) [21], 3-O-
α-L-arabinopyranosylhederagenin 28-O-β-D-glucopyranosyl(1 → 6)-β-D-
glucopyranoside (8) [21], 3-O-α-L-arabinopyranosyloleanolic acid 28-
O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (9) [22], 2′-O-
acetyl-3-O-α-L-arabinopyranosylhederagenin 28-O-β-D-glucopyranosyl-
(1 → 6)-β-D-glucopyranoside (10) [23], 3′-O-acetyl-3-O-α-L-arabinopyr-
anosylhederagenin 28-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyrano-
side (11) [24], 4′-O-acetyl-3-O-α-L-arabinopyranosylhederagenin 28-O-
β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (12) [25], elmalieno-
side B (13) [26], macranthoidin A (14) [15], 3-O-[β-D-xylopyranosyl-
(1 → 4)-β-D-glucopyranosyl-(1 → 4)][α-L-rhamnopyranosyl-(1 → 3)]-β-
D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyr-
anosylhederagenin (15) [27], 8′-hydroxypinoresinol-4′-O-β-D-gluco-
pyranoside (16) [28] and 8-hydroxypinoresinol-4′-O-β-D-glucopyrano-
side (17) [28] based on spectroscopic analyses and comparison of their
NMR data with those reported in the literature.
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(lung), HepG2 (liver) and MCF-7 (breast), while only compound 15
displayed significant and also selective inhibition toward the two lung
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0.24 (A549) and
9.06 0.12 μM (H157), which was consistent with our previous
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groups were necessary for the cytotoxicity of oleanane-type triterpenoid
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In summary, an intensive chemical investigation into the n-BuOH
partition of the ethanol extract from the roots of D. asper resulted in the
discovery of a rare IMIAH (1) featuring a 22-O-iridoid glucoside unit,
an oleanane triterpenoid saponin (2) and 15 known cometabolites. The
absolute structure of 1, a highly complex and flexible molecule, was
characterized by a combination of spectroscopic analyses, chemical
degradation and quantum chemical computation method. It was the
first example that whole absolute configuration was fully established
via solid evidences among this rare class of natural products with two
free iridoid moieties, which provided a beneficial experience for future
structure assignments of related analogues. Compounds 2 and 15 were
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