Diglycosidic indole alkaloid derivatives from an aqueous extract of Isatis indigotica roots

Article abstract of DOI:10.1080/10286020.2017.1320547

Six new indole alkaloid diglycosides named isatigotindolediosides A-F (1–6), along with three known analogs (7–9), were isolated from an aqueous extract of the Isatis indigotica roots (ban lan gen). Their structures including the absolute configurations were determined by comprehensive spectroscopic data analysis, combined with enzyme or acid hydrolysis, and comparison of experimental circular dichroism (CD) and calculated electronic circular dichroism (ECD) spectra. In the preliminary assays, compounds 3, 5, and 8 showed antiviral activity against Coxsackie virus B3.

Full text of DOI:10.1080/10286020.2017.1320547

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Diglycosidic indole alkaloid derivatives from an
aqueous extract of Isatis indigotica roots
Ling-Jie Meng, Qing-Lan Guo, Cheng-Bo Xu, Cheng-Gen Zhu, Yu-Feng Liu,
Ming-Hua Chen, Sheng Lin, Yu-Huan Li, Jian-Dong Jiang & Jian-Gong Shi
To cite this article: Ling-Jie Meng, Qing-Lan Guo, Cheng-Bo Xu, Cheng-Gen Zhu, Yu-Feng Liu,
Ming-Hua Chen, Sheng Lin, Yu-Huan Li, Jian-Dong Jiang & Jian-Gong Shi (2017): Diglycosidic
indole alkaloid derivatives from an aqueous extract of Isatis indigotica roots, Journal of Asian
Natural Products Research, DOI: 10.1080/10286020.2017.1320547
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1320547
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Date: 06 May 2017, At: 06:50

Journal of asian natural Products research, 2017
https://doi.org/10.1080/10286020.2017.1320547
Diglycosidic indole alkaloid derivatives from an aqueous
extract of Isatis indigotica roots
Ling-Jie Meng, Qing-Lan Guo, Cheng-Bo Xu, Cheng-Gen Zhu, Yu-Feng Liu,
Ming-Hua Chen, Sheng Lin, Yu-Huan Li, Jian-Dong Jiang and Jian-Gong Shi
state Key laboratory of Bioactive substance and function of natural Medicines, institute of Materia Medica,
chinese academy of Medical sciences and Peking union Medical college, Beijing 100050, china
ABSTRACT
ARTICLE HISTORY
received 18 January 2017
accepted 13 april 2017
Six new indole alkaloid diglycosides named isatigotindolediosides
A-F (1–6), along with three known analogs (7–9), were isolated from
an aqueous extract of the Isatis indigotica roots (ban lan gen). Their
structures including the absolute configurations were determined by
comprehensive spectroscopic data analysis, combined with enzyme
or acid hydrolysis, and comparison of experimental circular dichroism
(CD) and calculated electronic circular dichroism (ECD) spectra. In the
preliminary assays, compounds 3, 5, and 8 showed antiviral activity
against Coxsackie virus B3.
KEYWORDS
cruciferae; Isatis indigotica;
indole alkaloid diglycoside;
isatigotindolediosides a-f;
antiviral activity
CONTACT Jian-Gong shi
shijg@imm.ac.cn
supplemental data for this article can be accessed http://dx.doi.org/10.1080/10286020.2017.1320547.
© 2017 informa uK limited, trading as taylor & francis Group

2
L.-J. MENG ET AL.
1. Introduction
e dried roots of Isatis indigotica Fort. (Cruciferae), Chinese name “ban lan gen”, is a pop-
ular traditional medicine for the treatment of influenza and infection diseases. Considerable
studies have revealed that extracts of this herbal medicine had various pharmacological
effects including antiviral and anti-inflammatory activities [1]. Although around 100 chem-
ical constituents were isolated from the extracts, only a few showed marginal bioactivities
[2,3]. Literature shows that the previous chemical studies were mainly performed with
the ethanol and methanol extracts, whereas the herbal medicine is practically utilized by
decocting with water. erefore, an aqueous extract of “ban lan gen” was investigated as
part of our program to assess the chemical diversity of traditional Chinese medicines and
their biological activities, focusing on water-soluble and/or minor components [3–15].
Previously we reported more than 70 constituents from I. indigotica for the first time,
including 39 new alkaloids in free and monoglycosidic forms, some of them showed potent
antiviral and cell-damage protective activities [16–22]. With a continuation of our study on
the same extract, six new diglycosidic indole alkaloid derivatives named isatigotindoledi-
osides A-F (1–6, Figure 1), together with three known analogs (7–9), have been obtained.
is paper describes details of the isolation, structure elucidation, and bioactivity assay of
the new isolates.
2. Results and discussion
Compound 1 was isolated as a white amorphous powder with[ꢀ]2D0 − 27.2 (c 0.09, MeOH).
Its IR spectrum showed absorption bands for hydroxyl (3396 cm−1), conjugated carbonyl
(1706 and 1645 cm−1), and aromatic ring [1600 (sh) and 1516 cm−1] functionalities. e
molecular formula of 1 was determined as C21H27NO12 by (+)-HR-ESI-MS at m/z 508.1434
[M + Na]+ and NMR spectroscopic data (Tables 1 and 2). e 1H NMR spectrum of 1 in
MeOH-d4 showed diagnostic signals for a 3′-substituted 1′H-indole ring [21] at δH 8.28
(s, H-2′), 8.14 (d, J = 7.8 Hz, H-4′), 7.27 (t, J = 7.8 Hz, H-5′), 7.33 (t, J = 7.8 Hz, H-6′) and
Figure 1. the structures of compounds 1–9.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1h nMr spectral data (δ) for compounds 1–6.a
No
1b
2
3
4
5c
6
2a
2b
1′
2′
4′
5′
6′
7′
1″
2″
3″
4″
5″
4.20 dd (18.6, 0.6) 3.38 d (16.2)
4.16 dd (18.6, 0.6) 3.16 d (16.2)
4.33 d (16.8)
4.10 d (16.8)
11.23 s
8.28 s
8.07 s
8.10 dd (7.2, 1.8)
7.20 dt (7.2, 1.2) 6.82 dd (7.8, 0.6) 6.94 brd (8.4)
7.22 dt (7.2, 1.8) 7.08 t (7.8) 7.34 t (8.4)
7.45 dd (7.2, 1.2) 7.05 dd (7.8, 0.6) 6.65 brd (8.4)
5.70 d (7.8) 5.05 d (7.8) 5.00 d (7.8)
7.13 brs
8.14 d (7.8)
7.27 t (7.8)
7.33 t (7.8)
7.53 d (7.8)
5.69 d (7.8)
7.98 dd (7.2, 1.8)
7.10 dt (1.8, 7.2)
7.12 dt (1.8, 7.2)
7.31 dd (7.2, 1.8)
5.71 d (8.4)
6.72 d (7.8)
7.05 t (7.8)
6.99 d (7.8)
4.91 d (7.8)
3.53 dd (9.0, 7.8) 3.54 dd (9.0, 7.8) 3.60 dd (9.0, 7.8) 3.57 dd (9.0, 7.8) 3.40 dd (9.0, 7.8) 3.56 dd (9.0, 8.4)
3.50 t (9.0)
3.46 t (9.0)
3.63 m
3.51 t (9.0)
3.46 t (9.0)
3.64 m
3.49 t (9.0)
3.44 t (9.0)
3.65 m
3.47 t (9.0)
3.52 t (9.0)
3.63 m
3.29 brt (9.0)
3.21 brt (9.0)
3.32 m
3.45 t (9.0)
3.38 t (9.0)
3.41 m
6″a 4.13 dd (11.4, 1.8) 4.13 dd (11.4, 1.8) 4.09 dd (11.4, 1.8) 3.96 dd (10.8, 4.8) 3.73 dd (11.4, 2.4) 3.82 dd (12.0, 1.8)
6″b 3.75 dd (11.4, 5.4) 3.75 dd (11.4, 5.4) 3.79 (11.4, 6.0)
3.78 brd (10.8)
4.88 d (7.8)
3.49 dd (11.4, 5.4) 3.63 dd (12.0, 5.4)
4.36 d (9.6) 4.84 d (9.6)
1‴
2‴
3‴
4‴
4.30 d (6.6)
4.30 d (7.2)
4.31 d (7.8)
3.57 dd (9.0, 6.6) 3.57 dd (9.0, 7.2) 3.19 dd (9.0, 7.8) 3.39 dd (9.0, 7.8) 2.76 dd (9.6, 9.0) 3.25 dd (9.6, 9.0)
3.53 t (9.0)
3.76 m
3.52 t (9.0)
3.76 m
3.26 t (9.0)
3.47 m
3.67 t (9.0)
3.30 t (9.0)
3.15 t (9.0)
2.97 t (9.0)
3.13 m
3.38 t (9.0)
3.27 t (9.0)
3.49 m
5‴a 3.81 dd (12.6, 3.0) 3.81 dd (12.6, 3.0) 3.80 dd (11.4, 5.4) 3.64 m
5‴b 3.48 dd (12.6, 10.2) 3.48 dd (12.6, 10.2) 3.06 dd (11.4, 10.2)
6‴a
6‴b
3.75 brd (10.2)
3.62 dd (10.8, 5.4) 3.48 dd (10.4, 5.4) 3.66 dd (12.0, 5.4)
3.65 brd (10.4)
3.92 dd (12.0, 2.4)
a1h nMr data (δ) were measured at 600 Mhz in cd3od for 1–4 and 6 and in dMso-d6 for 5, respectively. Proton coupling
constants (J) in hz are given in parentheses. the assignments were based on 1h-1h cosY, hsQc, and hMBc experiments.
bdata for the methoxy group in 1: δh 4.18 (3h, s).
cdata for hydroxyl protons of 5 in dMso-d6: δh 5.32 (1h, d, J = 3.6 hz, oH-2″), 5.29 (1h, brs, oH-2‴), 5.15 (1h, brs, oH-4″),
5.06 (1h, brs, oH-3″), 5.06 (1h, brs, oH-3‴), 4.97 (1h, brs, oH-4‴), 4.61 (1h, brs, oH-6″), 4.54 (1h, brs, oH-6‴).
Table 2. 13c nMr spectral data (δ) for compounds 1–6.a
No
1b
2
3
4
5
6
1
2
164.5
165.5
121.3
16.2
117.1
25.5
120.0
14.6
165.6
2′
3′
3′a
4′
5′
6′
7′
7′a
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
4‴
5‴
6‴
131.0
103.2
124.4
122.5
123.7
124.8
109.9
133.6
95.5
74.1
78.0
71.3
77.7
134.3
107.5
127.5
122.0
122.8
123.9
113.1
138.2
95.3
74.1
78.0
71.3
77.7
123.6
105.3
118.6
152.9
105.4
124.1
107.6
140.0
102.7
75.2
78.1
71.4
77.4
69.6
178.4
74.7
122.4
110.7
116.9
151.3
103.6
123.7
105.7
138.0
101.2
73.6
76.8
69.8
77.2
60.9
87.2
72.1
77.9
69.5
81.1
60.8
140.5
107.3
127.8
121.9
122.9
123.9
112.2
138.3
95.6
74.2
78.3
71.2
78.9
62.5
86.7
74.3
79.4
71.4
82.3
62.5
117.9
156.6
112.5
133.6
106.6
144.4
103.2
75.0
77.8
71.3
76.8
67.4
100.1
73.6
75.2
71.7
73.7
69.2
104.7
72.4
74.2
69.5
69.2
104.7
72.4
74.2
69.5
105.2
75.0
77.6
71.2
66.8
66.7
66.7
62.6
a13c nMr data (δ) were measured at 150 Mhz in cd3od for 1–4 and 6 and in dMso-d6 for 5, respectively. the assignments
were based on dePt, 1h-1h cosY, hsQc, and hMBc experiments.
bdata for the methoxy group in 1: δc 67.4.

4
L.-J. MENG ET AL.
7.53 (d, J = 7.8 Hz, H-7′), a methoxy group at δH 4.18 (s, OCH3-N), and two sugar units, of
which anomeric protons resonated at δH 5.69 (d, J = 7.8 Hz, H-1″) and 4.30 (d, J = 6.6 Hz,
H-1‴), respectively, and partially overlapped hydroxyl-geminating protons between δH 4.13
and 3.46 (Table 1). e presence of the structural units was supported by the 13C NMR and
DEPT spectra of 1, which displayed 21 carbon resonances (Table 2) corresponding to the
3′-substituted 1′H-indole ring and methoxy units, along with resonances due to a pentosyl,
a hexosyl, and an additional ester carbonyl carbon at δC 164.5 (C-1). ese spectroscopic
data suggest that 1 is an uncommon diglycosidic 1′H-indole derivative containing an ester
carbonyl unit, of which the structure was further elucidated by 2D NMR data analysis.
e proton-bearing carbon and corresponding proton signals in the NMR spectra of 1
were assigned by analysis of the 1H-1H COSY and HSQC spectra. e HMBC spectrum of 1
exhibited two- and three-bond long range heteronuclear correlations (Figure 2) from H-2′ to
C-1, C-3′, C-3′a, and C-7′a; from H-4′ to C-3′, C-6′, and C-7′a; from H-5′ to C-3′a and C-7′;
from H-6′ to C-4′ and C-7′a, and from H-7′ to C-3′a and C-5′. ese correlations, together
with their chemical shifs and the 1H-1H COSY cross-peaks of H-4′/H-5′/H-6′/H-7′, revealed
the presence of a 1′H-indole-3′-carboxyl moiety in 1. e HMBC correlation from H-1″ to
C-1, in combination with the 1H-1H COSY cross-peaks of H-1″/H-2″/H-3″/H-4″/H-5″/H2-
6″ and the coupling constant values between the vicinal protons (J1″,2″ = 7.8 Hz and J2″,3″
=
J3″,4″ = J4″,5″ = 9.0 Hz), demonstrated that the 1′H-indole-3′-carboxyl connected via an ester
bond to the anomeric carbon (C-1″) of β-glucopyranosyl. In addition, the HMBC correla-
tions of H-1‴/C-5‴ and H2-5‴/C-1‴, along with the cross-peaks of H-1‴/H-2‴/H-3‴/H-4‴/
H2-5‴ and the coupling constant values (J1‴,2‴ = 6.6 Hz and J2‴,3‴ = J3‴,4‴ = 9.0 Hz), indicated
that there was a β-xylopyranosyl in 1. Meanwhile, the HMBC correlations of H-1‴/C-6″,
H2-6″/C-1‴ revealed a 1‴→6″ connection between β-xylopyranosyl and β-glucopyranosyl in
1. e remaining methoxy group was located at the nitrogen atom to satisfy the requirement
of the molecular formula of 1. is was supported by the chemical shif of the methoxy
carbon resonance (δc 67.4) [18,20]. From the enzymatic hydrolysate of 1, a sugar mixture
O
O
HO
HO
HO
HO
O
O
O
O
O
HO
HO
O
OH
OH
O
CN
HO
HO
OH
OH
N
H3CO
N
H
3
1
HO
HO
HO
HO
O
O
O
HO
O
CN
O
HO
HO
HO
O
OH
O
CN
O
OH
HO
HO
OH
OH
OH
OH
S
N
H
N
H
4
5
HO
HO
O
O
O
HO
OH
O
HO
HO
HO
S
N
OH
H
6
Figure 2. Main 1h-1h cosY (thick lines) and hMBc (arrows, from 1h to 13c) correlations of compounds 1
and 3–6.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
was isolated. Subsequent chiral derivatization and GC analysis of the mixture (Section 3),
as compared with derivatives of authentic sugar samples, proved liberation of d-xylose and
d-glucose by enzyme hydrolysis of 1. us, the structure of compound 1 was determined
as β-d-xylopyranosyl-(1‴→6″)-β-d-glucopyranosyl 1′-methoxy-1′H-indole-3′-carboxylate,
and given a trivial name isatigotindoledioside A.
Compound 2, a white amorphous powder with [ꢀ]2D0 − 25.7 (c 0.05, MeOH), showed
similar spectroscopic features as those of 1 and has the molecular formula C20H25NO11
as determined by HR-ESIMS at m/z 478.1326 [M + Na]+ and NMR spectroscopic data.
Comparison of the NMR spectroscopic data of 2 and 1 (Tables 1 and 2) indicated the absence
of the methoxy group in 2, suggesting that 2 is a demethoxy derivative of 1. e suggestion
was supported by shielded shifs of H-4′, H-5′, H-6′, and H-7′ (ΔδH −0.04 to −0.21) and
deshielded shifs of C-2′, C-3′, C-3′a, C-7′, and C-7′a (ΔδC +3.1 to +4.6) for 1′H-indole-
3′-carboxyl in 2, as compared with those in 1. is was further verified by 2D NMR data
analysis and enzyme hydrolysis of 2. Especially using the same protocols as described for
1, d-glucose and d-xylose were identified from the hydrolysate of 2. erefore, the struc-
ture of compound 2 was determined as β-d-xylopyranosyl-(1‴→6″)-β-d-glucopyranosyl
1′H-indole-3′-carboxylate and named isatigotindoledioside B.
Compound 3 was obtained as a white amorphous powder with [ꢀ]2D0 − 55.2 (c 0.12,
MeOH). e molecular formula of 3 was determined as C21H26N2O10 by HR-ESIMS at m/z
489.1491 [M + Na]+ and NMR spectroscopic data. Comparison of the NMR data between
3 and 2 (Tables 1 and 2) indicated replacement of the aglycone in 2 by a 4′-substituted
2-(1′H-indol-3′-yl)-acetonitrile in 3, while the sugar moiety was same in the two com-
pounds. Particularly the presence of the 2-substituted acetonitrile unit was indicated by a
characteristic absorption band (2254 cm−1) for the cyano group in the IR spectrum of 3, in
combination with the resonances due to an isolated methylene [δH 4.20 (1H, dd, J = 18.6,
0.6 Hz, H-2a) and 4.16 (1H, dd, J = 18.6, 0.6 Hz, H-2b) and δC 16.2 (C-2)] and a quaternary
cyano carbon at δC 121.3 (C-1) in the NMR spectra. is suggests that 3 is 2-(4′-hydroxy-
1′H-indol-3′-yl)-acetonitrile 4′-O-β-d-xylopyranosyl-(1‴→6″)-β-d-glucopyranoside, which
was further confirmed by 2D NMR spectroscopic data analysis and enzyme hydrolysis of
3. Especially, the HMBC correlations of H2-2/C-1, C-2′, C-3′, C-3′a; H-2′/C-2, C-3′, C-3′a,
and C-7′a; H-5′/C-3′a, C-4′, and C-7′; H-6′/C-4′ and C-7′a, H-7′/C-3′a and C-5′ confirmed
the aglycone while the correlations of H-1″/C-4′, H-1‴/C-6″, and H2-6″/C-1‴ proved the
linkage among the sugar and aglycone units. Enzyme hydrolysis of 3 also liberated d-glu-
cose and d-xylose as identified by application of the aforementioned method. erefore,
the structure of compound 3 was determined and named isatigotindoledioside C.
Compound 4, a white amorphous powder with [ꢀ]2D0 − 26.8 (c 0.02, MeOH), has the
molecular formula C22H28N2O13 as indicated by HR-ESIMS at m/z 551.1482 [M + Na]+.
e IR and NMR spectroscopic data showed that this compound is another diglycosidic
indole alkaloid differing from 1–3 in both the aglycone and sugar moieties. As compared
with those of 3, the IR absorption bands (2259 and 1728 cm−1) and NMR spectroscopic data
(Tables 1 and 2) demonstrated that 4 had an aglycone of 2-(3′,4′-dihydroxy-2′-oxoindo-
lin-3′-yl)acetonitrile. is was confirmed by the 1H-1H COSY cross-peaks of H-2a/H-2b and
H-5′/H-6′/H-7′ and the HMBC correlations of H-2/C-1, C-2′, C-3′, and C-3′a; H-5′/C-3′a,
and C-7′; H-6′/C-4′ and C-7′a; and H-7′/C-3′a, and C-5′, together with the chemical shifs
of these proton and carbon resonances. Meanwhile, the presence of two β-glucopyrano-
syls in 4 were indicated by the 1H-1H COSY cross-peaks of H-1″/H-2″/H-3″/H-4″/H-5″/

6
L.-J. MENG ET AL.
H2-6″ and H-1‴/H-2‴/H-3‴/H-4‴/H-5‴/H2-6‴, as well as by their chemical shifs and
coupling constants (Table 1). In addition, in the HMBC spectrum of 4, the correlations
from H-1″ to C-4′, from H2-6″ to C-1‴, and from H-1‴ to C-6″ revealed substitution of a
(1‴→6″)-di-β-glucopyranosyloxy at C-4′ of the aglycone.
1
Enzyme hydrolysis of 4 afforded the aglycone 4a and sugar. e H NMR spectrum
of 4a was completely consistent with that of (+)-(S)-2-(3,4-dihydroxy-2-oxoindolin-3-yl)
acetonitrile, which was previously isolated from the same extract and structurally proved
by single-crystal X-ray crystallographic analysis [16]. However, 4a gave a negative specific
rotation, while the circular dichroism (CD) spectra of 4 and 4a displayed positive Cotton
effects around 217 and 265 nm and a negative Cotton effect around 240 nm. ese Cotton
effects mirrored to those of (+)-(S)-2-(3,4-dihydroxy-2-oxoindolin-3-yl)acetonitrile [16],
suggesting the R configuration for 4 and 4a. e suggestion was further supported by
comparison of the experimental CD and calculated electronic circular dichroism (ECD)
spectra of 4a as shown in Figure 3. e sugar was identified as d-glucose by comparison
of the 1H NMR spectrum and specific rotation data ([ꢀ]D20 + 44.0 (c 0.02, H2O)) with those
of the authentic sugar sample. erefore, the structure of compound 4 was determined
as (–)-(R)-2-(3′,4′-dihydroxy-2′-oxoindolin-3′-yl)-acetonitrile 4′-O-β-d-glucopyranosyl-
(1‴→6″)-β-d-glucopyranoside and named isatigotindoledioside D.
Compound 5, a white amorphous powder with[ꢀ]20 − 43.5 (c 0.07, MeOH), showed IR
and NMR spectroscopic data similar to those of 3. eDmolecular formula C22H28N2O11S of
5 was assigned by HR-ESIMS at m/z 551.1305 [M + Na]+, suggesting that this compound is
a sulfur-containing derivative of 3. Comparison of the NMR spectroscopic data between 5
and 3 indicated that the sp2 hybridized methine of the aglycone in 3 was replaced by a sp2
hybridized quaternary carbon (δC 122.4, C-2′) in 5, and that the terminal β-xylopyranosyl
of 3 was replaced by a β-thioglucopyranosyl of 5 with characteristic anomeric proton
and carbon resonated at δH 4.36 (d, J = 9.6 Hz, H-1‴) and δC 87.2 (C-1‴) [18,20], respec-
tively. is suggests that 5 is 2-(4′-hydroxy-2′-mercapto-1′H-indol-3′-yl)-acetonitrile
Figure 3. the experimental cd spectra of 4a (blue) and 4 (red) and the calculated ecd spectra of 4a (blue,
dash line) and the enantiomer of 4a (green, dash line). the calculated ecd curves were blue-shifted by
10.5 nm.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
4′-O-β-d-glucopyranosyl-2′-S-β-d-glucopyranoside, which was confirmed by 2D NMR
data analysis (Figure 2 and Supporting Information) and acid hydrolysis. e aglycone
1
was verified by the H-1H COSY cross peaks of H-5′/H-6′/H-7′ and the HMBC corre-
lations of H-1′/C-2′, C-3′, C-3′a, and C-7′a; H-2/C-1, C-2′, C-3′, and C-3′a; H-5′/C-3′a,
C-4′, and C-7′; H-6′/C-4′ and C-7′a; and H-7′/C-5′ and C-3′a. e occurrence and loca-
1
tion of β-glucopyranosyl and β-thioglucopyranosyl were proved by the H-1H COSY
cross-peaks of H-1″/H-2″/H-3″/H-4″/H-5″/H2-6″ and H-1‴/H-2‴/H-3‴/H-4‴/H-5‴/
H2-6‴ and their chemical shifs and coupling constants (Table 1), along with the HMBC
correlations from H-1″ to C-4′ and from H-1‴ to C-2′. Acid hydrolysis of 5 produced a
1
sole sugar, which was identified as d-glucose by comparison of the H NMR spectrum
and specific rotation data ([ꢀ]2D0 + 47.5 (c 0.08, H2O)) with those of the authentic sugar
sample. erefore, the structure of compound 5 was determined as shown and named
isatigotindoledioside E.
Compound 6, a white amorphous powder with[ꢀ]2D0 − 23.3 (c 0.07, MeOH), is another sul-
fur-containing 1′H-indole diglucopyranoside having the molecular formula C21H27N2O12S
that was established by HR-ESIMS at m/z 540.1152 [M + Na]+ and the NMR spectroscopic
data. As comparison of the NMR spectroscopic data (Tables 1 and 2), the difference between
6 and 2 was similar to that between 5 and 3. Accordingly, compound 6 was deduced to be
β-d-glucopyranosyl 2′-β-d-thioglucopyranosyl-1′H-indole-3′-carboxylate. e deduction
was proved by subsequent 2D NMR data analysis and acid hydrolysis. e aglycone was
confirmed by the 1H-1H COSY cross peaks of H-4′/H-5′/H-6′/H-7′ and the HMBC correla-
tions of H-4′/C-3′, C-3′a, C-6′, and C-7′a; H-5′/C-3′a, C-4′, C-6′, and C-7′; H-6′/C-4′, C-5′,
C-7′, and C-7′a; and H-7′/C-3′a, C-5′, and C-7′a (Figure 2). Meanwhile, the occurrence of
β-glucopyranosyl and β-thioglucopyranosyl, as well as their locations, was demonstrated
by the 1H-1H COSY cross-peaks of H-1″/H-2″/H-3″/H-4″/H-5″/H2-6″ and H-1‴/H-2‴/H-
3‴/H-4‴/H-5‴/H2-6‴ and the HMBC correlations of H-1″/C-1 and H-1‴/C-2′. From the
1
acid hydrolysate of 6, d-glucose was isolated and identified by measurements of the H
NMR spectrum and specific rotation data ([ꢀ]2D0 + 45.3 (c 0.03, H2O)). us, the structure
of compound 6 was determined and named isatigotindoledioside F.
By comparing the spectroscopic data with the reported data in the corresponding litera-
ture, the known compounds were identified as cappariloside B (7) [23], calanthoside (8) [24],
and α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl 3-indolecarbonate (9) [25], respec-
tively. In the preliminary in vitro assays [16–22], compounds 3, 5, and 8 showed equally
inhibitory activity against Coxsackie virus B3 with an IC50 value of 33.3 μM and SI >3.0
(the positive control ribavirin gave an IC50 value of 0.0009 μM and a SI value of 17,122.2).
However, all the new isolates were inactive against influenza virus A/Hanfang/359/95
(H3N2) at a concentration of 10 μM.
In summary, six new indole alkaloid diglycosides, having diverse structure features,
were isolated from the aqueous extract of “ban lan gen”. Among them, three showed
antiviral activity against Coxsackie virus B3 but were inactive against influenza virus A/
Hanfang/359/95 (H3N2). is, together with our previous results [16–22], shows that
diverse indole alkaloid components have varied contributions to pharmacological efficacy
that supports the traditional applications of the herbal medicine. Especially, the diverse
structures provide a clue for in-depth investigation of structure-activity relationships of
the indole-derived analogs.

8
L.-J. MENG ET AL.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a P-2000 polarimeter (JASCO, Tokyo, Japan). UV spec-
tra were acquired on a V-650 spectrometer (JASCO, Tokyo, Japan). CD spectra were meas-
ured on a JASCO J-815 CD spectrometer (JASCO, Tokyo, Japan). IR spectra were obtained
on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission) (ermo
Electron Corporation, Madison, WI, U.S.A.). NMR spectra were recorded at 600 MHz for
1H and 150 MHz for 13C, respectively, on a SYS 600 instrument (Varian Associates Inc.,
Palo Alto, CA, U.S.A.) in CD3OD, DMSO-d6, or D2O with solvent peaks used as refer-
ences. ESIMS and HRESIMS data were taken on an Agilent 1100 Series LC-MSD-Trap-SL
and Agilent 6520 Accurate-Mass Q-TOFL CMS spectrometers (Agilent Technologies,
Ltd., Santa Clara, CA, U.S.A.), respectively. Column chromatography (CC) was carried
out on macroporous adsorbent resin (HPD-110, Cangzhou Bon Absorber Technology
Co. Ltd, Cangzhou, China), silica gel (200–300 mesh, Qingdao Marine Chemical Inc.,
Qingdao, China), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), CHP 20P
(Mitsubishi Chemical Inc., Tokyo, Japan), or reversed phase C-18 silica gel (W. R. Grace
& Co., Maryland, U.S.A.). HPLC separation was performed on an instrument equipped
with an Agilent ChemStation for LC system, an Agilent 1200 pump, and an Agilent 1100
single-wavelength absorbance detector (Agilent Technologies, Ltd.) using a Grace semi-
preparative column (250 × 10 mm i.d.) packed with C18 reversed phase silica gel (5 μm)
(W. R. Grace & Co., Maryland, U.S.A.). TLC was carried out on glass precoated silica gel
GF254 plates (Qingdao Marine Chemical Inc.). Spots were visualized under UV light or by
spraying with 7% H2SO4 in 95% EtOH followed by heating. Unless otherwise noted, all
chemicals were purchased from commercially available sources and were used without
further purification.
3.2. Plant material
e roots of Isatis indigotica were collected in December 2009 from Bozhou, Anhui Province,
China. Plant identity was verified by Mr. Lin Ma (Institute of Materia Medica, Beijing
100,050, China). A voucher specimen (No. ID-S-2385) was deposited at the herbarium of
Natural Medicinal Chemistry, Institute of Materia Medica.
3.3. Extraction and isolation
e air-dried and pulvarized plant material (50 kg) was decocted with H2O (150 L; 3 × 1 h).
e aqueous extracts were combined and evaporated under reduced pressure to yield a
dark-brown residue (32 kg). e residue was dissolved in H2O (122 L), loaded on a mac-
roporous adsorbent resin (HPD-110, 19 kg) column (20 × 200 cm), and eluted successively
with H2O (50 L), 50% EtOH (125 L), and 95% EtOH (100 L) to yield three corresponding
fractions A, B, and C. Afer removing the solvent under reduced pressure, fraction B (0.9 kg)
was separated by column chromatography (CC) over MCI gel CHP 20P (5 L), with suc-
cessive elution using H2O (10 L), 30% EtOH (30 L), 50% EtOH (20 L), 95% EtOH (10 L),
and Me2CO (8 L), to give fractions B1-B5. Fraction B2 (547 g) was subjected to silica gel
CC eluting with a gradient of increasing MeOH (0–100%) in EtOAc to yield B2-1–B2-5.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
Subfraction B2-4 (120 g) was separated by CC over Sephadex LH-20 (CHCl3–MeOH, 1:1) to
give B2-4-1–B2-4-3, of which B2-4-1 (40 g) was further fractionated by CC over Sephadex
LH-20 (H2O) to afford B2-4-1-1–B2-4-1-13. Subfraction B2-4-1-6 (20 g) was separated by
CC over Sephadex LH-20 (H2O) to yield B2-4-1-6-1–B2-4-1-6-5. Further fractionation of
B2-4-1-6-1 (8 g) by flash CC over C-18 silica gel (0–40% MeOH in H2O) gave B2-4-1-6-
1-1–B2-4-1-6-1-9, of which B2-4-1-6-1–4 (5 mg) was purified by RP-HPLC (6% MeOH
in H2O containing 0.2% acetic acid, v/v/v, 1.8 ml/min) to afford 4 (1.5 mg, tR = 40 min).
Subfraction B2-4-1-8 (2 g) was further isolated by silica gel CC (CHCl3–MeOH, 15:1~0:1)
to yield B2-4-1-8-1–B2-4-1-8-6, of which B2-4-1-8-2 (200 mg) was further fractionated
by CC over Sephadex LH-20 (MeOH) to afford B2-4-1-8-2-1–B2-4-1-8-2-5. Purification
of B2-4-1-8-2-4 (50 mg) by RP-HPLC (8% MeCN in H2O, 2.0 ml/min) to yield 3 (10.0 mg,
tR = 50 min). Subfraction B2-4-1-8-3 (550 mg) was fractionated by CC over Sephadex
LH-20 (MeOH) to yield B2-4-1-8-3-1–B2-4-1-8-3-4, of which B2-4-1-8-3-1 (220 mg) was
separated by HW-40C (H2O) to yield B2-4-1-8-3-1-1–B2-4-1-8-3-1-4. Subsequent isola-
tion of B2-4-1-8-3-1-4 (50 mg) by RP-HPLC (6% MeCN in H2O, 2.0 ml/min) obtained 5
(9.0 mg, tR = 62 min) and 7 (7.0 mg, tR = 52 min). Separation of B2-4-1-8-3-2 (90 mg) by
CC over HW-40C (MeOH–H2O, 4:6) afforded B2-4-1-8-3-2-1 and B2-4-1-8-3-2-2, of which
B2-4-1-8-3-2-1 (30 mg) was purified by RP-HPLC (10% MeCN in H2O, v/v, 2.0 ml/min)
to afford 8 (7.0 mg, tR = 19 min). Isolation of B2-4-1-8-3-2-2 (28 mg) by preparative TLC
(EtOAc:MeOH:H2O, 8:2:1, v/v/v) yielded B2-4-1-8-3-2-2-1 (6 mg) and B2-4-1-8-3-2-2-2
(10 mg), and further purification by RP-HPLC (20% MeOH in H2O, 2.0 ml/min) obtained
1 (3.0 mg, tR = 38 min) from the former and by RP-HPLC (12% MeCN in H2O, 2.0 ml/min)
yielded 2 (5.0 mg, tR = 37 min) from the latter. Subfraction B2-4-1-9 (3 g) was chromato-
graphed over silica gel and eluted by a gradient of increasing MeOH (0–100%) in CHCl3 to
yield B2-4-1-9-1–B2-4-1-9-4, of which B2-4-1-9-4 (1.8 g) was further separated by HW-40C
(H2O) to yield B2-4-1-9-4-1–B2-4-1-9-4-6. Separation of B2-4-1-9-4-6 (200 mg) by CC
over Sephadex LH-20 (H2O) yielded B2-4-1-9-4-6-1–B2-4-1-9-4-6-4. Successive purifica-
tion of B2-4-1-9-4-6-2 (8 mg) and B2-4-1-9-4-6-3 (10 mg) by RP-HPLC (10% MeCN in
H2O containing 0.2% acetic acid, v/v/v, 2.0 ml/min) afforded 6 (2.0 mg, tR = 32 min) and
9 (1 mg, tR = 58 min), respectively.
3.3.1. Isatigotindoledioside A (1)
White amorphous powder; [ꢀ]20 − 27.2 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 204
(2.66), 214 (sh, 2.53), 231 (sh, 2D.26), 291 (1.99) nm; IR νmax 3396, 3186, 3011, 2922, 2849,
1706, 1645, 1600 (sh), 1516, 1468, 1419, 1372, 1326, 1300, 1247, 1207, 1118, 1072, 1011,
816, 774, 745, 722, 646 cm−1; 1H NMR (CD3OD, 600 MHz) spectral data, see Table 1; 13
C
NMR (CD3OD, 150 MHz) spectral data, see Table 2; (+)-ESI-MS: m/z 508 [M + Na]+;
(−)-ESI-MS: m/z 484 [M − H]−, 520 [M + Cl]−; (+)-HR-ESI-MS: m/z 508.1434 [M + Na]+
(calcd for C21H27NO12Na, 508.1425).
3.3.2. Isatigotindoledioside B (2)
White amorphous powder;[ꢀ]20 − 25.7 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 204 (3.01),
214 (sh, 2.75), 229 (sh, 2.59), D285 (2.37) nm; IR νmax 3395, 3187, 3011, 2923, 2850, 1691,
1646, 1530, 1468, 1420, 1370, 1343, 1325, 1301, 1246, 1215, 1173, 1119, 1073, 1012, 817,
776, 753, 722, 648 cm−1; 1H NMR (CD3OD, 600 MHz) spectral data, see Table 1; 13C NMR
(CD3OD, 150 MHz) spectral data, see Table 2; (+)-ESI-MS: m/z 478 [M + Na]+; (−)-ESI-MS:

10
L.-J. MENG ET AL.
m/z 454 [M − H]−, 490 [M + Cl]−; (+)-HR-ESI-MS: m/z 478.1326 [M + Na]+ (calcd for
C20H25NO11Na, 478.1320).
3.3.3. Isatigotindoledioside C (3)
White amorphous powder;[ꢀ]2D0 − 55.2 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 219 (2.70),
267 (1.78) nm; IR νmax 3395, 3011, 2922, 2850, 2254, 1646, 1588, 1509, 1468, 1420, 1358,
1
1253, 1170, 1077, 1046, 783, 751, 651 cm−1; H NMR (CD3OD, 600 MHz) spectral data,
see Table 1; 13C NMR (CD3OD, 150 MHz) spectral data, see Table 2; (+)-ESI-MS: m/z 489
[M + Na]+, 505 [M + K]+; (−)-ESI-MS: m/z 465 [M − H]−, 501 [M + Cl]−; (+)-HR-ESI-MS:
m/z 489.1491 [M + Na]+ (calcd for C21H26N2O10Na, 489.1480).
3.3.4. Isatigotindoledioside D (4)
White amorphous powder;[ꢀ]20 − 26.8 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 202 (3.65),
216 (sh, 3.39), 297 (2.66) nm; DCD (MeOH) Δε215 nm + 2.59, Δε240 nm − 4.29, Δε265 nm + 1.06,
Δε298 nm + 0.17; IR νmax 3394, 2922, 2849, 2259, 1728, 1627, 1497, 1467, 1418, 1270, 1247, 1203,
1149, 1080, 1031, 923, 863, 818, 774, 708, 651 cm−1; 1H NMR (CD3OD, 600 MHz) spectral
data, see Table 1; 13C NMR (CD3OD, 150 MHz) spectral data, see Table 2; (+)-ESI-MS: m/z
551 [M + Na]+, 567 [M + K]+; (−)-ESI-MS: m/z 527 [M − H]−, 563 [M + Cl]−; (+)-HR-ESI-MS:
m/z 551.1482 [M + Na]+ (calcd for C22H28N2O13Na, 551.1484).
3.3.5. Isatigotindoledioside E (5)
White amorphous powder; [ꢀ]D20 − 43.5 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 202
(2.98), 223 (2.52), 278 (2.01) nm; IR νmax 3329, 2917, 2893, 2247, 1623, 1582, 1511, 1427,
1400, 1347, 1296, 1247, 1173, 1099, 1069, 1031, 921, 884, 855, 810, 774, 746, 582 cm−1; 1H
NMR (DMSO-d6, 600 MHz) spectral data, see Table 1; 13C NMR (DMSO-d6, 150 MHz)
spectral data, see Table 2; (+)-ESI-MS: m/z 551 [M + Na]+, 567 [M + K]+; (−)-ESI-MS:
m/z 527 [M − H]−, 563 [M + Cl]−; (+)-HR-ESI-MS: m/z 551.1305 [M + Na]+ (calcd for
C22H28N2O11SNa, 551.1306).
3.3.6. Isatigotindoledioside F (6)
White amorphous powder; [ꢀ]20 − 23.3 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 203
(3.00), 217 (sh, 2.70), 241 (sh, 2D.53), 3.05 (2.35) nm; IR νmax 3396, 3185, 3011, 2922, 2850,
1646, 1469, 1420, 1344, 1325, 1301, 1246, 1216, 1187, 1118, 1064, 1037, 817, 783, 752, 722,
1
648 cm−1; H NMR (CD3OD, 600 MHz) spectral data, see Table 1; 13C NMR (CD3OD,
150 MHz) spectral data, see Table 2; (+)-ESI-MS: m/z 540 [M + Na]+; (−)-ESI-MS: m/z 516
[M − H]−; (+)-HR-ESI-MS: m/z 540.1152 [M + Na]+ (calcd for C21H27NO12SNa, 540.1146).
3.3.7. Enzymatic hydrolysis of 1–4 and acidic hydrolysis of 5 and 6
Compounds 1–4 (1.0–3.0 mg) were separately hydrolyzed in H2O (3 ml) with snailase
(3.0 mg, CODE S0100, Beijing Biodee Biotech Co., Ltd., Beijing, China) at 37 °C for 24 h.
Compounds 5 and 6 (~1.5 mg) were separately hydrolyzed with 2 N HCl (3 ml) at 80 °C for
6 h. e hydrolysate was concentrated under reduced pressure and the residue was isolated
by CC over silica gel eluting with CH3CN–H2O (8:1), to afford two mixtures, respectively,
containing sugar and aglycone. e sugar mixtures from hydrolysis of 1–3, as well as the
authentic d- and L-glucose and d- and L-xylose (1 mg, each), were individually reacted
with L-cysteine methyl ester hydrochloride (2.5 mg) in pyridine (1.3 ml) at 60 °C for 2 h,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
then N-trimethylsilylimidazole (0.3 ml) was added and reacted at 60 °C for another 2 h.
e reaction mixtures were separately analyzed by GC under the following conditions:
injection temperature, 300 °C; detector temperature (FID), 300 °C; capillary column, HP-5
(60 m × 0.32 mm, Dikma); the carrier gas, N2; start temperature 200 °C raised to 260 °C
at a rate of 10 °C/min, and the final temperature maintained for 38.5 min. Retention times
(tR) for the derivatives of d-glucose, L-glucose, d-xylose, L-xylose were determined to be
29.82, 30.55, 18.90, and 20.03 min, respectively. For the derivatives of the sugar mixture
from the hydrolysate of 1, GC analysis displayed two peaks with the tR values of 29.44 min
and 19.01 min, which were consistent with those of the derivatives of d-glucose and d-xy-
lose, respectively, and similar peaks with the tR values of 29.46 and 19.01 min and 29.47
and 19.02 min were observed in the sugar mixtures from the hydrolysis of 2 and 3 (see in
Supporting information). e sugar (0.2–0.8 mg) from 4–6 showed retention factor (Rf ≈
0.38) on TLC (EtOAc-MeOH-AcOH-H O, 12:3:3:2), with [ꢀ]2D0 values of +44.0 to +47.5 (c
2
0.02–0.08, H2O), and 1H NMR (D2O) data in agreement with those of an authentic d-glu-
cose. Purification of the mixture containing the aglycone of 4 by Sephadex LH-20 (MeOH)
obtained 4a,[ꢀ]2D0 − 48.3 (c 0.02, MeOH). e 1H NMR spectrum of 4a was the same as that
of (+)-(S)-2-(3,4-dihydroxy-2-oxoindolin-3-yl)acetonitrile [16].
3.3.8. ECD Calculation of 4a
Briefly, conformational analysis was performed by Monte Carlo searching with the MMFF94
molecular mechanics force field using the Spartan 10 sofware 4a. e lowest-energy con-
formers with relative energies lower than 2 kcal/mol were re-optimized using the density
functional theory (DFT) at the B3LYP/6-31 + G (d, p) level via the Gaussian 09 program. e
conductor-like polarizable continuum model (CPCM) was employed to consider the solvent
effects using the dielectric constant of MeOH (ε = 32.6). e energies, oscillator strengths,
and rotational strengths of excitations were calculated using the TDDFT methodology at the
B3LYP/6-311++G (2d, 2p) level in vacuum. e re-optimized conformers having relative
Gibbs free energies (ΔG) within 2 kcal/mol were used to simulate the ECD spectra with
the Gaussian function (σ = 0.28 eV). To obtain the final spectrum, the simulated spectra of
the lowest energy conformers were averaged based on the Boltzmann distribution theory
and their relative Gibbs free energy (ΔG). All quantum computations were conducted using
Gaussian 09 program package on an IBM cluster machine located at the High Performance
Computing Center of Peking Union Medical College.
Acknlowledgements
We thank Chinese Academy of Medical Sciences and Peking Union Medical College High Performance
Computing Platform for supporting the calculation of the ECD spectra.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was supported by the National Natural Sciences Foundation of China (NNSFC) [grant
numbers 81373287, 81630094, 30825044].

12
L.-J. MENG ET AL.
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