Indole alkaloid glucosides from the roots of Isatis indigotica

Article abstract of DOI:10.1080/10286020.2015.1117452

Five new indole alkaloid glucosides named isatindigotindolosides A-E (1-5), along with three known analogs (6-8), were isolated from an aqueous extract of the Isatis indigotica roots. Their structures including the absolute configurations were determined based on comprehensive spectroscopic data analysis, combined with chemical methods and electronic circular dichroism spectra calculations. In the preliminary assays, compounds 1, 6 and 7 showed antiviral activity against influenza virus A/Hanfang/359/95 (H3N2) with IC₅₀ values of 14.6-33.3 μM. Compound 1 also exhibited inhibitory effect against nitric oxide (NO) production in microglial cell BV2 with an inhibition ratio of 93.0% at 10 μM.

Full text of DOI:10.1080/10286020.2015.1117452

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Indole alkaloid glucosides from the roots of Isatis
indigotica
Yu-Feng Liu, Ming-Hua Chen, Sheng Lin, Yu-Huan Li, Dan Zhang, Jian-Dong
Jiang & Jian-Gong Shi
To cite this article: Yu-Feng Liu, Ming-Hua Chen, Sheng Lin, Yu-Huan Li, Dan Zhang, Jian-Dong
Jiang & Jian-Gong Shi (2015): Indole alkaloid glucosides from the roots of Isatis indigotica,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1117452
To link to this article: http://dx.doi.org/10.1080/10286020.2015.1117452
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Date: 10 December 2015, At: 19:43

Journal of asian natural Products research, 2015
ꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2015.1117452
ꢀ
Indole alkaloid glucosides from the roots of Isatis indigotica
Yu-Feng Liua,b, Ming-Hua Chen , Sheng Lin , Yu-Huan Li , Dan Zhang ,
a,c
a
c
a
Jian-Dong Jiang and Jian-Gong Shia
a,c
ꢆ
sꢁꢆꢁꢋ Kꢋy lꢆbꢃꢅꢆꢁꢃꢅy ꢃꢌ Bꢄꢃꢆꢉꢁꢄvꢋ sꢍbꢊꢁꢆꢈꢉꢋ ꢆꢈꢂ fꢍꢈꢉꢁꢄꢃꢈ ꢃꢌ nꢆꢁꢍꢅꢆꢇ Mꢋꢂꢄꢉꢄꢈꢋꢊ, iꢈꢊꢁꢄꢁꢍꢁꢋ ꢃꢌ Mꢆꢁꢋꢅꢄꢆ Mꢋꢂꢄꢉꢆ,
b
cꢀꢄꢈꢋꢊꢋ aꢉꢆꢂꢋmy ꢃꢌ Mꢋꢂꢄꢉꢆꢇ sꢉꢄꢋꢈꢉꢋꢊ ꢆꢈꢂ Pꢋkꢄꢈg uꢈꢄꢃꢈ Mꢋꢂꢄꢉꢆꢇ cꢃꢇꢇꢋgꢋ, Bꢋꢄjꢄꢈg 100050, cꢀꢄꢈꢆ; dꢋpꢆꢅꢁmꢋꢈꢁ
ꢃꢌ Pꢀꢆꢅmꢆꢉy, Jꢄꢈꢄꢈg Mꢋꢂꢄꢉꢆꢇ uꢈꢄvꢋꢅꢊꢄꢁy, Jꢄꢈꢄꢈg, cꢀꢄꢈꢆ; iꢈꢊꢁꢄꢁꢍꢁꢋ ꢃꢌ Mꢋꢂꢄꢉꢄꢈꢆꢇ Bꢄꢃꢁꢋꢉꢀꢈꢃꢇꢃgy, cꢀꢄꢈꢋꢊꢋ aꢉꢆꢂꢋmy
ꢉ
ꢃꢌ Mꢋꢂꢄꢉꢆꢇ sꢉꢄꢋꢈꢉꢋꢊ ꢆꢈꢂ Pꢋkꢄꢈg uꢈꢄꢃꢈ Mꢋꢂꢄꢉꢆꢇ cꢃꢇꢇꢋgꢋ, Bꢋꢄjꢄꢈg 100050, cꢀꢄꢈꢆ
ABSTRACT
ARTICLE HISTORY
Five new indole alkaloid glucosides named isatindigotindolosides
A-E (1–5), along with three known analogs (6–8), were isolated from
an aqueous extract of the Isatis indigotica roots. Their structures
including the absolute configurations were determined based on
comprehensive spectroscopic data analysis, combined with chemical
methods and electronic circular dichroism spectra calculations. In the
preliminary assays, compounds 1, 6 and 7 showed antiviral activity
against influenza virus A/Hanfang/359/95 (H3N2) with IC50 values of
rꢋꢉꢋꢄvꢋꢂ 21 aꢍgꢍꢊꢁ 2015
aꢉꢉꢋpꢁꢋꢂ 4 nꢃvꢋmbꢋꢅ 2015
KEYWORDS
cꢅꢍꢉꢄꢌꢋꢅꢆꢋ; Isatis indigotica;
ꢄꢈꢂꢃꢇꢋ ꢆꢇkꢆꢇꢃꢄꢂ gꢇꢍꢉꢃꢊꢄꢂꢋ;
ꢄꢊꢆꢁꢄꢈꢂꢄgꢃꢁꢄꢈꢂꢃꢇꢃꢊꢄꢂꢋꢊ a–e;
ꢆꢈꢁꢄvꢄꢅꢆꢇ ꢆꢉꢁꢄvꢄꢁy; ꢄꢈꢀꢄbꢄꢁꢄꢃꢈ
ꢆꢉꢁꢄvꢄꢁy ꢆgꢆꢄꢈꢊꢁ ꢈꢄꢁꢅꢄꢉ ꢃxꢄꢂꢋ
pꢅꢃꢂꢍꢉꢁꢄꢃꢈ
1
4.6–33.3 μM. Compound 1 also exhibited inhibitory effect against
nitric oxide (NO) production in microglial cell BV2 with an inhibition
ratio of 93.0% at 10 μM.
1
. Introduction
“Ban lan gen ,” the dried roots of Isatis indigotica Fort. (Cruciferae), is an important
traditional Chinese medicine commonly used to treat influenza and infection diseases [1].
Decades of studies showed that extracts of this drug had extensive biological activities
(
antiviral, anti-endotoxic, antinociceptive, anti-inflammatory, antipyretic, and cytotoxic)
and contained various active constituents [2–4], of which indole alkaloids are considered as
the important ones [5]. However, previous chemical studies were mainly carried out on the
ethanol and methanol extracts of the drug materials, which is not in accordance with the
practical utilization of the herbal medicine by decocting with water. As part of a program
to assess the chemical and biological diversity of traditional Chinese medicines, focusing
on the minor components [6–18], we performed a detailed investigation of an aqueous
extract of “ban lan gen .” In our previous papers, the characterization of 28 new alkaloids
and 33 constituents firstly isolated from I. indigotica, as well as their antiviral and hepato-
cyte-protective activities were described [19–23]. e continuous investigation on the same
extract has afforded five new indole alkaloid glucosides named isatindigotindolosides A-E
CONTACT Jꢄꢆꢈ-Gꢃꢈg sꢀꢄ
ꢊꢀꢄjg@ꢄmm.ꢆꢉ.ꢉꢈ
tꢀꢋ ꢊꢍppꢇꢋmꢋꢈꢁꢆꢇ ꢂꢆꢁꢆ ꢌꢃꢅ ꢁꢀꢄꢊ ꢆꢅꢁꢄꢉꢇꢋ ꢉꢆꢈ bꢋ ꢆꢉꢉꢋꢊꢊꢋꢂ ꢆꢁ ꢀꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2015.1117452.
©
2015 tꢆyꢇꢃꢅ & fꢅꢆꢈꢉꢄꢊ

2
Y.ꢀF. LIꢁ ET AL.
Figure 1. tꢀꢋ ꢊꢁꢅꢍꢉꢁꢍꢅꢋꢊ ꢃꢌ ꢉꢃmpꢃꢍꢈꢂꢊ 1–8.
(
1–5) and three known analogs (6–8) (Figure 1). Herein reported are details of the isolation,
structure elucidation, and bioactivity assay of the new isolates.
2
. Results and discussion
20
Compound 1 was isolated as a white amorphous powder with[ꢀ] +17.9 (c 0.42, MeOH).
D
−1
Its IR spectrum indicated the presence of hydroxyl (3406 cm ), conjugated carbonyl
−
1
−1
(
1653 cm ), and aromatic ring (1616 and 1508 cm ) functional groups. e molecular
+
formula of 1 was elucidated as C H N O by (+)-HR-ESI-MS at m/z 459.1407 [M + H]
2
2
22
2
9
1
combined with NMR spectral data (Tables 1 and 2). e H NMR spectrum of 1 in DMSO-d
6
showed signals attributable to two ortho-disubstituted benzene rings [δ 8.52 (d, J = 8.5 Hz,
H
H-3), 7.29 (dt, J = 1.5, 8.5 Hz, H-4), 6.97 (t, J = 8.5 Hz, H-5), and 7.91 (dd, J = 1.5, 8.5 Hz,
H-6), and 7.70 (d, J = 8.0 Hz, H-4′), 6.94 (t, J = 8.0 Hz, H-5′), 7.14 (t, J = 8.0 Hz, H-6′),
and 7.36 (d, J = 8.0 Hz, H-7′)] and three exchangeable nitrogen-bearing and carboxylic
protons [δ 11.12 (s, NH-1′), 13.60 (s, NH-2), and 7.89 (COOH)], in addition to character-
H
1
3
istic signals for a β-glucopyranosyl moiety (Table 1). e CNMR and DEPT spectra of 1
exhibited carbon resonances corresponding to the above units (Table 2) and two additional
carbonyl carbons resonated at δ 159.0 (C-1″′) and 169.0 (C-7). ese spectroscopic data
C
indicate that 1 is an unusual aromatic alkaloid β-glucopyranoside, of which the structure
1
1
was further elucidated by 2D NMR data analysis. In the H- H COSY spectrum of 1, the
vicinal coupling correlations of H-3/H-4/H-5/H-6, H-4′/H-5′/H-6′/H-7′, and H-1″/H-
2
″/H-3″/H-4″/H-5″/H -6″, together with their chemical shif s and coupling constants,
2
confirmed the presence of the ortho-disubstituted benzene and β-glucopyranosyl moieties.

JOꢁꢂNAL OF ASIAN NATꢁꢂAL PꢂODꢁCTS ꢂESEAꢂCH
3
1
ꢆ
Table 1. h nMr ꢊpꢋꢉꢁꢅꢆꢇ ꢂꢆꢁꢆ (δ) ꢌꢃꢅ ꢉꢃmpꢃꢍꢈꢂꢊ 1–5 .
no
1b,c
2d
3
4
5e
1
1
1
4
5
6
7
1
2
3
4
5
6
6
ꢆ/2ꢆ
b/2b
/4.20 ꢂ (18.5)
/4.13 ꢂ (18.5)
/7.53 ꢊ
/5.80 ꢊ
/5.78 ꢊ
4.19 ꢂ (13.2)/
3.98 ꢂ (13.2)/
/7.52 ꢊ
ʹ/2ʹ
11.12 ꢊ/
7.70 ꢂ (8.0)
6.94 ꢁ (8.0)
7.14 ꢁ (8.0)
7.36 ꢂ (8.0)
ʹ
7.72 bꢅꢂ (7.8)
7.08 ꢂꢁ (1.2, 7.8)
7.18 ꢂꢁ (1.2, 7.8)
7.35 bꢅꢂ (7.8)
4.35 ꢂ (9.6)
2.87 ꢂꢂ (8.0, 9.6)
3.27 ꢁ (8.0)
3.05 ꢁ (8.0)
3.26 m
3.83 ꢂꢂ (2.4, 12.0)
3.66 ꢂꢂ (7.2, 12.0)
3.43 ꢊ
7.72 bꢅꢂ (7.8)
7.07 ꢂꢁ (1.2, 7.8)
7.18 ꢂꢁ (1.2, 7.8)
7.34 bꢅꢂ (7.8)
4.36 ꢂ (9.6)
2.98 ꢂꢂ (8.0, 9.6)
3.28 ꢁ (8.0)
3.21 m
3.21 m
3.83 ꢂꢂ (1.8, 12.0)
3.64 ꢂꢂ (4.2, 12.0)
3.43 ꢊ
7.70 bꢅꢂ (7.8)
7.07 ꢂꢁ (1.2, 7.8)
7.21 ꢂꢁ (1.2, 7.8)
7.40 bꢅꢂ (7.8)
4.28 ꢂ (9.6)
3.26–3.36 m
3.26–3.36 m
3.26–3.36 m
3.26–3.36 m
3.91 m
ʹ
7.07 ꢂ (8.0)
7.13 ꢁ (8.0)
6.76 ꢂ (8.0)
4.90 ꢂ (7.5)
3.37 m
3.28 m
3.19 m
ʹ
ʹ
ʺ
5.27 ꢂ (7.5)
3.41 ꢂꢂ (7.5, 9.5)
3.35 ꢁ (9.5)
3.28 ꢂꢂ (8.5, 9.5)
3.43 m
3.61 bꢅꢂ (11.0)
3.49 ꢂꢂ (4.5, 11.0)
ʺ
ʺ
ʺ
ʺ
3.32 m
ʺꢆ
ʺb
3.71 ꢂꢂꢂ (3.0, 6.6, 12.0)
3.49 ꢂꢂꢂ (6.0, 6.6, 12.0)
4.02 ꢊ
3.68 m
4.08 ꢊ
ocH
3
ꢆ1
h nMr ꢂꢆꢁꢆ (δ) wꢋꢅꢋ mꢋꢆꢊꢍꢅꢋꢂ ꢆꢁ 500 Mhz ꢄꢈ dMso-d ꢌꢃꢅ 1 ꢆꢈꢂ 2 ꢆꢈꢂ ꢆꢁ 600 Mhz ꢄꢈ cd od ꢌꢃꢅ 3–4 ꢆꢈꢂ ꢄꢈ ꢆꢉꢋꢁꢃꢈꢋ-d ꢌꢃꢅ
6
3
6
1
1
5
, ꢅꢋꢊpꢋꢉꢁꢄvꢋꢇy. Pꢅꢃꢁꢃꢈ ꢉꢃꢍpꢇꢄꢈg ꢉꢃꢈꢊꢁꢆꢈꢁꢊ (J) ꢄꢈ hz ꢆꢅꢋ gꢄvꢋꢈ ꢄꢈ pꢆꢅꢋꢈꢁꢀꢋꢊꢋꢊ. tꢀꢋ ꢆꢊꢊꢄgꢈmꢋꢈꢁꢊ wꢋꢅꢋ bꢆꢊꢋꢂ ꢃꢈ h- h cosY,
hsQc, ꢆꢈꢂ hMBc ꢋxpꢋꢅꢄmꢋꢈꢁꢊ.
b
ꢉ
dꢆꢁꢆ ꢌꢃꢅ pꢅꢃꢁꢃꢈꢊ ꢃꢌ ꢁꢀꢋ ꢆꢈꢁꢀꢅꢆꢈꢄꢇꢄꢉ ꢆꢉꢄꢂ mꢃꢄꢋꢁy ꢄꢈ 1: δ 13.60 (1h, ꢊ, nH-2), 8.52 (1h, ꢂ, J = 8.5 hz, h-3), 7.29 (1h, ꢂꢁ, J = 1.5,
h
8
.5 hz, h-4), 6.97 (1h, ꢁ, J = 8.5 hz, h-5), 7.91 (1h, ꢂꢂ, J = 1.5, 8.5 hz, h-6), 7.89 (1h, bꢅꢊ, cooH).
dꢆꢁꢆ ꢌꢃꢅ ꢀyꢂꢅꢃxyꢇ pꢅꢃꢁꢃꢈꢊ ꢄꢈ 1: δh 5.05 (2h, bꢅꢊ, oH-3″, oH-4″), 4.44 (1h, bꢅꢊ, oH-6″), 4.10 (1h, bꢅꢊ, oH-2″).
ꢂ
dꢆꢁꢆ ꢌꢃꢅ ꢀyꢂꢅꢃxyꢇ pꢅꢃꢁꢃꢈꢊ ꢄꢈ 2: δh 5.35 (1h, bꢅꢊ, oH-2″), 5.14 (1h, bꢅꢊ, oH-3″), 5.01 (1h, bꢅꢊ, oH-4″), 4.59 (1h, ꢁ, J = 6.6 hz,
oH-6″).
ꢋ
dꢆꢁꢆ ꢌꢃꢅ ꢀyꢂꢅꢃxyꢇ pꢅꢃꢁꢃꢈꢊ ꢄꢈ 5: δ 4.33 (1h, bꢅꢊ, oH-3″), 4.21 (1h, bꢅꢊ, oH-4″), 4.11 (1h, bꢅꢊ, oH-2″), 3.80 (1h, ꢂꢂ, J = 5.4,
h
6
.0 hz, oH-6″).
1
3
ꢆ
Table 2. c nMr ꢊpꢋꢉꢁꢅꢃꢊꢉꢃpꢄꢉ ꢂꢆꢁꢆ (δ) ꢌꢃꢅ ꢉꢃmpꢃꢍꢈꢂꢊ 1–5 .
no
1b
2
3
4
5
1
2
127.1
139.8
116.8
136.7
117.1
121.5
119.1
123.9
112.5
133.9
102.3
73.1
119.8
14.9
119.1
66.5
119.0
66.3
24.2
2
3
3
4
5
6
7
7
1
2
3
4
5
6
1
ʹ
121.8
100.8
112.9
152.2
104.5
124.1
102.7
133.5
101.5
73.6
76.7
69.8
77.2
60.8
125.9
116.0
126.7
120.4
121.5
124.7
112.6
138.5
88.7
73.7
79.4
71.5
82.7
126.9
115.5
126.6
120.2
121.4
124.6
112.6
138.6
89.2
73.8
79.5
70.9
82.1
123.6
108.9
124.4
120.4
120.4
123.2
109.0
133.5
84.8
74.3
79.7
71.8
81.7
ʹ
ʹꢆ
ʹ
ʹ
ʹ
ʹ
ʹꢆ
ʺ
ʺ
ʺ
75.6
69.4
77.0
60.6
ʺ
ʺ
ʺ
63.2
/57.2
62.5
/57.3
63.2
/66.1
ʹʺ/oCh
159.0/
/66.1
3
ꢆ13
c nMr ꢂꢆꢁꢆ (δ) wꢋꢅꢋ mꢋꢆꢊꢍꢅꢋꢂ ꢆꢁ 125 Mhz ꢄꢈ dMso-d ꢌꢃꢅ 1 ꢆꢈꢂ 2 ꢆꢈꢂ ꢆꢁ 150 Mhz ꢄꢈ cd od ꢌꢃꢅ 3–4 ꢆꢈꢂ ꢄꢈ ꢆꢉꢋꢁꢃꢈꢋ-d
ꢌꢃꢅ 5, ꢅꢋꢊpꢋꢉꢁꢄvꢋꢇy. tꢀꢋ ꢆꢊꢊꢄgꢈmꢋꢈꢁꢊ wꢋꢅꢋ bꢆꢊꢋꢂ ꢃꢈ dePt, h- h cosY, hsQc, ꢆꢈꢂ hMBc ꢋxpꢋꢅꢄmꢋꢈꢁꢊ.
6
3
6
1
1
b
dꢆꢁꢆ ꢌꢃꢅ ꢃꢁꢀꢋꢅ ꢉꢆꢅbꢃꢈ ꢅꢋꢊꢃꢈꢆꢈꢉꢋꢊ ꢃꢌ ꢁꢀꢋ ꢆꢈꢁꢀꢅꢆꢈꢄꢇꢄꢉ ꢆꢉꢄꢂ mꢃꢄꢋꢁy ꢄꢈ 1: δ 120.2 (c-3), 129.4 (c-4), 121.6 (c-5), 130.9 (c-6),
c
1
69.0 (c-7).

e HMBC spectrum of 1 showed long-range correlations from H-3 to C-1, C-2, and C-5;
from H-4 to C-2 and C-6; from H-5 to C-1, C-3, and C-6; from H-6 to C-2, C-4, and C-7;
and from NH-2 to C-1 and C-3. ese correlations, together with their chemical shif s,
revealed the presence of an N-substituted anthranilic acid moiety in 1. Meanwhile, the
HMBC correlations from H-4′ to C-3′, C-6′, and C-7′a; from H-5′ to C-3′a and C-7′;

4
Y.ꢀF. LIꢁ ET AL.
1
1
1
13
Figure 2. Mꢆꢄꢈ h- h cosY (ꢁꢀꢄꢉk ꢇꢄꢈꢋꢊ) ꢆꢈꢂ hMBc (ꢆꢅꢅꢃwꢊ, ꢌꢅꢃm h ꢁꢃ c) ꢉꢃꢅꢅꢋꢇꢆꢁꢄꢃꢈꢊ ꢃꢌ ꢉꢃmpꢃꢍꢈꢂꢊ
–3 ꢆꢈꢂ 5.
1
Figure 3. tꢀꢋ ꢋxpꢋꢅꢄmꢋꢈꢁꢆꢇ cd ꢊpꢋꢉꢁꢅꢆ (ꢌꢍꢇꢇ ꢇꢄꢈꢋ) ꢃꢌ 3 (bꢇꢍꢋ) ꢆꢈꢂ ꢁꢀꢋ ꢉꢆꢇꢉꢍꢇꢆꢁꢋꢂ ecd ꢊpꢋꢉꢁꢅꢆ (ꢂꢆꢊꢀꢋꢂ ꢇꢄꢈꢋ)
ꢃꢌ 3 (bꢇꢍꢋ) ꢆꢈꢂ ꢄꢁꢊ ꢋꢈꢆꢈꢁꢄꢃmꢋꢅ (ꢅꢋꢂ).
from H-6′ to C-4′ and C-7′a; from H-7′ to C-3′a and C-5′; and from NH-1′ to C-2′, C-3′,
C-3′a, and C-7′a; in combination with their chemical shif s, demonstrated that there was
a 2′,3′-disubstituted indole moiety in 1. e HMBC correlation from H-1″ to C-3′ indi-
cated that the β-glucopyranosyloxy was located at C-3′ of the indole moiety. In addition,
the HMBC correlation from NH-2 to the remaining carbonyl carbon (C-1″′) suggested
that this carbonyl group was not only linked with N-atom of the anthranilic acid moiety to
form an amide bond, but also connected to C-2′ of the 3′-O-β-glucopyranosyloxyindole
moiety to match requirement of the molecular composition of 1. Accordingly, the gross
structure of 1 was established as shown. A literature survey indicates that 1 is the acid form
of cephalandole C, which was isolated from a MeOH extract of Cephalanceropsis gracilis
(
Orchidaceae) [24]. e structure assignment of 1 was supported by comparison of its NMR
spectroscopic data with the reported data of cephalandole C, though the configuration of

JOꢁꢂNAL OF ASIAN NATꢁꢂAL PꢂODꢁCTS ꢂESEAꢂCH
5
Figure 4. tꢀꢋ ꢋxpꢋꢅꢄmꢋꢈꢁꢆꢇ cd ꢊpꢋꢉꢁꢅꢍm (ꢌꢍꢇꢇ ꢇꢄꢈꢋ) ꢃꢌ 4 (bꢇꢍꢋ) ꢆꢈꢂ ꢁꢀꢋ ꢉꢆꢇꢉꢍꢇꢆꢁꢋꢂ ecd ꢊpꢋꢉꢁꢅꢆ (ꢂꢆꢊꢀꢋꢂ
ꢇꢄꢈꢋ) ꢃꢌ 4 (bꢇꢍꢋ) ꢆꢈꢂ ꢄꢁꢊ ꢋꢈꢆꢈꢁꢄꢃmꢋꢅ (ꢅꢋꢂ).
β-glucopyranosyl in the latter was undetermined. From the hydrolysate of 1 with snailase,
ꢀ
-glucose was isolated and identified by comparison of the retention factor (R ) on TLC,
f
20
1
specific rotation {[ꢀ] }, and H NMR spectroscopic data with those of an authentic sugar
D
sample (Experimental Section and Supporting Information). erefore, compound 1 was
determined as (+)-2-[3′-β-ꢀ-glucopyranosyloxy-1′H-indol-2′-yl)carboxamido]benzoic acid
and named isatindigotindoloside A.
20
Compound 2 was obtained as a white amorphous powder with[ꢀ]D −36.9 (c 0.17, MeOH).
−
1
−1
Its IR spectrum showed absorption bands for hydroxyl (3301 cm ), cyano (2243 cm ),
−
1
and aromatic ring (1620 and 1503 cm ) functionalities. e (+)-HR-ESI-MS and the NMR
spectral data of 2 indicated that it has the molecular formula C H N O e NMR spec-
1
7
20
2
7.
tral data of 2 (Tables 1 and 2) showed signals assignable to an ortho-trisubstituted benzene
ring, one trisubstituted double bond, a cyano group, and an isolated methylene, as well as
a methoxy group and a β-glucopyranosyl moiety. e presence of these units was proved
by cross peaks of H-1a/H-1b, H-5′/H-6′/H-7′, and H-1″/H-2″/H-3″/H-4″/H-5″/H -6″ in
2
1
1
the H- H COSY spectrum of 2 (Figure 2), as well as by their chemical shif s and coupling
constants (Table 1). In the HMBC spectrum of 2, correlations from H -2 to C-1, C-2′, C-3′,
2
and C-3′a; from H-2′ to C-3′, C-3′a, 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′ (Figure 2), together with the chemical
shif s of these carbon resonances (Table 2), revealed a 4′-substituted 2-(1′H-indol-3′-yl)
acetonitrile nucleus. Furthermore, the HMBC correlation from H-1″ to C-4′ indicated
substitution of the β-glucopyranosyloxy group at C-4′. e remaining methoxy group was
located at the nitrogen atom to satisfy the requirement of the molecular formula of 2. is
was supported by the chemical shif of the methoxy carbon resonance (δ 66.1) and the
C
absence of a nitrogen-bearing proton resonance in the NMR spectrum of 2 in DMSO-d .
6
Using the same protocols as described for 1, ꢀ-glucose was isolated and identified from
the enzymatic hydrolysate of 2. erefore, 2 was determined as (−)-2-[(1′-methoxy-4′-β-ꢀ-
glucopyranosyloxy)-1′H-indol-3′-yl]acetonitrile and named isatindigotindoloside B.

6
Y.ꢀF. LIꢁ ET AL.
20
Compound 3, a white amorphous powder with[ꢀ]D −34.3 (c 0.2, MeOH), has the molec-
+
ular formula of C H N O S as indicated by (+)-HR-ESI-MS at m/z 381.1113 [M + H]
1
7
20
2
6
and the NMR spectral data (Tables 1 and 2). e NMR spectra of 3 in CD OD exhibited
3
typical resonances for a 2′,3′-disubstituted indole nucleus, a β-glucopyranosyl unit, a cyano,
and a methoxy group, in addition to signals due to an unusual oxymethine (δ 5.80 and
H
δ 66.5). e presence of the cyano group in 3 was confirmed by an absorption band at
C
−1
2
234 cm in the IR spectrum. is suggests that 3 is an abnormal sulfur-containing indole
β-glucopyranoside with cyano and methoxy substituents, of which the structure was further
1
1
deduced by 2D NMR data analysis. Detailed analysis of H- H COSY and HSQC spectra
of 3 not only proved the occurrence of the above structural units, but also unambiguously
assigned the resonances of protons and proton-bearing carbons in the NMR spectra. In the
HMBC spectrum of 3, besides the long-range correlations confirming the 2′,3′-disubstituted
indole nucleus and β-glucopyranosyl units, the correlations from H-2 to C-1, C-2′, C-3′,
C-3′a, and OCH demonstrated the oxymethine carbon (C-2) was connected with both
3
the methoxy and cyano groups, as well as with C-3′ of the indole nucleus. In addition, the
HMBC correlations from H-1″ to C-2′, C-2″, C-3″, and C-5″, together with their chemical
shif s and the molecular composition, indicated that the β-glucopyranosyl must be linked
through the sulfur atom with C-2′ of the indole nucleus to give a gross structure of β-thi-
oglucopyranoside for 3. is was confirmed by comparison of the NMR spectral data of
3
with those of the related co-occurring indole β-thioglucopyranosides [5]. Compound 3
was hydrolyzed with 2 N HCl and the sugar isolated from the hydrolysate was identified as
-glucose using the same protocols as described for 1. e circular dichroism (CD) spec-
trum of 3 displayed positive Cotton effects at 213 and 297 nm and negative ones at 229 and
70 nm. Using the time-dependent density functional theory (TDDFT) method [25], the
ꢀ
2
calculated electronic circular dichroism (ECD) spectrum of 3 with the 2R-configuration
gave the similar Cotton effects except for that at 270 nm, as compared with those in the
experimental CD spectrum (Figure 3). is suggests the 2R-configuration for 3. erefore,
compound 3 (isatindigotindoloside C) was determined as (−)-(2R)-2-methoxy-2-(2′-β-ꢀ-
thioglucopyranosyl-1′H-indol-3′-yl)acetonitrile.
20
Compound 4 was obtained as a white amorphous powder with[ꢀ]D +11.9 (c 0.2, MeOH).
e spectroscopic data of 4 indicated that it was an isomer of 3. 2D NMR spectroscopic data

analysis revealed that 4 had the same planar structure as that of 3. Because ꢀ-glucose was
isolated and identified from the acid hydrolysate, 4 must be the C-2 epimer of 3. is was
supported by comparison of the CD spectra of 4 and 3. e CD spectrum of 4 showed the
positive Cotton effects at 232 and 270 nm and negative ones at 218 and 292 nm, of which
the wavelengths are similar to those of 3, but the corresponding signs are opposite. In addi-
tion, comparison of the experimental CD and calculated ECD spectra of 4 (Figure 4) also
supported the 2S-configuration. erefore, the structure of compound 4 was determined
and named isatindigotindoloside D.
20
Compound 5, a white amorphous powder with [ꢀ] −34.8 (c 0.38, MeOH), has the
D
molecular formula of C H NO S as determined based on (+)-HR-ESI-MS at m/z 378.0980
1
6
21
6
+
[
1
M + Na] and the NMR spectral data. e NMR spectral data of 5 in acetone-d (Tables
6
and 2) indicated that this compound is another indole β-ꢀ-thioglucopyranoside with a
methoxy group and an isolated methylene unit. e presence of a 1′H-indol-3′-yl nucleus
1
1
was confirmed by the H- H COSY cross peaks of H-5′/H-6′/H-7′ and the HMBC
correlations from H-2′ to C-3′, C-3′a, and C-7′a; from H-4′ to C-3′, C-5′, and C-7′a; from

JOꢁꢂNAL OF ASIAN NATꢁꢂAL PꢂODꢁCTS ꢂESEAꢂCH
7
H-7′ to C-3′a and C-5′ (Figure 2). Additionally, the HMBC correlations from H -1 to C-1″,
2
C-2′, C-3′, and C-3′a and from H-1″ to C-1, in combination with their chemical shif s,
evidenced that the β-thioglucopyranosyl was connected via the isolated methylene to C-3′
of the indole nucleus in 5. e chemical shif of the methoxy carbon resonance (δ 66.1)
C
indicated that the methyl group was located at N-1′ of the nucleus to match the requirement
of the molecular composition. is was supported by the absence of a nitrogen-bearing
1
proton resonance in the H NMR spectrum of 5 in acetone-d . e ꢀ-configuration of
6
β-thioglucopyranosyl was verified by isolation and identification of ꢀ-glucose from the acid
hydrolysate of 5. erefore, compound 5 was determined as (−)-(1′-methoxy-1′H-indole-
3
′-yl)methylthiol S-β-ꢀ-glucopyranoside and named isatindigotindoloside E.
By comparing the spectroscopic data with the reported data, the known compounds
were identified as indole-3-acetonitrile-2-S-β-ꢀ-glucopyranoside (6), indole-3-acetonitrile-
4
-methoxy-2-S-β-ꢀ-gluco- pyranoside (7), and N-methoxy-indole-3-acetonitrile-2-S-β-ꢀ-
glucopyranoside (8) [5], respectively.
In the preliminary in vitro assays [22], compounds 1, 6, and 7 showed antiviral activ-
ity against influenza virus A/Hanfang/359/95 (H3N2) with IC values of 14.6, 33.3, and
5
0
3
3.3 μM and SI values of 6.8, 3.0, and 3.0 (the positive control ribavirin gave an IC value
50
of 1.4 μM and a SI value of 814.1), respectively. At 10 μM, compounds 1 and 2 reduced
DL-galactosamine-induced hepatocyte (WB-F344 cell) damage with the same 39% inhi-
bition as the positive control bicyclol, and compound 1 also exhibited inhibitory activity
against the release of NO induced by LPS in microglial cell BV2 with an inhibition ratio of
9
3.0%, while the positive control curcumin showed 67.7% inhibition.
In summary, five new indole alkaloid glycosides, having diverse structure features, were
isolated as the minor constituents from the aqueous extract of “ban lan gen .” Among them,
three showed antiviral activity against influenza virus A/Hanfang/359/95 (H3N2). is,
together with our previous results [19–23], shows that diverse minor indole alkaloid com-
ponents have contributions to pharmacological efficacy that supports the traditional appli-
cations of the herbal medicine. Especially, the bioactive new structures not only provide
diverse scaffolds, entities, and hits for drug discovery, but also provide important clues for
in-depth investigation of biomimetic and total syntheses, chemical transformation, struc-
tural modification, and structure–activity relationships, as well as for drug development
based on the traditional herbal medicine.
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 FTIR microscope instrument (FTIR microscope transmission) (ermo
Electron Corporation, Madison, WI, USA). NMR spectra were recorded at 500 or 600 MHz
1
13
for H, and 125 or 150 MHz for C, respectively, on Inova 500 or SYS 600 (Varian Associates
Inc., Palo Alto, CA, USA) in DMSO-d , MeOH-d , or acetone-d , with solvent peaks used
6
4
6
as references. ESIMS and HRESIMS data were taken on an AccuToFCS JMS-T100CS
spectrometer (Agilent Technologies, Ltd., Santa Clara, CA, USA). Column chromatography

8
Y.ꢀF. LIꢁ ET AL.
(
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
2
&
0P (Mitsubishi Chemical Inc., Tokyo, Japan), or reverse-phase C-18 silica gel (W. R. Grace
Co., Maryland, USA). 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 C reverse-phase silica gel (5 μm) (W.
1
8
R. Grace & Co., Maryland, USA) or a Chiralpak AD-H column (250 × 10 mm i.d.) packed
with amylose tris(3,5-dimethylphenylcarbamate) coated on 5-μm silica gel (Daicel Chiral
Technologies Co. Ltd. Shanghai, China). 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% H SO in 95% EtOH followed by heating. Unless otherwise noted, all
2
4
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
00050, China). A voucher specimen (No. ID-S-2385) was deposited at the herbarium of
Natural Medicinal Chemistry, Institute of Materia Medica.

1
3
.3. Extraction and isolation

e air-dried and pulvarized plant material (50 kg) was decocted with H O (150 L; 3 × 1 h).
2

e aqueous extracts were combined and evaporated under reduced pressure to yield a
dark-brown residue (32 kg). e residue was dissolved in H O (122 L), loaded on a mac-
2
roporous adsorbent resin (HPD-110, 19 kg) column (20×200 cm), and eluted successively
with H O (50 L), 50% EtOH (125 L), and 95% EtOH (100 L) to yield three correspond-
2
ing fractions A, B, and C. Af er removing the solvent under reduced pressure, fraction
B (0.9 kg) was separated by CC over MCI gel CHP 20P (5 L), with successive elution
using H O (10 L), 30% EtOH (30 L), 50% EtOH (20 L), 95% EtOH (10 L), and Me CO
2
2
(
8 L) to give fractions B1-B5. Fraction B3 (165 g) was chromatographed over silica gel
and eluted by a gradient of increasing MeOH (0–100%) in EtOAc to yield subfractions
B3-1–B3-16. Subfraction B3-3 (7.5 g) was separated by CC over Sephadex LH-20, eluting
with CHCl –MeOH (1:1) to yield subfractions B3-3-1−B3-3-4, of which subfraction B3-3-4
3
(
3.0 g) was further chromatographed over silica gel, eluted by a gradient of increasing
MeOH (0−100%) in CHCl to yield subfractions B3-3-4-1−B3-3-4-14. Subfraction B3-3-
3
4
-10 (243.5 mg) was further fractionated by CC over Sephadex LH-20 (MeOH) to afford
B3-3-4-10-1−B3-3-4-10-8, of which subfraction B3-3-4-10-1 (48.0 mg) was separated by
RP-HPLC (39% MeOH in H O, 2.0 ml/min) to afford 8 (6.0 mg, t 90 min) and a mixture
2
R
(
7.0 mg, t 80 min). e mixture was further separated by HPLC on a semipreparative
R
Chiralpak AD-H column using n-hexane-iPrOH (6:1) as the mobile phase (1.5 ml/min) to
yield 3 (2.0 mg, t = 36.0 min) and 4 (2.5 mg, t = 40 min). Subfraction B3-3-4-11 (150.0 mg)
R
R
was chromatographed over Sephadex LH-20 (MeOH) to yield B3-3-4-11-1−B3-3-4-11-7,

JOꢁꢂNAL OF ASIAN NATꢁꢂAL PꢂODꢁCTS ꢂESEAꢂCH
9
of which B3-3-4-11-1(88.0 mg) was isolated by PTLC (ethyl acetate:MeOH:H O, 8:1:0.5,
2
v/v/v) to yield B3-3-4-11-1-1−B3-3-4-11-1-4. Subfraction B3-3-4-11-1-4 (16.0 mg) was
purified by RP-HPLC (45% MeOH in H O, 1.5 ml/min) to afford 2 (3.8 mg, t = 25 min),
2
R
5
(1.6 mg, t = 51 min), and 8 (3.1 mg, t = 58 min). Subfraction B3-3-4-12 (481.0 mg) was
R R
chromatographed over Sephadex LH-20 (MeOH) to yield B3-3-4-12-1−B3-3-4-12-10, of
which B3-3-4-12-2 (380.0 mg) was further separated by PTLC (ethyl acetate:MeOH:H O,
2
8
:1:0.5, v/v/v) to yield B3-3-4-12-2-1−B3-3-4-12-2-3. Subfraction B3-3-4-12-2-2 (300.0 mg)
was further isolated by RP-HPLC (57% MeOH in H O, 1.5 ml/min) to afford 6 (64.0 mg,
2
t = 44 min) and 7 (202.0 mg, t = 63 min). Subfraction B3-4 (11.0 g) was fractionated by
R
R
CC over Sephadex LH-20 (MeOH) to yield B3-4-1−B3-4-6, of which B3-4-5 (2.5 g) was
separated by RP-MPLC and eluted with a gradient of increasing MeOH (20−100%) in H O
2
to afford subfractions B3-4-5-1−B3-4-5-30. Subfraction B3-4-5-17 (15.0 mg) was purified
by RP-HPLC (25% MeOH, 1.5 ml/min) to yield 1 (7.0 mg, t = 28 min).
R
3
.3.1. Isatindigotindoloside A (1)
20
White amorphous solid; [ꢀ] +17.9 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 208 (4.21),
D
2
33 (4.00, sh), 263 (3.71), 315 (3.93) nm; IR νmax 3406, 2931, 1653, 1616, 1585, 1508, 1453,
−1
1
431, 1379, 1346, 1291, 1246, 1158, 1066, 1045, 957, 891, 828, 764, 741, 706, 663, 624 cm ;
1
13
H NMR (DMSO-d , 500 MHz) spectral data, see Table 1; C NMR (DMSO-d , 125 MHz)
6
6
−
spectral data, see Table 2; (−)-ESI-MS: m/z 457 [M−H] ; (+)-HR-ESI-MS: m/z 459.1407
[
+
+
M + H] (calcd for C H N O , 459.1398), 481.1222 [M + Na] (calcd for C H N O Na,
22 23 2 9 22 22 2 9
4
81.1218).
3
.3.2. Isatindigotindoloside B (2)
2
0
White amorphous solid; [ꢀ] −36.9 (c 0.17, MeOH); UV (MeOH) λ (log ε) 220 (4.10),
D
max
2
69 (3.43), 298 (3.41) nm; IR νmax 3505, 3301, 2921, 2884, 2243, 1659, 1620, 1582, 1503,
−1
1
459, 1408, 1391, 1365, 1319, 1252, 1093, 1044, 999, 978, 887, 763, 729, 663, 640, 580 cm ;
1
13
H NMR (DMSO-d , 500 MHz) spectral data, see Table 1; C NMR (DMSO-d , 125 MHz)
6
6
+
−
spectral data, see Table 2; (+)-ESI-MS: m/z 387 [M + Na] ; (−)-ESI-MS: 399 [M + Cl] ;
+
(+)-HR-ESI-MS: m/z 387.1168 [M + Na] (calcd for C H N O Na, 387.1163).
1
7
20
2
7
3
.3.3. Isatindigotindoloside C (3)
20
White amorphous solid; [ꢀ] −34.3 (c 0.2, MeOH); UV (MeOH) λ (log ε) 220 (4.81),
D
max
2
81 (4.37), 290 (4.37) nm; CD (MeOH) Δε213 nm +2.29, Δε229 nm −3.78, Δε270 nm −1.53,
Δε
+0.42; IR ν 3417, 1990, 2927, 2825, 2575, 2519, 2454, 2234, 1632, 1589, 1538,
2
97 nm
max
1
8
447, 1417, 1357, 1335, 1291, 1245, 1189, 1133, 1103, 1077, 1031, 992, 960, 900, 871, 847,
−1 1
00, 743, 678, 613, 578, 550 cm ; H NMR (CD OD, 600 MHz) spectral data, see Table 1;
3
1
3
+
C NMR (CD OD, 150 MHz) spectral data, see Table 2; (+)-ESI-MS: m/z 403 [M + Na] ;
3
−
−
+
(
−)-ESI-MS: 379 [M − H] , 415 [M + Cl] ; (+)-HR-ESI-MS: m/z 381.1113 [M + H] (calcd
+
for C H N O S, 381.1115), 403.0939 [M + Na] (calcd for C H N O SNa, 403.0934).
1
7
21
2
6
17 20
2
6
3
.3.4. Isatindigotindoloside D (4)
20
White amorphous solid;[ꢀ]D +11.9 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 220 (4.81), 281
(
4.37), 290 (4.37) nm; CD (MeOH) Δε −1.98, Δε +2.12, Δε +0.13, Δε −1.23;
2
18 nm
232 nm
270 nm
292 nm
IR ν 3367, 2925, 2853, 2245, 1653, 1617, 1528, 1448, 1421, 1347, 1275, 1247, 1189, 1099,
max
−1 1
1
071, 957, 920, 890, 846, 799, 748, 687, 590 cm ; H NMR (CD OD, 600 MHz) spectral
3

10
Y.ꢀF. LIꢁ ET AL.
1
3
data, see Table 1; C NMR (CD OD, 150 MHz) spectral data, see Table 2; (+)-ESI-MS: m/z
3
+
+
−
−
4
03 [M + Na] , 419 [M + K] ; (−)-ESI-MS: 379 [M − H] , 415 [M + Cl] ; (+)-HR-ESI-MS:
+
+
m/z 381.1122 [M + H] (calcd for C H N O S, 381.1115), 403.0943 [M + Na] (calcd for
1
7
21
2
6
C H N O S Na, 403.0934).
1
7
20
2
6
3
.3.5. Isatindigotindoloside E (5)
20
White amorphous solid; [ꢀ] −34.8 (c 0.38, MeOH); UV (MeOH) λ (log ε) 205 (4.27,
D
max
sh), 223 (4.44), 275 (3.63), 291 (3.67) nm; CD (MeOH) Δε
−4.96, Δε
+0.23; IR
2
22 nm
268 nm
ν
3357, 2933, 2863, 1612, 1600, 1546, 1454, 1418, 1355, 1322, 1278, 1231, 1116, 1096,
max
−1 1
1
058, 1031, 984, 961, 925, 889, 832, 741, 652, 569 cm ; H NMR (Me CO-d , 600 MHz)
2 6
1
3
spectral data, see Table 1; C NMR (Me CO-d , 150 MHz) data, see Table 2; (+)-ESI-MS:
2
6
+
+
+
m/z 378 [M + Na] , 394 [M + K] ; (+)-HR-ESI-MS: m/z 378.0980 [M + Na] (calcd for
C H NO SNa, 378.0982).
1
6
21
6
3.3.6. Enzymatic hydrolysis of 1–2 and acidic hydrolysis of 3–5
Compounds 1 and 2 (~1.0 mg) were separately hydrolyzed in H O (3 ml) with snailase
2
(
3.0 mg, CODE S0100, Beijing Biodee Biotech Co., Ltd., Beijing, China) at 37 °C for 24 h.
Compounds 3–5 (~1.2 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 CH CN−H O (8:1) to afford sugar. e sugar (0.3–0.6 mg)

3
2
showed a retention factor (R ≈ 0.38) on TLC (EtOAc-MeOH-AcOH-H O, 12:3:3:2), with
f
2
20
1
[
ꢀ] values of +44.2–+47.5 (c 0.03–0.06, H O), and H NMR (D O) data in agreement with
D
2
2
those of an authentic ꢀ-glucose.
3
.3.7. ECD calculation of 3−4
For details, see Supporting Information. Briefly, conformational analysis was performed by
Monte Carlo searching method with the MMFF94 molecular mechanics force field using the
Spartan 10 sof ware for both 3 and 4. e lowest energy conformers with relative energies
lower than 2 kcal/mol were reoptimized 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 reoptimized 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 of each compound, the simulated spectra of its
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.
Acknowledgment
We thank Chinese Academy of Medical Sciences and Peking Union Medical College High Performance
Computing Platform for supporting the calculation of the ECD spectra.

JOꢁꢂNAL OF ASIAN NATꢁꢂAL PꢂODꢁCTS ꢂESEAꢂCH
11
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
number: 81373287], [grant number: 30825044]; the Beijing Excellent Talent Training Project [grant
number: 2013D009008000002]; and the National Science and Technology Project of China [grant
number: 2012ZX09301002-002], [grant number: 2011ZX0 9307-002-01].
Note
We dedicate this paper to Prof. Zhong-Jian Jia on the occasion of her 85th birthday.
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