Fitoterapia 137 (2019) 104189

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Lignans from Isatis indigotica roots and their inhibitory effects on nitric oxide T
production
Dongdong Zhanga, Jingyi Lia, Deqing Ruana, Zhaoqiang Chenb, Weiliang Zhub, Yanhong Shic,
⁎ ⁎
Kaixian Chena,b, Yiming Lia, , Rui Wanga,
a
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
b
Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China
c
Institute of TCM International Standardization of Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

A R T I C LE I N FO A B S T R A C T

Keywords: Seventeen lignans (1–17) were obtained from the roots of Isatis indigotica (I. indigotica). Among them, isa-
Isatis indigotica tindigosesquilignans A-C (1–3) were deduced as three undescribed sesquilignans, which possess unusual carbon
Lignans skeleton of aryltetralin unit connected with a C6-C3 moiety via a five-membered ring of C-3-C-8″-C-7″-O-C-4.
Structure determination Isatindigosesquilignans B and C were determined as the first examples of its glycosides from a natural source and
Anti-inflammatory activity
a plausible biosynthetic pathway was proposed. Moreover, all of the isolated lignans were assayed regarding
Nitric oxide production
their inhibitory effects on nitric oxide (NO) production in RAW 264.7 cells and compounds 1, 2 and 7 showed
inhibitory effects with IC50 values ranging from 19.46 μM to 64.82 μM.

1. Introduction dimethoxyphenyl)-methylene]-2(3H)-furanone (7) [11], lariciresinol

The roots of Isatis indigotica Fort. (Cruciferae), named Ban Lan Gen
in China, is a famous traditional Chinese medicine (TCM) [1,2]. It is
widely distributed and cultivated in the North of the Yangtze River in
China and is usually used for the treatment of influenza, cold, fever,
epidemic hepatitis and infections [3–5]. In industrial manufacture, I.
indigotica is usually used as an important source for indirubin and in-
digo exaction, which are momentous dyes [6]. Lignans have been re-
ported as one of the most important chemical constituents in I. in-
digotica, which possess anti-inflammatory, antiviral, antibacterial,
antitumor and antioxidant activities [6,7]. In order to explore more
bioactive lignans from I. indigotica, seventeen lignans were obtained
through the chemical constituents and pharmacological investigations.
Among them, isatindigosesquilignans A-C (1–3) were deduced as three
new sesquilignans, whose structures and absolute configurations were
determined by extensive spectroscopic data analysis, including 1D, 2D
NMR, HR-ESI-MS data, optical rotation data, and comparison of their
experimental CD and calculated ECD spectra. The known ones (4–17,
Fig. 1) were identified by comparison of their spectroscopic and optical
rotation data with those of reported literatures as (−)-isolariciresinol
(4) [8], spicatolignan B (5) [9], 2-(3′-hydroxy-5′-methoxyphenyl)-3-
hydroxylmethyl-7-methoxy-2,3-dihydrobenzofuran-5-carboxylic acid
(6) [10], (E)-5-(4-hydroxy-3,5-dimethoxyphenyl)-3-[(4-hydroxy-3,5-
(8) [12], lariciresinol-4-β-D-glucoside (9) [13], (−)-justiciresinol (10)
[14], conicaoside (11) [15], (+)-epipinoresinol-4-β-D-glucoside (12)
[16], (1S,2R,5S,6R)-2-(4-hydroxyphenyl)-6-(3-methoxy-4-hydro-
xyphenyl)-3,7-dioxabicyclo[3.3.0]octane (13) [17], (+)-pinoresinol
(14) [18], (+)-pinoresinol-4-β-D-glucopyranoside (15) [19], syringar-
esinol (16) [20] and syringaresinol-4-β-D-glucoside (17) [21]. The ni-
tric oxide (NO) inhibitory activities of the isolated compounds (1–17)
were also evaluated. Herein, we reported the isolation and structure
elucidation, putative biosynthetic pathway, and the NO inhibitory ac-
tivities of these compounds.
2. Experimental section
The general experimental procedures are listed in the Supporting
information. The air-dried roots of Isatis indigotica (45 kg) were ex-
tracted with 80% EtOH for three times. After removing the solvent
under reduced pressure, the concentrated residue was successively
partitioned with petroleum ether (PE), dichloromethane (CH2Cl2) and
n-BuOH. The detailed process of isolation is described in the supporting
information.

⁎
Corresponding authors.
E-mail addresses: ymlius@163.com (Y. Li), wr@shutcm.edu.cn (R. Wang).

https://doi.org/10.1016/j.fitote.2019.104189
Received 25 April 2019; Received in revised form 29 May 2019; Accepted 30 May 2019
Available online 31 May 2019
0367-326X/ © 2019 Published by Elsevier B.V.
D. Zhang, et al.
Fig. 1. Structures of compounds 1–17.
2.1. Plant material
The Isatis indigotica roots (Ban Lan Gen) was collected on July 2017
from the Daqing city, HeiLongJiang Province of China, and it was au-
thenticated by one of our Co-authors Rui Wang professor (School of
Pharmacy, Shanghai University of Traditional Chinese Medicine). A
voucher specimen (herbarium No. 20170716SL) has been deposited in
the Medicinal Plants Herbarium (MPH), School of Pharmacy, Shanghai
University of Traditional Chinese Medicine, Shanghai, China.
2.2. Physical and spectroscopic data of isatindigosesquilignans A–C
Isatindigosesquilignan A (1), white amorphous power; [α]D20 43.5°
(c 0.16, MeOH); IR (KBr) νmax: 3428, 2937, 1598, 1514, 1460, 1261,
1139, 1070, 1024, 859, 813, 760 cm−1; m/z 539.2290 [M + H]+
(calcd for 539.2276 [M + H]+); 1H NMR (DMSO‑d6, 600 MHz) and 13C
NMR (DMSO‑d6, 150 MHz) see Table 1.
Isatindigosesquilignan B (2), white amorphous power; [α]D20 40.0°
(c 0.10, MeOH); IR (KBr) νmax: 3445, 2921, 1616, 1513, 1462, 1265,
1121, 1071, 1031, 855, 802, 721 cm−1; m/z 723.2609 [M + Na]+
(calcd for 723.2623 [M + Na]+); 1H NMR (DMSO‑d6, 600 MHz) and
13
C NMR (DMSO‑d6, 150 MHz) see Table 1.
Isatindigosesquilignan C (3), white amorphous power; [α]D20 31.4°
2
Fitoterapia 137 (2019) 104189
(c 0.11, MeOH); IR (KBr) νmax: 3415, 2936, 1598, 1512, 1460, 1423,
1123, 1070, 1029, 824, 761 cm−1; m/z 723.2633 [M + Na]+ (calcd for
723.2623 [M + Na]+); 1H NMR (DMSO‑d6, 600 MHz) and 13C NMR
(DMSO‑d6, 150 MHz) see Table 1.
2.3. ECD calculation of compounds 1–3
The conformers of compounds 1–3 were obtained using the MM2
force field with ChemBio3D software. Gaussian 09 software was utilized
for the semiempirical PM3 quantum mechanical geometry optimiza-
tions and the time-dependent density functional theory (TDDFT) ECD
was calculated at the b3lyp/6-31 g(d) level [22,23]. The ECD spectra
conformers of 1–3 were obtained using SpecDis 1.62 and compared
with the experimental data, the calculation details are listed in the
supporting information (Figs. S28–S36).
2.4. Acid hydrolysis of compounds 2–3 and absolute configuration
determination of sugars
Solutions of 2 and 3 (2.0 mg each) were hydrolyzed in 2 M hydro-
chloric acid (4 mL) at 80 °C for 2 h. After cooling, each solution was
concentrated under vacuum, dissolved with water, and extracted twice
with dichloromethane (CH2Cl2). The aqueous parts were treated via a
D. Zhang, et al. Fitoterapia 137 (2019) 104189

Table 1 spectrum (Table 1) of 1 showed signals of two ABX spin system (1,3,4-
1
H and 13C NMR data for compounds 1–3 (δ in ppm, J in Hz). trisubstituted benzene ring) at δH 6.56 (1H, d, J = 2.2 Hz, H-2′), 6.58
No. 1a 2a 3a (1H, d, J = 8.1 Hz, H-5′) and 6.45 (1H, dd, J = 8.1, 2.2 Hz, H-6′) and
6.55 (1H, d, J = 2.2 Hz, H-2″), 6.54 (1H, d, J = 8.1 Hz, H-5″) and 6.25
δH δC δH δC δH δC (1H, dd, J = 8.1, 2.2 Hz, H-6″); a 1,2,3,4,5-pentasubstituted aromatic
rings at δH 6.63 (1H, s, H-6), two exchangeable proton at δH 8.67 (1H,
1 129.2 129.3 128.1
2 130.6 130.8 130.8 brs, OH-4′) and 8.92 (1H, brs, OH-4″) and three methoxy groups at δH
3 127.7 127.5 127.5 3.54, 3.64 and 3.72 (each 3H, s). The 13C NMR spectrum (Table 1)
4 146.2 146.2 146.1 displayed 30 carbon signals, based on the DEPT 135° and HSQC ex-
5 142.4 142.5 142.6 periments, eleven quaternary carbons at δC (147.7, 147.6, 146.7, 146.2,
6 6.63, s 112.5 6.63, s 112.5 6.77, s 112.5
144.8, 142.4, 137.1, 133.4, 130.6, 129.2, 127.7), twelve tertiary car-
7 2.60, m; 2.72, 33.5 2.58, m; 2.67, 33.5 3.32, m; 3.38, 31.8
m m m bons at δC (121.1, 118.7, 115.5, 115.4, 113.1, 112.5, 111.1, 86.6, 51.9,
8 1.66, ov 47.7 1.66, ov 47.5 2.06, m 47.5 47.7, 43.4, 38.5), four secondary carbons at δC (64.2, 62.2, 60.1, 33.5),
9 3.29, m; 3.41, 60.1 3.32, m; 3.38, 60.1 3.06, m; 3.24, 66.4 as well as three methoxy carbons at δC (55.8, 55.8, 56.0) were also
m m m
observed. When comparing these NMR data with the reported sesqui-
1′ 137.1 137.0 137.0
2′ 6.56, d (2.2) 113.1 6.59, d (2.2) 113.2 6.59, ov 112.6
lignan of majusalignan A [26], they showed almost similar NMR
3′ 147.6 147.6 147.8 spectroscopic features except the 7′-(5-hydroxy-3-methoxyphenyl)
4′ 144.8 144.8 144.8 benzene ring in majusalignan A was replaced by 7′-(4-hydroxy-3-
5′ 6.58, d (8.1) 115.5 6.58, d (8.1) 115.5 6.20, d (7.7) 115.6 methoxyphenyl) benzene ring in isatindigosesquilignans A (1). This
6′ 6.45, dd (8.1, 121.1 6.43, dd (8.1, 121.1 6.59, ov 119.8
inference was confirmed by detailed analysis of the 1H NMR data of the
2.2) 2.2)
7′ 4.03, d (7.0) 43.4 4.03, d (7.0) 43.4 4.18, d (2.4) 42.1 7′-aromatic rings at δH [6.56 (1H, d, J = 2.2 Hz, H-2′), 6.58 (1H, d,
8′ 1.66, ov 38.5 1.66, ov 38.5 2.60, m 41.2 J = 8.1 Hz, H-5′) and 6.45 (1H, dd, J = 8.1, 2.2 Hz, H-6′)] and 2D NMR
9′ 3.42, m; 3.55, 64.2 3.42, m; 3.56, 64.2 2.85, m; 3.21, 65.3 data of 1 including HSQC, HMBC, 1He1H COSY experiments (Fig. 2).
m m m
The proton and protonated carbon resonances in the NMR spectra of 1
1″ 133.4 136.2 136.7
2″ 6.55, d (2.2) 111.1 6.53, d (2.2) 111.1 6.84, d (1.6) 110.2
were unambiguously assigned by the HSQC experiment. 1He1H COSY
3″ 147.7 149.2 149.3 correlations of H-7/H-8/H-9 and HMBC correlations of H-7/C-1, C-2
4″ 146.7 146.5 146.4 and C-6 and 1He1H COSY correlations of H-7’/H-8’/H-9′ and HMBC
5″ 6.54, d (8.1) 115.4 6.84, d (8.1) 115.2 7.04, d (8.3) 115.6 correlations of H-7’/C-1′, C-2′ and C-6′ determined two C6-C3 units in
6″ 6.25, dd (8.1, 118.7 6.38, dd (8.1, 118.2 6.77, ov 117.9
1. The HMBC correlations of H-7’/C-1, C-2 and C-3, along with the
2.2) 2.2) 1
7″ 5.38, d (2.5) 86.6 5.38, d (2.5) 86.2 5.63, d (3.4) 86.5 He1H COSY correlations of H-7/H8/H8’/H7’ deduced the two C6-C3
8″ 2.73, m 51.9 2.72, m 52.3 3.41, ov 53.1 units connected via a six-membered ring of C-1-C-2-C-7’-C-8’-C-8-C-7
9″ 3.26, m; 3.74, 62.2 3.28, m; 3.74, 62.1 2.85, m; 2.98, 63.6 (Fig. 1). Then an aryltetralin unit was observed by the analysis above.
m m m
The third C6-C3 unit was observed by the 1He1H COSY correlations of
5-OMe 3.72, s 55.8 3.73, s 55.9 3.80, s 56.1
3′-OMe 3.54, s 55.8 3.51, s 55.9 3.66, s 56.0
H-7″/H-8″/H-9″ and HMBC correlations of H-7”/C-1″, C-2″ and C-6″.
4′-OH 8.67, brs 8.64, brs 8.69, brs The HMBC correlations of H-8″/C-2, C-3 and C-4 and correlations of H-
3″-OMe 3.64, s 56.0 3.64, s 56.1 3.69, s 56.2 7″/C-3 and C-4 deduced the third C6-C3 unit connected with the ar-
4″-OH 8.92, brs yltetralin unit via a five-membered ring of C-3-C-8″-C-7″-O-C-4. Fur-
Glc-1 4.81, d (7.3) 100.4 4.88, d (7.3) 100.4
thermore, the HMBC correlations of 5-OMe/C-5, 3’-OMe/C-3′ and 3″-
2 3.23, m 73.6 3.23, m 73.7
3 3.25, ov 77.4 3.22, ov 77.4 OMe/C-3″ confirmed the 5-OMe, 3′-OMe and 3″-OMe in 1. The planar
4 3.14, m 70.1 3.16, m 70.1 structure of 1 was thus deduced as depicted in Fig. 1.
5 3.25, ov 77.4 3.27, ov 77.3 In the ROESY spectrum (Fig. S9, Supporting information) of 1, ROE
6 3.43, ov; 3.64, 61.0 3.43, ov; 61.1 correlations observed from H-8/H-7a and H-7′ and from H-8′/H-7b
ov 3.64, ov
along with the coupling constants (J7′, 8′ = 7.0 Hz) confirmed the trans-
a 1
H NMR and 13C NMR were measured at 600 MHz and 150 MHz in orientation of H8′/H7′ and H8′/H8. ROE correlations observed from H-
DMSO‑d6; ov: overlap signals. 7″/H-8″ along with the coupling constants (J7″, 8″ = 2.5 Hz) confirmed
the cis-orientation of H-7″/H-8″ [27]. In the CD spectrum, the positive
similar method which we described previously [24]. The D configura- cotton effects (CEs) in 240-250 nm and the negative CEs in 290-300 nm
tions of the β-glucopyranosyl glucose moiety in 2 and 3 were confirmed (Fig. 2) disclosed the 8S,7′R,8′S absolute configuration of 1 [28].
through comparison their retention times (tR) with those of authentic Therefore, the absolute configurations of 1 would be 8S,7′R,8′S,7″R,8″R
sugar samples (tR D-glucose 45.23 min, tR L-glucose 45.38 min). or 8S,7′R,8′S,7″S,8″S. To further determine its absolute configurations,
the ECD curves (Fig. 3) were simulated for the two epimers of 1
[(8S,7′R,8′S,7″R,8″R)-1 and (8S,7′R,8′S,7″S,8″S)-1]. The experimental
2.5. Inhibitory assay of NO production
and calculated ECD curves of (8S,7′R,8′S,7″R,8″R)-1 matched well.
Accordingly, the structure of isatindigosesquilignans A was elucidated
Compounds 1–17 were tested for their inhibitory effects on the NO
as depicted (Fig. 1). To the best of our knowledge, isatindigosesqui-
production in the LPS activated RAW 264.7 cells using the same method
lignans A is the second example, which possesses a novel carbon ske-
as we previously reported [25]. The IC50 values of compounds 1–17
leton as majusalignan A from natural source.
were calculated (see Table 2).
Isatindigosesquilignan B (2) was obtained as a white amorphous
power, with a molecular formula as C36H44O14, (15 IHD), which was
3. Results and discussion deduced from the NMR data and the HR-ESI-MS positive ion at m/z
723.2609 [M + Na]+ (calcd for 723.2623 [M + Na]+). When com-
Isatindigosesquilignan A (1) was obtained as a white amorphous paring the NMR data (Table 1) of 2 and 1, they showed almost the same
power, with [α]D20 43.5° (c 0.16, MeOH). Its molecular formula was spectroscopic features of the aglycone moiety. The differences were the
assigned as C30H34O9, (implying an index of hydrogen deficiency (IHD) appearance of a glucose unit observed in 2 (δC 100.4, 73.6, 77.4, 70.1,
of 14), by the 1D NMR data and the HR-ESI-MS positive ion at m/z 77.4, 61.0) [13–16] and the disappearance of the phenolic hydroxyl
539.2290 [M + H]+ (calcd for 539.2276 [M + H]+). The 1H NMR signal of 4-OH. These differences may deduce compound 2 as the 4″-

3
D. Zhang, et al. Fitoterapia 137 (2019) 104189

Table 2
NO inhibitory activities of compounds 1–17 in RAW 264.7 cell line.
Comopounds IC50a Cytotoxicity Comopounds IC50a Cytotoxicity

1 18.53 ± 2.21 > 100 10 > 100 > 100

2 28.83 ± 3.50 > 100 11 > 100 > 100
3 > 100 > 100 12 > 100 > 100
4 > 100 > 100 13 > 100 > 100
5 > 100 > 100 14 > 100 > 100
6 > 100 > 100 15 > 100 > 100
7 64.28 ± 3.17 > 100 16 > 100 > 100
8 > 100 > 100 17 > 100 > 100
9 > 100 > 100 AGb 22.67 ± 0.39 > 100

a
IC50 values were expressed as mean ± SD (n = 3).
b
AG = aminoguanidine hydrochloride was used as the positive control.

glucoside of compound 1. Analysis of the 2D NMR data including
HSQC, HMBC (Fig. 2) and 1He1H COSY (Fig. 2) and MS data confirmed
the above conclusion. Acid hydrolysis of 2 resulted in the product of D-
glucose which was confirmed by GC analysis of derivatives of the hy-
drolysate of 2 and the authentic sugars (tR D-glucose 45.23 min, tR L-
glucose 45.38 min). The large coupling constant of Glc-H1 (J = 7.3 Hz)
revealed the β-glucopyranosyl linkage in 2 [29,30]. The ROE correla-
tions (as the same description as 1, Fig. S18, supporting information)
and the CD spectrum of 2 revealed that the absolute configurations of 2
would be 8S,7′R,8′S,7″R,8″R or 8S,7′R,8′S,7″S,8″S. Comparison of ex-
perimental and calculated ECD curves (Fig. 3) of 2 confirmed the
(8S,7′R,8′S,7″R,8″R) absolute configurations of 2. Accordingly, the
structure of isatindigosesquilignans B was elucidated as depicted
(Fig. 1). This is the first example of a glucoside of isatindigosesqui-
lignans A from natural source and a plausible biosynthetic pathway was
proposed (Fig. 4).
Isatindigosesquilignan C (3), a white amorphous powder, has the
same molecular formula C36H44O14 as compound 2, which was in-
dicated by the positive HR-ESI-MS ion at m/z 723.2609 [M + Na]+
(calcd for 723.2623 [M + Na]+) and NMR data. When comparing the
NMR data (Table 1) of 3 and 2, they showed almost identical NMR
spectroscopic features. Detailed analysis of the NMR data showed the
different spectroscopic features around C-7 (downfield of C-9, C-8′, C-
9′, C-8″ and C-9″, and upfield of C-1, C-7, C-2′ and C-6′). The above
differences along with the ROE correlations (Fig. 2) of H-8′/H-7′ and H-
7a and correlations of H-8/H-7b supported isatindigosesquilignan C
would be the 7′-enantiomers of isatindigosesquilignan B. Comparison of
experimental and calculated ECD curves (Fig. 3) of 3 confirmed the
(8S,7′S,8′S,7″R,8″R) absolute configurations of 3. The β-configuration
for the D-glucose moiety in 3 was confirmed by the same method as 2.
Accordingly, the structure of isatindigosesquilignans C was elucidated
as depicted (Fig. 1). This is the first example of epimers of isatindigo-
sesquilignans B and a plausible biosynthetic pathway was proposed
(Fig. 4).
As a traditional Chinese medicine, Ban Lan Gen is used for the
treatments of influenza, cold, fever, epidemic hepatitis and infections
and is reported with potent anti-inflammatory effects. Hence, com-
pounds 1–17 were tested for their inhibitory effects on the NO pro-
duction of LPS activated RAW 264.7 cells. The results (Table 2) sug-
gested that almost all of the monoepoxylignans and the bisepoxylignans
showed on inhibitory activities, and only two sesquilignans (1 and 2)
and a lignanolids (7) exhibited inhibitory activities with IC50 values of
19.46 μM, 28.83 μM and 64.28 μM, respectively.
Isatindigosesquilignan A is the second example of 4′-hydroxy-3′-
methoxyphenyl analogue of majusalignan A which is firstly reported to
possess a novel carbon skeleton of aryltetralin unit linked with a C6-C3
unit and a five-membered ring by a C-7″ and C-4 linkage pattern via an
oxygen atom [26]. Isatindigosesquilignans B and C are isolated as the

Fig. 2. Key 1He1H COSY, HMBC and ROESY correlations of compounds 1–3.

4
D. Zhang, et al.
Fig. 3. Experimental and calculated ECD spectra of 1–3.
Fig. 4. Plausible biosynthetic pathway of compounds 1–3.
first examples of the glucoside of isatindigosesquilignan A from a nat-
ural source. Based on the unique structural feature, a plausible bio-
synthetic pathway for isatindigosesquilignans A-C are proposed in
Fig. 4. The biosynthetic precursors of 1–3 are proposed from L-pheny-
lanaline [7]. First, L-phenylanaline was modified by sequential or si-
multaneous enzymatic catalysis to give 1a and 1b [7], and then 1b with
a dimerization step via an enzyme-catalyzed reaction to give 1c and 1d
[15]. 1e and 1f were obtained by enzymatic catalysis of 1c and 1d [26],
respectively. 1e connected with 1b by steps of free radicals coupling
and nucleophilic reactions [26] to give 1 and then changed via a step of
glycosylation [5] to give 2. Compound 3 was obtained by 1f via the
same reactions of 1e.
Declaration of competing interests
The authors declare no competing financial interest.
Acknowledgments
This work was supported by National Natural Science Foundation of
China (81573571, 81673570), Excellent Academic Leaders Program of
Shanghai (16XD1403500), the programs of High level University
Innovation Team and Shanghai E-Research Institute of Bioactive
Constituents in Traditional Chinese Medicine and Shanghai Scientific
and Technological Innovation Program (18401931100). The authors
thank Songshan Shi (S.S.) in the Institute of Chinese Materia Medica,
Shanghai University of Traditional Chinese Medicine for his kind help
on the NMR spectra collected.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2019.104189.
5
Fitoterapia 137 (2019) 104189
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