A new chlorine-containing iridoid glycoside from Plantago maxima

Article abstract of DOI:10.1080/14786419.2019.1655410

A phytochemical investigation on the whole plant of Plantago maxima Juss. ex Jacq led to the isolation of a new and rare chlorinated iridoid glycoside named plantomoside (1), along with three known compounds, geniposidic acid (2), 10-deoxygeniposidic acid (3), and viteoid II (4). The structure of 1 was determined through 1 D and 2 D NMR spectroscopic data analysis, HR-ESI-MS, and acid hydrolysis.

Full text of DOI:10.1080/14786419.2019.1655410

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new chlorine-containing iridoid glycoside from
Plantago maxima
Liang-Liang Ma, Lu-Lu Wang, Yue-Feng Zhang, Xiang-Fu Jiang, Xin-Liu Zhu, Ke
Pan, Chuan-Xing Wan & Zhong-Bo Zhou
To cite this article: Liang-Liang Ma, Lu-Lu Wang, Yue-Feng Zhang, Xiang-Fu Jiang, Xin-Liu Zhu,
Ke Pan, Chuan-Xing Wan & Zhong-Bo Zhou (2019): A new chlorine-containing iridoid glycoside
from Plantagoꢀmaxima, Natural Product Research, DOI: 10.1080/14786419.2019.1655410
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1655410
A new chlorine-containing iridoid glycoside from
Plantago maxima
a
ꢀ
b
ꢀ
a
a
Liang-Liang Ma , Lu-Lu Wang , Yue-Feng Zhang , Xiang-Fu Jiang ,
b
b
a
a
Xin-Liu Zhu , Ke Pan , Chuan-Xing Wan and Zhong-Bo Zhou
a
Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological
b
Resources in Tarim Basin, Tarim University, Alaer, PR China; School of Traditional Chinese Pharmacy,
China Pharmaceutical University, Nanjing, PR China
ABSTRACT
ARTICLE HISTORY
A phytochemical investigation on the whole plant of Plantago
maxima Juss. ex Jacq led to the isolation of a new and rare chlori-
nated iridoid glycoside named plantomoside (1), along with three
known compounds, geniposidic acid (2), 10-deoxygeniposidic acid
Received 27 April 2019
Accepted 2 August 2019
KEYWORDS
Plantaginaceae; Plantago
maxima; iridoid glycoside
(
3), and viteoid II (4). The structure of 1 was determined through
D and 2 D NMR spectroscopic data analysis, HR-ESI-MS, and acid
1
hydrolysis.
1. Introduction
The genus Plantago L. (Plantaginaceae) comprises more than 200 species distributed
with a cosmopolitan distribution, many of which have been used as folk remedies for
the treatment of wounds, insect stings, eye irritation and inflammation of mouth and
throat (Ronsted et al. 2003; Goncalves and Romano 2016; Zubair et al. 2016; Adom
et al. 2017). Previous phytochemical studies on Plantago species have revealed
the presence of several classes of bioactive metabolites including phenylethanoid
CONTACT Chuan-Xing Wan
Joint first authors
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1655410.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
wanchuanxing@163.com; Zhong-Bo Zhou
zzb7855@163.com
?

2
L.-L. MA ET AL.
Figure 1. Structures of compounds 1 –4.
glycosides, iridoids, as well as flavonoids, phenolic acids and polysaccharides (Jensen
et al. 1996; Franzyk et al. 1998; Zaghloul and Zaghloul 2000; Jankovic et al. 2012; Yin
et al. 2019). Iridoids are a type of monoterpenoids possessing a cyclopentanopyran
ring system and usually occur as glycosides (El-Naggar and Beal 1980). These
compounds are plant protectants and useful as chemotaxonomic markers of several
genera in various plant families such as Plantago (Plantaginaceae), Galium (Rubiaceae),
Scrophularia (Scrophulariaceae) (Andrzejewska-Golec et al. 1993), and Cornus
(Cornaceae) (He et al. 2017). Plantago maxima Juss. ex Jacq is a perennial herb
distributed mainly in the Western Siberia and in the European part of Russia, but it
also has some localities in Xinjiang Uygur Autonomous Region of China. In the search
for novel bioactive constituents of the whole plants of P. maxima, a new chlorine-
containing iridoid glycoside named plantomoside (1), together with three known
analogues (2–4), were isolated. Herein, we describe the isolation and structure
elucidation of these compounds.
2. Results and discussion
Compound 1 was obtained as white amorphous solid. In the HR-ESI-MS of 1, the
-
molecular ion peaks [M-H] at m/z 423.1068 (calcd for C H ClO , 423.1063) and
17
24
10
425.1034 with an abundance ratio of 3:1 implied the existence of a chlorine atom in 1,
and established its molecular formula as C H ClO . The UV spectrum showed
17
25
10
absorption maxima at 233 nm. The IR spectrum exhibited absorptions for hydroxyl
ꢁ
1
ꢁ1
ꢁ1
(
3379 cm ) and carbonyl (1694 cm ) groups and an enol ether system (1592 cm ).
1
The H NMR spectrum of 1 displayed signals characteristic of an iridoid skeleton,
including an olefinic proton at dH 7.45 (s), a methoxy group at dH 3.71 (3 H, s), and
a hemiacetal proton at d 5.34 (d, J ¼ 7.5 Hz). In addition, the signals of an anomeric
H
proton at d 4.72 (d, J ¼ 7.8 Hz) and a set of oxygenated protons in the range of d
H
H
13
3
.24 ꢁ 3.92 were clearly observed, suggesting that 1 was an iridoid glycoside. The
C
NMR data of 1 were highly similar to those of 6,7-dihydrogardenoside (Damtoft et al.

NATURAL PRODUCT RESEARCH
3
1
981), except for an obvious upfield shift of C-10 signal (d 52.5, 1; d 66.0, 6,7-dihy-
C C
drogardenoside). This observation, together with HR-ESI-MS analysis, suggested the
only difference between 6,7-dihydrogardenoside and 1 was the replacement of the
hydroxyl group at C-10 in 6,7-dihydrogardenoside by a chlorine atom in compound 1.
Interpretation of 2 D NMR data including HSQC and HMBC spectra (Figure S1) con-
firmed the planar structure of 1 as depicted in Figure 1, and allowed an unambiguous
assignment of all proton and carbon signals. The relative configuration of 1 was estab-
13
lished by analysing the ROESY (Figure S1) and C NMR data. ROESY correlation
between H-5 and H-9, together with the chemical shift of C-1 at d 95.4 (Dinda et al.
C
2007), suggested that 1 possessed a bicyclic H-5/H-9 b,b-cis-fused cyclopentanopyran
ring system, which is the most common structural feature of naturally occurring
iridoids (Krull et al. 1998; He et al. 2014). Moreover, ROESY correlations between H-1
and H-6a/H-7a/H -10 indicated the a-orientation of H-1 and H -10. Acid hydrolysis of 1
2
2
furnished D-glucose, as identified by a comparison of the HPLC retention times of
its thiocarbamoyl-thizolidine derivative (tR 12.9 min) with those of sugar standards
(Tanaka et al. 2007). Accordingly, the structure of 1 was assigned and named
plantomoside. After rehmaglutin D (Gen c¸ et al. 2010), plantomoside (1) represents the
second chlorinated iridoid glucoside identified from the genus Plantago.
The known compounds were identified as geniposidic acid (2) (Jensen et al. 1996), 10-
deoxygeniposidic acid (3) (Park et al. 2014), and viteoid II (4) (Ono et al. 1997), by com-
parison of their spectroscopic data with previously reported values. Geniposidic acid (2)
is recognized as the biosynthetic precursor of aucubin which has been reported to be
universally present in plantago species. Despite that aucubin was not isolated in the cur-
rent study, an HPLC analysis using commercial standard clearly showed that this iridoid
glucoside was present in the crude methanol extract (Figure S11). Viteoid II (4) has not
been identified from Plantago, but it is known from Vitex (Lamiaceae) (Ono et al. 1997;
Santos et al. 2001; Leitao et al. 2011). The identification of 4 in the present study is thus
consistent with the literature indicating that P. maxima with an amazing structural diver-
sity of the iridoid glucosides was a unique species within subgenus Plantago section
Plantago (Rønsted et al. 2003). Chlorinated iridoid glucosides are rarely encountered and
have been only identified from Plantago (Gen c¸ et al. 2010), Globularia (Es-Safi et al. 2006;
Kırmızıbekmez et al. 2009) and Veronica (Harput et al. 2003; Jensen et al. 2010) in the
Plantaginaceae family. Therefore, the isolation of plantomoside (1) provides further sup-
port for the close relationship of the genera Plantago, Globularia, and Veronica from a
chemotaxonomic perspective.
All isolated compounds were tested for the cytotoxic activities on human cancer
cell lines (HeLa and SH-SY5Y), the DPPH free radical scavenging activity, the inhibitory
activitie of LPS-induced NO production in RAW 264.7 macrophages and quorum
sensing (QS) inhibition against Chromobacterium violaceum CV026. Unfortunately,
the isolated compounds exhibited no activity in all biological evaluations.
3. Conclusion
A new chlorinated iridoid glycoside named plantomoside (1), along with three known
analogues, geniposidic acid (2), 10-deoxygeniposidic acid (3), and viteoid II (4),

4
L.-L. MA ET AL.
were isolated from the whole plants of Plantago maxima Juss. ex Jacq. To our know-
ledge, plantomoside (1) is the second chlorinated iridoid glucoside identified from the
genus Plantago, while compounds 3 and 4 were isolated from P. maxima for the
first time.
4. Experimental
4.1. General
Optical rotation was measured using a Rudolf AUTOPOL IV polarimeter. The UV spec-
trum was measured on a Shimadzu UV-1800 spectrophotometer. A Thermo Scientific
Nicolet iN10 Microscope was used to acquire the IR spectrum. NMR spectra were
performed at 303 K on a Bruker AVANCE III-500 NMR spectrometer with TMS as
internal standard. HRESIMS data was obtained on a Thermo Scientific Q Exactive mass
spectrometer. Preparative HPLC was accomplished over a Waters Xbridge C-18 column
(
5 lm, 19 mm ꢂ 150 mm) on a Shimadzu HPLC system consisted of a LC-6AD pump
and a Shimadzu SPD-20A detector. Macroporous resin (Nankai Chemical Company;
AB-8) was used for column chromatography (CC). All solvents were of analytical grade.
4.2. Plant material
The whole plants of Plantago maxima Juss. ex Jacq were collected at Yining, Xinjiang
Uygur Autonomous Region, People’s Republic of China, in June 2016, and identified
by one of the authors (C.X. Wan). A voucher specimen (No. TLM-201601) was
deposited in Xinjiang Production & Construction Corps Key Laboratory of Protection
and Utilization of Biological Resources in Tarim Basin, Tarim University.
4.3. Extraction and isolation
The dried and powdered whole plants (10.0 kg) of P. maxima were extracted with 80%
aqueous MeOH (3 ꢂ 100 L) for 2 h under reflux. A crude extract (3.1 kg) was obtained
after concentration under reduced pressure. A portion (1.0 kg) of the extract was sub-
jected to a column of macroporous resin eluting with MeOH ꢁ H O (0:1!7:3, v/v) to
2
afford four fractions (A ꢁ D). Fraction C (4.5 g) was separated by preparative HPLC
(16% CH CN in H O containing 0.5% formic acid, 10 mL/min) to give eight subfractions
3 2
(
C1–C8). Fraction C1 (3.1 g) was separated by preparative HPLC (9% CH CN in H O con-
3
2
taining 0.5% formic acid, 10 mL/min) to give eight subfractions (C1a–C1h). Fraction
C1b (1.5 g) was subjected to preparative HPLC (11% MeOH in H O containing 0.5%
2
formic acid, 10 mL/min) to yield 2 (t 11.9 min, 204.2 mg) and 3 (t 13.2 min, 5.3 mg).
R
R
Fraction C4 (123.6 mg) was further chromatographed on preparative HPLC (20%
CH CN in H O containing 0.5% formic acid, 10 mL/min) to afford four subfractions
3
2
(
C4a–C4d). Fraction C4a (76.3 mg) was separated by preparative HPLC (16% CH CN in
3
H2O containing 0.5% formic acid, 10 mL/min) to give six subfractions (C4a1–C4a6).
Fraction C4a2 (24.6 mg) was purified by preparative HPLC (28% MeOH in H O contain-
2
ing 0.5% formic acid, 10 mL/min) to afford 1 (t 9.5 min, 3.4 mg). Fraction C4c (36.9 mg)
R
was chromatographed on preparative HPLC (18% CH CN in H O containing 0.5%
3
2

NATURAL PRODUCT RESEARCH
5
formic acid, 10 mL/min) to yield two subfractions (C4c1–C4c2). Fraction C4c2 (11.5 mg)
was purified by preparative HPLC (14% CH CN in H O containing 0.5% formic acid,
3
2
1
0 mL/min) to obtain 4 (t 12.5 min, 6.8 mg).
Plantomoside (1): white amorphous solid. [a]
R
25
-91.5 (c ¼ 0.11, MeOH); UV (MeOH)
D
kmax (log e) 233 (2.38) nm; IR ꢀ : 3379, 2954, 1694, 1592, 1414, 1296, 1188, 1157,
max
ꢁ
1 1
1
7
1
099, 1077, 769, 738 cm ; H NMR (500 MHz, MeOH-d ): d 5.34 (1 H, d, J ¼ 7.5 Hz, H-1),
4
.45 (1 H, s, H-3), 3.31 (1 H, overlapped, H-5), 1.46 (1 H, m, H-6a), 2.37 (1 H, m, H-6b),
.96 (1 H, m, H-7a), 1.84 (1 H, m, H-7b), 2.21 (1 H, dd, J ¼ 7.6, 7.5 Hz, H-9), 3.85 (2 H,
ABq, J ¼ 11.6 Hz, H -10), 3.71 (3 H, s, OCH ), 4.72 (1 H, d, J ¼ 7.8 Hz, H-1’), 3.24 (1 H,
2
3
overlapped, H-2’), 3.37 (1 H, overlapped, H-3’), 3.24 (1 H, overlapped, H-4’), 3.32
1 H, overlapped, H-5’), 3.92 (1 H, br d, J ¼ 11.7 Hz, H-6’a), 3.63 (1 H, dd, J ¼ 11.7, 7.0 Hz,
(
1
3
H-6’b); C NMR (500 MHz, MeOH-d ): d 95.4 (C-1), 152.4 (C-3), 112.6 (C-4), 34.5 (C-5),
4
3
0.6 (C-6), 35.6 (C-7), 83.2 (C-8), 51.7 (C-9), 52.5 (C-10), 169.3 (C-11), 51.7 (OCH ), 100.1
3
(
[
C-1’), 74.8 (C-2’), 78.0 (C-3’), 71.8 (C-4’), 78.6 (C-5’), 63.1 (C-6’); HR-ESI-MS: m/z 423.1068
ꢁ
M-H] (calcd for C H ClO , 423.1063).
17
24
10
4.4. Determination of the absolute configuration of sugar moiety
ꢃ
Compound 1 (1.0 mg) was hydrolyzed with 10% aqueous HCl (0.2 ml) at 80 C for 3 h
and then neutralized by aqueous ammonia. After drying in vacuo, the residue was
ꢃ
dissolved in pyridine (0.2 ml) and mixed with L-cysteine methyl ester (1.0 mg) at 60 C
for 1 h. Phenylisothiocyanate (1.0 ll) was added to the mixture, and then heated
ꢃ
at 60 C for an additional 1 h. The reaction mixture was directly analysed by HPLC
[
column: Waters Xbridge C-18 (5 lm, 4.6 mm ꢂ 250 mm); mobile phase: CH CN–H O–HOAc
3
2
(
25: 75: 0.1); detection: UV at 250 nm; flow rate: 1.0 ml/min] to identify the derivative
of D-glucose by comparison of its retention time with those of authentic samples
tR 13.0 min for D-glucose and 12.1 min for L-glucose).
(
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financial supported by the National Natural Science Foundation of China [grant
number 31560101] and the Innovation Team Project in Key Field of Xinjiang Production &
Construction Corps [grant number 2017CB014].
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