A new anti-inflammatory lignan from Lonicerae Japonicae flos

Article abstract of DOI:10.1080/14786419.2019.1587430

One new lignan, loniceralanside A (1), together with three known compounds (2–4), were purified from Lonicerae Japonicae Flos. Their structures were determined by HR-ESI-MS, 1 D and 2 D NMR data. Compounds 2–4 were separated from the plant for the first time. The biological test showed that compound 1 had significant anti-inflammatory activity with inhibiting the release of β-glucuronidase induced by platelet-activating factor (PAF) in rat polymorphonuclear leukocytes (PMNs) (IC₅₀ = 3.05 μM).

Full text of DOI:10.1080/14786419.2019.1587430

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new anti-inflammatory lignan from Lonicerae
Japonicae flos
Ran Yang, Lei Fang, Jia Li & Yong-Qing Zhang
To cite this article: Ran Yang, Lei Fang, Jia Li & Yong-Qing Zhang (2019): A new anti-
inflammatory lignan from LoniceraeꢀJaponicae flos, Natural Product Research, DOI:
10.1080/14786419.2019.1587430
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1587430
A new anti-inflammatory lignan from Lonicerae
Japonicae flos
Ran Yanga , Lei Fanga,b , Jia Lia and Yong-Qing Zhanga
aLaboratory of Natural Pharmaceutical Chemistry, Shandong University of Traditional Chinese
b
Medicine, Jinan, China; School of Biological Science and Technology, University of Jinan,
Jinan, China
ABSTRACT
ARTICLE HISTORY
Received 26 January 2019
Accepted 22 February 2019
One new lignan, loniceralanside A (1), together with three known
compounds (2–4), were purified from Lonicerae Japonicae Flos.
Their structures were determined by HR-ESI-MS, 1 D and 2 D NMR
data. Compounds 2–4 were separated from the plant for the first
time. The biological test showed that compound 1 had significant
anti-inflammatory activity with inhibiting the release of b-glucuro-
nidase induced by platelet-activating factor (PAF) in rat poly-
morphonuclear leukocytes (PMNs) (IC50 ¼ 3.05 mM).
KEYWORDS
Lonicerae Japonicae Flos;
lignan; anti-inflammatory
activity; b-glucuronidase
1. Introduction
The genus Lonicera Linn. consists of more than 200 species and is widely distributed
in temperate and subtropical regions (Sun 2012). Lonicerae Japonicae Flos is a very
common species with highly economic and medicinal values. It has been widely used
in the pharmaceutical, cosmetic and functional food industries with varieties
of biological properties, such as antioxidant, anti-inflammatory, anti-virus, antitumor
and antihypertensive activities (Han et al. 2016; Yu et al. 2016; Chaowuttikul et al.
2017; Ding et al. 2017; Wang et al. 2017). To date, more than 210 compounds have
been isolated from L. Japonicae Flos including flavonoids, triterpenoids, iridoids,
volatile oils and organic acids (Wang et al. 2014; Wang et al. 2016). As part of our
ongoing project to study the bioactive constituents in L. Japonicae Flos, in the present
CONTACT Yong-Qing Zhang
zyq622003@163.com
Laboratory of Natural Pharmaceutical Chemistry, Shandong
University of Traditional Chinese Medicine, Jinan, China.
Supplementary material of this article can be accessed at https://doi.org/10.1080/14786419.2019.1587430.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

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R. YANG ET AL.
study, one new lignan, loniceralanside A (1), together with three known compounds,
forsythialanside C (2), (7S, 8S)-3-methoxy-3’,7-epoxy-8,4’-oxyneoligna-4,9,9’-triol (3) and
eugenyl-a-L-arabinopyranosyl-(1!6)-b-D-glucopyranoside (4), were isolated from
L. Japonicae Flos. The bioassays showed that compounds 1–4 expressed anti-
inflammatory activities with IC50 values ranging from 3.05 to 27.80 mM, and the activity
of 1 was comparable to the positive control. In addition, compounds 2–4 were firstly
separated from L. Japonicae Flos. Herein, we report the isolation, structural elucidation
and biological assays of these compounds.
2. Results and discussion
Compound 1 was obtained as a yellow colloid. Its molecular formula was assigned
as C31H42O14. The IR spectral data indicated the presence of hydroxyl (3428 cmꢀ1) and
aromatic ring (1624, 1506 and 1459 cmꢀ1) groups. Its 1H NMR spectrum showed
signals for a 1, 3, 4, 5-tetrasubstituted aromatic ring with two phenyl protons [dH 6.76
(1H, br s, H-2’), 6.76 (1H, br s, H-6’)], and a 1, 3, 4-trisubstituted aromatic ring with
three phenyl protons [dH 7.08 (1H, s, H-2), 7.09 (1H, d, J ¼ 8.3 Hz, H-5), 6.96 (1H, dd,
J ¼ 1.8, 8.3 Hz, H-6)]. Signals for two methoxyl groups [dH 3.89 (3H, s, OCH3-3’), 3.83
(3H, s, OCH3-3)], two oxymethylene groups [dH 3.88 (1H, m, H-9a), 3.76 (1H, m, H-9b),
3.89 (1H, m, H-9’a), 3.57 (1H, m, H-9’b)], two methylene groups [dH 2.64 (2H, m, H-7’),
1.82 (2H, m, H-8’)], a oxymethine group [dH 5.67 (1H, d, J ¼ 5.8 Hz, H-7)], and a methine
group [dH 3.63 (1H, m, H-8)] were also observed. The 13C NMR spectrum exhibited the
presence of six signals for a rhamnosyl group and five signals for an arabinosyl group.
The key 1H-1H COSY correlations showed the presence of CH(7)-CH(8)-CH2(9) and
CH2(7’)-CH2(8’)-CH2(9’) groups (Supplementary Material Figure S1). In the HMBC
spectrum, the correlations from H-7 to C-2 and C-6, and from H-8 to C-1 indicated
that the 1, 3, 4-trisubstituted aromatic ring was located at C-7. The correlations from
H-7’ to C-1’, C-6 ’ and C-2’ confirmed that the propanol group was linked to C-1’. The
HMBC correlations from H-7 to C-4’, and from H-8 to C-4’ and C-6’ indicated the
presence of a dihydrobenzofuran group. In addition, the rhamnosyl and arabinosyl
groups were located at C-4 and C-9 respectively, according to the HMBC correlations
from H-1’’ to C-4 and from H-1’’’ to C-9 (Supplementary Material Figure S1).
The relative configuration of C-7 and C-8 was determined as a relative-trans form
by the chemical shift and coupling constant value of H-7 [dH 5.67 (1H, d, J ¼ 5.8 Hz, H-
7)], and the NOESY correlations of H-7/H-9, H-8/H-2 and H-8/H-6 (Supplementary
Material Figure S2). Then, in the experimental ECD data, 1 showed a negative cotton
effect at 282 nm (-1.02) (Supplementary Material Figure S11), so the configuration of
the dihydrofuran ring was determined as 7 R, 8S (Antus et al. 2001; Huang et al. 2015).
The relative configuration of glycosyl groups in 1 were determined to be a for both
rhamnosyl and arabinosyl units on the basis of chemical shifts and coupling constant
values [dH 5.35 (1H, d, J ¼ 1.6 Hz, H-1’’), 4.34 (1H, d, J ¼ 7.3 Hz, H-1’’’)] (Wang et al. 2016;
Yan et al. 2017). Acid hydrolysis of 1 yielded L-rhamnose and L-arabinose as sugar
units (Supplementary Material Figure S12). Thus, the structure of 1 was determined to
be (7 R, 8S)-dihydrodehydrodiconiferyl alcohol-4-O-a-L-rhamnopyranosyl-9-O-a-L-arabi-
nopyranoside, and named as loniceralanside A (Figure 1).

NATURAL PRODUCT RESEARCH
3
Figure 1. Chemical structures of compounds 1–4.
The three known compounds were identified as forsythialanside C (2) (Li et al.
2014), (7S, 8S)-3-methoxy-3’,7-epoxy-8,4’-oxyneoligna-4,9,9’-triol (3) (Chang et al. 2014)
and eugenyl-a-L-arabinopyranosyl-(1!6)-b-D-glucopyranoside (4) (Nakamura et al.
2011) by comparing their spectroscopic data with the literature.
As far as we know, lignans have varieties of structures such as benzofuran, bicy-
clooctene, futoenone and biphenyl derivative lignans. In addition, they display a wide
range of significant biological properties, such as anti-inflammatory, antitumor, anti-
microbial and antioxidant activities (Geethangili and Ding 2018; Xu et al. 2018). In the
present study, the anti-inflammatory activities of compounds 1–4 were evaluated
(Supplementary Material Table S2). Compounds 1 and 2 were benzofuran lignan glyco-
sides, and showed comparable activities (1: IC50 ¼ 3.05 mM; 2: IC50 ¼ 4.31 mM) to that
of the positive control (IC50 ¼ 2.21 mM). Their activities appeared to be related with
the structures. Comparing the structures and activities of 1 and 2 with those of the
reported lignan hawthornnin H which possessed two hydroxyl groups at C-70 and 80
(Huang et al. 2015), it seemed that the two methylene groups of C-7’ and 8’ in 1 and
2 might lead to better anti-inflammatory activities. Comparing 1 and 2 with the
known benzofuran lignan difengpiol B which possessed a partially hydrogenated aro-
matic ring (Fang et al. 2010), it seemed that the 1, 3, 4-trisubstituted aromatic rings
and glycosyl substituents in 1 and 2 might be important for higher activities.
Moreover, compound 3 was an oxyneolignan and 4 was a phenylpropanoid glycoside,
both with mild anti-inflammatory activities (3: IC50 ¼ 15.30 mM; 4: IC50 ¼ 27.80 mM).
3. Experimental
3.1. General experimental procedures
Optical rotations were obtained on a Rudolph VI automatic polarimeter (Rudolph
Research Analytical, Hackettstown, USA). IR spectra were recorded on an FT-IR VERTEX
70 spectrometer (Bruker BioSpin AG, Fremont, USA). NMR experiments were performed
with a Bruker AVANCE DRX (600 MHz) spectrometer (Bruker BioSpin Corporation,
Billerica, USA). ESIMS data were measured on an Agilent 1260-6460 Triple Quad LC-MS

4
R. YANG ET AL.
spectrometer (Agilent Technologies, Santa Clara, USA). HRESIMS data were measured
on an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, USA).
HPLC preparation was carried out on an Agilent 1260 instrument (Agilent
Technologies, Santa Clara, USA). Sugar analysis was carried out using a Shodex
Asahipak NH2P-50 column (5 lm, 4.6 ꢁ 250 mm; Shodex Denko, Tokyo, Japan). All sol-
vents used for column chromatography were obtained with analytical grade (Fuyu
Chemical Factory, Tianjin, China).
3.2. Plant material
The initial flowers of L. Japonicae were obtained from Shandong University of
Traditional Chinese Medicine (Jinan, China), and were identified by Prof. Jia Li
(Shandong University of Traditional Chinese Medicine, Jinan, China). A voucher speci-
men (JYH-201705) was stored in the Laboratory of Pharmaceutical Chemistry,
University of Jinan (Jinan, China).
3.3. Extraction and isolation
The freeze-dried flowers (6.0 kg) were extracted with 95% ethanol (30.0 L) to obtain
crude extract (900.5 g). The extract was then extracted with ethyl acetate (10.0 L) and
n-butanol (12.0 L). The n-butanol fraction (65.2 g) was chromatographed over a macro-
porous resin column (EtOH-H2O, 0:100 to 90:10, v/v) to yield six fractions (A-F).
Fraction B (10.4 g) was chromatographed over a silica gel column (CH2Cl2-MeOH, 10:1
to 1:1, v/v) to afford fractions B1-B7. Fr.B4 (1.1 g) was subjected to silica gel column
chromatography eluted with EtOAc-MeOH (130:1 to 30:1, v/v) to obtain night subfrac-
tions Fr.B4-1–B4-9. Fr.B4-3 (38.5 mg) was purified through preparative HPLC (MeOH-
H2O, 40:60, v/v), yielding compounds 1 (3.4 mg), 2 (2.5 mg) and 4 (12.1 mg). Fr.B7
(2.0 g) was separated by a silica gel column eluted with EtOAc-MeOH (150:1 to 30:1, v/
v) to obtain five subfractions Fr.B7-1–B7-5. Fr.B7-4 (41.5 mg) was purified by prepara-
tive HPLC eluted with MeOH-H2O (50:50, v/v) to afford compound 3 (17.0 mg).
3.3.1 Compound 1
25
Yellow colloid; [a]D -60.1 (c 0.7, CH3OH); IR (KBr) ꢀmax: 3428, 2927, 1624, 1506, 1459,
1066 cmꢀ1 1H NMR (methanol-d4, 600 MHz) data, see Supplementary Material Table
;
S1; 13C NMR (methanol-d4, 150 MHz) data, see Supplementary Material Table S1; ESIMS
m/z 661.2 [M þ Na]þ; HRESIMS m/z 673.2266 [M þ Cl]– (calcd for C31H42ClO14,
673.2263).
3.4. Acid hydrolysis of compound 1
The absolute configurations of glycosyl units in compound 1 were determined by the
reported method (Yan et al. 2017). Briefly, the sample solution of 1 was firstly hydro-
lyzed by hydrochloric acid at 80 ꢂC for 4 h. Then the reaction system was neutralized
and extracted with CH2Cl2. The aqueous layer was analyzed by HPLC with acetonitrile
and water (75: 25) at a flow of 1.0 mL/min. Compound 1 gave L-rhamnose

NATURAL PRODUCT RESEARCH
5
(tR ¼ 13.30 min) and L-arabinose (tR ¼ 6.98 min) as sugar residues on the basis of
the same retention times with those of authentic L-rhamnose (tR ¼ 13.30 min) and
L-arabinose (tR ¼ 6.98 min).
3.5. Anti-inflammatory activity assay
The anti-inflammatory activities were assayed by the method that we described previ-
ously (Yang et al. 2019).
4. Conclusions
In conclusion, one new lignan, loniceralanside A (1), along with three known com-
pounds (2-4), were separated from L. Japonicae Flos. Compounds 2-4 were purified
from the plant for the first time. Compound 1 expressed decent anti-inflammatory
activity with an IC50 of 3.05 mM. This work provided valuable support for the further
study of L. Japonicae Flos.
Acknowledgements
We are grateful to Shandong University of Traditional Chinese Medicine for the donation of
plant materials.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Standardization Project Fund of Administration of Traditional
Chinese Medicine [grant number: ZYBZH-Y-SD-32], Postdoctoral Science Foundation of China
[grant number: 2017M622268], Specialized Fund for Dependent Innovation of Shandong
Province [grant number: 2016GSF202009].
ORCID
Ran Yang
Lei Fang
http://orcid.org/0000-0002-7152-7040
http://orcid.org/0000-0003-3999-7170
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