The caffeoyl phenylethanoid glycosides from Lindernia ruellioides and their anti-HBV effects

Article abstract of DOI:10.1080/10286020.2017.1357549

Five caffeoyl phenylethanoid glycosides, including two new ones linderruelliosides A (1) and B (2), were isolated from the leaves and stems of Lindernia ruellioides. Their structures were elucidated on the basis of spectroscopic methods, including extensive NMR and MS spectra. In addition, all these compounds were tested for their anti-HBV activity. Compounds 1, 3, and 4 showed anti-HBV activities, with IC₅₀ values of 54.87, 30.74, and 69.02?μM for HBsAg and 26.70, 5.17, and 7.08?μM for HBeAg, respectively.

Full text of DOI:10.1080/10286020.2017.1357549

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
The caffeoyl phenylethanoid glycosides from
Lindernia ruellioides and their anti-HBV effects
Jing-Chen Wei, Peng-Cheng Wang, Xian-Li Zhou, Kun Tang, Qin Luo, Qin Xu,
Xue-Jiao Chen, Cheng-Qin Liang & Xu Chen
To cite this article: Jing-Chen Wei, Peng-Cheng Wang, Xian-Li Zhou, Kun Tang, Qin Luo, Qin Xu,
Xue-Jiao Chen, Cheng-Qin Liang & Xu Chen (2017): The caffeoyl phenylethanoid glycosides from
Lindernia ruellioides and their anti-HBV effects, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2017.1357549
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Date: 30 September 2017, At: 03:49

Journal of asian natural Products research, 2017
https://doi.org/10.1080/10286020.2017.1357549
The caffeoyl phenylethanoid glycosides from Lindernia
ruellioides and their anti-HBV effects
Jing-Chen Weia,b†, Peng-Cheng Wanga†, Xian-Li Zhoua, Kun Tangc, Qin Luoa, Qin Xua,b
Xue-Jiao Chena, Cheng-Qin Lianga and Xu Chena
,
acollege of Pharmacy, Guilin Medical university, Guilin 541004, china; bGuangxi Key laboratory of Molecular
Medicine in liver injury and repair, Guilin Medical university, Guilin 541001, china; cdepartment of Pharmacy,
affiliated hospital of Guilin Medical university, Guilin 541004, china
ARTICLE HISTORY
received 1 May 2017
accepted 16 July 2017
ABSTRACT
Five caffeoyl phenylethanoid glycosides, including two new ones
linderruelliosides A (1) and B (2), were isolated from the leaves and
stems of Lindernia ruellioides. Their structures were elucidated on
the basis of spectroscopic methods, including extensive NMR and
MS spectra. In addition, all these compounds were tested for their
anti-HBV activity. Compounds 1, 3, and 4 showed anti-HBV activities,
with IC50 values of 54.87, 30.74, and 69.02 μM for HBsAg and 26.70,
5.17, and 7.08 μM for HBeAg, respectively.
KEYWORDS
scrophulariaceae; Lindernia
ruellioides; caffeoyl
phenylethanoid glycoside;
anti-hBV
OH
O
OH
R3O
O
R1
R2 R3
O
HO
OR1
OR2
OH
Caff
H
1
2
Api
Api
H
OH
Caff
Caff
1. Introduction
Lindernia ruellioides, belonging to the family Scrophulariaceae, is widely distributed in the
areas of the southern parts of China [1]. e whole plant is used by local people for the
treatment of detumescence, detoxification, menoxenia, dysmenorrhea, amenorrhea, mas-
titis, injuries, and snake or dog’s biting [2,3]. Our previous investigation on L. ruellioides
found that the 75% EtOH extract of L. ruellioide could inhibit hepatitis B virus (HBV) sur-
face antigen (HBsAg) and e antigen (HBeAg) secretion effectively in Hep G2.2.15 cells. In
order to clarify the constituents responsible for anti-HBV activity in L. ruellioides, further
investigation on this plant was carried out and resulted in the isolation of five caffeoyl phe-
nylethanoid glycosides (Figure 1). Among them, linderruelliosides A (1) and B (2) are new
compounds, and desrhamnosylverbascoside (3) [4], plantainoside A (4) [5], and acteoside
(5) [6] are known ones. Anti-HBV assay in vitro showed that five compounds possessed
CONTACT cheng-Qin liang
cqliang@glmc.edu.cn; Xu chen
chenxu@glmc.edu.cn
†these authors contribute to the paper equally.
© 2017 informa uK limited, trading as taylor & francis Group

2
J.-C. WEI ET AL.
R1 R2
R3 R4
O
OH
Caff
H
1
2
3
4
5
H
H
Api
Api
H
OH
Caff
R4O
O
O
H
H
Caff
H
HO
OR2
OR3
OH
H
Caff
H
H
H
H
OR1
Glc
Caff
Caff
Figure 1. structures of compounds 1–5.
inhibitory activities on HBsAg and HBeAg. e most active compound 3 could inhibit the
secretions of HBsAg and HBeAg with IC50 values of 30.74 and 5.17 μM, respectively. is
article reports the isolation and structure elucidation of compounds 1 and 2, as well as the
anti-HBV activities of the isolates.
2. Results and discussion
Compound 1 was obtained as a white amorphous powder, with[ꢀ]18 − 31.8 (c 0.92, MeOH).
UV spectrum showed absorption maxima at 202 (3.85), 217 (3.57)D, 329 (3.46) nm; while IR
spectrum showed absorption bands at 3423, 2885, 1694, 1630, 1606, 1520, 1445, 1364, 1281,
1114, and 1043 cm−1. e HR-ESIMS of 1 exhibited an [M + Na]+ ion peak at m/z 633.1790,
consistent with the molecular formula C28H34O15. e IR absorption bands indicated the
presence of hydroxyl (3423 cm−1), ester carbonyl (1694 cm−1), and aromatic rings (1630,
1606, 1520, and 1445 cm−1) moieties. e 1H-NMR spectrum (Table 1) showed the pres-
ence of two 1,3,4-trisubstituted aromatic rings at δH 6.52 (1H, dd, J = 8.0, 1.6 Hz, H-6), 6.62
(1H, d, J = 8.0 Hz, H-5), 6.65 (1H, d, J = 1.6 Hz, H-2), 6.91 (1H, dd, J = 8.1, 1.4 Hz, H-6′),
6.74 (1H, d, J = 8.1 Hz, H-5′) and 7.01 (1H, d, J = 1.4 Hz, H-2′), and a pair of trans-olefinic
protons at δH 6.29 (1H, d, J = 15.9 Hz, H-8′) and δH 7.55 (1H, d, J = 15.9 Hz, H-7′). ese
characteristic proton signals suggested that there may be a caffeoyl unit in 1. In addition,
two doublet signals due to anomeric protons at δH 4.37 (1H, d, J = 7.8 Hz, H-1′′) and δH
4.97 (1H, d, J = 2.2 Hz, H-1′′′) suggested a diglycosidic structure for 1. e 13C NMR and
DEPT spectra (Table 1) exhibited 28 carbon signals, including seven quaternary carbons (six
aromatic and one carbonyl), 16 methines (six aromatic, two olefinic and eight oxygenated),
and five methylenes (four oxygenated). ese data suggested the presence of four fragments,
including a phenylethanol, a caffeoyl, a glucosyl, and an apiosyl unit. e phenylethanoid
unit was established unambiguously by the HMBC (Figure 2) correlations of H2-7 (δH 2.75,
m) with C-1 (δC 131.4), C-2 (δC 117.1), C-6 (δC 121.3) and C-8 (δC 72.3), H-2 (δH 6.65, d,
J = 1.6 Hz) with C-1 (δC 131.4), C-3 (δC 146.0) and C-4 (δC 144.6), and H-6 (δH 6.52, dd,
J = 8.0, 1.6 Hz) with C-1 (δC 131.4), C-4 (δC 144.6) and C-5 (δC 116.3), together with the
1
spin systems of H-7/H-8 and H-5/H-6 in the H-1H COSY spectrum. e caffeoyl unit
was established by the HMBC correlations of H-7′ (δH 7.55, d, J = 15.9 Hz) with C-1′ (δC
127.8), C-2′ (δC 115.1), C-6′ (δC 122.9), C-8′ (δC 115.4) and C-9′ (δC 169.0), H-2′ (δH 7.01,
d, J = 1.4 Hz) with C-1′ (δC 127.8), C-3′ (δC 147.0) and C-4′ (δC 149.5), and H-6′ (δH 6.91,
dd, J = 8.1, 1.4 Hz) with C-1′ (δC 127.8), C-4′ (δC 149.5) and C-5′ (δC 116.5), together with
the spin systems of H-7′/H-8′ and H-5′/H-6′ in the 1H-1H COSY spectrum. e glucosyl
unit was established by the HMBC correlations of H-1′′ (δH 4.37, d, J = 7.8 Hz) with C-2′′

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1h and 13c nMr spectroscopic data for compounds 1–2 (cd3od, tMs, δ in ppm, J in hz)a,b
.
1
2
Position
δH
δC
δH
δC
aglycone 1
131.4
117.1
146.0
144.6
116.3
121.3
36.6
131.4
117.1
146.1
144.7
116.3
121.3
36.6
2
3
4
5
6
7
8
6.65 (1h, d, 1.6)
6.66 (1h, d, 1.7)
6.62 (1h, d, 8.0)
6.52 (1h, dd, 8.0, 1.6)
2.69–2.80 (2h, m)
3.95–3.97 (1h, m)
3.64–3.71 (1h, m)
6.64 (1h, d, 8.0)
6.53 (1h, dd, 8.0, 1.7)
2.70–2.81 (2h, m)
3.93–3.99 (1h, m)
3.66–3.69 (1h, m)c
72.3
72.4
caffeicacid moiety 1′
2′
3′
4′
5′
6′
7′
8′
127.8
115.1
147.0
149.5
116.5
122.9
146.7
115.4
169.0
104.2
73.4
127.7
114.8
146.8
149.7
116.5
123.2
147.7
115.2
168.4
104.4
75.2
7.01 (1h, d, 1.4)
7.02 (1h, d, 1.4)
6.74 (1h, d, 8.1)
6.91 (1h, dd, 8.1, 1.4)
7.55 (1h, d, 15.9)
6.29 (1h, d, 15.9)
6.75 (1h, d, 8.1)
6.92 (1h, dd, 8.1, 1.4)
7.55 (1h, d, 15.8)
6.26 (1h, d, 15.8)
9′
Glc 1′′
2′′
3′′
4′′
5′′
6′′
4.37 (1h, d, 7.8)
3.35 (1h, dd, 9.3, 8.0)
4.99–5.02 (1h, m)
3.47–3.50 (1h, m)c
3.47–3.50 (1h, m)c
3.95–3.97 (1h, m)c
3.60 (1h, dd, 11.1, 5.0)
4.97 (1h, d, 2.2)
4.32 (1h, d, 7.7)
3.25–3.29 (1h, m)
3.57–3.60 (1h, m)
4.82–4.85 (1h, m)
3.61–3.64 (1h, m)
3.69–3.71 (1h, m)
3.46 (1h, dd, 11.2, 6.2)
4.88 (1h, m)c
78.8
69.9
76.6
68.3
75.8
72.7
74.7
68.6
api-1′′′
2′′′
3′′′
110.9
78.0
80.5
75.0
111.0
78.1
80.6
75.0
3.89 (1h, d, 2.2)
3.84 (1h, d, 1.7)
4′′′
3.93 (1h, d, 9.6)
3.73 (1h, d, 9.6)
3.54 (2h, br s)
3.89 (1h, d, 9.6)
3.69–3.71 (1h, m)c
3.51 (2h, br s)
5′′′
65.5
65.6
a1h and 13c nMr data were recorded at 400 and 100 Mhz, respectively.
bthe assignments were based on hsQc, hMBc and 1h-1h cosY experiments.
coverlapping signals.
O
O
OH
5'''
3
HO
2'''
7
1
3'''
OH
OH
6''
O
OH
O
5
O
OH
2''
HO
3''
O
1'
8'
5'
3'
HO
OH
Figure 2. selected hMBc correlations of 1.
(δC 73.4), C-3′′ (δC 78.8) and C-5′′ (δC 76.6), and H-6′′ (δH 3.60, dd, J = 11.1, 5.0 Hz) with
1
C-4′′ (δC 69.9) and C-5′′ (δC 76.6), and the H-1H COSY correlations of H-1′′/H-2′′, and
the signals (δ 110.9, 78.0, 80.5, 75.0 and 65.5) were attributed to the apiosyl unit which was
established by the HMBC correlations of H-1′′′ (δH 4.97, d, J = 2.2 Hz) with C-2′′′ (δC 78.0),
C-3′′′ (δC 80.5) and C-5′′′ (δC 65.5), as well as the 1H-1H COSY spin system H-1′′′/H-2′′′.
e glucose and apiose were further confirmed by comparison with authentic samples afer

4
J.-C. WEI ET AL.
acid hydrolysis of 1. e configuration of the anomeric proton of glucose was proposed
as β on the basis of its large coupling constant at δH 4.37 (1H, d, J = 7.8 Hz, H-1′′), and the
apiose unit was also determined to have a β-configuration at C-1′′′ due to the chemical shif
of its anomeric carbon signal at δC 110.9 [7]. e HMBC correlations of H-1′′ (δH 4.37, d,
J = 7.8 Hz) with C-8 (δC 72.3) indicated that the glucopyranosyl unit was located at C-8.
e HMBC correlations of H-3′′ (δH 5.00 m) with C-9′ (δC 169.0) indicated that the caffeoyl
group was connected to C-3′′. In the HMBC spectrum, the anomeric proton signal of the
apiose at δH 4.97 (1H, d, J = 2.2 Hz, H-1′′′) correlated with the carbon signal at δC 68.3 (C-6′′)
indicated that the apiose attached to C-6′′ of the glucose.
Compound 2 was obtained as an amorphous powder, with[ꢀ]1D8 − 37.6 (c 1.06, MeOH).
It was determined to possess the same molecular formula as 1 from its HR-ESI-MS data
(m/z 633.1792 [M + Na]+). Extensive analysis of the IR, UV, and NMR spectra (Table
1) revealed that 2 was structurally similar with 1 except that the caffeoyl group of 2 was
connected to C-4′′ of the glucose, rather than C-3′′. Unfortunately, the HMBC correlation
between H-4′′ (δH 4.83, m) and C-9′ (δC 168.4) was not found, but this could be confirmed
by the appearance of greatly downshifed signal H-4′′ (δH 4.83, m) compared with that of
1. e downfield-shifed signal of H-4′′ was obviously due to the deshielding effect of the
α, β-unsaturated carbonyl group of caffeoyl unit which suggested the unit was connected
to C-4′′ [7,8]. Certainly, the location of C-4′′ and the structure of glucosyl unit can be
1
deduced by the obvious spin systems of H-1′′/H-2′′/H-3′′/H-4′′/H-5′′/H-6′′ in the H-1H
COSY spectrum. In addition, the signals of olefinic and anomeric protons and anomeric
carbons suggested this compound to have the same relative configuration as that of 1. us,
the structure of 2 (linderruellioside B) was elucidated as shown.
Compounds 3–5 were identified by comparison of their spectral data with those in the
literature [4–6].
In addition, since the extracts of the plant have been shown anti-HBV activities in our
previous studies, all isolates were evaluated for their in vitro anti-HBV activities against
HepG 2.2.15 cell lines according to a previously described procedure [9]. e results are
given in Table 2. As a result, compounds 1–5 exhibited potential anti-HBV activities with
IC50 values between 30.74 and 84.76 μM on HBsAg secretion, while IC50 values between
5.17 and 69.84 μM on HBeAg secretion, respectively. And at the highest concentration used,
none of the tested compounds exhibited cytotoxic activities.
3. Experimental
3.1. General experimental procedures
Optical rotations were recorded on a JASCO P-1020 digital polarimeter (Horiba, Kyoto,
Japan). UV spectra were recorded on a UV-2401PC spectrometer (Shimadzu, Kyoto, Japan).
IR spectra were measured with a Bruker Tensor 27 infrared spectrometer with KBr pellets
(Bruker, Karlsruhe, Germany). 1D and 2D NMR spectra were performed on Bruker AM-400
and DRX-500 MHz spectrometers (Bruker Optics, Ettlingen, Germany). High-resolution
electrospray-ionization mass spectra (HR-ESI-MS) was obtained on a VG Autospec–3000
spectrometer (VG, Micromass, England). Column chromatography (CC) was carried out on
silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China),
MCI gel (75–150 μM, Mitsubishi Chemical Corporation, Tokyo, Japan), Sephadex LH-20
(Amersham Pharmacia Biotech, Uppsala, Sweden), and Lichroprep RP-18 gel (40–63 μM,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 2. anti-hBV activities of compounds 1–5a.
HBsAgb
HBeAgc
Compounds
CC50 (μM)
IC50 (μM)
SId
IC50 (μM)
SI
1
>163.93
>163.93
>209.21
>209.21
>160.26
149.71
54.87
74.08
30.74
69.02
84.76
97.83
>2.99
>2.21
>6.81
>3.03
>1.89
1.53
26.70
36.08
5.17
7.08
69.84
84.28
>6.14
>4.54
>40.47
>29.55
>2.29
1.78
2
3
4
5
3tce
aall values are the mean of three independent experiments.
bhBsag: hBV surface antigen.
chBeag: hBV e antigen.
dcc50 = 50% cytotoxic concentration, ic50 = 50% inhibitory concentration, si (selectivity index) = cc50/ic50
e3tc (lamivudine), an antiviral agent used as a positive control.
.
Merck, Merck & Co Inc, Darmstadt, Germany). Semi-preparative HPLC was carried out
on an Agilent 1260 liquid chromatography (LC) with a Zorbax SB-C18 column (5 μM,
9.4 mm × 25 cm) (Agilent, Santa Clara, CA, USA). Glucose and apiose were obtained from
J&K Scientific Ltd. (Beijing, China).
3.2. Plant material
e leaves and stems of Lindernia ruellioides (4.0 kg) were collected in Pingnan County of
Guangxi Province, China, in September 2013, and was identified by Mr. De-Qing Huang,
College of Pharmacy, Guilin Medical University. e voucher specimen (No. 201309002)
has been deposited in the College of Pharmacy, Guilin Medical University, China.
3.3. Extraction and isolation
e powdered dried leaves and stems of L. ruellioides (4.0 kg) were extracted with 75%
EtOH (10 L × 3) at room temperature (each 72 h). Afer concentrated in vacuo to get a
residue, the extract was partitioned between H2O and EtOAc. e EtOAc extract (55.0 g)
was chromatographed using silica gel column (100–200 mesh, 8 × 70 cm, 0.5 kg), eluting
with a CHCl3–Me2CO gradient system (1:0, 9:1, 8:2, 2:1, 1:1, and 0:1, v/v), to give fractions
I–VI. Afer decolorized on MCI gel (10 × 60 cm, 90% MeOH/H2O), fraction II (15 g) was
subjected to RP-18 column chromatography (5.5 × 45 cm, 10, 30, 50 and 100% gradient
MeOH–H2O, each 5 L) to yield four subfractions (II-2-a–II-2-d). Subfraction VI-2-a was
subjected to semipreparative HPLC (eluted with 23–27% MeOH-H2O, 3 ml/min) to afford
compounds 1 (33.4 mg, tR 35.0 min), 2 (33.6 mg, tR 45.0 min), and 4 (13.3 mg, tR 31.8 min).
Fraction II-2-b (8.5 g) was purified by semipreparative HPLC (eluted with 30–35% MeOH-
H2O, 3 ml/min) to give 3 (4.8 mg, tR 10.5 min) and 5 (18.0 mg, tR 27.0 min).
3.3.1. Linderruellioside A (1)
Amorphous powder;[ꢀ]1D8 − 31.8 (c 0.92, MeOH); UV (MeOH) λmax (log ε): 202 (3.85), 217
(3.57), 329 (3.46) nm; IR (KBr) νmax 3423, 2885, 1694, 1630, 1606, 1520, 1445, 1360, 1281,
1114, 1042 cm−1; 1H and 13C NMR spectral data, see Table 1; ESI-MS: m/z 633 [M + Na]+;
HR-ESI-MS: m/z 633.1790 [M + Na]+ (calcd for C28H34O15Na, 633.1790).

6
J.-C. WEI ET AL.
3.3.2. Linderruellioside B (2)
Amorphous powder;[ꢀ]1D8 − 37.6 (c 1.06, MeOH); UV (MeOH) λmax (log ε): 202 (3.84), 217
(3.56), 333 (3.46) nm; IR (KBr) νmax 3424, 2884, 1698, 1631, 1606, 1519, 1445, 1367, 1281,
1114, 1018 cm−1; 1H and 13C NMR spectral data, see Table 1; ESI-MS: m/z 633 [M + Na]+;
HR-ESI-MS: m/z 633.1792 [M + Na]+ (calcd for C28H34O15Na, 633.1790).
3.4. Acid hydrolysis of compounds 1 and 2
Compounds 1 (4.0 mg) and 2 (4.0 mg) were each refluxed with 2 N HCl (5 ml) at 90 °C for
2 h. Afer neutralization with NaHCO3 and extraction with ethyl acetate, the aqueous layer
was concentrated and detected by TLC over silica gel. e presence of glucose and apiose
was confirmed by comparison with authentic samples [7]. Using semipreparative HPLC
with gradient aqueous MeOH, pure sugars were obtained from the concentrate, and they
were identified as D-glucose and D-apiose by measuring their optical rotations [glucose:
[ꢀ]2D2 + 30.5 (c 0.12, H O); apiose: [ꢀ]2D2 + 10.9 (c 0.08, H O)].
2
2
3.5. Anti-HBV assay on HepG 2.2.15 cell line in vitro
e anti-HBV activity was evaluated according to the previous report [9] using lamivudine
(3TC) (GlaxoSmithKline Pharmaceuticals Co, Ltd., Brentford, U.K.) as the positive control.
e toxicity of the test reagents was assayed by the MTT method [10].
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was supported by the National Natural Science Foundation of China [grant number
31560100 and 8156130225]; the Science Research and Technology Development Program of Guangxi
[grant number Guikeneng 14123006-21 and 1598025-18]; and the Science Research and Technology
Development Program of Guilin [grant number 20130120-2].
References
[1] Chinese Herbalism Editorial Board of State Administration of Traditional Chinese Medicine
of the People’s Republic of China, Chinese Herbalism, (Shanghai Scientific and Technical Press,
Shanghai, 1999), p. 351.
[2] Y.R. Tang, P.H. Yan, X.Y. Ceng, and Y. Cai, Lishizhen. Med. Mater. Med. Res. 26, 1938 (2015).
[3] Y.R. Tang, P.H. Yan, X.Y. Zeng, and Y. Cai, West China. J. Pharm. Sci. 31, 386 (2016).
[4] W.J. Chen, Y.X. Wen, Y.Y. Chen, Y.L. Huang, and D.P. Li, Chin. Tradit. Pat. Med. 32, 1000 (2010).
[5] S. Damtof and S.R. Jensen, Phytochemistry 37, 441 (1994).
[6] L.M. Gao, X.M. Wei, and Y.Q. He, China J. Chin. Mater. Med. 28, 948 (2003).
[7] I. Calis and H. Kirmizibekmez, Phytochemistery 65, 2619 (2004).
[8] J. Ma, C.J. Li, J.Z. Yang, Y. Li, X.Q. Bao, N.H. Chen, and D.M. Zhang, J. Asian. Nat. Prod. Res.
19, 320 (2017).
[9] L.Q. Li, J. Li, Y. Huang, Q. Wu, S.P. Deng, X.J. Su, R.Y. Yang, J.G. Huang, Z.Z. Chen, and S. Li,
Fitoterapia 83, 303 (2012).
[10] Z.W. Yang, X. Zu, and W.W. Li, Pract Prev. Med. 14, 1552 (2007).