Phenylethanoid glycosides from the Schnabelia nepetifolia (Benth.)P.D.Cantino promote the proliferation of osteoblasts

Article abstract of DOI:10.1016/j.phytochem.2019.05.003

An investigation of the n-BuOH fraction of Schnabelia nepetifolia (Benth.)P.D.Cantino led to the isolation and identification of 12 undescribed phenylethanoid glycosides (nepetifosides A–L)and one undescribed phenylmethanoid glycoside (nepetifoside M), together with 23 known compounds. The structures of these compounds were determined by spectroscopic analyses including two-dimensional nuclear-magnetic-resonance (2D-NMR)spectroscopy and chemical-hydrolysis methods. Nepetifoside F exhibited strong activity that significantly increased osteoblast proliferation at three concentrations of 0.1, 1, and 10 μM. Moreover, nepetifoside C and nepetifoside D exhibited moderate activities in promoting the proliferation of osteoblasts at medium and high concentrations of 1 μM and 10 μM, respectively.

Full text of DOI:10.1016/j.phytochem.2019.05.003

Phytochemistry164(2019)111–121
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phenylethanoid glycosides from the Schnabelia nepetifolia (Benth.)
P.D.Cantino promote the proliferation of osteoblasts
Hong-Tao Xua, Cheng-Gang Zhanga, Yu-Qiong Hec, Song-Shan Shia, Yong-Li Wanga,
Gui-Xin Choua,b,∗
a The MOE Key Laboratory of Standardization of Chinese Medicines and SATCM Key Laboratory of New Resources and Quality Evaluation of Chinese Medicines, Institute
of Chinese Materia Medica (ICMM), Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, 201203, PR China
b Shanghai R&D Center for Standardization of Chinese Medicines, Shanghai, 201203, PR China
c School of Pharmacy, Second Military Medical University, Shanghai, 200433, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
An investigation of the n-BuOH fraction of Schnabelia nepetifolia (Benth.) P.D.Cantino led to the isolation and
identification of 12 undescribed phenylethanoid glycosides (nepetifosides A–L) and one undescribed phe-
nylmethanoid glycoside (nepetifoside M), together with 23 known compounds. The structures of these com-
pounds were determined by spectroscopic analyses including two-dimensional nuclear-magnetic-resonance (2D-
NMR) spectroscopy and chemical-hydrolysis methods. Nepetifoside F exhibited strong activity that significantly
increased osteoblast proliferation at three concentrations of 0.1, 1, and 10 μM. Moreover, nepetifoside C and
nepetifoside D exhibited moderate activities in promoting the proliferation of osteoblasts at medium and high
concentrations of 1 μM and 10 μM, respectively.
Schnabelia nepetifolia (Benth.) P.D.Cantino
(lamiaceae)
NMR spectroscopy
Phenylethanoid glycosides
Nepetifosides A–M
Osteoblast
1. Introduction
extract of the whole plant of S. nepetifolia revealed the presence of
thirteen undescribed compounds (1–13).
Schnabelia is a genus of plants in the Lamiaceae family that was first
described in 1921 (World Checklist of Selected Plant Families [WCSP].,
2019). The entire genus is endemic to China (Cantino et al., 1999). Five
species are currently in the genus Schnabelia according to the World
Checklist (WCSP, 2019) and among them is Schabelia nepetifolia
(Benth.) P.D.Cantino. S. nepetifolia is a folk medicine used to treat in-
flammatory diseases in eastern China (Yu et al., 1982). Recently, we
reported the isolation of 10 undescribed and 7 previously known
abietane diterpenes from the dichloromethane fraction of this plant
(Zhang et al., 2017). In the present study, as a component of our con-
tinuing search for natural bioactive products from this species, a phy-
tochemical study on the n-BuOH fraction of S. nepetifolia was conducted
using isolation and characterization to obtain 32 phenylethanoid gly-
cosides and 4 phenylmethanoid glycosides including 12 undescribed
2.1. Structural analysis
Compound was obtained as
1
a yellowish-brown amorphous
powder, and its molecular formula C29H34O15 was determined by
HRESIMS at m/z 621.1821 [M–H]-, (calcd. for 621.1819). The 1H NMR
spectrum (Table 1) of 1 showed the presence of three aromatic protons
δH 6.58 (2H, br d, J ≈ 7.0 Hz, H-5″ and H-6″) and δH 6.61 (1H, br d,
J = 1.2 Hz, H-2″), of caffeoyl moiety; two aromatic protons, δH 6.52
(1H, s, H-5) and δH 6.75 (1H, s, H-2), are assumed to be located in the
para position to each other on the other benzene ring. Two anomeric
protons at δH 4.47 (1H, d, J = 2.9 Hz, H-1′) and 4.94 (1H, s, H-1‴), as
well as one methyl group of rhamnose at δH 1.24 (3H, d, J = 6.2 Hz, H-
6‴), were consistent with the presence of α-glucopyranosyl and α-
rhamnopyranosyl units, which were also demonstrated by acid hydro-
lysis through GC-MS affording D-glucose and L-rhamnose. The 1H and
13C nuclear-magnetic-resonance (NMR) spectroscopic data of 1 sug-
gested the presence of general structural features similar to those of
isoverbascoside (Scarpati et al., 1963; Olivier et al., 2010), except for
the absence of the trans-olefinic proton at C-8″ and the aromatic proton
phenylethanoid glycosides (nepetifosides A–L [1–12]) and
described phenylmethanoid glycoside (nepetifoside M [13]).
1 un-
2. Results and discussion
The phytochemical study on the n-BuOH fraction of the ethanolic
∗ Corresponding author. The MOE Key Laboratory of Standardization of Chinese Medicines and SATCM Key Laboratory of New Resources and Quality Evaluation of
Chinese Medicines, Institute of Chinese Materia Medica (ICMM), Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai, 201203, PR China.
E-mail address: chouguixinzyb@126.com (G.-X. Chou).
https://doi.org/10.1016/j.phytochem.2019.05.003
Received 14 January 2019; Received in revised form 7 May 2019; Accepted 7 May 2019
0031-9422/©2019ElsevierLtd.Allrightsreserved.

H.-T. Xu, et al.
Phytochemistry164(2019)111–121
Table 1
1H NMR spectroscopic data of compounds 1–7 from S. nepetifolia.a
No
1
2
3
4
5
6
7
Aglycone
2
3
4
5
6
7
8
6.75 s
6.52 s
6.72 d (1.8)
6.74 d (1.8)
6.77 d (1.9)
6.69 d (2.3)
6.78 d (1.9)
7.27 d (4.3)
7.27 d (4.3)
7.18 dd (8.7, 4.3)
7.27 d (4.3)
7.27 d (4.3)
2.95 t (6.6)
4.08 m
6.81 d (8.2)
6.68 dd (8.2, 1.8)
2.81 td (7.4, 2.0)
4.00 m
6.83 d (8.2)
6.70 dd (8.2, 1.8)
2.82 td (7.3, 1.9)
4.02 m
6.77 d (8.0)
6.68 d (8.6)
6.78 d (8.0)
6.67 dd (8.2, 1.8)
4.46c
6.67 dd (8.1, 1.9)
4.38 dd (8.9, 3.0)
3.78c
6.57 dd (8.1, 1.8)
2.79 td (7.8, 2.6)
4.00 dd (16.4, 8.1)
3.74c
2.61 m
3.93c
3.76c
3.54c
3.72 m
3.74 m
3.79c
4-OCH3
3.80 s
3.82 s
7-OCH3
3.25 s
Glc
1′
2′
3′
4′
4.47 d (2.9)
3.60c
4.35 d (7.9)
3.36c
4.37 d (7.9)
3.39c
4.41 d (7.9)
3.43c
4.37 d (7.9)
3.40c
4.45c
3.41c
3.81c
4.94 m
3.71c
3.72c
3.47c
4.38 d (7.9)
3.38c
3.47c
3.78c
3.82c
3.83c
3.82 t (9.2)
3.80c
3.49c
4.93c
4.94c
4.96c
4.94c
4.94 m
5′
6′
3.60c
3.68c
3.71c
3.72c
3.76c
3.70c
4.84c
3.71c
3.71c
3.74c
3.70c
3.72c
4.01 dd (11.6, 3.2)
3.45c
3.48c
3.49c
3.47c
3.48c
Ester moiety
2''
5''
6''
7''
6.61 d (1.2)
6.58 d (≈7.0)
6.58 d (≈7.0)
7.64 s
7.04 d (1.7)
6.76 d (8.2)
6.94 dd (8.2, 1.7)
7.58 d (15.9)
6.26 d (15.9)
7.06 d (1.6)
6.79 d (8.2)
6.97 dd (8.2, 1.6)
7.58 d (15.8)
6.24 d (15.9)
7.21 d (1.6)
6.81 d (8.2)
7.09 dd (8.2, 1.6)
7.66 d (15.9)
6.38 d (15.9)
7.06 d (1.6)
6.79 d (8.2)
6.97 dd (8.2, 1.7)
7.58 d (15.9)
6.24 d (15.9)
7.06 d (1.8)
6.74 d (8.1)
6.97 dd (8.2, 1.9)
7.60 d (15.9)
6.28 d (15.9)
7.06 d (1.6)
6.78 d (8.2)
6.96 dd (8.2, 1.6)
7.60 d (15.9)
6.28 d (15.9)
8''
CO
3″-OCH3
3.89 s
Rha
1'''
2'''
3'''
4'''
5'''
4.94c
5.16 s
5.23 s
5.22 d (1.3)
3.92c
5.23 s
5.21 d (1.3)
3.92c
5.18 s
3.87c
3.89c
3.74c
3.76c
3.91c
3.74c
3.55c
3.91c
3.56c
3.91c
3.57c
3.57c
3.39c
3.27c
3.44c
3.30c
3.43c
3.29c
3.29c
3.81c
3.53c
3.63c
3.59c
3.63 m
1.13 d (6.2)
3.55c
3.54c
6'''
1.24 d (6.2)
1.07 d (6.2)
1.14 d (6.2)
1.10 d (6.2)
1.09 d (6.2)
1.09 d (6.2)
Api
1IV
4.88c
3.85 d (2.0)
3.92c, 3.73c
3.52 s
4.90c
3.87 d (2.0)
3.92c, 3.73c
3.54 s
4.90c
3.86 d (2.3)
3.93c, 3.74c
3.52 s
4.87c
3.87 d (2.0)
3.91c, 3.74c
3.54 s
4.88c
3.85 d (2.3)
3.92c, 3.72c
3.53 s
4.90 d (2.1)
3.86 d (2.0)
3.91c, 3.73c
3.53 s
2IV
4IV
5IV
Xyl/n-butoxy
1V
2V
3V
4V
5V
4.38 d (7.6)
3.00 m
4.38 d (7.6)
3.00 m
3.38c
1.56 m
1.37 m
0.90 t (7.4)
3.35c
3.34c
3.24 t (9.0)
3.24 t (9.0)
3.73c
3.72c
3.07 t (10.8)
3.07 t (10.8)
a
500 MHz in CD3OD. c: Overlapped signal. (δ in ppm, J values in Hz).
at C-6 of phenylethyl moieties (see Table 1). In the HMBC spectrum
(Fig. 2), correlations between H-5 (δH 6.52) with C-8'' (δC 129.7) and H-
7″ (δH 7.64) with C-6 (δC 128.6) were observed, which proved that the
C-8″ of the caffeoyl moiety was connected to the C-6 of the phenylethyl
moiety. Accordingly, the structure of 1 was determined (Fig. 1) and was
named nepetifoside A.
(Fig. 2) because a correlation between methoxy protons at δH 3.80 and
C-4 at δC 147.5 was evident. The methoxy group was assigned to C-4,
which was aided by the correlation between the protons of methoxy (δH
3.80) and H-5 (δH 6.81) in NOESY (Fig. 2). The remaining correlations
in the HMBC spectrum were consistent with the presence of a for-
sythoside B moiety. Based on these findings, the structure of 2 was
established as 3-hydroxy-4-methoxy-β-phenylethyl [α-L-rhamnopyr-
anosyl-(1 → 3)]- [β-D-apiofuranosyl-(1 → 6)]-4-O-caffeoyl-β-D-gluco-
pyranoside and it was named nepetifoside B.
Compound 2 was obtained as a light-yellowish amorphous powder,
and its HRESIMS results suggested that the molecular formula of 2 is
C
35H46O19 [m/z 769.2595 [M–H]-, (calcd. for 769.2555)]. The 1H NMR
spectrum (Table 1) of 2 showed characteristic signals of (E)-caffeoyl
and 3-hydroxy-4-methoxy-phenylethyl moieties. Resonances of three
anomeric protons at δH 4.35 (1H, d, J = 7.9 Hz, H-1′), 4.88 (1H, ov, H-
1IV), and 5.16 (1H, s, H-1‴), as well as one methyl group of rhamnose at
δH 1.07 (3H, d, J = 6.2 Hz, H-6‴) were consistent with the presence of
β-glucopyranosyl, β-apiofuranosyl and α-rhamnopyranosyl units, which
were also demonstrated by acid hydrolysis through GC-MS affording D-
glucose, D-apiose and L-rhamnose. The 1H and 13C NMR spectroscopic
data of 2 suggested the presence of general structural features similar to
those of forsythoside B (Delazar et al., 2005), except for the presence of
an additional methoxy group (see Tables 1 and 2). The position of the
methoxy group was deduced from analysis of the HMBC spectrum
Compound 3 was obtained as a light-yellowish amorphous powder,
and its HRESIMS results suggested that the molecular formula of 3 is
C
40H54O23 [m/z 901.2996 [M–H]-, (calcd. for 901.2978)]. The 1H NMR
spectrum (Table 1) showed characteristic signals of (E)-caffeoyl and 3-
hydroxy-4-methoxy-phenylethyl moieties. The resonances of four
anomeric protons at δH 4.37 (1H, d, J = 7.9 Hz, H-1′), 4.38 (1H, d,
J = 7.6 Hz, H-1V), 4.90 (1H, d, J = 2.2 Hz, H-1IV), and 5.23 (1H, s, H-
1‴), as well as one methyl group of rhamnose at δH 1.14 (3H, d,
J = 6.2 Hz, H-6‴), were consistent with the presence of β-glucopyr-
anosyl, β-xylopyranosyl, β-apiofuranosyl and α-rhamnopyranosyl units.
The 1H and 13C NMR spectroscopic data of 3 were similar to those of
compound 2, except for the presence of an additional β-xylopyranosyl
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H.-T. Xu, et al.
Phytochemistry164(2019)111–121
Fig. 1. Structures of compounds 1–36 isolated from the S. nepetifolia.
113

H.-T. Xu, et al.
Phytochemistry164(2019)111–121
Fig. 2. Key HMBC and NOESY correlations of 1, 2, 4, 9, 12 and 13.
unit (see Tables 1 and 3). The chemical shifts of C-4IV in the rhamno-
pyranosyl moiety were shifted downfield from δC 73.8 to δC 83.7
compared with that of compound 2, and the position of the β-xylo-
pyranosyl unit was deduced from analysis of the HMBC spectrum, be-
cause a correlation between H-1V and C-4‴ was evident. The remaining
correlations in the HMBC spectrum were consistent with the presence of
a compound 2 moiety. Thus, the structure of compound 3 was estab-
lished as 3-hydroxy-4-methoxy-β-phenylethyl [β-D-xylopyranosyl-(1 →
caffeoyl-β-D-glucopyranoside and it was named nepetifoside E.
Compound 6 was obtained as a light-yellowish amorphous powder,
and its HRESIMS results suggested that the molecular formula of 6 is
C
38H52O20 [m/z 851.2960 [M+Na]+, (calcd. for 851.2950)]. The 1H
and 13C NMR spectroscopic data of 6 suggested the presence of general
structural features similar to those of forsythoside B (Delazar et al.,
2005), except for the presence of an additional n-butoxy unit (see
Tables 1 and 3). In the HMBC spectrum, a cross-peak was present be-
tween the C-1V methylene protons at δH 3.38 of n-butoxy and C-7 at δC
83.1 of the phenylethyl moiety, which proved that the position of the n-
butoxy unit was at the C-7 of the phenylethyl moiety. The correlations
between the caffeoyl, phenylethyl, rhamnosyl, and apiosyl to glucosyl
moieties were similar to those in forsythoside B. These findings sup-
ported that compound 6 was established as 7-O-n-butoxy-3, 4-dihy-
4)-α-L-
rhamnopyranosyl-(1 → 3)]-[β-D-apiofuranosyl-(1 → 6)]-4-O-
caffeoyl-β-D-glucopyranoside and it was named nepetifoside C.
Compound 4 was obtained as a light-yellowish amorphous powder,
and its HRESIMS results suggested that the molecular formula of 4 is
C
36H48O20 [m/z 823.2639 [M+Na]+, (calcd. for 823.2637)]. The 1H
and 13C NMR spectral data were closely related to those of 3‴-O-me-
thylcampneoside I (Wu et al., 2016), except for the presence of an
additional β-apiofuranosyl unit (see Tables 1 and 3). In the HMBC
spectrum (Fig. 2), two cross-peaks were present between the C-6′ me-
thylene protons at δH 3.74 and 3.49 of glucosyl and C-1IV at δC 111.0 of
the apiosyl, indicating that the position of the apiofuranosyl unit was at
the C-6′ of the glucopyranosyl unit. Thus, compound 4 was established
as β-methoxy-3, 4-dihydroxy-β-phenylethyl [α-L-rhamnopyranosyl-
(1 → 3)]-[β-D-apiofuranosyl-(1 → 6)]-4-O-feruloyl-β-D-glucopyranoside
and it was named nepetifoside D.
droxy-β-phenylethyl
[α-L-rhamnopyranosyl-(1 → 3)]-[β-D-apiofur-
anosyl-(1 → 6)]-4-O-caffeoyl-β-D-glucopyranoside and it was named
nepetifoside F.
Compound 7 was obtained as a light-yellowish amorphous powder,
and its HRESIMS results suggested that the molecular formula of 7 is
C
34H44O17 [m/z 747.2479 [M+Na]+, (calcd. for 747.2476)]. The 1H
and 13C NMR spectroscopic data of 7 suggested the presence of general
structural features similar to those of jionoside C (Hiroshi et al., 1989),
except for the presence of an additional β-apiofuranosyl unit (see Tables
1 and 3). In the HMBC spectrum, two cross-peaks were present between
the C-6′ methylene protons at δH 3.72 and 3.48 of glucosyl and C-1IV at
Compound 5 was obtained as a light-yellowish amorphous powder,
and its HRESIMS results suggested that the molecular formula of 5 is
C
39H52O23 [m/z 911.2793 [M+Na]+
,
(calcd. for 911.2797)].
δC 111.1 of the apiosyl, indicating that the position of the apiofuranosyl
According to the 1H and 13C NMR (see Tables 1 and 3) and 2D NMR
spectroscopic data, it could be concluded that compound 5 consisted of
six subunits of (E)-caffeoyl, one 3, 4-dihydroxy-phenylethyl, one β-
glucopyranosyl, one β-apiofuranosyl, one α-rhamnopyranosyl and one
β-xylopyranosyl, which were also demonstrated by acid hydrolysis
through GC-MS affording D-glucose, D-apiose, L-rhamnose and D-xy-
lose. Compared with the spectrum of compound 3, the only difference
for compound 5 was the missing methyl group in the 3,4-dihydroxy-
phenylethyl moiety. These findings suggested that compound 5 was
established as 3, 4-dihydroxy-β-phenylethyl [β-D-xylopyranosyl-(1 →
4)-α-L-rhamnopyranosyl-(1 → 3)]-[β-D-apiofuranosyl-(1 → 6)]-4-O-
unit was at the C-6′ of the glucopyranosyl unit. These findings sup-
ported that compound 7 was established as β-phenylethyl [α-L-rham-
nopyranosyl-(1 → 3)]-[β-D-apiofuranosyl- (1 → 6)]-4-O- caffeoyl-β-D-
glucopyranoside and it was named nepetifoside G.
Compound 8 was obtained as a colorless amorphous powder, and its
HRESIMS results suggested that the molecular formula of
8 is
C
35H46O18 [m/z 777.2592 [M+Na]+, (calcd. for 777.2582)]. The 1H
and 13C NMR spectroscopic data of 8, compared with those of leu-
cosceptoside B (Saracoglu et al., 2002), suggested the presence of an
(E)-coumaroyl moiety instead of a feruloyl moiety. Thus, the 1H NMR
spectrum (Table 1) of 8 showed the presence of an AA'BB’ system of
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Phytochemistry164(2019)111–121
Table 2
1H NMR spectroscopic data of compounds 8–13 from S. nepetifolia.a.
No
8
9
10
11
12
13
Aglycone
2
3
4
5
6
7
6.74 d (1.9)
6.70c
6.69 d (2.1)
6.68 d (1.5)
6.69 d (2.0)
7.43 d (7.3)
7.35 t (7.3)
7.29 d (7.2)
7.35 t (7.3)
7.43 d (7.3)
4.91c
6.83 d (8.2)
6.70 dd (8.2, 2.0)
2.83 t (7.2)
6.68c
6.67 d (8.4)
6.56 dd (8.0, 1.9)
2.80c
6.64 d (8.0)
6.54 dd (8.1, 1.7)
2.78 t (7.1)
6.68 d (7.8)
6.57 dd (8.1, 1.8)
2.80 td (7.4,2.9)
6.57 dd (8.0, 1.9)
2.79c
4.68 d (11.8)
8
4.01 dd (15.7, 8.7)
3.99c, 3.71c
4.00c, 3.72c
3.95c
3.70 m
3.97 m
3.71c
3.73c
3.82c
4-OCH3
Glc
1′
2′
3′
4′
5′
6′
4.37 d (7.9)
3.38c
4.34 d (7.9)
3.37c
4.33 d (7.9)
3.38c
4.33 d (7.9)
3.31c
4.38 dd (7.8,2.5)
3.39c
4.44 d (7.9)
3.45c
3.80c
3.77c
3.80c
3.58c
3.82c
3.80c
4.93c
4.91c
4.86c
3.39c
5.00 t (9.7)
4.96c
3.72c
3.61c
3.59c
3.54c
3.69c
3.71c
3.72c, 3.48c
3.75c, 3.40c
3.60c, 3.33c
4.49 d (10.4),4.34c
3.74c, 3.47c
3.75c, 3.52c
Ester moiety
2''
3''
5''
6''
7''
8''
7.47 d (8.7)
6.80 d (8.8)
6.80 d (8.8)
7.47 d (8.7)
7.66 d (15.9)
6.34 d (15.9)
6.77 d (1.3)
6.77 d (2.0)
7.04 s
7.06 d (1.7)
7.07 d (1.1)
6.76 d (8.3)
6.76 d (7.9)
6.77 d (8.2)
6.79 d (8.2)
6.79 d (8.3)
6.66c
6.66 dd (8.0, 2.0)
4.49 dd (9.0, 5.1)
2.76 d (9.2),
6.88 dd (8.2, 1.6)
7.56 d (15.8)
6.28 d (15.8)
6.96 dd (8.2, 1.7)
7.58 d (15.8)
6.24 d (15.9)
6.97 dd (8.1, 1.4)
7.60 d (15.9)
6.28 d (15.8)
4.47 dd (9.9, 3.9)
2.74 dd (15.2, 10.0), 2.64 dd (15.1, 3.9)
2.71 dd (15.5, 5.2)
CO
7″- OCH3
Rha
1'''
2'''
3'''
4'''
5'''
6'''
3.16 s
3.17 s
5.18 d (1.4)
3.91c
5.23 d (1.4)
3.87c
5.30 s
5.20 s
3.95c
3.95c
3.54c
4.08 m
1.26c
5.23 s
5.17 s
3.87c
3.75c
3.92c
3.57c
3.55c
3.57c
3.92 s
3.57c
3.29c
3.32c
3.36c
3.44c
3.29c
3.56c
3.53c
3.66c
3.63c
3.54c
1.08 d (6.2)
1.18 d (6.2)
1.26 d (6.2)
1.13 d (6.2)
1.08 d (6.1)
Api/Rha
1IV
4.90c
3.87c
4.92c
3.91 d (2.1)
4.91c
3.87 d (2.0)
4.62 s
4.94c
3.90c
2IV
3.68c
3IV
3.84c
4IV
3.91c, 3.72c
3.53c
3.93c, 3.76c
3.59 s
3.92c, 3.73c
3.57 s
3.34c
3.95c,3.75c
3.57c
5IV
3.60c
6IV
1.20 d (6.2)
Xyl
1V
4.54 d (7.5)
3.18c
4.38 dd (7.8, 2.5)
3.00 dd (16.3, 8.4)
3.46 m
2V
3V
3.46c
4V
3.32c
3.24 t (9.0)
5V
3.84 dd (11.3, 5.3)
3.73c
3.18c
3.09 d (10.9)
a
500 MHz in CD3OD. c: Overlapped signal. (δ in ppm, J values in Hz).
aromatic protons δH 6.80 (2H, d, J = 8.8 Hz, H-3″, H-5″) and δH 7.47
(2H, d, J = 8.7 Hz, H-2″, H-6″) (see Tables 2 and 3). In the HMBC
spectrum, a correlation was found between the H-4′ of the glucosyl
group and the carbonyl carbon of the trans-coumaroyl moiety, which
proved that the position of the trans-coumaroyl moiety was at the C-4′
of the glucose. These findings supported that compound 8 was estab-
lished as 3-hydroxy-4-methoxy- β-phenylethyl [α-L-rhamnopyranosyl-
moiety, indicating that the position of the methoxy unit was at the C-7″
of the ester moiety. Based on the above-mentioned evidence, compound
9 was elucidated as shown in Fig. 2 and it was named nepetifoside I.
Compound 10 was obtained as
a light-yellowish amorphous
powder, and its HRESIMS results suggested that the molecular formula
of 10 is C35H48O20 [m/z 787.2665 [M–H]-, (calcd. for 787.2661)]. The
1H and 13C NMR spectroscopic data of 10 suggested the presence of
general structural features similar to those of compound 9, except for
the coupling constant of H-7″ of the ester moiety protons at δH 4.49
(1H, dd, J = 9.0, 5.1 Hz, H-7″) and protons at δH 4.47 (1H, dd, J = 9.9,
3.9 Hz, H-7″). Compound 10 had the same planar structure as that of
compound 9, and the C-7″ also had a chiral carbon. Therefore, it is
deduced that the two compounds are epimers. Based on the above-
mentioned evidence, compound 10 was determined as shown in Fig. 2
and it was named nepetifoside J.
(1 → 3)]-[β-D-apiofuranosyl-(1 → 6)]-4-O-
coumaroyl-β-D-glucopyr-
anoside and it was named nepetifoside H.
Compound 9 was obtained as a light-yellowish amorphous powder,
and its HRESIMS results suggested that the molecular formula of 9 is
C
35H48O20 [m/z 787.2677 [M–H]-, (calcd. for 787.2661)]. The 1H and
13C NMR spectroscopic data of 9 suggested the presence of general
structural features similar to those of forsythoside B (Delazar et al.,
2005), except for additional resonances for a methoxy group and an
extra methylene group instead of two trans-olefinic protons. The as-
signments were confirmed by two-dimensional (2D) NMR spectroscopic
data, and in the HMBC spectrum (Fig. 2), a cross-peak was present
between the methoxy protons at δH 3.16 and C-7″ at δC 81.5 of the ester
Compound 11 was obtained as
a light-yellowish amorphous
powder, and its HRESIMS results suggested that the molecular formula
of 11 is C34H44O19 [m/z 755.2410 [M–H]-, (calcd. for 755.2399)]. The
1H and 13C NMR spectroscopic data of 11 suggested the presence of
115

H.-T. Xu, et al.
Phytochemistry164(2019)111–121
Table 3
13C NMR spectroscopic data of compounds 1–13 from S. nepetifolia.b.
No
1
2
3
4
5
6
7
8
9
10
11
12
13
Aglycone
1
2
3
4
5
6
7
129.4
119.8
145.3
145.1
118.0
128.6
34.8
132.9
117.1
147.3
147.5
112.9
121.2
36.6
132.9
117.1
147.4
147.6
112.9
121.2
36.6
130.7
115.0
146.5
146.6
116.4
119.9
84.4
131.4
117.1
146.1
144.7
116.3
121.3
36.6
131.9
115.2
146.7
146.5
116.7
120.0
83.1
140.0
130.0
129.4
127.2
129.4
130.0
37.2
132.9
117.1
147.4
147.6
112.9
121.2
36.6
131.4
117.1
146.5
144.7
116.3
121.3
36.6
131.4
117.1
146.5
144.7
116.4
121.3
36.6
131.4
117.0
146.1
144.6
116.3
121.3
36.7
131.4
117.1
146.1
144.7
116.4
121.3
36.6
138.9
129.3
129.3
128.8
129.3
129.3
72.1
8
66.8
72.2
72.2
75.3
72.1
75.7
72.0
72.4
72.4
72.4
72.4
72.4
4-OCH3
56.5
56.5
56.5
7-OCH3
56.7
Glc
1′
2′
3′
4′
5′
6′
102.0
75.0
84.6
67.9
74.8
63.7
104.3
76.1
81.6
70.9
74.6
68.4
104.2
76.3
80.6
70.8
74.6
68.4
104.6
76.3
81.2
70.8
74.6
68.5
104.2
76.3
80.6
70.8
74.6
68.4
104.9
76.5
81.7
71.0
74.9
68.7
104.3
76.2
81.6
70.9
74.6
68.5
104.3
76.2
81.6
70.9
74.6
68.5
104.1
76.0
80.2
70.9
74.5
67.8
104.1
76.2
79.4
70.9
74.6
67.8
104.4
75.8
83.4
70.2
75.4
64.7
104.3
76.3
80.6
70.3
74.6
67.5
103.2
76.2
81.7
70.9
74.7
68.5
Ester moiety
1''
2''
3''
4''
5''
6''
7''
8''
128.0
118.5
148.7
146.7
116.0
125.1
141.7
129.7
170.8
127.7
115.2
146.8
149.8
116.5
123.2
148.0
114.8
168.1
127.7
115.4
146.8
149.8
116.5
123.4
148.3
114.6
168.2
127.7
111.8
149.4
150.8
116.5
124.4
147.9
115.2
168.1
56.4
127.7
115.4
146.8
149.8
116.5
123.4
148.2
114.6
168.2
127.9
115.5
147.1
150.0
116.5
123.5
148.3
115.0
168.4
127.7
115.2
146.9
149.9
116.5
123.2
148.0
114.7
168.1
126.9
131.4
117.0
161.8
117.0
131.4
147.7
114.7
168.2
132.8
114.6
146.1
146.7
116.3
119.5
81.5
132.6
114.6
146.1
146.7
116.3
119.7
81.1
127.4
114.9
147.0
150.1
116.5
123.3
147.4
114.6
169.3
127.7
115.4
146.8
149.8
116.5
123.4
148.3
114.6
168.1
127.6
115.3
146.9
149.9
116.5
123.2
148.1
114.7
168.2
44.7
172.2
44.8
171.2
CO
3″-OCH3
7″-OCH3
56.7
56.6
Rha
1'''
2'''
3'''
4'''
5'''
6'''
100.6
72.6
72.1
74.1
70.0
17.9
103.1
72.3
72.0
73.8
70.4
18.4
102.4
72.2
72.1
83.7
68.7
18.4
102.9
72.3
72.0
73.7
70.4
18.4
102.3
72.2
72.1
83.7
68.7
18.4
103.2
72.6
72.3
74.0
70.6
18.6
103.1
72.4
72.0
73.8
70.4
18.4
103.0
72.2
72.1
73.8
70.4
18.4
102.3
72.2
72.0
73.7
70.2
18.1
102.1
72.2
72.1
73.9
70.2
18.2
102.4
72.3
72.2
83.5
68.6
17.9
102.4
72.2
72.1
83.6
68.7
18.4
103.1
72.3
72.0
73.8
70.4
18.4
Api/Rha
1IV
111.0
78.1
80.6
75.1
65.7
111.0
78.1
80.6
75.1
65.7
111.0
78.1
80.6
75.1
65.2
111.0
78.1
80.6
75.1
65.7
111.3
78.3
80.8
75.3
65.9
111.1
78.1
80.6
75.1
65.7
111.1
78.1
80.6
75.1
65.7
111.2
78.0
80.6
75.0
65.8
111.0
78.0
80.6
75.0
65.7
102.2
72.3
72.0
73.9
69.9
18.0
111.1
78.2
80.6
75.1
65.7
2IV
3IV
4IV
5IV
6IV
Xyl/n-butoxy
1V
2V
3V
4V
5V
106.6
75.7
71.0
78.0
67.1
106.5
75.7
71.0
78.0
67.0
69.8
33.1
20.6
14.5
106.4
75.9
71.1
78.1
67.2
106.5
75.7
71.0
78.0
67.0
b
125 MHz in CD3OD.
general structural features similar to those of isoverbascoside
(Kanchanapoom et al., 2002), except for the presence of an additional
β-xylopyranosyl unit (see Tables 2 and 3), as also demonstrated by acid
hydrolysis through GC-MS affording D-glucose, L-rhamnose and D-xy-
lose. In the HMBC spectrum, a cross-peak was present between the C-4‴
proton at δH 3.54 of rhamnosyl and C-1IV at δC 106.4 of the xylosyl,
indicating that the position of the xylopyranosyl unit was at the C-4‴ of
the rhamnopyranosyl unit. These findings supported that compound 11
was established as 3, 4-dihydroxy-β-phenylethyl [β-D-xylopyranosyl-
(1 → 4)-α-L-rhamnopyranosyl-(1 → 3)]-6-O-caffeoyl-β-D-glucopyrano-
side and it was named nepetifoside K.
through GC-MS affording one D-glucose, two L-rhamnose and one D-
xylose. In the HMBC spectrum (Fig. 2), a cross-peak was present be-
tween the C-1V anomeric proton at δH 4.38 of xylosyl and C-4'''at δC
83.6 of the rhamnosyl, indicating that the position of the xylopyranosyl
unit was at the C-4‴ of the rhamnopyranosyl unit. These findings
supported that compound 12 was established as 3, 4-dihydroxy-β-
phenylethyl
[β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 →
3)]-[α-L-rhamnopyranosyl-(1 → 6)]-4-O-caffeoyl-β-D-glucopyranoside
and it was named nepetifoside L.
Compound 13 was obtained as
a light-yellowish amorphous
powder, and its HRESIMS results suggested that the molecular formula
of 13 is C33H42O17 [m/z 709.2363 [M–H]-, (calcd. for 709.2344)]. The
1H and 13C NMR spectroscopic data of 13 suggested the presence of
general structural features similar to those of salsaside B (Nan et al.,
2013), except for the presence of an additional β-apiofuranosyl unit (see
Tables 2 and 3). In the HMBC spectrum (Fig. 2), two cross-peaks were
present between the C-6′ methylene protons at δH 3.75 and 3.52 of
glucosyl and C-1IV at δC 111.1 of the apiosyl, indicating that the
Compound 12 was obtained as
a light-yellowish amorphous
powder, and its HRESIMS results suggested that the molecular formula
of 12 is C40H54O23 [m/z 925.2957 [M+Na]+, (calcd. for 925.2954)].
The 1H and 13C NMR spectroscopic data of 12 suggested the presence of
general structural features similar to those of poliumoside (Akdemir
et al., 2004), except for the presence of an additional β-xylopyranosyl
unit (see Tables 2 and 3), as also demonstrated by acid hydrolysis
116

H.-T. Xu, et al.
Phytochemistry164(2019)111–121
position of the apiofuranosyl unit was at the C-6′ of the glucopyranosyl
unit. These findings supported that compound 13 was established as
phenmethyl [α-L-rhamnopyranosyl-(1 → 3)]-[β-D-apiofuranosyl- (1 →
6)]-4-O-caffeoyl-β-D-glucopyranoside and it was named nepetifoside M.
In addition, 23 known compounds were isolated, including 20
phenylethanoid glycosides, leucosceptoside B (14) (Saracoglu et al.,
2002), alyssonoside (15) (Ma et al., 2014), β-methoxyforsythoside B
(16) (Shi et al., 2013), forsythoside B (17) (Frezza et al., 2018;
Saracoglu et al., 2002), betonyoside E (18) (Miyase et al., 1996), ac-
teoside (19) (Scarpati et al., 1963; Wu et al., 2003), trichosanthosides A
(20) (Calis et al., 1999), leucosceptoside A (21) (Kim et al., 2001;
Venditti et al., 2016), ligupurpuroside C (22) (He et al., 1992), mar-
tynoside (23) (Kim et al., 2001), jionoside D (24) (Hiroshi et al., 1989),
isoverbascoside (25) (Kanchanapoom et al., 2002), 3-hydroxy-4-meth-
oxyphenyl-ethyl-O-[(α-L-rhamnosyl)-(1 → 3)]-6-O-caffeoyl- β-D-gluco-
pyranoside (26) (Hiroshi et al., 1989), plantainoside C (27) (Kim et al.,
2001), 3-O-methyl poliumoside (28) (Mostafa et al., 2007), 2-(3-hy-
that the xylopyranosyl unit was connected to the C-4"' of the rhamno-
pyranosyl unit. Therefore, the xylopyranosyl unit on these compounds
was likely to be responsible for the effect on osteoblasts. Similarly,
compounds 4 and 6 promoted the proliferation of osteoblasts, probably
due to the substituents of the methoxy group and n-butoxy unit at the C-
7 of the phenylethyl moiety.
3. Conclusions
Twelve undescribed phenylethanoid glycosides (nepetifosides A–L
[1–12]) and one undescribed phenylmethanoid glycoside (nepetifoside
M [13]), together with 23 known compounds, were isolated from the n-
BuOH fraction of S. nepetifolia. Their structures were elucidated via a
combination of 1D (1H and 13C MNR) NMR spectroscopy, 2D (HSQC,
HMBC and NOESY) NMR spectroscopy, mass spectrometry (HRESIMS),
HPLC, GC-MS, and chemical-hydrolysis methods. It is noteworthy that
the undescribed feature of compound 1—with a 12-membered macro-
cyclic structure linked through the C-5 of phenylethyl moiety and the C-
8″ of the caffeoyl moiety—has not been reported previously.
Twelve undescribed substances were examined for their effects on
the proliferation of LPS-stimulated MC3T3-E1 cells. From the data ob-
tained, nepetifoside F exhibited strong activity by significantly in-
creasing osteoblast proliferation at three continuous concentrations of
0.1, 1, and 10 μM. Moreover, nepetifoside C and nepetifoside D ex-
hibited moderate activities at medium and high concentrations of 1 μM
and 10 μM by promoting the proliferation of osteoblasts. However,
further studies are required to elucidate the mechanistic basis for pro-
motion of osteoblast proliferation associated with these compounds.
droxy-4-methoxyphenyl)-ethyl-O-(α-L-rhamnosyl)-(1 → 3)-O-
(α-L-
rhamnosyl) -(1 → 6)-4-O-E-feruloyl-β-D-glucopyranoside (29) (Zhou
et al., 1998), poliumoside (30) (Venditti et al., 2017; Zhou et al., 1998),
decaffeoylverbascoside (31) (Kanchanapoom et al., 2002), 2-pheny-
lethyl-O-β-D-glucopyranoside (32) (Kong et al., 2013), and p-hydro-
xyphenethyl-O-β-D-glucopyranoside (33) (Ouyang et al., 2003). Ad-
ditionally, three phenylmethanoid glycosides, salsaside B (34) (Nan
et al., 2013), 2-hydroxybenzyl β-D-glucopyranoside (35) (Kanho et al.,
2005), and benzyl-O-β-D-glucopyranoside (36) (De Rosa et al.,
1996)—had 1H NMR and 13C NMR data that were consistent with those
reported in the literature.
To determine whether compounds 4, 6 and 16 were the natural or
artificial products, the H2O extract of plants (degreased with CH2Cl2) of
S. nepetifolia was compared to the isolated compounds 4, 6 and 16 by
LC-MS. The results (Figs. S107–109 in supporting information) showed
that the H2O extract contained compounds 4, 6 and 16, indicating that
the compounds 4, 6 and 16 were the metabolites of the plant.
Artifacts are common in natural-product studies, which mostly oc-
curred through unwanted chemical reactions but would also occur
simply by the introduction of contaminants present in trace amounts in
the organic solvents (Venditti, 2018a). Therefore, in the study of nat-
ural products, a particular attention should be paid in terms of the
possible production of artifacts (Venditti, 2018b).
In Flora of China, the S. nepetifolia was assigned to the Verbenaceae
plant (Yu et al., 1982). However, modern-science research has shown
that this species should belong to Lamiaceae (Cantino et al., 1999;
Alvarenga et al., 2001). In the present study, 32 phenylethanoid gly-
cosides were isolated and identified from S. nepetifolia. It is worth
noting that this is the first report on the occurrence of phenylethanoid
glycosides in S. nepetifolia. The majority of the phenylethanoid glyco-
sides reported to date are found in the Scrophulariaceae, Oleaceae,
Plantaginaceae, Lamiaceae, and Orobanchaceae families (Fu et al.,
2008). Therefore, the presence of specific phenylethanoids such as
forsythoside B (17) and leucosceptoside A (21), have already been
observed in several Lamiaceae species and is in accordance with the
current systematic classification of the studied species.
4. Experimental
4.1. General
An Autopol VI polarimeter (No. 90079, Rudolph Research
Analytical) was used to determine the optical rotations. Infrared spectra
were measured on a PerkinElmer FT-IR spectrometer. A Bruker AV-400
instrument was applied for 1D and 2D NMR spectroscopy with TMS
used as the internal standard. HRESIMS data were acquired on a LCQ
DECA XP Plus ESI-MS mass spectrometer. Column chromatography
(CC) was performed on silica gel (200–300 mesh; Qingdao Marine
Chemical Factory, China) or Sephadex LH-20 (Amersham Pharmacia
Biotech) column. TLC was conducted on silica-gel GF254 plates
(Qingdao Marine Chemical Factory, China). MPLC was performed on a
GRACE system. Preparative HPLC was performed on the Puri Master-
7000 model from Shanghai cozhe, equipped with a Shiseido Capcellpak
Prep C18 ODS column (250 × 20 mm, 5 μm, flow rate: 16 mL min−1).
Spots were visualized under UV light or by spraying with 10% sulfuric
acid in EtOH followed by heating.
4.2. Plant material
A sample of S. nepetifolia was collected at Jilong Mountain (latitude
31°81′ N; longitude 118°21′ E; altitude 172 m), Hexian County, Anhui
Province, China, in the summer (wet season) of July 2014. The plant
was identified by Mr. Qing-Shan Yang (College of Pharmacy, Anhui
University of Traditional Chinese Medicine). A voucher specimen
(DHY2014072301) was deposited in the herbarium of the Institute of
Chinese Materia Medica, Shanghai University of Traditional Chinese
Medicine.
2.2. Effects on the proliferation of osteoblasts
Compounds 1 and 3–13 were screened for their effects on the pro-
liferation of osteoblasts (Fig. 3). Compound 6 exhibited strong activity
that significantly increasing osteoblast proliferation at three continuous
concentrations of 0.1, 1, and 10 μM. Moreover, compounds 3 and 4
exhibited moderate activities at medium and high concentrations of
1 μM and 10 μM to promote the proliferation of osteoblasts. In contrast,
compounds 5, 7, and 9 promoted the proliferation of osteoblasts only at
the low concentration of 0.1 μM. Similarly, compound 12 promoted the
proliferation of osteoblasts only at the medium concentration of 1 μM.
Structurally, the compounds 3, 5, and 12 were characterized such
4.3. Extraction and isolation
The dried and powdered plants of S. nepetifolia (8.1 kg) were ex-
tracted with 95% ethanol (3 × 60 L, seven days each) at room tem-
perature. The combined extracts (872 g) were evaporated under re-
duced pressure, subsequently suspended in H2O (2 L, 60 °C) and
117

H.-T. Xu, et al.
Phytochemistry164(2019)111–121
Fig. 3. The effects of compounds 1 and 3–13 isolated from the S. nepetifolia on the proliferation of osteoblasts.
successively partitioned with CH2Cl2 (4 × 2 L), EtOAc (4 × 2 L), and n-
BuOH (6 × 2 L) successively to yield three corresponding portions
(232, 16, and 314 g). The n-BuOH fraction (314 g) was adsorbed on
silica gel (100–200 mesh, 320 g) and was subjected to silica-gel CC
(200–300 mesh, 3.0 kg) eluted with an EtOAc-MeOH-H2O gradient
[1:0:0 (12 L); 15:2:0 (40 L); 15:2:1 (10 L); 10:2:1 (28 L); 8:2:1 (20 L);
6:2:1 (15 L); 4:2:1 (16 L), v/v/v] to collect 141 fractions of 1 L each,
which were combined into six fractions (A-F): fractions A (12.8 g), B
(16.5 g), C (21.4 g), D (84.0 g), E (65.0 g), and F (42.0 mg).
were purified by preparative HPLC using CH3CN-H2O (20:80, v/v) as a
mobile phase to yield compound 17 (115.0 mg, Rt 8.7 min, L: 16.4 min).
Compound 24 (15.0 mg, Rt 28.3 min, L: 41.0 min) was obtained by
Sephadex LH-20 in MeOH-H2O (50:50, v/v) and preparative HPLC
using CH3CN-H2O (20:80, v/v) from fraction B33 (133.0 mg). Fraction
B36 (489.0 mg) was purified by preparative HPLC using CH3CN-H2O
(20:80, v/v) as a mobile phase to obtain compounds 8 (6.7 mg, Rt
29.2 min, L: 54.0 min), 26 (7.0 mg, Rt 34.9 min, L: 54.0 min), and 34
(65.0 mg, Rt 42.3 min, L: 54.0 min). Compounds
7 (11.2 mg, Rt
Fraction B (16.5 g) was subjected to MPLC on an ODS-AQ column
and eluted with a MeOH-H2O gradient (0:100; 10:90; 20:80; 30:70;
40:60; 50:50; 60:40; 100:0, the volume of each solvent mixture: 4 L, v/
v) to afford 53 fractions, namely fractions B1 (157.0 mg), B2
(618.0 mg), B3 (222.0 mg), B4 (517.0 mg), B5 (150.0 mg), B6
(34.0 mg), B7 (84.0 mg), B8 (96.0 mg), B9 (120.0 mg), B10 (23.0 mg),
B11 (59.0 mg), B12 (84.0 mg), B13 (55.0 mg), B14 (137.0 mg), B15
(60.0 mg), B16 (57.0 mg), B17 (70.0 mg), B18 (240.0 mg), B19
(110.0 mg), B20 (108.0 mg), B21 (110.0 mg), B22 (83.0 mg), B23
(112.0 mg), B24 (555.0 mg), B25 (966.0 mg), B26 (370.0 mg), B27
(1372.0 mg), B28 (330.0 mg), B29 (580.0 mg), B30 (308.0 mg), B31
(362.0 mg), B32 (93.0 mg), B33 (133.0 mg), B34 (120.0 mg), B35
(380.0 mg), B36 (489.0 mg), B37 (386.0 mg), B38 (480.0 mg), B39
(100.0 mg), B40 (260.0 mg), B41 (133.0 mg), B42 (205.0 mg), B43
(173.0 mg), B44 (177.0 mg), B45 (200.0 mg), B46 (160.0 mg), B47
(12.0 mg), B48 (43.0 mg), B49 (75.0 mg), B50 (24.0 mg), B51
(48.0 mg), B52 (68.0 mg), and B53 (112.0 mg). Fraction B12 (84.0 mg)
was subjected to Sephadex LH-20 in MeOH-H2O (30:70, v/v) and pre-
parative HPLC using CH3CN-H2O (6:94, v/v) to yield compounds 33
(8.0 mg, Rt 22.5 min, L (length of chromatographic run): 39.0 min) and
35 (8.0 mg, Rt 24.6 min, L: 39.0 min). Compound 36 (11.2 mg, Rt
17.8 min, L: 22.0 min) was isolated through Sephadex LH-20 in MeOH-
H2O (10:90, v/v) with the preparative HPLC using CH3CN-H2O (15:85,
v/v) and was purified by preparative HPLC using CH3CN-H2O (10:90,
v/v) from fraction B14 (137.0 mg). Fraction B20 (108.0 mg) was sub-
jected to Sephadex LH-20 in MeOH-H2O (10:90, v/v) to afford three
subfractions. B20-3 (17.0 mg) was further purified by preparative HPLC
using CH3CN-H2O (10:90, v/v) to gain compound 32 (7.0 mg, Rt
38.1 min, L: 53.0 min). Fractions B28 (330.0 mg) and B29 (580.0 mg)
45.6 min, L: 75.0 min), 23 (70.0 mg, Rt 55.9 min, L: 80.0 min), and 6
(8.1 mg, Rt 65.3 min, L: 80.0 min) were isolated from fraction B37
(386.0 mg) by Sephadex LH-20 in MeOH-H2O (50:50, v/v) and pre-
parative HPLC using CH3CN-H2O (20:80, v/v). Fraction C (21.4 g) was
subjected to Sephadex LH-20 and eluted with a MeOH-H2O gradient
[10:90 (5 L); 20:80 (9 L); 30:70 (6 L); 40:60 (4 L); 50:50 (4 L); 60:40
(3 L); 70:30 (7 L), v/v] to afford 50 subfractions, namely fractions C1
(2450.0 mg), C2 (455.0 mg), C3 (246.0 mg), C4 (138.0 mg), C5
(125.0 mg), C6 (82.0 mg), C7 (135.0 mg), C8 (104.0 mg), C9 (98.0 mg),
C10 (256.0 mg), C11 (55.0 mg), C12 (55.0 mg), C13 (532.0 mg), C14
(780.0 mg), C15 (777.0 mg), C16 (593.0 mg), C17 (1360.0 mg), C18
(2930.0 mg), C19 (2430.0 mg), C20 (1306.0 mg), C21 (380.0 mg), C22
(90.0 mg), C23 (84.0 mg), C24 (93.0 mg), C25 (91.0 mg), C26
(87.0 mg), C27 (107.0 mg), C28 (120.0 mg), C29 (126.0 mg), C30
(120.0 mg), C31 (70.0 mg), C32 (54.0 mg), C33 (100.0 mg), C34
(890.0 mg), C35 (526.0 mg), C36 (222.0 mg), C37 (70.0 mg), C38
(55.0 mg), C39 (72.0 mg), C40 (60.0 mg), C41 (47.0 mg), C42
(50.0 mg), C43 (35.0 mg), C44 (48.0 mg), C45 (32.0 mg), C46
(60.0 mg), C47 (44.0 mg), C48 (290.0 mg), C49 (354.0 mg), and C50
(67.0 mg). C13 (532.0 mg), C28 (330.0 mg), and C48 (290.0 mg) were
further purified by preparative HPLC using CH3CN-H2O (18:82, v/v) to
obtain compounds 13 (7.6 mg, Rt 39.5 min, L: 100.0 min), 29 (6.0 mg,
Rt 61.9 min, L: 100.0 min); 21 (30.0 mg, Rt 28.7 min, L: 43.0 min) and
11 (13.0 mg, Rt 37.7 min, L: 61.0 min). C33 (150.0 mg) was separated
by MCI and eluted with a MeOH-H2O gradient (40:60; 50:50; 60:40;
70:30, the volume of each solvent mixture: 200 mL, v/v) to afford four
subfractions, namely subfractions C33-1 (56.0 mg), C33-2 (23.0 mg),
C33-3 (19.0 mg) and C33-4 (15.0 mg). C33-2 (23.0 mg) was further
purified by preparative HPLC using CH3CN-H2O (18:82, v/v) to yield
118

H.-T. Xu, et al.
Phytochemistry164(2019)111–121
compound 22 (3.0 mg, Rt 36.2 min, L: 47.0 min). Compound 27
(15.0 mg, Rt 28.5 min, L: 40.0 min) was obtain by preparative HPLC
using CH3CN-H2O (20:80, v/v) from C33-3 (19.0 mg). C40 (284.0 mg)
was further separated by MCI and eluted with a MeOH-H2O gradient
(50:50; 60:40; 70:30, the volume of each solvent mixture 200 mL, v/v)
to afford seven subfractions, namely subfractions C40-1 (19.0 mg), C40-
2 (14.0 mg), C40-3 (15.0 mg), C40-4 (70.0 mg), C40-5 (50.0 mg), C40-6
(50.0 mg), and C40-7 (10.0 mg). C40-1 (19.0 mg) was further purified
by preparative HPLC using CH3CN-H2O (15:85, v/v) to yield compound
1 (9.2 mg, Rt 17.6 min, L: 42.0 min). Fraction D (84.0 g), was subjected
to Sephadex LH-20 and eluted with a MeOH-H2O gradient [60:40
(3.5 L); 70:30 (2 L); 80:20 (2 L); 90:10 (3 L); 100:0 (4 L), v/v] to afford
nine subfractions, namely subfractions D1 (245.0 mg), D2 (11.9 g), D3
(12.0 g), D4 (50.0 g), D5 (6.0 g), D6 (100.0 mg), D7 (152.0 mg), D18
(11.0 mg), and D9 (46.0 mg). D4 (50.0 g) was applied to Sephadex LH-
20 in MeOH-H2O (80:20, v/v), to afford five subfractions, namely
subfractions D4-1 (20.0 mg), D4-2 (33.0 mg), D4-3 (440.0 mg), D4-4
(3.2 g), and D4-5 (44.0 g). D4-4 (3.2 g) was subjected to Sephadex LH-
20 in MeOH-H2O (30:70, v/v), to afford 14 subfractions, namely sub-
fractions D4-4-1 (247.0 mg), D4-4-2 (110.0 mg), D4-4-3 (660.0 mg),
D4-4-4 (170.0 mg), D4-4-5 (170.0 mg), D4-4-6 (175.0 mg), D4-4-7
(180.0 mg), D4-4-8 (390.0 mg), D4-4-9 (280.0 mg), D4-4-10
(155.0 mg), D4-4-11 (205.0 mg), D4-4-12 (53.0 mg), D4-4-13
(55.0 mg), and D4-4-14 (105.0 mg). D4-4-2 (110.0 mg), D4-4-3
(660.0 mg), and D4-4-4 (160.0 mg) were subjected to separation on
preparative HPLC using CH3CN-H2O (19:81, v/v) to yield compounds 3
(20.0 mg, Rt 22.7 min, L: 86.0 min), 14 (15.0 mg, Rt 46.3 min, L:
86.0 min); 5 (20.0 mg, Rt 14.3 min, L: 68.0 min), 4 (21.1 mg, Rt
16.5 min, L: 68.0 min), 15 (10.0 mg, Rt 19.5 min, L: 68.0 min), 28
(7.0 mg, Rt 27.9 min, L: 68.0 min), 16 (9.0 mg, Rt 12.7 min, L:
100.0 min), 30 (20.0 mg, Rt 14.6 min, L: 100.0 min), and 2 (20.0 mg, Rt
25.6 min, L: 100.0 min). D4-4-5 (170.0 mg) was subjected to Sephadex
LH-20 in MeOH-H2O (10:90, v/v), to afford five subfractions, namely
subfractions D4-4-5-1 (15.0 mg), D4-4-5-2 (7.0 mg), D4-4-5-3
(35.0 mg), D4-4-5-4 (35.0 mg), and D4-4-5-5 (38.0 mg). D4-4-5-3
(35.0 mg) was further purified by preparative HPLC using CH3CN-H2O
(8:92, v/v) to yield compound 31 (13.0 mg, Rt 12.1 min, L: 47.0 min).
Compounds 19 (170.0 mg, Rt 15.4 min, L: 42.0 min), 25 (140.0 mg, Rt
22.1 min, L: 42.0 min), and 20 (70.0 mg, Rt 25.8 min, L: 42.0 min) were
obtained from D5 (6.0 g) by preparative HPLC using CH3CN-H2O
(16:84, v/v). Fraction E (65.0 g) was divided into 81 subfractions,
namely fractions E1 (4.0 mg), E2 (8.0 mg), E3 (21.0 mg), E4 (1.7 g), E5
(12.0 g), E6 (14.0 g), E7 (2.0 g), E8 (790.0 mg), E9 (712.0 mg), E10
(800.0 mg), E11 (640.0 mg), E12 (586.0 mg), E13 (564.0 mg), E14
(574.0 mg), E15 (456.0 mg), E16 (525.0 mg), E17 (980.0 mg), E18
(1.2 g), E19 (1.3 g), E20 (990.0 mg), E21 (657.0 mg), E22 (1.4 g), E23
(3.2 g), E24 (3.0 g), E25 (2.4 g), E26 (1.8 g), E27 (834.0 mg), E28
(140.0 mg), E29 (242.0 mg), E30 (111.0 mg), E31 (128.0 mg), E32
(125.0 mg), E33 (132.0 mg), E34 (150.0 mg), E35 (212.0 mg), E36
(191.0 mg), E37 (250.0 mg), E38 (180.0 mg), E39 (173.0 mg), E40
(152.0 mg), E41 (86.0 mg), E42 (80.0 mg), E43 (80.0 mg), E44
(74.0 mg), E45 (110.0 mg), E46 (122.0 mg), E47 (220.0 mg), E48
(220.0 mg), E49 (180.0 mg), E50 (93.0 mg), E51 (77.0 mg), E52
(68.0 mg), E53 (62.0 mg), E54 (46.0 mg), E55 (56.0 mg), E56
(10.0 mg), E57 (46.0 mg), E58 (95.0 mg), E59 (60.0 mg), E60
(164.0 mg), E61 (125.0 mg), E62 (170.0 mg), E63 (140.0 mg), E64
(146.0 mg), E65 (107.0 mg), E66 (120.0 mg), E67 (197.0 mg), E68
(247.0 mg), E69 (177.0 mg), E70 (130.0 mg), E71 (94.0 mg), E72
(63.0 mg), E73 (40.0 mg), E74 (44.0 mg), E75 (44.0 mg), E76
(47.0 mg), E77 (35.0 mg), E78 (30.0 mg), E79 (20.0 mg), E80
(50.0 mg), and E81 (11.0 mg), by Sephadex LH-20 and eluted with a
MeOH-H2O gradient [0:100 (14 L); 10:90 (5 L); 20:80 (4 L); 30:70 (3 L);
40:60 (3 L); 50:50 (5 L); 60:40 (3 L); 70:30 (3 L); 80:20 (2 L), v/v]; E13
(564.0 mg) was separated by MCI and eluted with a MeOH-H2O gra-
dient (20:80; 30:70; 40:60; 50:50; 60:40; 70:30, the volume of each
solvent mixture 300 mL, v/v) to obtain 10 subfractions, namely
subfractions E13-1 (70.0 mg), E13-2 (22.0 mg), E13-3 (75.0 mg), E13-4
(66.0 mg), E13-5 (77.0 mg), E13-6 (21.0 mg), E13-7 (47.0 mg), E13-8
(22.0 mg), E13-9 (7.0 mg), and E13-10 (23.0 mg). E13-3 (75.0 mg) was
further purified by preparative HPLC using CH3CN-H2O (13:87, v/v) to
gain compounds 9 (14.0 mg, Rt 59.0 min, L: 75.0 min) and 10 (11.0 mg,
Rt 62.2 min, L: 75.0 min). E18 (2.48 g) was separated by MCI and eluted
with a MeOH-H2O gradient (20:80; 30:70; 40:60; 50:50; 60:40; 70:30;
80:20; 90:10; 100:0, the volume of each solvent mixture 500 mL, v/v)
to afford 18 subfractions, namely subfractions E18-1 (5.0 mg), E18-2
(55.0 mg), E18-3 (65.0 mg), E18-4 (770.0 mg), E18-5 (260.0 mg), E18-6
(195.0 mg), E18-7 (366.0 mg), E18-8 (100.0 mg), E18-9 (17.0 mg), E18-
10 (127.0 mg), E18-11 (40.0 mg), E18-12 (68.0 mg), E18-13 (54.0 mg),
E18-14 (58.0 mg), E18-15 (20.0 mg), E18-16 (32.0 mg), E18-17
(68.0 mg), and E18-18 (50.0 mg). E18-2 (55.0 mg) was further purified
by preparative HPLC using CH3CN-H2O (13:87, v/v) to yield compound
18 (26.0 mg, Rt 25.6 min, L: 42.0 min). Compound 12 (15.0 mg, Rt
34.6 min, L: 47.0 min) was obtained by preparative HPLC using CH3CN-
H2O (23:77, v/v) from E18-7 (366.0 mg).
4.4. Determination of the absolute configuration of sugars
Compounds 1, 2, 5, 11 and 12 (each 0.5 mg) in the 10-mL ampoules
were dissolved in 2-M trifluoroacetic acid (1 mL) and hydrolysed for
2 h at 120 °C. The hydrolysate was evaporated under a vacuum to yield
a residue. The residue was evaporated three times with methanol, and
was dried overnight in a vacuum desiccator with phosphorus pentoxide.
Then S-(+)-1-amino-2-propanol-methanol (1:8), acetic acid-methanol
(1:4), and 3% sodium cyanoborohydride in methanol solution (each
25 μL) were added to the residue, and heated for 1.5 h at 65 °C. After
cooling to room temperature, methanol-acetic acid (5:1) (2 mL) was
added to the mixture and evaporated twice. The residue was evaporated
three times with methanol. After drying, 0.4 mL of pyridine-acetic an-
hydride (1:1) was added to the residue, heated for 45 min at 100 °C, and
removed from the heat. An amount of 3-mL of distilled water was added
and mixed, at room temperature for 30 min. The solution was extracted
with 3 mL chloroform, and the chloroform layer was washed three
times with distilled water, and dried with anhydrous sodium sulphate
(Cases et al., 1995). The extracts were subjected to GC-MS analysis
under the following conditions: Agilent7000QQQ GC-MS, capillary
column: HP-5 ms (30 m × 0.25 mm, 0.25 μm), detection, FID; the
temperature of the injector and detector were both at 280 °C; the initial
temperature of 140 °C, was raised to 220 °C at a rate of 2 °C/min, 220 °C
was maintained for 40 min, and subsequently raised to 280 °C at a rate
of 6 °C/min; the final temperature was maintained for 10 min, and He
was used as the gas carrier at a flow rate of 25 mL min−1. The D-glucose
(tR = 92.923 min),
D-apiose
(tR = 80.694 min),
D-xylose
(tR = 81.242 min), and L-rhamnose (tR = 77.564 min) were determined
via comparison with the retention time of the standards (Supplemen-
tary data, Fig. S1).
4.5. Proliferation assay via the MTT method
The MC3T3-E1 osteoblast-like cell line was purchased from the
Chinese Academy of Medical Sciences and the Institute of Basic Medical
Sciences Cell Resource Center in Shanghai. In the experiment (He et al.,
2018), MC3T3-E1 cells were cultured in α-modified minimal-essential
medium (α-MEM) supplemented with 10% (v/v) fetal bovine serum
(FBS), and 1% (v/v) penicillin-streptomycin solution, and incubated at
37 °C in 5% CO2 humidified air. The culture medium was changed every
three days.
To examine the effects of the substances on proliferation in LPS-
stimulated MC3T3-E1 cells, cells were plated in 96-well culture plates
at a density of 5 × 103 cells per well and allowed to attach overnight.
The cells were treated with various concentrations (0.01, 0.1, 1.0, or
10 μM) of constituents, as well as with or without 10 μg/mL LPS for
48 h. At the end of the treatment, 20 μLMTT (5 mg/mL) in phosphate-
119

H.-T. Xu, et al.
Phytochemistry164(2019)111–121
buffered solution (pH 7.4) was added. After an additional 4 h of in-
cubation, the supernatant was discarded and 150 μL of dimethyl sulf-
oxide (DMSO) was added to each well to dissolve the formazan crystals
that formed in the cells. The absorbance was measured at 570 nm.
IR ν
3346, 2920, 1733, 1607, 1520, 1447, 1372, 1282, 1046, and
max
1016 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 2 and 3;
HRESIMS m/z 787.2665 [M–H]- (calcd. for C35H47O20−, 787.2661).
4.5.11. Nepetifoside K (11)
4.5.1. Nepetifoside A (1)
Light-yellowish amorphous powder; [α]20 D-27.1 (c 0.077, MeOH);
IR ν max 3357, 2920, 1592, 1260, and 1041 cm−1; for 1H and 13C NMR
spectroscopic data, see Tables 2 and 3; HRESIMS m/z 755.2410 [M–H]-
(calcd. for C34H43O19−, 755.2399).
Yellowish-brown amorphous powder; [α]20 D-48.1 (c 0.036,
MeOH); IR ν
3357, 2921, 1598, 1228, and 1033 cm−1; for 1H and
max
13C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z
621.1821 [M–H]- (calcd. for C29H33O15−, 621.1819).
4.5.12. Nepetifoside L (12)
4.5.2. Nepetifoside B (2)
Light-yellowish amorphous powder; [α]20 D-72.6 (c 0.093, MeOH);
Light-yellowish amorphous powder; [α]20 D-72.3 (c 0.047, MeOH);
IR ν max 3393, 2920, 1703, 1646, 1515, 1269, and 1073 cm−1; for 1H
and 13C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z
769.2595 [M–H]- (calcd. for C35H45O19−, 769.2555).
IR ν
3332, 2918, 1713, 1593, 1517, 1442, 1260, 1116, and
max
1033 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 2 and 3;
HRESIMS m/z 925.2957 [M+Na]+ (calcd. for C40H54O23Na+
,
925.2954).
4.5.3. Nepetifoside C (3)
4.5.13. Nepetifoside M (13)
Light-yellowish amorphous powder; [α]20 D-76.1 (c 0.067, MeOH);
Light-yellowish amorphous powder; [α]20 D-48.8 (c 0.080, MeOH);
IR ν
3353, 2922, 1714, 1600, 1615, 1270, and 1024 cm−1; for 1H
IR ν
3394, 2920, 2850, 1646, 1596, 1469, 1419, 1261, and
max
max
and 13C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z
901.2996 [M–H]- (calcd. for C40H53O23−, 901.2978).
1056 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 2 and 3;
HRESIMS m/z 709.2363 [M–H]- (calcd. for C33H41O17−, 709.2344).
4.5.4. Nepetifoside D (4)
Acknowledgements
Light-yellowish amorphous powder; [α]20 D-79.2 (c 0.132, MeOH);
IR ν
3290, 2921, 1712, 1631, 1599, 1516, 1280, 1157, and
This project was funded by the National Natural Science Foundation
of China (21672152).
max
1021 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 3;
HRESIMS m/z 823.2639 [M+Na]+ (calcd. for C36H48O20Na+
,
823.2637).
Appendix A. Supplementary data
4.5.5. Nepetifoside E (5)
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.05.003.
Light-yellowish amorphous powder; [α]20 D-90.9 (c 0.119, MeOH);
IR ν 3350, 2922, 1697, 1603, 1518, 1446, 1261, 1158, and
max
1025 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 3;
References
HRESIMS m/z 911.2793 [M+Na]+ (calcd. for C39H52O23Na+
,
911.2797).
Akdemir, Z.S., Tatli, I.I., Bedir, E., Khan, I.A., 2004. Iridoid and phenylethanoid glyco-
sides from Verbascum lasianthum. Turk. J. Chem. 28, 227–234.
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V.D.P., 2001. A computer-assisted approach for chemotaxonomic studies–diterpenes
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4.5.6. Nepetifoside F (6)
Light-yellowish amorphous powder; [α]20 D-58.0 (c 0.044, MeOH);
Calis, I., Kirmizibekmez, H., Rüegger, H., Sticher, O., 1999. Phenylethanoid glycosides
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max
from Globularia trichosantha. J. Nat. Prod. 62 (8), 1165–1168.
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conflict between phylogenetic and pragmatic considerations in botanical nomen-
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HRESIMS m/z 851.2960 [M+Na]+ (calcd. for C38H52O20Na+
,
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4.5.7. Nepetifoside G (7)
Light-yellowish amorphous powder; [α]20 D-88.8 (c 0.079, MeOH);
IR ν
3351, 2925, 1707, 1604, 1516, 1261, and 1023 cm−1; for 1H
max
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747.2479 [M+Na]+ (calcd. for C34H44O17Na+, 747.2476).
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4.5.8. Nepetifoside H (8)
Colorless amorphous powder; [α]20 D-63.0 (c 0.046, MeOH); IR ν
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tential leads for new therapeutics. Curr. Med. Chem. 15 (25), 2592–2613.
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pathway. 2018. Chem. Biol. Interact. 291, 128–136.
3355, 2921, 2852, 1715, 1633, 1604, 1515, 1444, 1133, and
max
1022 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 2 and 3;
HRESIMS m/z 777.2592 [M+Na]+ (calcd. for C35H46O18Na+
,
777.2582).
He, Z.D., Liu, Q.Y., Ren, Y.C., 1992. Glycosides from Ligustrum purpurascen. Acta Bot.
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4.5.9. Nepetifoside I (9)
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esters of phenethylalcohol glycosides from Rehmannia glutinosa, var. purpurea.
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Light-yellowish amorphous powder; [α]20 D-45.6 (c 0.079, MeOH);
IR ν max 3325, 2933, 1737, 1593, 1519, 1442, 1374, 1282, 1098, 1048,
Kanchanapoom, T., Kasai, R., Yamasaki, K., 2002. Phenolic glycosides from Markhamia
and 1002 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 2
stipulata. Phytochemistry 59, 557–563.
−
and 3; HRESIMS m/z 787.2677 [M–H]- (calcd. for C35H47O20
,
Kanho, H., Yaoya, S., Kawahara, N., Nakane, T., Takase, Y., Masuda, K., Kuroyanagi, M.,
2005. Biotransformation of benzaldehyde-type and acetophenone-type derivatives by
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inhibitory phenylpropanoid glycosides from Clerodendron trichotomum. Arch Pharm.
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