Firmosides A and B: two new sucrose ferulates from the aerial parts of Silene firma and evaluation of radical scavenging activities

Article abstract of DOI:10.1007/s11418-020-01426-5

Abstract: Two new tri-ferulates of sucrose, firmosides A and B (1 and 2, respectively), together with 18 known compounds (3–20), were isolated from the aerial parts of Silene firma. The structures of the isolated compounds were elucidated by various spectroscopic methods, including 1D, 2D NMR, and high-resolution electro-spray ionization–mass spectrometry (HR-ESI–MS). All the isolated compounds were evaluated for their free radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. As a result, two new compounds (1, 2) and 11 demonstrated significant radical scavenging activity, implying the usefulness as antioxidant agents. Graphic Abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11418-020-01426-5

Journal of Natural Medicines
https://doi.org/10.1007/s11418-020-01426-5
NOTE
Firmosides A and B: two new sucrose ferulates from the aerial parts
of Silene frma and evaluation of radical scavenging activities
Nguyen Hoang Uyen1 · Retno Widyowati1,2 · Melanny Ika Sulistyowaty1,3 · Sachiko Sugimoto1 · Yoshi Yamano1 ·
Susumu Kawakami4 · Hideaki Otsuka4 · Katsuyoshi Matsunami1
Received: 16 May 2020 / Accepted: 23 June 2020
© The Japanese Society of Pharmacognosy 2020
Abstract
Two new tri-ferulates of sucrose, frmosides A and B (1 and 2, respectively), together with 18 known compounds (3–20),
were isolated from the aerial parts of Silene frma. The structures of the isolated compounds were elucidated by various spec-
troscopic methods, including 1D, 2D NMR, and high-resolution electro-spray ionization–mass spectrometry (HR-ESI–MS).
All the isolated compounds were evaluated for their free radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical. As a result, two new compounds (1, 2) and 11 demonstrated signifcant radical scavenging activity, implying
the usefulness as antioxidant agents.
Graphic Abstract
R
OH
HO
O
O
O
O
HO
O
O
O
O
O
OH
OH
OMe
R
OH
1: OH
MeO
OMe
2: OAc
OH
Keywords Silene frma · Ferulic acid · Radical scavenging activities · DPPH · Sucrose · Firmoside
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11418-020-01426-5) contains
supplementary material, which is available to authorized users.
3
* Katsuyoshi Matsunami
Department of Pharmaceutical Chemistry, Faculty
matunami@hiroshima-u.ac.jp
of Pharmacy, Universitas Airlangga, Gedung Nanizar Zaman
Joenoes, Kampus C Unair, Surabaya 60115, Indonesia
1
Graduate School of Biomedical and Health Sciences,
4
Faculty of Pharmacy, Yasuda Women’s University, 6-13-1
Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan
Hiroshima University, 1-2-3 Kasumi, Minami-ku,
Hiroshima 734-8553, Japan
2
Department of Pharmacognosy and Phytochemistry, Faculty
of Pharmacy, Universitas Airlangga, Gedung Nanizar Zaman
Joenoes, Kampus C Unair, Surabaya 60115, Indonesia
Vol.:(0123456789)
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Journal of Natural Medicines
Introduction
The genus Silene (family: Caryophyllaceae) comprises
more than 700 species mainly distributed in the temper-
ate zone of the northern hemisphere. The genus Silene
includes several taxa previously treated as diferent genera,
such as Coronaria, Cucubalus, Lychnis, Melandrium, Pet-
rocopsis, and Viscaria [1]. Melandrium frmum Rohrbach
is a synonym of Silene frma and is widely distributed in
China, Korea, Russia, and Japan. S. frma is an annual or
biennial herb. The stems erect and reach 30–100 cm in
height, and the knots are sometimes dark violet. Leaves are
opposite, lanceolate to ovate–lanceolate, 3–10 cm long,
1–3 cm wide, apex acute, and hairs in the margins. Flowers
bloom from June to September, and the petals are white.
The seeds are black, renal shape, 0.7 to 1 mm, with spines,
and ripen from July to August [2, 3]. The dried aerial parts
have been used for the treatment of anuria, breast cancer,
gonorrhea, and diseases of lactation in Korea [4], and, as
Chinese traditional medicine, of acute nephritis, liver cir-
rhosis, and ascites in China [5]. The methanolic extract of
this plant inhibited the development of benign prostatic
hyperplasia using the testosterone propionate induced rat
model [6]. Previous phytochemical investigation of this
plant reported cytotoxic anthraquinone dimers, triterpenes,
β-carboline alkaloids, favonoids, and mannitol [7–9]
Reactive oxygen species (ROS) play an important
role in human physiological processes. However, exces-
sive ROS accumulation leads to oxidative damage of cell
membranes, proteins, and DNA, which causes a variety of
diseases, including the above-mentioned diseases such as
cancer, nephritis, liver cirrhosis, and prostatic hyperplasia
[10]. Therefore, the traditional medicinal usage of Silene
frma may be related to the supplementation of antioxi-
dants as an efective measure for preventing and repairing
the damages caused by ROS.
Fig. 1 Structures of new compounds 1 and 2
4-hydroxybenzaldehyde (15) [23], (+)-dehydrovomifoliol
(16) [24], α,β-dihydroferulic acid (17) [25], indole-3-car-
boxaldehyde (18) [26, 27], isovanillic acid (19) [28], and
grasshopper ketone (20) [29] (Fig. 2). These compounds
were isolated by performing chromatography such as silica
gel, ODS, and HPLC. The chemical structures of the iso-
lated compounds were elucidated by spectroscopic analy-
ses of 1D and 2D NMR spectra (1H, 13C NMR, DEPT,
COSY, HSQC, and HMBC) (Figs S1–S12 in supplemen-
tary data) in combination with high-resolution electro-
spray ionization–mass spectrometry (HR-ESI–MS). This
paper mainly deals with the structural elucidation of two
new compounds and their 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging activity.
Results and discussion
The methanolic extract of the aerial parts of S. frma was
partitioned with n-hexane, EtOAc, 1-butanol, and H2O, suc-
cessively. The n-hexane- and EtOAc-soluble fractions were
combined and fractionated by repeated chromatography to
aford two new (1 and 2) and 18 known compounds (3–20)
(Figs. 1 and 2).
Thus, we aimed to clarify the chemical constituents by
the extensive fractionation and purifcation procedures.
Our investigation of the n-hexane- and EtOAc-soluble
fractions of the aerial parts of S. frma revealed the pres-
ence of two new ferulic acid sucrose esters named frmo-
sides A and B (1 and 2, respectively) (Fig. 1) together with
18 known compounds identifed to be vanillin (3) [11],
(3R,6R,7E)-3-hydroxy-4,7-megastigmadien-9-one (4) [12],
scopoletin (5) [13], (9S,12S,13S)-E-9,12,13-trihydroxy-
10-octadecaenoic acid (6) [14], 20-hydroxy-ecdysone (7)
[15], luteolin 3′-O-methyl-6-C-β-D-glucopyranoside (8)
[16], apigenin-6-C-β-D-glucopyranoside (9) [17], maltol
β-D-glucopyranoside 6’-O-benzoate (10) [18], helo-
nioside A (11) [19], 22-O-acetyl 20-hydroxy-ecdysone
(12) [20], 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)
propan-1-one (13) [21], (6S,9R)-vomifoliol (14) [22],
Compound 1 was obtained as a white amorphous powder
with a molecular formula of C42H46O20 determined from
HR-ESI–MS at m/z 869.2501 [M–H]− (calcd for C42H45O20:
869.2501). The presence of multiple carbonyl and hydroxy
groups was estimated from the IR absorptions at 1716, 1697,
1686, and 3344 cm−1, respectively. The UV absorptions at
237 (4.18), 264 (3.69), 300 (4.17), and 327 (4.34) were
indicative of the presence of aromatic rings.
1
The H NMR spectrum displayed three ABX coupling
systems [δH 7.20 (1H, d, J = 1.9 Hz, H-2ʹʹ), 7.13 (1H, d,
J=1.9 Hz, H-2ʹʹʹ), 6.98 (1H, d, J=1.9 Hz, H-2ʹʹʹʹ), 6.73 (1H,
1 3

Journal of Natural Medicines
Fig. 2 Structures of known compounds 3–20
d, J = 8.3 Hz, H-5ʹʹ), 6.79 (1H, d, J = 8.2 Hz, H-5ʹʹʹ), 6.70
(1H, d, J = 8.2 Hz, H-5ʹʹʹʹ), 7.08 (1H, dd, J = 8.3, 1.9 Hz,
H-6ʹʹ), 7.02 (1H, dd, J=8.2, 1.9 Hz, H-6ʹʹʹ), 6.92 (1H, dd,
J=8.2, 1.9 Hz, H-6ʹʹʹʹ)], three methoxy groups at δH 3.86,
3.75, and 3.84 (each 3H, s, 3ʹʹ, 3ʹʹʹ, 3ʹʹʹʹ-OCH3, respectively),
an anomeric proton at δH 5.65 (1H, d, J = 3.7 Hz, H-1ʹ),
and three trans-olefnic protons pairs with large coupling
constants at δH 7.65 (1H, d, J=15.9 Hz, H-8ʹʹ), 6.45 (1H,
d, J=15.9 Hz, H-7ʹʹ), 7.61 (1H, d, J=15.9 Hz, H-8ʹʹʹ), 6.34
(1H, d, J=15.9 Hz, H-7ʹʹʹ), 7.70 (1H, d, J=15.9 Hz, H-8ʹʹʹʹ),
6.31 (1H, d, J=15.9 Hz, H-7ʹʹʹʹ), which suggested the pres-
ence of three feruloyl functions on sugar moiety (Table 1).
The 13C NMR spectrum of 1 showed 42 carbon reso-
nances classifed by comparing its chemical shift values,
DEPT, and HSQC spectra, as three carbonyl carbons at δC
168.6 (C-9ʹʹ), 169.2 (C-9ʹʹʹ), and 168.9 (C-9ʹʹʹʹ), two ano-
meric carbons at δC 105.9 (C-2) and 91.4 (C-1ʹ), six olefnic
carbons at δC 115.0 (C-7ʹʹ), 115.28 and 115.34 (C-7ʹʹʹ or
C-7ʹʹʹʹ), 148.0 (C-8ʹʹ), 147.3 (C-8ʹʹʹ), 147.6 (C-8ʹʹʹʹ), three
oxygenated methylene carbons at δC 64.4 (C-1), 66.1 (C-6),
and 62.6 (C-6ʹ), seven oxygenated methine carbons at δC
78.7 (C-3), 74.9 (C-4), 81.2 (C-5), 74.8 (C-2ʹ), 72.4 (C-3ʹ),
71.8 (C-4ʹ), 74.2 (C-5ʹ), three methoxy carbons at δC 56.5,
56.6, and 56.7, and 18 characteristic carbon signals attrib-
utable to three benzene ring (δC 111.9–150.9) (Table 1).
Chemical shift values of feruloyl and isoferuloyl functions
have been reported as follows: feruloyl: [δC ca. 112.0 (C-2),
ca. 116.5 (C-5) in CD3OD] and isoferuloyl: [δC ca. 115.0
(C-2), ca. 112.5 (C-5) in CD3OD [30]. The chemical shifts
and ΔδC-2,5 values (1: ΔδC-2,5 ≈4.5–4.71 ppm) of three acyl
moieties of 1 were in good agreement with those of feruloyl
function (i.e., feruloyl: ΔδC-2,5 ≈4.5 ppm, and isoferuloyl:
ΔδC-2,5 ≈2.5 ppm). These fndings strongly suggested that
the structure of 1 was a disaccharide having three ferulic
acid functions.
The 1H and 13C NMR spectroscopic data of 1 were simi-
lar to quiquesetinerviuside A [31], except for the presence of
p-coumaroyl function and chemical shift diferences on the
sugar moiety probably caused by a diference in the attach-
ment position (Fig. 1 and Table 1). The HMBC spectrum
showed the correlations of methoxy protons (3H each, δH
3.86, 3.75, and 3.84) with C-3ʹʹ, C-3ʹʹʹ (δC 149.45/149.48),
and C-3ʹʹʹʹ (δC 149.3), respectively. Besides, the correlations
of H-2ʹ (δH 4.68) with C-9ʹʹʹʹ (δC 168.9), H-3 (δH 5.54) with
C-9ʹʹ (δC 168.6), and H-6 (δH 4.46) with C-9ʹʹʹ (δC 169.2)
revealed the position of three feruloyl groups on C-2ʹ, C-3,
and C-6, respectively (Fig. 2). The two sugars were con-
nected between C-1’ and C-2 determined by the HMBC
correlation from H-1ʹ (δH 5.65) to C-2 (δC 105.9) (Fig. 3).
Mild alkaline hydrolysis of 1 with metha-
nolic 100 mM NaOCH3 liberated D-sucrose and
methyl ferulate [32] by HPLC analyses with opti-
cal rotation detector and spectroscopic data (HR-
1
ESI–MS and H NMR). Based on these results, the
structure of 1 was characterized as (3,6-O-diferuloyl)-
β-D-fructofuranosyl-(2 → 1)-(2-O-feruloyl)-α-D-
glucopyranoside, designated as frmoside A.
Compound 2 was obtained as a white amorphous powder
with a molecular formula of C44H48O21 determined by HR-
ESI–MS at m/z 935.2581 [M+Na]+ (calcd for C44H48O21Na:
1 3

Journal of Natural Medicines
Table 1 1H and 13C NMR
spectroscopic data for
compound 1
No.
1
δH (J in Hz)
δC
No.
δH (J in Hz)
δC
3.49 (1H, d, 12.0)
3.62 (1H, d, 12.0)
–
64.4
9’’
OCH3
1’’’
2’’’
3’’’
4’’’
5’’’
6’’’
7’’’
–
168.6
3.86 (3H, s)
–
7.13 (1H, d, 1.9)
–
56.5d
2
3
4
5
105.9
78.7
74.9
81.2
66.1
91.4
74.8
72.4
71.8
74.2
62.6
127.79e
111.9
5.54 (1H, d, 8.3)
4.42 (1H, t, 8.3)
4.12 (1H, m)
4.46 (2H, d, 5.4)
5.65 (1H, d, 3.7)
4.68 (1H, dd, 10.0, 3.7)
3.94 (1H, m)
3.48 (1H, t, 9.1)
3.95 (1H, m)
3.76 (1H, m)
3.87 (1H, m)
–
7.20 (1H, d, 1.9)
–
–
6.73 (1H, d, 8.3)
7.08 (1H, dd, 8.3, 1.9)
6.45 (1H, d, 15.9)
7.65 (1H, d, 15.9)
149.48a
150.7
–
6
6.79 (1H, d, 8.2)
7.02 (1H, dd, 8.2, 1.9)
6.34 (1H, d, 15.9)
7.61 (1H, d, 15.9)
–
3.75 (3H, s)
–
6.98 (1H, d, 1.9)
–
–
6.70 (1H, d, 8.2)
6.92 (1H, dd, 8.2, 1.9)
6.31 (1H, d, 15.9)
7.70 (1H, d,15.9)
–
116.61b
124.46c
115.28f
147.3
1’
2’
3’
4’
5’
6’
8’’’
9’’’
169.2
OCH3
1’’’’
2’’’’
3’’’’
4’’’’
5’’’’
6’’’’
7’’’’
8’’’’
9’’’’
OCH3
56.6d
127.83e
112.0
1’’
2’’
3’’
4”
5’’
6”
127.7
149.3
112.1
150.9
149.45a
150.7
116.64b
124.2
116.58b
124.37c
115.0
115.34f
147.6
7”
8’’
168.9
148.0
3.84 (3H, s)
56.7d
1H (600 MHz) and 13C NMR (150 MHz) in methanol-d4, m: multiplet or overlapped signals, a–f: inter-
changeable
Fig. 3 Important HMBC and COSY correlations of 1 and 2
1
935.2580), indicating 42 mass unit larger than 1. The pres-
ence of multiple carbonyl and hydroxy groups was suggested
from the IR spectrum (1717, 1699, and 1684 cm−1, and the
signal at 3344 cm−1, respectively).
The H NMR and 13C NMR spectra (Table 2) were
closely similar to 1, except for a methyl proton at δH 2.08
(3H, s) and two carbons at δC 21.0 and 173.0 ppm, suggest-
ing the structure of 2 was an acetyl derivative of 1.
1 3

Journal of Natural Medicines
Table 2 1H and 13C NMR
spectroscopic data for
compound 2
No.
1
δH (J in Hz)
δC
No.
δH (J in Hz)
δC
3.45 (1H, d, 12.0)
3.64 (1H, d, 12.0)
64.8
8’’
9”
OCH3
1’’’
2’’’
3’’’
4’’’
5’’’
6’’’
7.71 (1H, d, 15.8)
148.1
168.6
56.6
2
3
4
5
105.6
78.5
74.3
81.4
65.4
90.7
74.6
3.83 (3H, s)
5.58 (1H, d, 8.5)
4.50 (1H, t, 8.5)
4.12 (1H, m)
128.0
112.04c
149.55e
150.8
116.65d
124.4
115.5
147.3
169.1
56.7
7.21 (1H, d, 1.8)
6
4.50 (2H, m)
1’
2’
3’
4’
5’
6’
5.70 (1H, d, 3.8)
4.69 (1H, dd, 10.1, 3.8)
3.92 (1H, m)
3.38 (1H, t, 9.3)
4.19 (1H, m)
4.16 (1H, m)
4.56 (1H, m)
6.82 (1H, d, 8.2)
7.00 (1H, dd, 8.2, 1.8)
6.40 (1H, d, 16.0)
7.67 (1H, d, 16.0)
72.3
7’’’
8’’’
9’’’
72.12a
72.15a
65.4
OCH3
1’’’’
2’’’’
3’’’’
4’’’’
5’’’’
6’’’’
7’’’’
8’’’’
9’’’’
OCH3
3.90 (3H, s)
127.80b
112.11c
149.57e
150.9
116.66d
124.3
115.2
147.7
168.8
56.7
OAc-6’
173.0
21.0
7.01 (1H, d, 1.8)
2.08 (3H, s)
1”
2’’
3”
127.78b
111.95c
149.5
151.0
116.65d
124.6
114.9
7.27 (1H, d, 1.8)
6.82 (1H, d, 8.2)
7.10 (1H, m)
6.36 (1H, d, 15.9)
7.64 (1H, d, 15.9)
4”
5’’
6’’
7’’
6.76 (1H, d, 8.2)
7.13 (1H, dd, 8.2, 1.8)
6.48 (1H, d, 15.8)
3.90 (3H, s)
1H (600 MHz) and 13C NMR (150 MHz) in methanol-d4, m: multiplet or overlapped signals, a–e: inter-
changeable
The HMBC correlations of H-2ʹ (δH 4.69) with C-9ʹʹʹʹ
(δC 168.8), H-3 (δH 5.58) with C-9ʹʹ (δC 168.6), H-6 (δH
4.50) with C-9ʹʹʹ (δC 169.1), and H-6ʹ (δH 4.56 and 4.16)
with the acetyl carbonyl (δC 173.0) suggested three feruloyl
groups were on C-2ʹ, C-3, and C-6, and the acetyl group on
C-6ʹ, respectively. The glycosidic linkage was confrmed by
the HMBC correlation between H-1ʹ (δH 5.70) and C-2 (δC
105.6) to form sucrose.
any significant activity at 100 µg/mL (Fig. S13 in sup-
plementary data); however, further fractionation by ODS
column chromatography unveiled active components. All
compounds (1–20) were isolated from the active fractions.
However, from some active fractions, such as HE-7-3 and 4,
no compound was isolated in this study, because of the high
complexity and instability of the constituents.
In conclusion, chemical investigation of the n-hexane-
and ethyl acetate-soluble fractions of the aerial parts of S.
frma provided 20 compounds (1–20), including two new
ferulic acid esters, frmosides A and B (1 and 2), and 18
known compounds (3–20). The isolated compounds were
tested for their DPPH radical scavenging activities, and 1,
2, and 11 exhibited strong radical scavenging activity com-
parable to Trolox, implying the use of them as antioxidant
agents.
The absolute stereochemistry was determined by
mild alkaline hydrolysis of 2, as above mentioned, to
liberate D-sucrose and methyl ferulate by analyzing
with HPLC equipped with the optical rotation detec-
1
tor, HR-ESI–MS, and H NMR. Based on these data, the
structure of 2 was determined as (3,6-O-diferuloyl)-β-
D-fructofuranosyl-(2→1)-(2-O-feruloyl-6-O-acetyl)-α-D-
glucopyranoside, named frmoside B.
All isolated compounds were examined on DPPH radical
scavenging activity (Fig. 4). As a result, frmosides A and
B (1 and 2, respectively) and known compound 11 showed
signifcant DPPH free radical scavenging activities (IC50
33.4, 39.1, and 37.9 µM, respectively) comparable to the
positive control, Trolox (IC50 36.4 µM). It is noteworthy
that the hexane- and EtOAc-soluble mixture and the frst
silica gel column chromatographic fractions did not show
Experimental
General experimental procedures
1H and 13C NMR spectra were measured on a Bruker Avance
III spectrometer at 600 MHz and 150 MHz, respectively,
1 3

Journal of Natural Medicines
Fig. 4 DPPH radical scaveng-
ing activity. a % inhibition
of isolated compounds 1–15,
and 18–20 (100 µg/mL). b
Concentration-dependent inhibi-
tion of 1, 2, 11. Tro: Trolox as a
positive control
100
80
60
40
20
0
(a)
(%)
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 18 19 20 Tro
(b)
100
80
60
40
20
0
(%)
1
2
11
6.125 (µg/ml)
Tro
100.00
50
25
12.5
with the residual solvent signal as the reference. IR and UV
spectra were recorded on a HORIBA FT-720 and a JASCO
V-520 UV/Vis spectrophotometer, respectively. Optical
rotations were measured using a JASCO P-1030 spectropo-
larimeter. Positive- and negative-ion HR-ESI–MS was
performed on an LTQ Orbitrap XL spectrometer (Thermo
Fisher Scientifc).
deposited in the Herbarium of the Department of Pharma-
cognosy, Graduate School of Biomedical Sciences, Hiro-
shima (Accession No. 05-SR-09-Ehime).
Extraction and isolation
The air-dried plants (10.6 kg) were extracted three times
with methanol (45 L). The methanol solution was concen-
trated to 6 L and then partitioned with an equal volume of
n-hexane. The remaining layer was evaporated, resuspended
in water (6 L), and partitioned with ethyl acetate (6L) and
with 1-butanol (6 L), successively.
Silica gel column chromatography (CC) and octadecyl-
silyl silica gel (ODS) CC were performed on silica gel 60
(E. Merck, Darmstadt, Germany) and Cosmosil 75C18-OPN
(Nacalai Tesque, Kyoto, Japan; Φ =35 mm, L=350 mm),
respectively. HPLC was performed on Inertsil ODS-3 col-
umn (GL Science, Tokyo, Japan; Φ = 10 mm, L = 25 cm,
fow rate 2.00 mL/min), and the eluate was monitored with
a refractive index monitor. TLC was performed on precoated
silica gel 60 F254 plates (E. Merck; 0.25 mm in thickness)
by spraying with a 10% solution of H2SO4 in ethanol and
heated on a hotplate around 150 ºC. Sugars were analyzed by
HPLC on an amino column using a chiral detector (JASCO
OR-2090plus) [Shodex Asahipak NH2P-50, CH3CN-H2O
(3:1), 1.0 mL/min].
The n-hexane and ethyl acetate fractions were combined
(164.8 g), and fractionated by silica gel (1.45 kg) CC with
increasing amounts of MeOH in CHCl3 [(CHCl3, 6L),
CHCl3-MeOH (30:1, 6 L), (20:1, 6 L), (10:1, 6 L), (7:1,
6 L), (5:1, 6 L), (3:2, 6 L), (2:1, 6 L), and (MeOH, 6 L)],
yielding nine fractions (SF-HE 1–SF-HE 9). The fraction
SF-HE 1 (46.5 g) was subjected to ODS CC with MeOH-
H2O (30%, 0.5 L), (40%, 0.5 L), (50%, 0.5 L), (60%, 0.5 L),
(70%, 0.5 L), (80%, 0.5 L), (90%, 0.5 L), (100%, 0.5 L), and
(acetone, 0.5 L), led nine fractions (SF-HE 1-1–SF-HE 1-9).
The fraction SF-HE 1-1 (577 mg) was purifed by HPLC
(25% acetone) to give 3 (vanillin, 6.0 mg), 4 ((3R,6R,7E)-
3-hydroxy-4,7-megastigmadien-9-one, 2.3 mg), and 5
(scopoletin, 2.7 mg). The fraction SF-HE 2 (22.7 g) was
Plant material
Aerial parts of S. frma were collected in September 2005
in Imabari, Ehime, Japan, and a voucher specimen was
1 3

Journal of Natural Medicines
subjected to ODS CC with MeOH-H2O (20% → 100% of
MeOH, 10% step gradient, 0.5 L each), and (acetone, 0.5 L),
as mentioned above, led nine fractions (SF-HE 2-1–SF-HE
2-9). The residue of fraction SF-HE 2-1 (1.67 g) was puri-
fed by HPLC (25% acetone) to obtain 13 (3-hydroxy-1-(4-
hydroxy-3-methoxyphenyl) propan-1-one, 11.4 mg), 14
((6S,9R)-vomifoliol, 17.5 mg), 15 (4-hydroxybenzaldehyde,
20.0 mg), and mixture of 16 and 17 ((+)-dehydrovomifo-
liol and 3-(4-hydroxy-3-methoxyphenyl) propanoic acid,
18.3 mg), respectively. The fraction SF-HE 2-2 (689.1 mg)
was purifed by HPLC (35% acetone) to give 18 (indole-
3-carboxaldehyde, 3.8 mg). The fraction SF-HE 3 (14.0 g)
was subjected to ODS CC with MeOH-H2O (30%→100%
of MeOH, 10% step gradient, 0.5 L each), and (acetone,
0.5 L), as mentioned above, led nine fractions (SF-HE
3-1–SF-HE 3-9). The fraction SF-HE 3-1 (1.15 g) was puri-
fed by HPLC (25% acetone) to give 19 (isovanillic acid,
26.2 mg), 20 (grasshopper ketone, 11.9 mg). The fraction
SF-HE 3-2 (854 mg) was purifed by HPLC (35% acetone)
to give 10 (maltol β-D-glucopyranoside 6’-O- benzoate,
300 mg). The fraction SF-HE 3-3 (855 mg) was purifed by
HPLC (40% acetone) to obtain new compound 2 (16.2 mg).
The fraction SF-HE 4 (13.4 g) was subjected to ODS CC
with MeOH-H2O (20%→100% of MeOH, 10% step gradi-
ent, 0.5 L each), and (acetone, 0.5 L), as mentioned above,
led ten fractions (SF-HE 4-1–SF-HE 4-10). The fraction
SF-HE 4-4 (1.29 g) was purifed by HPLC (60% acetone)
to give new compound 1 (70.0 mg) and 11 (helonioside A,
12 mg). The fraction SF-HE 4-5 (929 mg) was purifed by
HPLC (50% acetone) to give 6 ((9S,12S,13S)-E-9,12,13-
trihydroxy-10-octadecaenoic acid, 20.0 mg). The fraction
SF-HE 5 (10.3 g) was subjected to ODS CC with MeOH-
H2O (20% → 100% of MeOH, 10% step gradient, 0.5 L
each), and (acetone, 0.5 L), as mentioned above, led ten
fractions (SF-HE 5-1–SF-HE 5-10). The fraction SF-HE
5-2 (878 mg) was purifed by HPLC (30% acetone) to give
7 (20-hydroxy-ecdysone, 15.0 mg). The fraction SF-HE 5-3
(630 mg) was purifed by HPLC (35% acetone) to give 12
(22-O-acetyl 20-hydroxy-ecdysone, 7.6 mg). The fraction
SF-HE 6 (9.23 g) was subjected to ODS CC with MeOH-
H2O (20% → 100% of MeOH, 10% step gradient, 0.5 L
each), and (acetone, 0.5 L), as mentioned above, led ten
fractions (SF-HE 6-1–SF-HE 6-10). The fraction SF-HE
6-3 (715 mg) was purifed by HPLC (25% acetone) to give
8 (luteolin 3′-O-methyl-6-C-β-D-glucopyranoside, 8.8 mg)
and 9 (apigenin-6-C-β-D-glucopyranoside, 4.5 mg).
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), as
shown in Table 1; negative-ion HR-ESI–MS m/z 869.2501
[M–H]− (calcd for C42H45O20: 869.2501)
Firmoside B (2)
White amorphous powder; [α]2D6 +46.9 (c=1.03, MeOH); IR
(flm) νmax cm−1: 3344, 2921, 2851, 1717, 1699, 1684, 1520,
1507, 1270, 1161, 1030; UV λ max (MeOH) nm (log ε):
236 (4.06), 264 (3.62), 300 (sh, 4.12), 327 (4.25); 1H NMR
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), as
shown in Table 2; positive-ion HR-ESI–MS m/z 935.2581
[M+Na]+ (calcd for C44H48O21Na: 935.2580)
Mild alkaline hydrolysis of 1 and 2
Compounds 1 and 2 (2 mg each) were dissolved in MeOH
(1.8 mL), and 200 μL of 1 M NaOCH3 methanol solution
was added to start the reaction. After stirring for 30 min
at 25 °C, the reaction mixture was neutralized with ion
exchange resin (Organo IR120B, H+-form) and evaporated.
The residue was partitioned with EtOAc and H2O. The
organic and aqueous layers were analyzed to identify methyl
ferulate and sugar, respectively. The methyl ferulate was iso-
lated by preparative TLC and identifed by HR-ESI–MS and
1H NMR compared to those reported values. The sugar was
analyzed by HPLC by comparing their retention time and
optical rotation sign with authentic sucrose; tR: 10.8 min
(positive optical rotation).
DPPH free radical scavenging activity
The samples were dissolved in 100 µL of MeOH in 96-well
microtiter plates at various concentrations. The initial
absorbance was measured at 515 nm, as AS0. DPPH solution
(100 µL, 200 µM) was added to each well and incubated in
a dark place at room temperature. After the incubation for
30 min, the absorbance was measured again, as AS30. The %
inhibition of free radicals was calculated according to the
following equation:
[
(
) (
)]
% Inhibition = 1 − AS30− AS0 ∕ AD30− AD0 × 100
where AD is the absorbance of the control reaction mixture
containing DMSO and all reagents without test compounds
[33]. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-car-
boxylic acid) is an analog of vitamin E and widely used for
antioxidant reagent.
Firmoside A (1)
White amorphous powder; [α]2D6 +54.4 (c=0.93, MeOH); IR
(flm) νmax cm−1: 3344, 2923, 2851, 1716, 1697, 1686, 1519,
1508, 1270, 1161, 1028; UV λ max (MeOH) nm (log ε):
237 (4.18), 264 (3.69), 300 (sh, 4.17), 327 (4.34); 1H NMR
Acknowledgment The authors are grateful for access to the super-
conducting NMR instrument (Bruker Avance 600, JEOL ECA600)
and Thermo Fisher Scientifc LTQ Orbitrap XL spectrometer at the
Analysis Center of Life Science, Natural Science Center for Basic
Research and Development, Hiroshima University. This work was
1 3

Journal of Natural Medicines
17. Rayyan S, Fossen T, Nateland HS, Andersen OM (2005) Isolation
and identifcation of favonoids, including favone rotamers, from
the herbal drug Crataegi folium cum fore (hawthorn). Phytochem
Anal 16(5):334–341
supported by JSPS KAKENHI (18K06740, 17K15465, 17K08336),
the Cosmetology Research Foundation, and the Japan Food Chemical
Research Foundation.
18. Nakano T, Sugimoto S, Matsunami K, Otsuka H (2011) Diantho-
saponins A-F, triterpene saponins, favonoid glycoside, aromatic
amide glucoside and γ-pyrone glucoside from Dianthus japonicas.
Chem Pharm Bull 59(9):1141–1148
Compliance with ethical standards
Conflict of interest The authors declare no conficts of interest associ-
ated with this manuscript.
19. Yan LL, Gao WY, Zhang YJ, Wang Y (2008) A new phenylpro-
panoid glycosides from Paris polyphylla var. yunnanensis. Fito-
terapia 79(4):306–307
20. Odinokov VN, Galyautdinov IV, Nedopekin DV, Khalilov LM,
Shashkov AS, Kachala VV, Dinan L, Lafont R (2002) Phyto-
ecdysteroids from the juice of Serratula coronate L. (Asteraceae).
Insect Biochem Mol Biol 32(2):161–165
References
1. Mamadalieva NZ, Lafont R, Wink M (2014) Diversity of second-
ary metabolites in the genus Silene L. (Caryophyllaceae)—struc-
tures, distribution, and biological properties. Diversity 6:415–499.
https://doi.org/10.3390/d6030415
21. Lui S, Que S, Cheng W, Zhang Q, Liang H (2013) Chemical
constituents from whole plants of Carduus acanthoides. China J
Chin Materia Med 38(14):2334–2337
2. Satake Y, Ohwi J, Kitamura S, Watari S, Tominari T (eds) (1982)
Wild fowers of Japan. Herbaceous plants II, Heibon-sha, p 21
3. Makino T (2008) New Makino’s illustrated fora of Japan. Tokyo,
Hokuryukan, p 584
22. Chang YC, Chang FR, Wu YC (2000) The Constituents of Lindera
glauca. J Chin Chem Soc 47(2):373–380
23. Wang G, Zhu L, Zhao Y, Gao S, Sun D, Yuan J, Huang Y,
Zhang X, Yao X (2017) A natural product from Cannabis sativa
subsp. sativa inhibits homeodomain-interacting protein kinase 2
4. Perry LM, Metzger J (1980) Medical plants of east and southeast
Asia. Attributed properties and uses. MIT Press, Cambridge, p 74
5. Cui SN (1995) Chaoyaozhi. Yanbian People’s Press, Yanji, pp
77–79
+-induced apoptosis in human neuro-
(HIPK2), attenuating MPP
blastoma SH-SY5Y cells. Bioorg Chem 72:64–73
24. Kai H, Baba M, Okuyama T (2007) Two new megastigmanes from
the leaves of Cucumis sativus. Chem Pharm Bull 55(1):133–136
25. Kreye O, Toth T, Meier MAR (2011) Copolymers derived from
rapeseed derivatives via ADMET and thiol-ene addition. Eur
Polym J 47(9):1804–1816
6. Lee MY, Shin IS, Seo CS, Lee NH, Ha HK, Son JK, Shin HK
(2012) Effect of methanolic extract on testosterone-induced
benign prostatic hyperplasia in Wistar rats. Asian J Androl
14:320–324
7. Chang HZ, Da LY, Cheng SL, Jie L, Mei J, Ming SZ, Zhen HL,
Tie FJ, Gao L (2015) Cytotoxic anthraquinone dimers from Mel-
andrium frmum. Arch Pharmacal Res 38:1033–1037
8. Chang HZ, Jie L, Tian L, Yong C, Mei J, Da LY, Ming SZ,
Zhen HL, Jiong MC, Gao L (2015) Chemical constituents from
the aerial parts of Melandrium frmmum. Arch Pharmacal Res
38:1746–1751
26. Rong GQ, Geng CA, Ma YB, Huang XY, Wang HL, Zhao Y,
Zhang XM, Chen JJ (2014) Chemical constituents from ethyl
acetate extract of fower of Albizia julibrissin. Zhongguo Zhong
Yao ZaZhi 39(10):1845–1851
27. Ashour MA, Elkhayat ES, Ebel R, Edrada R, Proksch P (2007)
Indole alkaloid from the Red Sea sponge Hyrtios erectus.
ARKIVOC 15:225–231
9. Seo CS, Shin HK (2016) Simultaneous determination of the fve
marker compounds in Melandrium frmum using high-perfor-
mance liquid chromatography with photodiode-array detection.
Nat Prod Commun 11(11):1934578X1601101111. https://doi.
org/10.1177/1934578x1601101111
28. Zou Y, Zhang L, Xu JK, Cheng Q, Ye XS, Li P, Zhang WK, Li
YJ (2015) A new benzaldehyde from aerial part of Rehmannia
glutinosa. Zhongguo Zhong Yao ZaZhi 40(7):1316–1319
29. Kuang HX, Yang BY, Xia YG, Feng WS (2008) Chemical con-
stituents from the fower of Datura metel L. Arch Pharm Res
31(9):1094–1097
10. Pham-Huy LA, He H, Pham-Huy C (2008) Free radicals, antioxi-
dants in disease and health. Int J Biomed Sci 4:89–96
11. Bao K, Fan A, Dai Y, Zhang L, Zhang W, Cheng M, Yao X (2009)
Selective demethylation and debenzylation of aryl ethers by mag-
nesium iodide under solvent-free conditions and its application
to the total synthesis of natural products. Org Biomol Chem
7(24):5084–5090
30. Takahira M, Kusano A, Shibano M, Kusano G, Miyase T (1998)
Piscidic acid and fukiic acid esters from Cimicifuga simplex. Phy-
tochemistry 49(7):2115–2119
31. Chang CL, Zhang LJ, Chen RY, Kuo LMY, Huang JP, Huang HC,
Lee KH, Wu YC, Kuo YH (2010) Antioxidant and anti-infam-
matory phenylpropanoid derivatives from Calamus quiquesetin-
ervius. J Nat Prod 73(9):1482–1488
12. Peng WW, Song WW, Huang MB, Tan NH (2014) Monoterpenes
and sesquiterpenes from Clausena excavate. Zhongguo Zhong
Yao ZaZhi 39(9):1620–1624
32. Putt KS, Nesterenko V, Dothager RS, Hergenrother PJ (2006)
The compound 13-D selectively induces apoptosis in white
blood cancers versus other cancer cell types. ChemBioChem
7(12):1916–1922
13. Wang L, Yu MM, Chi YQ, Ouyang WB, Zang Z, Zhao Y (2014)
Chemical constituents of Euphorbia dracunculoides. Zhongguo
Zhong Yao ZaZhi 39(20):3969–3973
33. Matsunami K, Takamori I, Shinzato T, Aramoto M, Kondo K,
Otsuka H, Takeda Y (2006) Radical-scavenging activities of new
megastigmane glucosides from Macaranga tanarius (L.) MULL.-
ARG. Chem Pharm Bull 54(10):1403–1407
14. Shirahata T, Sunazuka T, Yoshida K, Yamamoto D, Harigaya
Y, Kuwajima I, Nagai T, Kiyohara H, Yamada H, Omura S
(2006) Total synthesis, elucidation of absolute stereochemis-
try, and adjuvant activity of trihydroxy fatty acids. Tetrahedron
62(40):9483–9496
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
15. Roussel PG, Sik V, Turner NJ (1997) Dinan LN (1997) Synthe-
sis and biological activity of side-chain analogs of ecdysone and
20-hydroxyecdysone. J Chem Soc Perkin 15:2237–2246
16. Senatore F, D’Agostino M, Dini I (2000) Flavonoid glycosides
of Barbarea vulgaris L. (Brassicaceae). J Agric Food Chem
48(7):2659–2662
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