A new phenolic glycoside from Spiraea prunifolia var. simpliciflora twigs

Article abstract of DOI:10.1007/s12272-015-0610-y

The phytochemical investigation of the methanol extract from the twigs of Spiraea prunifolia var. simpliciflora (Rosaceae) using column chromatography led to the isolation of a new phenol glycoside, 1-O-(E)-caffeoyl-2-O-p-(E)-coumaroyl-β-d-glucopyranose (1), together with 16 known phenolic compounds (2-17). The structure of this new compound was elucidated by analysis of spectroscopic data including 1D, 2D nuclear magnetic resonance and HR-FAB-MS data. The isolated compounds were tested for cytotoxicity against four human tumor cell lines in vitro using the sulforhodamine B bioassay.

Full text of DOI:10.1007/s12272-015-0610-y

Arch. Pharm. Res.
DOI 10.1007/s12272-015-0610-y
RESEARCH ARTICLE
A new phenolic glycoside from Spiraea prunifolia var. simpliciflora
twigs
Sung Wan Jang1 Won Se Suh1 Chung Sub Kim1 Ki Hyun Kim1
Kang Ro Lee1
•
•
•
•
Received: 14 December 2014 / Accepted: 25 April 2015
Ó The Pharmaceutical Society of Korea 2015
Abstract The phytochemical investigation of the
methanol extract from the twigs of Spiraea prunifolia var.
simpliciflora (Rosaceae) using column chromatography led
to the isolation of a new phenol glycoside, 1-O-(E)-caf-
feoyl-2-O-p-(E)-coumaroyl-b-D-glucopyranose (1), togeth-
er with 16 known phenolic compounds (2–17). The
structure of this new compound was elucidated by analysis
of spectroscopic data including 1D, 2D nuclear magnetic
resonance and HR-FAB-MS data. The isolated compounds
were tested for cytotoxicity against four human tumor cell
lines in vitro using the sulforhodamine B bioassay.
terpenoids (Oh et al. 2013; Park et al. 2009), alkaloids (Lui
et al. 2007, 2009), flavonoids (Uzma et al. 2012) and phe-
nolic compounds (Yoshida et al. 2010), and the methanol
extract of this species have been reported to exhibit anti-
inflammatory (Jun et al. 2007; Oh et al. 2003) and antioxidant
activities (Park et al. 2013). As part of our ongoing research
of Korean medicinal plants, we have studied the methanol
extract of twigs of S. prunifolia var. simpliciflora, and iso-
lated a new phenolic glycoside and 16 known phenolic
constituents (Fig. 1). The structure of the new compound,
1-O-(E)-caffeoyl-2-O-p-(E)-coumaroyl-b-D-glucopyranose
(1) was elucidated by spectroscopic methods, including 1D,
2D nuclear magnetic resonance (NMR) and HR-FAB-MS.
All isolated compounds (1–17) were evaluated for their cy-
totoxic activities against four human cancer cell lines.
Keywords Spiraea prunifolia var. simpliciflora Á
Phenolic glycoside Á Cytotoxicity
Introduction
Materials and methods
Spiraea prunifolia var. simpliciflora (Nakai) Nakai (Rosa-
ceae) is distributed throughout China and Korea. The twigs
and roots of this source have been used to treat colds, sore
throat, phlegm, neuralgia and malaria in Chinese folk med-
icine remedies (Lee 2003). Previous phytochemical inves-
tigations on Spiraea species reported the isolation of
General experimental procedure
Thin layer chromatography (TLC) was performed using
Merck precoated silica gel F254 plates and RP-18 F254s
plates. Spots were detected on TLC under ultraviolet (UV)
light or by heating after spraying with 10 % H2SO4 in
C2H5OH (v/v). Silica gel 60 (70–230 and 230–400 mesh,
Merck, Germany) and RP-C18 silica gel (230–400 mesh,
Merck, Germany) was used for open column chromatog-
raphy. Sep-PakÒ (Vac 12 cm3, Waters, MA, USA) was
also used for column chromatography. Optical rotations
were measured on a Jasco P-1020 polarimeter (Jasco,
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-015-0610-y) contains supplementary
material, which is available to authorized users.
Easton, MD, USA). NMR spectra, including H–1H cor-
1
& Kang Ro Lee
krlee@skku.edu
relation spectroscopy (COSY), heteronuclear multiple
quantum coherence (HMQC) and hetero nuclear multiple
bond correlation (HMBC) were recorded on an AvanceIII
1
Natural Product Laboratory, School of Pharmacy,
Sungkyunkwan University, Suwon 440-746, Korea
123

S. W. Jang et al.
NMR spectrometer operating at 700 MHz (1H) and
175 MHz (13C) (Bruker, Germany), with chemical shifts
given in ppm (d). HR-FAB-MS data were obtained on a
JEOL JMS700 mass spectrometer (JEOL, Japan). Pre-
parative HPLC was performed using a Gilson 306 pump
(Gilson, Germany) with a Shodex refractive index detector
(Shodex, New York, NY, USA) and Apollo Silica 5l
column (250 9 22 mm, Alltech, Nicholasville, KY, USA)
or EconosilÒ RP-18 10l column (250 9 22 mm, Alltech,
Nicholasville, KY, USA). Silica gel 60 (230–400 mesh,
Merck, Germany) was used for column chromatography.
(3:1:1) as the eluent to give eight subfractions (C511–518).
Subfraction C512 (106 mg) was purified with an RP-C18
semi prep. HPLC (35 % MeOH) to yield compound 7
(15 mg, Rt = 19.4 min). The EtOAc soluble fraction (10 g)
was separated over a silica gel column with CHCl3:-
MeOH:H2O (4:1:0.1) as the eluent to yield seven fractions
(E1–7). Fraction E3 (750 mg) was subjected to RP-C18 silica
gel column chromatography (40 % MeOH) to give nine
subfractions (E31–39). Subfraction E35 (228 mg) was
separated over a silica gel column chromatography with a
solvent system of n-hexane:EtOAc:MeOH (4:1:1) as the
eluent to give four subfractions (E351–354). Subfraction
E353 (50 mg) was purified with an RP-C18 semi prep. HPLC
(20 % ACN) to yield compound 11 (12 mg, Rt = 24.2 min).
Subfraction E4 (1.4 g) was subjected to RP-C18 silica gel
column chromatography (50 % MeOH) to give seven sub-
fractions (E41–47). Subfraction E44 (118 mg) was purified
with an RP-C18 semi prep. HPLC (40 % MeOH) to yield
Plant materials
Spiraea prunifolia var. simpliciflora (Rosaceae) (7.0 kg)
was collected from Goesan-gun in Chungchoengbuk-do,
Korea in March 2013. The plants were authenticated by
one of the authors (K.R. Lee). A voucher specimen
(SKKU-NPL-1301) of the plant was deposited at the
School of Pharmacy, Sungkyunkwan University, Suwon,
Korea.
compounds
3
(7 mg, Rt = 19.2 min),
4
(11 mg,
Rt = 21.5 min), 13 (4 mg, Rt = 22.4 min), 14 (8 mg,
Rt = 25.3 min), and 17 (6 mg, Rt = 28.3 min). Subfraction
E45 (1.0 mg) was separated over a silica gel column chro-
matography with a solvent system of CHCl3:MeOH:H2O
(4:1:0.1) as the eluent to give four subfractions (E451–454).
Subfraction E451 was purified with an RP-C18 semi-prep.
HPLC (30 % ACN) to yield compounds 1 (4 mg,
Rt = 15.1 min) and 2 (6 mg, Rt = 16.2 min).
Extraction and isolation
The twigs of S. prunifolia var. simpliciflora (7.0 kg) were
extracted at 85 °C with 80 % MeOH (3 3 5 L every 3 days)
and evaporated under reduced pressure to give a residue
(380 g), which was dissolved in water (800 mL) and solvent-
partitioned to give n-hexane (28 g), CHCl3 (29 g), EtOAc
(12 g) and n-BuOH fractions (47 g). The CHCl3 soluble
fraction (19 g) was separated over a silica gel column with
CHCl3:MeOH (30:1–1:1 gradient) as the eluent to yield 10
fractions (C1–10). Fraction C3 (616 mg) was subjected to
RP-C18 silica gel column chromatography (50 % MeOH) as
eluent to give seven subfractions (C31–37). Subfraction C32
(60 mg) was purified with a silica semi prep. HPLC (n-
hexane:EtOAc:MeOH = 5:1:1) to yield compounds 5
(15 mg, Rt = 15.1 min), 6 (7 mg, Rt = 17.2 min), 9
(14 mg, Rt = 23.8 min), and 10 (8 mg, Rt = 27.1 min).
Fraction C4 (1.3 g) was subjected to RP-C18 silica gel col-
umn chromatography (50–100 % MeOH gradient) as eluent
to give six subfractions (C41–46). Subfraction C41 (316 mg)
was separated over silica gel column chromatography with a
solvent system of CHCl3:MeOH (20:1) as the eluent to give
four subfractions (C411–414). Subfraction C414 (123 mg)
was purified with an RP-C18 semi prep. HPLC (25 % ACN)
to yield compounds 8 (12 mg, Rt = 14.8 min), 12 (4 mg,
Rt = 15.0 min), 15 (6 mg, Rt = 19.7 min), and 16 (10 mg,
Rt = 23.2 min). Fraction C5 (3.5 g) was subjected to RP-
C18 silica gel column chromatography (50–100 % MeOH
gradient) to give six subfractions (C51–56). Subfraction C51
(985 mg) was separated over silica gel column chromatog-
raphy with a solvent system of n-hexane:EtOAc:MeOH
1-O-(E)-caffeoyl-2-O-p-(E)-coumaroyl-b-D-
glucopyranose (1)
25
D
Yellow gum, ½aŠ -14.0° (c 0.1, MeOH); UV (MeOH)
kmax (log e) 203, 227, 314 nm; IR (KBr) mmax 3385, 1703,
1631, 1604, 1515, 1262, 1170, 1075, 832 cm-1; HR-FAB-
MS m/z 487.1236 [M-H]2 (calc. C24H23O11, 487.1235);
1H- (CD3OD, 700 MHz) and 13C-NMR (CD3OD,
175 MHz), see Table 1.
2-O-(E)-caffeoyl-1-O-p-(E)-coumaroyl-b-D-
glucopyranose (2)
25
Yellow gum, ½aŠ -0.8° (c 0.25, MeOH); FAB-MS m/
D
z 487.12 [M-H]-; 1H- (CD3OD, 700 MHz) and 13C-NMR
(CD3OD, 175 MHz), see Table 1.
1,2-Di-O-(E)-caffeoyl-b-D-glucopyranose (3)
25
Yellow gum, ½aŠ -15.3° (c 0.7, MeOH); FAB-MS m/
D
1
z 503.10 [M-H]-; H-NMR (CD3OD, 700 MHz): d 3.49
(1H, m, H-500), 3.53 (1H, t, J = 9.0 Hz, H-400), 3.72 (1H, t,
J = 9.0 Hz, H-300), 3.74 (1H, dd, J = 5.0, 12.0 Hz, H-600b),
3.94 (1H, dd, J = 2.0, 12.0 Hz, H-600a), 5.06 (1H, dd,
123

A new phenolic glycoside from Spiraea prunifolia…
Table 1 1H- and 13C-NMR
Positions
Compound 1
Positions
Compound 2
spectral data of compounds 1
and 2
dH
dC
dH
dC
1
127.2
131.4
116.9
150.2
116.9
131.4
147.5
113.9
167.3
127.6
115.4
147.0
161.5
116.6
123.5
147.0
114.9
168.5
93.8
1
126.8
131.4
116.8
161.5
116.8
131.4
147.7
114.6
167.2
127.5
115.2
146.6
149.5
116.4
123.1
148.5
115.2
168.3
93.8
2
7.45 d (8.5)
6.80 d (2.0)
2
7.42 d (9.0)
6.78 d (9.0)
3
3
4
4
5
6.80 d (2.0)
7.45 d (8.5)
7.67 d (16.0)
6.36 d (16.0)
5
6.78 d (9.0)
7.43 d (9.0)
7.65 d (16.0)
6.25 d (16.0)
6
6
7
7
8
8
9
9
10
20
30
40
50
60
70
80
90
100
200
300
400
500
600-a
600-b
1
10
20
30
40
50
60
70
80
90
100
200
300
400
500
600-a
600-b
7.04 d (2.0)
7.01 d (2.0)
6.79 d (2.0)
6.74 d (8.0)
6.95 dd (2.0, 8.0)
7.61 d (16.0)
6.21 d (16.0)
6.91 dd (2.0, 8.0)
7.58 d (16.0)
6.27 d (7.5)
5.81 d (8.5)
5.08 dd (8.5, 9.5)
3.76 ma
3.54 ma
3.54 ma
5.82 d (8.0)
74.0
3.73 dd (8.0, 9.0)
5.09 dd (8.0, 9.0)
3.56 t (9.0)
74.1
75.4
75.9
71.0
71.1
78.9
3.54 m
78.9
3.76 ma
62.0
3.76 dd (5.0, 12.0)
3.90 dd (2.0, 12.0)
62.1
3.93 m
H- and 13C-NMR run at 700 and 175 MHz (CD3OD), respectively. Proton coupling constants (J) in Hz
are given in parentheses
a
Overlapped signals
J = 8.0, 9.0 Hz, H-200), 5.79 (1H, d, J = 8.0 Hz, H-100), 6.18
J = 9.0 Hz, H-300), 3.66 (2H, t, J = 6.5 Hz, H-40), 3.67
(1H, m, H-500), 4.33 (1H, dd, J = 5.5, 12.0 Hz, H-600a),
4.48 (1H, dd, J = 2.0, 12.0 Hz, H-600b), 5.56 (1H, d,
J = 7.5 Hz, H-100), 5.69 (1H, d, J = 1.5 Hz, H-50), 6.24
(1H, d, J = 1.5 Hz, H-50), 6.30 (1H, d, J = 16.0 Hz, H-8),
6.78 (1H, d, J = 8.5 Hz, H-5), 6.97 (1H, dd, J = 2.0,
8.5 Hz, H-6), 7.06 (1H, d, J = 2.0 Hz, H-2), 7.66 (1H, d,
J = 16.0 Hz, H-7); 13C-NMR (CD3OD, 175 MHz): d 34.9
(C-30), 60.2 (C-40), 63.1 (C-600), 69.9 (C-400), 72.5 (C-200),
74.7 (C-500), 76.4 (C-300), 94.3 (C-100), 112.8 (C-2), 115.1
(C-8), 121.9 (C-9), 126.1 (C-4), 126.7 (C-50), 113.8 (C-5),
137.1 (C-20), 145.4 (C-6), 147.0 (C-3), 148.5 (C-7), 166.2
(C-1), 166.7 (C-10).
(1H, d, J = 8.0 Hz, H-8), 6.27 (1H, d, J = 8.0 Hz, H-80),
6.75 (1H, d, J = 8.0 Hz, H-5), 6.76 (1H, d, J = 8.0 Hz,
H-50), 6.91 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.93 (1H, dd,
J = 2.0, 8.0 Hz, H-60), 7.04 (1H, d, J = 2.0 Hz, H-1), 7.57
(1H, d, J = 16.0 Hz, H-90), 7.58 (1H, d, J = 16.0 Hz, H-9);
13C-NMR (CD3OD, 175 MHz): d 62.2 (C-600), 71.1 (C-400),
74.2 (C-200), 75.9 (C-300), 78.9 (C-500), 93.9 (C-100), 113.7 (C-
8), 114.6 (C-80), 115.2 (C-2), 115.3 (C-20), 116.5 (C-5,50),
123.1 (C-6), 123.4 (C-60), 127.2 (C-10) 127.4 (C-1), 147.7 (C-
7), 148.9 (C-70), 149.6 (C-40), 149.7 (C-3,30), 149.9 (C-40),
167.2 (C-9), 168.3 (C-90).
1-Caffeoyl-6-tuliposide A (4)
(2)-Nortrachelogenin (5)
25
Yellow gum, ½aŠ ?4.5° (c 0.1, MeOH); FAB-MS m/
D
25
Yellow gum, ½aŠ -29.8° (c 0.3, MeOH); EI-MS m/z 374
1
z 439.1 [M-H]-; H-NMR (CD3OD, 700 MHz): d 2.53
D
1
(2H, t, J = 6.5 Hz, H-30), 3.41 (1H, t, J = 9.0 Hz, H-400),
3.45 (1H, dd, J = 9.0, 7.5 Hz, H-200), 3.48 (1H, t,
[M]?; H-NMR (CDCl3, 700 MHz): d 2.43 (1H, m, H-80),
2.46 (1H, dd, J = 4.5, 13.5 Hz, H-70b), 2.78 (1H, dd,
123

S. W. Jang et al.
J = 4.5, 13.5 Hz, H-70a), 2.84 (1H, d, J = 13.5 Hz, H-7b),
3.11 (1H, d, J = 13.0 Hz, H-7a), 3.77 (3H, s, 30-OCH3),
3.80 (3H, s, 3-OCH3), 3.97 (2H, d, J = 7.5 Hz, H-90), 6.56
(1H, dd, J = 2.0, 8.0 Hz, H-6), 6.58 (1H, dd, J = 2.0,
8.0 Hz, H-60), 6.66 (1H, d, J = 2.0 Hz, H-20), 6.68 (1H, d,
J = 2.0 Hz, H-2), 6.69 (1H, d, J = 2.0 Hz, H-50), 6.77
(1H, dd, J = 2.0, 8.0 Hz, H-5); 13C-NMR (CDCl3,
175 MHz): d 31.7 (C-70), 42.2 (C-7), 43.9 (C-80), 55.9 (3-
OCH3), 56.0 (30-OCH3), 70.1 (C-90), 76.4 (C-8), 111.4 (C-
20), 112.6 (C-2), 114.3 (C-5), 114.5 (C-50), 121.5 (C-60),
123.2 (C-6), 126.0 (C-1), 130.2 (C-10), 144.4 (C-40), 145.1
(C-4), 146.6 (C-3), 146.6 (C-30), 178.3 (C-9).
3-OCH3), 3.89 (1H, d, J = 11.5 Hz, H-9b), 3.90 (3H, s, 30-
OCH3), 4.17 (1H, dd, J = 6.5, 8.5 Hz, H-90b), 4.85 (1H, s,
H-7), 6.70 (1H, d, J = 2.0 Hz, H-20), 6.79 (1H, dd,
J = 2.0, 8.0 Hz, H-6), 6.85 (1H, d, J = 8.0 Hz, H-50), 6.90
(1H, d, J = 2.0 Hz, H-2), 6.92 (1H, d, J = 8.0 Hz, H-5),
6.69 (1H, dd, J = 2.0, 8.0 Hz, H-60); 13C-NMR (CD3OD,
175 MHz): d 40.7 (C-70), 48.0 (C-8), 56.6 (30,300-OCH3),
60.8 (C-9), 78.0 (C-90), 82.6 (C-80), 85.7 (C-7), 112.0 (C-
20), 115.7 (C-2), 115.8 (C-5), 115.9 (C-50), 120.9 (C-60),
124.0 (C-6), 130.5 (C-10), 135.5 (C-10), 146.3 (C-40), 147.3
(C-4), 148.7 (C-3), 149.2 (C-30).
(1)-1-Hydroxypinoresinol (9)
Lariciresinol (6)
25
Yellow gum, ½aŠ ?11.5° (c 0.1, MeOH); FAB-MS m/
D
Yellow gum, ½aŠ25 ?17.1° (c 0.4, MeOH); FAB-MS m/z 359
z 373 [M-H]-; 1H-NMR (CD3OD, 700 MHz): d 3.03 (1H,
m, H-5), 3.75 (1H, dd, J = 6.0, 9.0 Hz, H-4b), 3.83 (1H, d,
J = 9.5 Hz, H-8b), 3.84 (6H, s, 30,300-OCH3), 4.02 (1H, d,
J = 9.5 Hz, H-8a), 4.45 (1H, dd, J = 9.0, 9.0 Hz, H-4a),
4.66 (1H, s, H-2), 4.83 (1H, d, J = 5.0 Hz, H-6), 6.77 (1H,
d, J = 8.0 Hz, H-500), 6.78 (1H, d, J = 8.0 Hz, H-50), 6.86
(1H, dd, J = 2.0, 8.0 Hz, H-600), 7.03 (1H, d, J = 2.0 Hz,
H-20); 13C-NMR (CD3OD, 175 MHz): d 33.4 (C-7), 42.4
(C-8), 52.6 (C-80), 55.9 (3,30-OCH3), 60.9 (C-90), 72.9 (C-
9), 82.8 (C-70), 108.2 (C-20), 111.6 (C-2), 114.1 (C-50),
114.4 (C-5), 118.7 (C-60), 121.2 (C-6), 131.6 (C-10), 134.8
(C-1), 144.0 (C-4), 145.0 (C-40), 146.5 (C-3), 146.6 (C-30).
D
1
[M-H]-; H-NMR (CD3OD, 700 MHz): d 2.44 (1H, dd,
J = 2.0, 8.0 Hz, H-6), 2.73 (1H, dd, J = 2.0, 8.0 Hz, H-60),
2.92 (2H, d, J = 7.5 Hz, H-90), 3.76 (3H, s, 30-OCH3), 3.80
(3H, s, 3-OCH3), 4.06 (1H, d, J = 2.0 Hz, H-20), 4.81 (1H, d,
J = 2.0 Hz, H-2), 5.49 (1H, d, J = 2.0 Hz, H-50), 6.85 (1H,
m, H-5); 13C-NMR (CD3OD, 175 MHz): d 33.3 (C-70), 42.5
(C-7), 52.6 (C-80), 55.9 (3,30-OCH3), 61.1 (C-9), 72.9 (C-90),
82.8 (C-8), 109.1 (C-20), 111.2 (C-2), 114.4 (C-5), 118.0 (C-
50), 121.2 (C-60), 132.3 (C-6), 135.6 (C-1), 144.0 (C-10),
146.6 (C-40), 148.5 (C-4), 149.2 (C-3).
(2)-Olivil (7)
(1)-Fraxiresinol (10)
Yellow gum, ½aŠ25 -60.8° (c 0.3, MeOH); FAB-MS m/z 375
D
25
1
[M-H]-; H-NMR (CD3OD, 700 MHz): d 2.29 (1H, ddd,
Yellow gum, ½aŠ ?22.1° (c 0.1, MeOH); EI-MS m/z 403
D
J = 5.5, 6.0, 7.0 Hz, H-8), 2.98 (1H, d, J = 14.0 Hz, H-70a),
2.91 (1H, d, J = 14.0 Hz, H-70b), 3.59 (1H, d, J = 9.0 Hz,
H-90a), 3.72 (1H, dd, J = 6.0, 11.5 Hz, H-9a), 3.80 (1H, d,
J = 9.0 Hz, H-90a), 3.83 (1H, dd, J = 6.0, 11.5 Hz, H-9b),
3.84 (6H, s, 3,30-OCH3), 4.71 (1H, d, J = 7.0 Hz, H-7), 6.73
(1H, d, J = 8.0 Hz, H-50), 6.75 (1H, d, J = 8.0 Hz, H-5),
6.77 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.93 (1H, d, J = 2.0,
8.0 Hz, H-60), 7,17 (1H, d, J = 2.0 Hz, H-2); 13C-NMR
(CD3OD, 175 MHz): d 40.6 (C-70), 48.0 (C-8), 56.6 (3,30-
OCH3), 60.8 (C-9), 78.0 (C-90), 82.6 (C-80), 85.7 (C-7), 112.0
(C-20), 115.7 (C-5), 116.0 (C-50), 116.0 (C-2), 121.0 (C-60),
124.1 (C-6), 130.6 (C-1), 135.5 (C-10), 146.4 (C-40), 147.4
(C-4), 148.7 (C-3), 149.1 (C-30).
[M]?; 1H-NMR (CD3OD, 700 MHz): d 3.06 (1H, ddd,
J = 5.5, 6.5, 8.5 Hz, H-5), 3.79 (1H, dd, J = 6.5, 9.0 Hz,
H-4b), 3.88 (6H, s, 30,50-OCH3), 3.89 (3H, s, 300-OCH3),
3.90 (1H, d, J = 9.0 Hz, H-8b), 4.09 (1H, d, J = 9.0 Hz,
H-8a), 4.49 (1H, dd, J = 8.5, 9.0 Hz, H-4a), 4.70 (1H, s,
H-2), 4.87 (1H, d, J = 6.0 Hz, H-6), 6.75 (2H, s, H-20,60),
6.79 (1H, d, J = 8.0 Hz, H-500), 6.89 (1H, dd, J = 2.0,
8.0 Hz, H-600), 7.07 (1H, d, J = 2.0 Hz, H-200); 13C-NMR
(CD3OD, 175 MHz): d 33.3 (C-7), 42.4 (C-8), 52.6 (C-80),
55.9 (3,30-OCH3), 60.9 (C-90), 72.9 (C-9), 82.8 (C-70),
108.2 (C-20), 111.6 (C-2), 114.4 (C-5), 114.1 (C-50), 118.7
(C-60), 121.21 (C-6), 134.8 (C-1), 131.6 (C-10), 145.0 (C-
40), 144.0 (C-4), 146.5 (C-3), 146.6 (C-30).
(2)-Berchemol (8)
(1)-1-Hydroxypinoresinol 1-O-b-D-glucopyranoside
(11)
25
Yellow gum, ½aŠ -8.3° (c 0.1, MeOH); FAB-MS m/z 375
D
25
D
1
[M-H]-; H-NMR (CD3OD, 700 MHz): d 2.44 (1H, d,
Yellow gum, ½aŠ -9.6° (c 0.05, MeOH); FAB-MS m/
J = 12.5, 13.0 Hz, H-70a), 2.61 (1H, m, H-80), 3.07 (1H, d,
J = 4.0, 13.0 Hz, H-70b), 3.64 (1H, d, J = 11.5 Hz, H-9a),
3.72 (1H, dd, J = 5.0, 8.5 Hz, H-90a), 3.88 (3H, s,
z 535 [M-H]-; 1H-NMR (CD3OD, 700 MHz): d 2.87 (1H,
m, H-500), 3.03 (1H, dt, J = 2.5, 5.0 Hz, H-200), 3.14 (2H,
m, H-300,400), 3.40 (1H, m, H-8), 3.51 (1H, dd, J = 6.0,
123

A new phenolic glycoside from Spiraea prunifolia…
12.0 Hz, H-600a), 3.68 (dd, J = 2.0, 12.0 Hz, H-600b), 3.80
(1H, dd, J = 6.0, 9.0 Hz, H-9b), 3.86 (3H, s, 30-OCH3),
3.87 (3H, s, 3-OCH3), 3.96 (1H, d, J = 10.0 Hz, H-90b),
4.37 (1H, d, J = 7.5 Hz, H-100), 4.49 (1H, dd, J = 7.0,
9.0 Hz, H-9a), 4.52 (1H, d, J = 10.0 Hz, H-90a), 4.71 (1H,
s, H-7), 4.82 (1H, s, H-70), 6.75 (1H, d, J = 8.0 Hz, H-50),
6.83 (1H, d, J = 8.0 Hz, H-5), 6.89 (1H, dd, J = 2.0,
8.0 Hz, H-60), 6.91 (1H, dd, J = 2.0, 8.0 Hz, H-6), 7.06
(1H, d, J = 2.0 Hz, H-2), 7.12 (1H, d, J = 2.0 Hz, H-20);
13C-NMR (CD3OD, 175 MHz): d 55.3 (3-OCH3), 55.4 (30-
OCH3), 59.0 (C-8), 61.3 (C-600), 70.9 (C-400), 72.1 (C-9),
73.6 (C-90), 76.8 (C-200), 77.1 (C-500), 78.2 (C-300), 85.6 (C-
7), 88.8 (C-70), 98.0 (C-80), 98.8 (C-100), 109.5 (C-2), 112.9
(C-20), 114.0 (C-50), 115.1 (C-5), 118.7 (C-6), 121.2 (C-60),
127.4 (C-10), 131.9 (C-1), 146.1 (C-40), 146.3 (C-4), 147.2
(C-30), 148.1 (C-3).
(1)-9-O-b-D-glucopyranosyl isolariciresinol (14)
25
Yellow gum, ½aŠ ?3.9° (c 0.1, MeOH); FAB-MS m/z 521
D
1
[M-H]-; H-NMR (CD3OD, 700 MHz): d 1.80 (2H, t,
J = 3.0, 10.0 Hz, H-80), 2.18 (2H, m, H-8), 2.84 (2H, m,
H-1), 3.21 (1H, dd, J = 8.0, 9.0 Hz, H-200), 3.39 (1H, dd,
J = 3.0, 12.0 Hz, H-90a), 3.66 (1H, dd, J = 6.0, 12.0 Hz,
H-9a), 3.68 (1H, t, J = 5.0, 10.0 Hz, H-9a), 3.73 (1H, t,
J = 3.0, 12.0 Hz, H-90b), 3.77 (3H, s, 3-OCH3), 3.80 (3H,
s, 30-OCH3), 3.86 (1H, brd, J = 8.0 Hz, H-70), 3.87 (1H,
dd, J = 2.0, 12.0 Hz, H-600b), 4.00 (1H, dd, J = 4.0,
10.0 Hz, H-9b), 4.29 (1H, d, J = 8.0 Hz, H-100), 6.19 (1H,
s, H-5), 6.61 (1H, dd, J = 2.0, 8.0 Hz, H-60), 6.65 (1H, s,
H-2), 6.68 (1H, d, J = 2.0 Hz, H-20), 6.74 (1H, d,
J = 8.0 Hz, H-50); 13C-NMR (CD3OD, 175 MHz): d 33.9
(C-7), 37.5 (C-8), 48.0 (C-70), 48.3 (C-80), 56.5 (3,30-
OCH3), 61.8 (C-90), 62.9 (C-100), 71.8 (C-400), 73.9 (C-9),
75.3 (C-200), 78.1 (C-500), 78.2 (C-300), 104.7 (C-100), 112.6
(C-2), 114.1 (C-20), 116.1 (C-50), 117.5 (C-5), 123.3 (C-60),
129.2 (C-1), 134.3 (C-6), 138.7 (C-10), 146.0 (C-40), 145.4
(C-4), 147.3 (C-3), 149.1 (C-30).
(2)-Secoisolariciresinol (12)
25
Yellow gum, ½aŠ -21.7° (c 0.1, MeOH); FAB-MS m/
D
z 361 [M-H]-; 1H-NMR (CD3OD, 700 MHz): d 1.92 (2H,
m, H-8,80), 2.57 (2H, m, H-7b,70b), 2.68 (2H, dd, J = 8.0,
14.0 Hz, H-7a,70a), 3.60 (4H, m, H-9,90), 3.75 (6H, s, 3, 30-
OCH3), 6.56 (2H, dd, J = 2.0, 8.0 Hz, H-6,60), 6.61 (2H, d,
J = 2.0 Hz, H-2,20), 6.67 (2H, d, J = 8.0 Hz, H-5,50); 13C-
NMR (CD3OD, 175 MHz): d 36.2 (C-7,70), 44.2 (C-8,80),
56.1 (3,30-OCH3), 62.3 (C-9,90), 114.2 (C-2,20), 116.1 (C-
5,50), 122.8 (C-6,60), 134.0 (C-1,10), 145.6 (C-4,40), 148.9
(C-3,30).
7R,8S-dihydrodehydrodiconiferyl alcohol (15)
25
Yellow gum, ½aŠ -19.0° (c 0.1, MeOH); FAB-MS m/
D
1
z 383 [M?Na]?; H-NMR (CD3OD, 700 MHz): d 2.78
(1H, dd, J = 4.5, 13.5 Hz, H-70a), 2.84 (1H, d,
J = 13.5 Hz, H-7b), 3.77 (3H, s, 30-OCH3), 3.11 (1H, d,
J = 13.0 Hz, H-7a), 3.80 (3H, s, 3-OCH3), 3.97 (2H, d,
J = 7.5 Hz, H-90), 6.56 (1H, dd, J = 2.0, 8.0 Hz, H-6),
6.58 (1H, dd, J = 2.0, 8.0 Hz, H-60), 6.66 (1H, d,
J = 2.0 Hz, H-20), 6.68 (1H, d, J = 2.0 Hz, H-2), 6.69
(1H, d, J = 2.0 Hz, H-50), 6.77 (1H, dd, J = 2.0, 8.0 Hz,
H-5); 13C-NMR (CD3OD, 175 MHz) d: 32.7 (C-7), 35.9
(C-8), 55.1 (C-8), 55.6 (3-OCH3), 56.8 (30-OCH3), 61.5 (C-
9), 64.4 (C-90), 89.1 (C-70), 110.6 (C-2), 114.2 (C-2), 116.5
(C-50), 117.6 (C-6), 119.8 (C-60), 130.0 (C-1), 134.9 (C-10),
137.0 (C-5), 145.3 (C-3), 147.6 (C-30,4), 149.2 (C-40).
(1)-9-O-b-D-glucopyranosyl lyoniresinol (13)
25
Yellow gum, ½aŠ ?36.2° (c 0.1, MeOH); FAB-MS m/
D
z 581 [M-H]-; 1H-NMR (CD3OD, 700 MHz): d 1.80 (2H,
t, J = 3.0, 10.0 Hz, H-80), 2.18 (2H, m, H-8), 2.84 (2H, m,
H-1), 3.21 (1H, dd, J = 8.0, 9.0 Hz, H-200), 3.39 (1H, dd,
J = 3.0, 12.0 Hz, H-9a), 3.66 (1H, dd, J = 6.0, 12.0 Hz,
H-90a), 3.68 (1H, t, J = 5.0, 10.0 Hz, H-9a), 3.73 (1H, t,
J = 3.0, 12.0 Hz, H-9b), 3.77 (3H, s, 3-OCH3), 3.80 (3H,
s, 30-OCH3), 3.86 (1H, brd, J = 8.0 Hz, H-70), 3.87 (1H,
dd, J = 2.0, 12.0 Hz, H-600b), 4.00 (1H, dd, J = 4.0,
10.0 Hz, H-90b), 4.29 (1H, d, J = 8.0 Hz, H-100), 6.19 (1H,
s, H-5), 6.61 (1H, dd, J = 2.0, 8.0 Hz, H-60), 6.65 (1H, s,
H-2), 6.68 (1H, d, J = 2.0 Hz, H-20), 6.74 (1H, d,
J = 8.0 Hz, H-50); 13C-NMR (CD3OD, 175 MHz): d 33.9
(C-7), 39.6 (C-8), 47.9 (C-70), 46.0 (C-80), 56.5 (3,30-
OCH3), 62.8 (C-600), 65.3 (C-9), 69.6 (C-90), 71.7 (C-400),
75.2 (C-200), 77.9 (C-500), 78.2 (C-300), 105.2 (C-100), 112.6
(C-2), 114.1 (C-20), 116.1 (C-50), 117.5 (C-5), 123.3 (C-60),
129.2 (C-1), 134.3 (C-6), 138.7 (C-10), 146.1 (C-40), 145.3
(C-4), 147.1 (C-3), 149.0 (C-30).
7R,8S-5-methoxydihydrodehydroconiferyl alcohol
(16)
25
Yellow gum, ½aŠ -11.4° (c 0.1, MeOH); FAB-MS m/
D
z 389 [M-H]-; 1H-NMR (CD3OD, 700 MHz): d 2.43 (1H,
m, H-80), 2.46 (1H, dd, J = 4.5, 13.5 Hz, H-70b), 2.78 (1H,
dd, J = 4.5, 13.5 Hz, H-70a), 2.84 (1H, d, J = 13.5 Hz,
H-7b), 3.11 (1H, d, J = 13.0 Hz, H-7a), 3.77 (3H, s, 30-
OCH3), 3.80 (3H, s, 3-OCH3), 3.97 (2H, d, J = 7.5 Hz,
H-90), 6.56 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.58 (1H, dd,
J = 2.0, 8.0 Hz, H-60), 6.66 (1H, d, J = 2.0 Hz, H-20),
6.68 (1H, d, J = 2.0 Hz, H-2), 6.69 (1H, d, J = 2.0 Hz,
H-50), 6.77 (1H, dd, J = 2.0, 8.0 Hz, H-5); 13C-NMR
123

S. W. Jang et al.
(CD3OD, 175 MHz): d 32.0 (C-70), 34.6 (C-80), 53.8 (C-8),
56.0 (3-OCH3), 56.3 (30-OCH3), 62.2 (C-90), 63.7 (C-9),
88.1 (C-7), 103.1 (C-2,6), 112.4 (C-20), 115.9 (C-60), 127.7
(C-50), 132.3 (C-1), 134.6 (C-4), 135.5 (C-10), 144.2 (C-30),
146.5 (C-40), 147.1 (C-3, 5).
Results and discussion
Compound 1 was obtained as a yellow gum. From the HR-
FAB-MS m/z 487.1236 [M-H]2 (calc. C24H23O1À1;
1
487.1235) and H- and 13C-NMR spectral data, the mole-
cular formula was deduced to be C24H24O11. The 1H-NMR
spectrum showed the presence of seven aromatic protons
signals at dH 7.45 (2H, d, J = 8.0 Hz), 7.04 (1H, d,
J = 1.5 Hz), 6.96 (1H, dd, J = 2.0, 8.0 Hz), 6.80 (2H, d,
J = 8.0 Hz) and 6.79 (1H, d, J = 8.0 Hz), four olefinic
proton signals dH 7.68 (1H, d, J = 16.0 Hz), 7.62 (1H, d,
J = 16.0 Hz), 6.37 (1H, d, J = 16.0 Hz) and 6.20 (1H, d,
J = 16.0 Hz). The 13C-NMR spectrum exhibited 18 carbon
signals of each aglycone composed of the signals at dC
168.5 (C-9), 150.2 (C-4), 149.0 (C-7), 147.0 (C-3), 127.6
(C-1), 123.5 (C-6), 116.6 (C-5), 115.4 (C-2), and 113.9 (C-
8) for the caffeoyl moiety, as well as other signals at dC
167.3 (C-90), 161.5 (C-40), 147.5 (C-70), 131.4 (C-20,60),
127.2 (C-10), 117.0 (C-30,50) and 114.9 (C-80) for coumar-
oyl moiety. These spectral data suggested that compound 1
was a phenolic glycoside (Jiang et al. 2001). Overall NMR
spectral data were similar to those of 2-O-(E)-caffeoyl-1-
O-p-(E)-coumaroyl-b-D-glucopyranose (2) (Jiang et al.
2001), except for slight shift of H-8 (dH 6.36) and H-80 (dH
6.21), and aromatic protons at dH 6.80 (H-3,5) and 6.79 (H-
50). The D-glucose position was established by a HMBC
experiment, in which correlations were observed between
the H-100 (dH 5.82) of D-glucopyranose and C-90 (dC
168.46) of caffeoyl moiety, and between H-200 (dH 5.10) of
D-glucopyranose and C-9 (dC 167.33) of coumaroyl moiety.
The coupling constant (J = 8.5 Hz) of the anomeric proton
of D-glucose suggested that it was the b form. An analysis
Dihydrodehydrodiconiferyl alcohol 4-O-b-D-
glucopyranoside (17)
25
Yellow gum, ½aŠ -8.2° (c 0.3, MeOH); FAB-MS m/z 545
D
1
[M?Na]?; H-NMR (CD3OD, 700 MHz): d 1.81 (2H, m,
H-80), 2.62 (2H, t, J = 6.5 Hz, H-70), 3.56 (2H, t,
J = 6.5 Hz, H-90), 3.83 (3H, s, 3-OCH3), 3.86 (3H, s,
3-OCH3), 5.56 (1H, d, J = 6.0 Hz, H-7), 6.72 (2H, d,
J = 2.0 Hz, H-20,60), 6.93 (1H, dd, J = 2.0, 8.5 Hz, H-6),
7.03 (1H, d, J = 2.0 Hz, H-2), 7.14 (1H, d, J = 8.5 Hz,
13
H-5); C-NMR (CD3OD, 175 MHz): d 32.9 (C-70), 35.8
(C-80), 55.6 (C-8), 56.7 (3-OCH3), 56.8 (30-OCH3), 62.2
(C-90), 62.5 (C-600), 65.1 (C-9), 71.3 (C-400), 74.4 (C-100),
77.8 (C-500), 78.2 (C-300), 88.5 (C-7), 102.8 (C-100), 112.2
(C-2), 114.3 (C-20), 118.0 (C-60), 118.2 (C-5), 119.4 (C-6),
129.6 (C-10), 137.1 (C-50), 138.4 (C-1), 145.2 (C-30), 147.5
(C-4), 147.6 (C-40), 150.9 (C-3).
Acid hydrolysis of compound 1
Compound 1 (1.5 mg) was heated in an ampoule with
1 mL of 2 N HCl (aq.) at 90 °C for 3 h. After cooling, the
reaction mixture was extracted with CHCl3. The CHCl3
solvent was evaporated in vacuo, and identified as cou-
maroyl acid and caffeoyl acid by co-TLC [CHCl3:MeOH
(10:1, Rf 0.45 of coumaroyl acid, Rf 0.6 of caffeoyl acid)]
with an authentic sample (Sigma, St. Louis, MO, USA).
The H2O layer yielded D-glucose which was identified with
an authentic sample (Sigma, St. Louis, MO, USA) using
silica gel co-TLC with a solvent system of CHCl3:-
MeOH:H2O at a 2:1:0.1 ratio with an Rf value of 0.3 (Cho
et al. 2014; Lee et al. 2014).
1
of the H–1H COSY, HMQC and HMBC correlations led
to the establishment of the structure for 1 (Fig. 2).
Acid hydrolysis of 1 yielded the caffeic acid, coumaric
acid and D-glucopyranose. The caffeic acid and coumaric
acid were identified by co-TLC confirmation with authentic
samples [CHCl3:MeOH (10:1, Rf 0.45 of coumaric acid, Rf
0.6 of caffeoyl acid)]. D-Glucopyranose was identified
through co-TLC [CHCl3:MeOH:H2O (2:1:0.1, Rf 0.3 D-
glucopyranoside)]
25
and
specific
optical
rotation
Test for cytotoxicity in vitro
{½aŠ ? 62.2 (c = 0.05, H2O)}. Thus, structure of 1 was
D
Sulforhodamine B (SRB) bioassays were used to screen the
compounds for cytotoxicity (Skehan et al. 1990). Cyto-
toxicity assays for each compound were performed in vitro
against four cultured human tumor cell lines (National
Cancer Institute, Bethesda, MD, USA) at the Korean Re-
search Institute of Chemical Technology. The cell lines
used were A549 (non-small cell lung adenocarcinoma),
SK-OV-3 (ovarian cancer cells), A498 (kidney carcinoma
cells), and HCT15 (colon cancer cells). Etoposide was used
as a positive control.
identified as 1-O-(E)-caffeoyl-2-O-p-(E)-coumaroyl-b-D-
glucopyranose.
The structures of the known compounds were identified to
be 2-O-(E)-caffeoyl-1-O-p-(E)-coumaroyl-b-D-glucopyra-
nose (2) (Jiang et al. 2001), 1,2-di-O-(E)-caffeoyl-b-D-glu-
copyranose (3) (Jiang et al. 2001), 1-caffeoyl-6-tuliposide A
(4) (Park et al. 2013), (-)-nortrachelogenin (5) (Woo et al.
2011), lariciresinol (6) (Fonseca et al. 1978), (-)-olivil (7)
(Hans et al. 1983), (-)-berchemol (8) (Sakurai et al. 1989),
(?)-1-hydroxypinoresinol (9), (?)-fraxiresinol (10) (Ando
123

A new phenolic glycoside from Spiraea prunifolia…
OH
OH
4'
3'
OH
HO
HO
8'
O
2''O
7'
HO
O
O
O
9'
HO
O
HO
1''
HO
O
9
8
O
1
HO
O
4
HO
HO
O
7
2
1
OH
HO
HO
O
O
O
HO
O
HO
OH
OH
O
O
O
HO
O
HO
HO
OH
O
HO
3
4
R1
O
R1
R2
OH
H
OH
H3CO
HO
H3CO
HO
O
O
O
O
OCH3
H
R2
OH
HO
H3CO
OCH3
OCH3
OH
OH
R1
H
R2
OH
R1
H
OH
H
R2
H
9
6
7
8
5
10 OCH3 OH
H
11
H
OGlc
OH
OCH3
H3CO
HO
R2
H3CO
HO
R1
R2
OH
OH
O
R1
HO
R3
OCH3
R3
OCH3
OCH3
OH
OH
R1
R2
H
R3
R1
13 OGlc OH
14 OH
R2
R3
H
15 OH
16 OH
17 OGlc
OH
12
OCH3 OH
OGlc OCH3
H
OH
Fig. 1 Structures of compounds 1–17 from the twigs of S. prunifolia var. simpliciflora
et al. 2007), (?)-1-hydroxypinoresinol 1-O-b-D-glucopyra-
noside (11) (Yang et al. 2007), (-)-secoisolariciresinol (12)
(Park et al. 2009), (?)-9-O-b-D-glucopyranosyl lyoniresinol
(13) (Tanaka et al. 2004), (?)-9-O-b-D-glucopyranosyl iso-
lariciresinol (14) (Otsuka et al. 2000), 7R,8S-dihydrodehy-
drodiconiferyl alcohol (15) (Fang et al. 1992), 7R,8S-5-
methoxydihydrodehydroconiferyl alcohol (16) (Wang et al.
2010), and dihydrodehydrodiconiferyl alcohol 4-O-b-D-
glucopyranoside(17) (Matsuda et al. 1996) by comparing the
spectroscopic data with those in the literature.
The isolated compounds 1–17 were tested for cyto-
toxicity against four human tumor cells in vitro using the
123

S. W. Jang et al.
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HO
HO
HO
OH
OH
O
O
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A549
SK-OV-3
A498
HCT15
4
8.92
[10.0
[10.0
[10.0
1.65
8.14
[10.0
[10.0
9.56
4.05
9.27
9.14
6.27
0.72
[10.0
[10.0
[10.0
6.44
5
10
17
Etoposideb
1.73
1.80
a
IC50 value of compounds against cancer cell lines, defined as the
concentration (lM) that cause 50 % inhibition of cell growth in vitro
b
Etoposide as positive control
SRB assay. Of them the compounds 4, 5, 10, and 17 ex-
hibited significant cytotoxicities against A549, SK-OV-3,
A498, and HCT15 cell lines with the IC50 values (com-
pound 4: 8.92, 8.14, 4.05, and [10.0 lM, compound 5:
[10.0, [10.0, 9.27, and [10.0 lM, compound 10: [10.0,
[10.0, 9.14, and [10.0 lM, and compound 17: [10.0,
9.56, 6.27, and 6.44 lM, respectively) and the other
compounds showed little cytotoxicity (IC50 [10 lM). IC50
values for the cytotoxicity of the control compound, eto-
poside, against A549, SK-OV-3, A498, and HCT15 were
1.65, 1.73, 0.72, and 1.80 lM, respectively (see Table 2).
Interestingly, although the structures of 1–3 and 4 are quite
similar except of the presence of a 4-hydroxy-2-methylene-
butyrate instead of a phenylpropanoid, they differed sub-
stantially with respect to their cytotoxic effects. The ob-
tained data suggest that the presence of the 4-hydroxy-2-
methylene-butyrate at C-600 in glucose unit is important for
the cytotoxic activity on A549, SK-OV-3, and A498 cell
lines though more phenolic glycosides need to be tested to
confirm this hypothesis.
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Acknowledgments This research was supported by the Basic Sci-
ence Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science and
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