Phenolic constituents from the twigs of Betula schmidtii collected in Goesan, Korea

Article abstract of DOI:10.1016/j.phytochem.2019.112085

Six undescribed phenolic derivatives along with thirty two known compounds were isolated from the twigs of Betula schmidtii. The chemical structures were characterized through extensive spectroscopic analysis and chemical methods. All known compounds were first isolated in this plant. The anti-inflammatory effect of the isolates was tested by measuring nitric oxide production in lipopolysaccharide-activated BV-2 cells. Isotachioside, 4-allyl-2-hydrophenyl 1-O-β-D-apiosyl-(1 → 6)-β-D-glucopyranoside, genistein 5-O-β-D-glucoside, and prunetinoside showed a slight potency to lower the NO production against LPS-activated microglia with IC₅₀ values of 23.9, 25.3, 28.8, and 34.0 μM, respectively.

Full text of DOI:10.1016/j.phytochem.2019.112085

Phytochemistry 167 (2019) 112085
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phenolic constituents from the twigs of Betula schmidtii collected in Goesan,
Korea
a
a
b,c
b,c
a,⁎
Kyoung Jin Park , Joon Min Cha , Lalita Subedi , Sun Yeou Kim , Kang Ro Lee
a
Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon, 16419, Republic of Korea
Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, 21936, Republic of Korea
College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon, 21936, Republic of Korea
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Betula schmidtii Regel
Betulaceae
Phenolic derivatives
Anti-inflammatory activity
Six undescribed phenolic derivatives along with thirty two known compounds were isolated from the twigs of
Betula schmidtii. The chemical structures were characterized through extensive spectroscopic analysis and che-
mical methods. All known compounds were first isolated in this plant. The anti-inflammatory effect of the
isolates was tested by measuring nitric oxide production in lipopolysaccharide-activated BV-2 cells.
Isotachioside, 4-allyl-2-hydrophenyl 1-O-β-D-apiosyl-(1 → 6)-β-D-glucopyranoside, genistein 5-O-β-D-glucoside,
and prunetinoside showed a slight potency to lower the NO production against LPS-activated microglia with IC50
values of 23.9, 25.3, 28.8, and 34.0 μM, respectively.
1. Introduction
et al., 1998). Hence, a phytochemical research of B. schmidtii twigs was
conducted. Following our previous report of triterpenoids from the
Inflammation in the central nervous system (CNS) is termed as
3
CHCl layer of a B. schmidtii MeOH extract (Park et al., 2018), thirty
neuroinflammation. Neuroinflammation cause degeneration and death
of neurons characterizing by neurodegenerative conditions like
Alzheimer's disease (AD), Parkinson's disease (PD), ischemia, and
multiple sclerosis (MS) (Gelders et al., 2018). CNS immune cells spe-
cially microglia play a key role for the neuroinflammation through its
chronic activation and subsequent overproduction of inflammatory
mediators including nitric oxide (NO) (Lull and Block, 2010). Excessive
production of NO by activated microglia as a characteristic feature of
neuroinflammation is known to be a major reason behind pathogenesis
of neurodegenerative disorders like AD, PD, MS, and ischemia etc.
eight phenolic derivatives including four undescribed phenolic glyco-
sides (1–4), an undescribed lignan glycoside (20), and an undescribed
isoflavonoid glycoside (28) were further isolated and identified from
the EtOAc and n-BuOH layers (Fig. 1). Herein, the isolation and struc-
tural elucidation of compounds 1–38 and their inhibitory activity of NO
generation are presented.
2. Results and discussion
Compound 1, a colorless gum, was established as C19
20 9
H O on the
basis of its HRESIMS data (m/z 415.1004, [M + Na]+). The H NMR
1
(
Yuste et al., 2015).
Betula genus, the largest group of Betulaceae, consists of 137 species
data displayed the presence of one monosubstituted aromatic ring [δ
H
distributed widely in areas of mild climate in the northern hemisphere.
They have been commonly used as traditional medicines to treat ar-
thritis, rheumatism, and renal disease in different regions of the world.
Previous studies of this genus have reported chemical constituents in-
cluding triterpenoids, diarylheptanoids, phenylbutanoids, flavonoids,
lignans, and phenolic compounds associated with anticancer, anti-in-
flammatory, arthritis, rheumatism, antioxidant, antimicrobial, and an-
tiviral activities. However, Betula schmidtii Regel has rarely been in-
vestigated for potential bioactive chemical constituents due to its
localized distribution in Northeast Asia (Rastogi et al., 2015; Fuchino
8.08 (2H, dd, J = 8.3, 1.2 Hz, H-2″ and H-6″), 7.61 (1H, t, J = 7.5 Hz,
H-4″), and 7.49 (2H, brt, J = 7.5 Hz, H-3″ and H-5″)], one 1,3,5-tri-
substituted aromatic ring [δ
H
6.13 (2H, d, J = 2.1 Hz, H-2 and H-6) and
4.92
6.00 (1H, t, J = 2.1 Hz, H-4)], and one glucopyranosyl unit [δ
H
(1H, d, J = 7.6 Hz, H-1′), 4.76 (1H, dd, J = 11.8, 2,2 Hz, H-6′a), 4.34
(1H, dd, J = 11.8, 7.2 Hz, H-6′b), 3,79 (1H, ddd, J = 9.5, 7.2, 2.2 Hz,
H-5′), 3.52 (1H, m, H-3′), 3.48 (1H, m, H-2′), and 3.46 (1H, m, H-4′)].
The 13C NMR data showed 15 peaks for 19 carbons, including one
carbonyl carbon [δ
C
168.2 (C-7″)], two aromatic rings [δ
C
160.9 (C-1),
160.3 (C-3 and C-5), 134.3 (C-4″), 131.3 (C-1″), 130.9 (C-2″ and C-6″)
⁎
Corresponding author. Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, 16419, Republic of
Korea.
E-mail address: krlee@skku.edu (K.R. Lee).
https://doi.org/10.1016/j.phytochem.2019.112085
Received 11 June 2019; Received in revised form 6 August 2019; Accepted 6 August 2019
0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

K.J. Park, et al.
Phytochemistry 167 (2019) 112085
Fig. 1. Chemical structures of compounds 1–38.
1
29.7 (C-3″ and C-5″), 98.1 (C-4), and 96.8 (C-2 and C-6)], and one
anomeric carbon [δ 102.0 (C-1′)]. Analysis of these 1D NMR data of 1
Table 1) indicated similarities with those of 3,4-dihydroxyphenyl-(6′-
C
J = 9.7 Hz, H-4″b), and 3.57 (2H, brs, H-5″); δ 111.1 (C-1″), 80.7 (C-
C
3″), 78.1 (C-2″), 75.1 (C-4″), and 65.7 (C-5″)] and glucose signals [δ
H
(
4.83 (1H, d, J = 7.4 Hz, H-1′), 3.99 (1H, dd, J = 11.1, 1.7 Hz, H-6′a),
3.61 (1H, dd, J = 11.1, 6.6 Hz, H-6′b), 3.54 (1H, m, H-5′), 3.48 (1H, m,
H-2′), 3.43 (1H, m, H-3′), and 3.35 (1H, m, H-4′); δ 103.0 (C-1′), 77.9
C
O-benzoyl)-O-β-D-glucopyranoside (Verotta et al., 1999). The major
difference was in the presence of a 1,3,5-trisubstituted benzene ring in
1
, instead of a 1,3,4-trisubstituted benzene ring in 3,4-dihydrox-
yphenyl-(6′-O-benzoyl)-O-β-D-glucopyranoside. The planar structure of
was determined from 2D NMR (COSY, HSQC, and HMBC) spectro-
(C-3′), 77.2 (C-5′), 75.0 (C-2′), 71.7 (C-4′), and 68.8 (C-6′)]. The large
coupling constant of H-1′ (J = 7.4 Hz) and the small coupling constant
of H-1′′ (J = 2.4 Hz) indicated a β-glucopyranose and a β-apiofuranose,
respectively (Kim et al., 2015; Kanchanapoom et al., 2002). On the
basis of the HMBC data, the correlation of H-1′′/C-6′ was confirmed,
indicating that β-apiofuranose was connected with C-6′ of β-glucopyr-
anose. The disaccharide moiety of 2 was established to be located at C-4
by the HMBC correlation of H-1′/C-4. Additionally, the location of a
methoxy group at C-3 was corroborated from the HMBC cross-peak
1
scopic data (Fig. 2). The HMBC correlations of H-1'/C-1 and H-6'/C-7″
indicated the location of the glucopyranosyl moiety and benzoic acid,
respectively. The coupling constant of the anomeric proton (J = 7.6 Hz)
confirmed as β-glucopyranose (Kim et al., 2015) and acid hydrolysis of
1
gave a D-glucopyranose, which was identified by co-TLC confirmation
and GC/MS analysis (Hara et al., 1987). Thus, the structure of 1 was
elucidated as 3,5-dihydroxyphenyl-(6′-O-benzoyl)-1-O-β-D-glucopyr-
anoside.
3
between OCH -3 and C-3 (Fig. 2). D-Glucopyranose and D-apiofuranose
acquired via acid hydrolysis of 2 were established by co-TLC con-
firmation and GC/MS analysis (Hara et al., 1987). The relative con-
figuration at C-7 and C-8 of 2 was confirmed by their coupling constant.
The large coupling constant of the H-7 (J = 6.8 Hz) indicated that C-7
and C-8 were to be threo-form (Balboul et al., 1996). The absolute
configuration of 2 was determined through comparison of the optical
Compound 2, colorless gum, gave the molecular formula C21
based on the pseudomolecular ion peak at m/z 515.1735 (calcd for
13Na, 515.1741). The 1D NMR data (Table 1) were closely si-
32 13
H O ,
21 32
C H O
milar to those of 1-(4-hydroxy-3-methoxyphenyl)propan-1,2-diol iso-
lated from Zingiber officinale (Ma et al., 2004), except for the additional
presence of apiose signals [δ
d, J = 9.7 Hz, H-4″a), 3.88 (1H, d, J = 2.4 Hz, H-2″), 3.74 (1H, d,
25
4.96 (1H, d, J = 2.4 Hz, H-1″), 3.95 (1H,
rotation of 2a {[α]D –47.4 (c 0.05, MeOH)} as 7R, 8R (Lee and Ley,
2003). Hence, the structure of 2 was established as (7R,8R)-1-(4-
H
2

K.J. Park, et al.
Phytochemistry 167 (2019) 112085
Table 1
1
H NMR [ppm, mult, (J in Hz)] and13C NMR data of compounds 1–4 in CD
3
OD.
Position
1
2
3
4
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
1
2
3
4
5
6
7
8
9
160.9
96.8
160.3
98.1
160.3
96.8
138.5
112.6
150.8
147.5
117.9
121.2
80.0
72.9
19.4
103.0
75.0
77.9
132.1
119.8
146.8
146.7
117.1
125.3
39.8
140.9
107.4
151.4
133.8
151.4
107.4
65.0
6.13 d (2.1)
6.00 t (2.1)
6.13 d (2.1)
7.03 d (1.8)
7.08 d (1.8)
6.42 brs
7.13 d (8.3)
6.91 dd (8.3, 1.8)
4.31 d (6.8)
3.80 m
0.99 d (6.4) (3H)
4.83 d (7.4)
3.48 m
3.43 m
3.35 m
3.54 m
6.76 d (8.2)
6.79 dd (8.2, 1.8)
2.73 t (7.2) (2H)
3.71 t (7.2) (2H)
6.42 brs
4.43 s (2H)
64.6
1′
2′
3′
4′
5′
6′
4.92 d (7.6)
3.48 m
3.52 m
3.46 m
3.79 ddd (9.5, 7.2, 2.2)
4.76 dd (11.8, 2.2)
102.0
74.9
78.1
72.5
75.6
65.7
4.71 d (7.5)
3.48 m
3.45 m
3.35 m
3.56 m
4.05 dd (10.5, 1.4)
3.60 dd (10.5, 6.7)
5.00 d (2.5)
3.96 d (2.5)
104.6
75.0
77.7
71.7
77.3
68.8
4.54 d (7.8)
3.48 m
3.41 m
3.39 m
3.51 m
3.98 dd (11.1, 2.2)
3.67 dd (11.1, 6.1)
4.99 d (2.3)
3.93 d (2.3)
107.9
75.0
77.7
71.4
77.5
68.6
71.7
77.2
68.8
3.99 dd (11.1, 1.7)
3.61 dd (11.1, 6.6)
4.96 d (2.4)
3.88 d (2.4)
4
.34 dd (11.8, 7.2)
1″
2″
3″
4″
131.3
130.9
129.7
134.3
111.1
78.1
80.7
75.1
111.1
78.2
80.6
75.1
111.2
78.3
80.7
75.2
8.08 dd (8.3, 1.2)
7.49 brt (7.5)
7.61 t (7.5)
3.95 d (9.7)
3.98 d (9.7)
3.77 d (9.7)
3.59 d (1.1) (2H)
3.97 d (9.7)
3.76 d (9.7)
3.60 brs (2H)
3
.74 d (9.7)
5
6
7
3
″
″
″
7.49 brt (7.5)
8.08 dd (8.3, 1.2)
129.7
130.9
168.2
3.57 brs (2H)
65.7
56.8
65.6
65.9
-OCH
3
3.88 s (3H)
Fig. 2. Key COSY (bold) and HMBC (arrow) correlations of 1–4, 20, and 28.
hydroxy-3-methoxyphenyl)propan-7,8-diol-4-O-β-D-apiofuranosyl-
26
formula of C18H O13. The molecular formula was deduced by the po-
+
1
13
(
1 → 6)-O-β-D-glucopyranoside. Compound 3 was obtained as a color-
sitive ion peak [M + Na] in the HRESIMS. The H and C NMR data
of 4 (Table 1) were very similar to those of 6′-O-β-D-apiofur-
anosylcalleryanin (Itoh et al., 2008). The major difference was found to
less gum, and its molecular formula was confirmed as C19
H
28
O
12 based
+
on the HRESIMS, displaying a pseudomolecular ion peak [M + Na] at
1
13
m/z 471.1473 (calcd for C19
spectra of 3 were quite similar to those of 2-hydroxy-5-(2-hydro-
xyethyl)phenyl O-α-D-apiofuranosyl-(1 → 6)-O-β-D-glucopyranoside
Lei et al., 2008), except for the coupling constant of H-1′′ (J = 2.5 Hz)
H
28
O
12Na, 471.1478). The H and C NMR
be the presence of a hydroxyl group at C-5 of aromatic ring [δ
H
6.42
(2H, brs, H-2 and H-6); δ 151.4 (C-3 and C-5), 140.9 (C-1), 133.8 (C-
C
4), and 107.4 (C-2 and C-6)], indicating that 4 has a 1,3,4,5-tetra-
substituted aromatic ring. The HMBC cross-peaks of H-1′/C-4 and H-1′′/
C-6′ confirmed the connection between glucopyranosyl moiety with the
aglycone and the apiofuranosyl moiety (Fig. 2). Acid hydrolysis of 4
yielded two sugar moieties, which were determined by the identical
manner to that of 2. Therefore, compound 4 was established as 3,5-
dihydroxy-1-(hydroxymethyl)phenyl-4-O-β-D-apiofuranosyl-(1 → 6)-O-
β-D-glucopyranoside.
(
and chemical shifts of C-1′′ (δ 111.1) (Table 1), indicating the β con-
C
figuration of apiofuranosyl unity in 3. The HMBC correlation from H-1″
to C-6′ revealed the linkage between two sugars and correlation from H-
1
′ to C-3 established the position of the disaccharide moiety (Fig. 2).
The sugar analysis of 3 was confirmed using a same approach to that of
. Thus, the structure of 3 was determined as 4-hydroxy-1-(2-hydro-
2
xyethyl)phenyl-3-O-β-D-apiofuranosyl-(1 → 6)-O-β-D-glucopyranoside.
The molecular formula of compound 20 was confirmed as
33 46
C H O
17 from positive ion HRESIMS and C NMR data. The 1H and
13
Compound 4 was purified as a colorless gum with the molecular
3

K.J. Park, et al.
Phytochemistry 167 (2019) 112085
2
5
Table 2
NMR and positive specific rotation {[α] +58.2 (c 0.03, MeOH)} (Li and
D
1
H NMR [ppm, mult, (J in Hz)] and13C NMR data of compounds 20 and 28 in
Seeram, 2010). The D configuration of glucopyranosyl and xylopyr-
anosyl units in compound 20 was determined using the method de-
scribed for 2, respectively. The relative configuration was confirmed via
NOESY correlations of H-2/H-4 and H-3/H-2′ and 6′, and the 8R, 7′S,
8′R configuration was deduced from the positive Cotton effects at 241
and 272 nm by comparison of the CD spectrum (Fig. 3 and Fig. S32,
Supplementary material) (Suh et al., 2015). Therefore, compound 20
was elucidated as (+)-(8R,7′S,8′R)-6-O-β-D-glucopyranosyl lyonir-
esinol-9′-O-β-D-xylopyranoside.
Compound 28 was isolated as a yellowish gum with a molecular
30
formula of C27H O15 established using HRESIMS data. Inspection of
the NMR data of 28 (Table 2) was quite similar to those of sphaer-
obioside (29) (Laupattarakasem et al., 2004), with the main difference
3
CD OD.
Position
20
28
δ
H
δ
C
δ
H
δ
C
1
2
3
4
5
6
7
134.8
125.5
151.6
137.2
151.6
107.9
32.6
8.18 brs
155.8
125.1
182.6
163.5
101.4
164.7
6.70 s
2.78 dd (15.4, 4.7)
6.50 brs
6.74 brs
2
.67 dd (15.4, 11.6)
8
9
1.71 m
3.64 m
38.9
64.5
96.0
159.3
being that the signals of a 1′,3′,4′-trisubstituted aromatic ring [δ
H
7.05
3
.56 dd (10.9, 6.5)
(
1H, brs, H-2′), 6.88 (1H, brd, J = 7.4 Hz, H-6′), and 6.82 (1H, brd,
1
1
2
3
4
5
6
7
8
9
0
′
′
′
′
′
′
′
108.2
123.9
117.5
146.4
147.0
116.4
121.8
C
J = 7.4 Hz, H-5′); δ 147.0 (C-4′), 146.4 (C-3′), 123.9 (C-1′), 121.8 (C-
6′), and 116.4 (C-5′)], instead of a 1′,4′-disubstituted aromatic ring
137.5
105.5
147.6
133.2
147.6
105.5
41.4
6.44 brs
7.05 brs
H
found in 29. The coupling constants of anomeric protons [δ 4.98 (1H,
d, J = 6.2 Hz, H-1″) and 3.90 (1H, brs, H-1‴)] indicated the config-
uration of β-glucopyranose and α-rhamnopyranose, respectively
6.82 brd (7.4)
6.88 brd (7.4)
6.44 brs
4.44 d (6.3)
2.12 m
(
Laupattarakasem et al., 2004).The HMBC cross-peaks of H-1‴/C-6″
′
′
45.1
69.5
and H-1′′/C-7 confirmed the position and presence of a rutinose [α-
rhamnopyranosyl-(1 → 6)-β-glucopyranoside] moiety (Fig. 2). Acid
hydrolysis of 28 yielded the aglycone, which was identified as orobol
3.83 dd (9.6, 5.6)
3
.44 m
1″
2″
3″
4″
5″
4.24 d (7.5)
104.0
73.5
76.6
69.8
65.5
4.98 d (6.2)
3.50 brs
3.49 brs
3.34 m
101.8
74.8
78.2
71.8
77.3
(
28a) (Anhut et al., 1984) by comparing its 1H NMR spectrum data,
3.24 dd (9.0, 7.6)
3.33 overlap
3.49 m
together with D-glucopyranose and L-rhamnopyranose, which were
confirmed by GC/MS analysis, respectively (Hara et al., 1987). Thus,
compound 28 was determined as orobol-7-O-α-L-rhamnopyranosyl-
3.84 dd (11.4, 5.4)
3.64 m
3
.18 dd (11.4, 10.3)
(
1 → 6)-O-β-D-glucopyranoside.
6″
4.08 d (10.6)
68.0
The isolated 32 known compounds were identified as 3,5-di-
3
.56 m
1
2
3
4
5
6
‴
‴
‴
‴
‴
‴
4.91 d (7.4)
3.46 m
3.18 m
3.40 m
3.41 m
103.3
74.3
76.8
69.8
76.4
61.0
4.70 brs
3.90 brs
3.78 m
3.37 m
3.67 m
102.4
72.2
72.6
74.3
70.0
18.1
methoxy-4-hydroxybenzyl alcohol 4-O-β-D-glucopyranoside (5)
(
(
Kitajima et al., 1998), vanillyl alcohol 4-O-β-D-glucopyranoside (6)
Kanho et al., 2005), apionikoenoside (7) (Matsuoka et al., 2011),
3
,4,5-trimethoxyphenol O-β-D-glucopyranoside (8) (Le et al., 2012),
3.72 dd (12.0, 2.4)
1.22 d (6.2) (3H)
3,4,5-trimethoxyphenol O-β-D-apiofuranosyl-(1 → 6)-β-D-glucopyrano-
side (9) (Fuchino et al., 1995), isotachioside (10) (Zhong et al., 1999),
3
.64 m
5
7
3
-OCH
-OCH
′,5′-OCH
3
3
3.40 s
3.88 s
3.77 s
60.0
55.5
55.4
4
-hydroxy-3-methoxyphenol O-β-D-apiopyranosyl (1 → 6)-O-β-D-glu-
copyranoside (11) (Kitajima et al., 2003), tachioside (Zhong et al.,
999) (12), canthoside D (13) (Kanchanapoom et al., 2002), benzyl 6-
3
1
O-β-D-apiofuranosyl-β-D-glucopyranoside (14) (Suzuki et al., 1988),
prunasin (15) (Seigler et al., 2002), amygdalin (16) (Backheet et al.,
2
003), benzyl O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside
(
(
17) (Kawahara et al., 2005), 4-allyl-2-hydrophenyl 1-O-β-D-apiosyl-
1 → 6)-β-D-glucopyranoside (18) (Yamauchi et al., 2007), 2,6-dihy-
droxy-4-O-β-D-glucopyranosylbenzophenone (19) (Fu et al., 2011),
lyoniside (21) (Smite et al., 1995), (+)-lyoniresinol-3α-O-β-D-gluco-
pyranoside (22) (Lee et al., 2005), (+)-lyoniresinol (23) (Li and
Seeram, 2010), nudiposide (24) (Smite et al., 1995), (−)-lyoniresinol-
3
α-O-β-D-glucopyranoside (25) (Achenbach et al., 1992), ssioriside
(
26) (Yoshinari et al., 1989), (−)-(7R,8S)-4,7,9,3′,9′-pentahydroxy-3-
methoxy-8-4′-oxyneolignan-3′-O-β-D-glucopyranoside (27) (Gan et al.,
2008), sphaerobioside (29) (Laupattarakasem et al., 2004), genistein 5-
O-β-D-glucoside (30) (Geibel et al., 1990), prunetinoside (31) (Khalid
et al., 1989), (−)-epicatechin (32) (Abdel Kader Ahmed and Al-Refai,
Fig. 3. Key NOESY correlations of 20.
13C NMR spectra (Table 2) closely resembled those of lyoniside (21)
Smite et al., 1995), except for the presence of glucose signals [δ 4.91
H
1H, d, J = 7.4 Hz, H-1‴), 3.72 (1H, dd, J = 12.0, 2.4 Hz, H-6‴a), 3.64
1H, m, H-6‴b), 3.46 (1H, m, H-2‴), 3.41 (1H, m, H-5‴), 3.40 (1H, m,
2
1
014), (−)-epicatechin 5-O-β-D-glucopyranoside (33) (Cui et al.,
(
(
(
992b), (+)-catechin (34) (Abdel Kader Ahmed and Al-Refai, 2014),
(
+)-catechin 5-O-β-D-glucopyranoside (35) (Raab et al., 2010),
proanthocyanidin A-2 (36) (Lou et al., 1999), proanthocyanidin A-1
37) (Lou et al., 1999), and procyanidin B-5 (38) (Cui et al., 1992a) by
H-4‴), and 3.18 (1H, m, H-3‴); δ
C
103.3 (C-1‴), 76.8 (C-3‴), 76.4 (C-
‴), 74.3 (C-2‴), 69.8 (C-4‴), and 61.0 (C-6‴)]. The configurations of
(
5
comparison with NMR and MS data in the literature.
sugars indicated β-xylopyranose and β-glucopyranose based on the J
values of anomeric protons [J = 7.5 Hz (H-1″) and J = 7.4 Hz (H-1‴)],
respectively (Smite et al., 1995). The HMBC correlation between H-1‴
and C-4 indicated that the β-glucopyranosyl unit is connected to C-4.
In this study, NO production assay was performed to evaluate the
anti-inflammatory effect of isolates (1–38) against LPS-activated
murine microglia (Table 3). Compound 10 (IC50 23.9 μM) showed more
G
Acid hydrolysis of 20 gave (+)-lyoniresinol (20a), identified by 1
potent effect than the positive control, N -mono-methyl-L-arginine (L-
H
NMMA) (IC50 25.2 μM). Besides, compounds 18, 30, and 31 displayed
4

K.J. Park, et al.
Phytochemistry 167 (2019) 112085
Table 3
Inhibitory effect of compounds 1–38 on NO production in LPS-activated BV-2 cells.
a
Cell Viability (%)b
a
Cell Viability (%)b
Comp.
IC50 (μM)
Comp.
IC50 (μM)
1
2
3
4
5
6
7
8
9
66.7
55.6
130.6
44.8
58.4
62.0
45.1
54.1
51.5
23.9
46.2
53.1
39.0
49.1
72.0
103.1
45.6
25.3
44.4
93.8
89.6 ± 2.0
106.5 ± 15.6
101.9 ± 5.5
103.0 ± 12.8
101.9 ± 9.0
108.9 ± 9.7
90.6 ± 2.9
93.4 ± 7.2
93.8 ± 9.2
114.1 ± 5.4
95.7 ± 15.7
99.8 ± 6.0
93.8 ± 5.0
86.4 ± 4.8
93.4 ± 5.4
88.2 ± 2.5
102.4 ± 6.5
96.0 ± 6.4
99.5 ± 14.6
100.9 ± 15.4
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
85.3
132.6
48.8
94.3 ± 3.4
98.0 ± 5.2
103.9 ± 6.0
97.6 ± 2.3
104.0 ± 5.5
95.6 ± 4.9
130.3 ± 3.9
111.1 ± 9.5
91.6 ± 7.1
93.6 ± 9.5
98.7 ± 6.9
104.7 ± 11.5
89.9 ± 6.5
102.9 ± 3.8
94.2 ± 2.8
88.0 ± 3.8
94.0 ± 5.7
101.0 ± 11.7
105.2 ± 2.6
39.9
> 500
201.4
96.6
> 500
37.9
28.8
34.1
> 500
81.0
69.7
41.2
> 500
62.6
74.8
25.2
10
11
12
13
14
15
16
17
18
19
20
37
38
L-NMMA
c
a
IC50 value of each compound was defined as the concentration (μM) that caused 50% inhibition of NO production in LPS-activated BV-2 cells.
b
Cell viability after treatment with 20 μM of each compound was determined by the MTT assay and is expressed as a percentage (%). Results are means of three
independent experiments, and the data are expressed as mean ± SD.
c
L-NMMA as a positive control.
moderate effects with IC50 values of 25.3, 28.8, and 34.0 μM, respec-
tively. Other compounds showed weak or no activity to lower the NO
production against LPS-activated microglia. None of the treated com-
pounds showed any significant toxicity to the microglial cell up to the
concentration of 20 μM.
performed with silica gel 60 (70–230 and 230–400 mesh; Merck,
Darmstadt, Germany) and RP-C18 silica gel (230–400 mesh; Merck,
Darmstadt, Germany). Merck precoated silica gel F254 plates and RP-18
F254s plates (Merck, Darmstadt, Germany) were used for TLC. Spots
were detected on TLC under UV light or by heating after spraying the
samples with anisaldehyde–sulfuric acid.
3. Conclusion
4.2. Plant material
A phytochemical study on the MeOH extract of the twigs of B.
schmidtii led to the isolation of 38 phenolic derivatives including six
previously undescribed compounds (1–4, 20, and 28). Their chemical
structures were elucidated by spectroscopic and spectrometric methods,
and all the known compounds (5–19, 21–27, and 28–38) were first
isolated from this plant. These compounds (1–38) were evaluated for
their anti-inflammatory effects on inhibiting NO production against
LPS-activated microglia. Among the isolated compounds, compound 10
exhibited a slight activity (IC50 23.9 μM), while compounds 18, 30, and
The twigs of Betula schmidtii Regel were collected in Goesan, Korea
in March 2013, and the plant was identified by one of the authors
(K.R.L.). A voucher specimen (SKKU-NPL 1303) was deposited in the
herbarium of the School of Pharmacy, Sungkyunkwan University,
Suwon, Korea.
4.3. Extraction and isolation
3
1 showed moderate activities (IC50 25.3, 28.8, and 34.0 μM, respec-
The twigs of B. schmidtii (7.0 kg) were extracted with 80% aqueous
MeOH under reflux at 70 °C for 9 h and filtered. The filtrate was con-
centrated under a reduced pressure to obtain a MeOH extract (410 g),
which was suspended in deionized water and successively partitioned
tively) to lower NO production against LPS-activated microglia, sug-
gesting their possible role to lower neuroinflammation and neurode-
generative complications.
3
with n-hexane, CHCl , EtOAc, and n-BuOH, yielding 15 g, 18 g, 19 g,
4. Experimental
and 116 g, respectively. The n-BuOH-soluble layer (15 g of 116 g) was
3 2
subjected to silica gel column (CHCl –MeOH–H O, 5:1:0.1 → 1:1:0.1)
4.1. General experimental procedures
to yield five fractions (B1–B5).
Fraction B1 (2.9 g) was chromatographed on an RP-C18 silica gel
column with 40% aqueous MeOH to give 6 subfractions (B1A–B1F).
Compounds 8 (7 mg), 21 (24 mg), and 26 (20 mg) were isolated from
subfractions B1A (90 mg), B1B (1.4 g), and B1C (574 mg) using semi-
preparative HPLC (13–27% aqueous CH CN). Fraction B2 (1.3 g) was
3
separated over an RP-C18 silica gel column with 30% aqueous MeOH to
Optical rotations and IR spectra were measured with a JASCO P-
020 polarimeter (JASCO, Easton, MD, USA) and JASCO FT/IR-4600
1
spectrometer (JASCO, Easton, MD, USA), respectively. UV spectra were
obtained on Shimadzu UV-1601 UV–visible spectrophotometer
Shimadzu, Tokyo, Japan). CD spectra were recorded with a JASCO J-
a
(
1500 CD spectrometer (JASCO, Tokyo, Japan). NMR spectra were re-
give 5 subfractions (B2A–B2E). Fraction B2A was fractionated by silica
corded on a Bruker AVANCE III 700 NMR spectrometer (Bruker,
gel column (CHCl
fractions (B2A1–B2A3). Fraction B2A2 (54 mg) was purified by semi-
preparative HPLC (7% aqueous CH CN) to give compounds 5 (3 mg), 6
(3 mg), 10 (12 mg), and 12 (7 mg). Compound 7 (3 mg) was isolated
from fraction B2C (217 mg) using semipreparative HPLC with 30%
3 2
–MeOH–H O, 4:1:0.1 → 1:1:0.1) to afford three
1
13
Karlsruhe, Germany) operating at 500 MHz ( H) and 125 MHz ( C).
HRESI mass spectra were obtained with a Waters SYNAPT G2 (Waters,
Milford, MA, USA) mass spectrometer. Semi-preparative HPLC system
was conducted utilizing a Gilson 306 pump (Gilson, Middleton, WI,
USA) and a Shodex refractive index detector (Shodex, New York, NY,
USA) with a YMC-Triart C18 column (10 × 250 mm, YMC, Kyoto,
3
aqueous CH
tions (B2D1–B2D4) on a silica gel column, eluted with a gradient sol-
vent system of CHCl –MeOH–H O (4:1:0.1 → 1:1:0.1). Compounds 15
3
CN. Fraction B2D (141 mg) was separated into 4 subfrac-
Japan) at
a
flow rate 2 mL/min. Column chromatography was
3
2
5

K.J. Park, et al.
Phytochemistry 167 (2019) 112085
(
(
CH
4 mg), 22 (7 mg), and 25 (5 mg) were purified from subfractions B2D1
15 mg) and B2D3 (38 mg) by semipreparative HPLC (17% aqueous
4.3.2. (7R,8R)-1-(4-hydroxy-3-methoxyphenyl)propan-7,8-diol-4-O-β-D-
apiofuranosyl-(1 → 6)-O-β-D-glucopyranoside (2)
25
3
CN). Fraction B3 (0.9 g) was fractionated by an RP-C18 silica gel
Colorless gum; [α]D –107.9 (c 0.10, MeOH); UV (MeOH) λmax (log ε)
−1
column with 30% aqueous MeOH to obtain 8 subfractions (B3A–B3H).
Fraction B3B (106 mg) was subjected to Lobar-A Si gel 60
276 (2.42), 226 (2.96), 202 (3.50) nm; IR (KBr) νmax cm : 3702, 3670,
1
3399, 2956, 2868, 2835, 1352, 1054, 1011; H NMR (CD
3
OD,
500 MHz) and 13C NMR (CD
OD, 125 MHz) data, see Table 1; HRESIMS
(positive-ion mode) m/z 515.1735 [M + Na] (calcd. for C21H O13Na,
32
(
240 × 10 mm) column (CHCl
3
–MeOH–H
2
O, 3.8:1:0.1 → 1:1:0.1) to
3
+
acquire 3 subfractions (B3A1–B3A3). Compounds 9 (19 mg), 16 (3 mg),
and 20 (3 mg) were isolated upon purification of B3A1 (36 mg) and
515.1741).
B3A3 (13 mg) by semipreparative HPLC (13% aqueous CH
Fraction B3C (151 mg) was separated on Lobar-A Si gel 60
240 × 10 mm) column (CHCl –MeOH–H O, 3:1:0.1) to give 3 sub-
fractions (B3C1–B3C3). Fractions B3C1 (65 mg), B3C2 (27 mg), and
B3C3 (12 mg) were purified by semipreparative HPLC (13% aqueous
3
CN).
a
4
6
.3.3. 4-Hydroxy-1-(2-hydroxyethyl)phenyl-3-O-β-D-apiofuranosyl-(1 →
(
3
2
)-O-β-D-glucopyranoside (3)
25
Colorless gum; [α] –78.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε)
D
−1
2
3
5
79 (2.44), 222 (sh), 201 (3.37) nm; IR (KBr) νmax cm : 3703, 3670,
492, 3398, 2961, 2868, 2835, 1351, 1054, 1012; H NMR (CD
00 MHz) and C NMR (CD OD, 125 MHz) data, see Table 1; HRESIMS
3
CH
8 mg), 18 (4 mg), 23 (13 mg), and 29 (4 mg) were isolated upon pur-
ification of fractions B4 (27 mg), B5 (100 mg), and B6 (25 mg) through
semipreparative HPLC (17–20% aqueous CH CN), respectively.
3
CN) to yield compounds 14 (30 mg) and 27 (5 mg). Compounds 17
1
3
OD,
(
13
+
(
28
positive-ion mode) m/z 471.1473 [M + Na] (calcd. for C19H O12Na,
3
4
71.1478).
Fraction B4 (2.6 g) was subjected to RP-C18 silica gel column with 20%
aqueous MeOH to yield 10 subfractions (B4A–B3J). B4B (82 mg) was
4
.3.4. 3,5-Dihydroxy-1-(hydroxymethyl)phenyl-4-O-β-D-apiofuranosyl-
separated to silica gel column (CHCl
3 2
–MeOH–H O, 3:1:0.1) to afford 2
(1 → 6)-O-β-D-glucopyranoside (4)
subfractions (B4A–B4B) and further purified by semipreparative HPLC
2
5
Colorless gum; [α] –17.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε)
D
(
(
(
6% aqueous CH
4 mg), respectively. B4D (104 mg) was purified to give 3 (3 mg) and 35
5 mg) by semipreparative HPLC (12% aqueous CH CN) after fractio-
3
CN) to afford 2 (5 mg), 4 (5 mg), 11 (7 mg), and 13
−1
2
3
74 (2.08), 226 (sh), 203 (3.35) nm; IR (KBr) νmax cm : 3702, 3669,
1
491, 3397, 2970, 2946, 2868, 2835, 1352, 1054, 1022; H NMR
3
13
(
3 3
CD OD, 500 MHz) and C NMR (CD OD, 125 MHz) data, see Table 1;
nation on
a
Lobar-A Si gel 60 (240 × 10 mm) column
+
HRESIMS (positive-ion mode) m/z 473.1267 [M + Na] (calcd. for
13Na, 473.1271).
(
(
CH
CHCl –MeOH–H O, 2:1:0.1). Fractions B4E (100 mg) and B4G
3 2
18 26
C H O
159 mg) were purified by semipreparative HPLC (9–20% aqueous
CN) to acquire compounds 33 (3 mg) and 28 (9 mg), respectively.
The EtOAc-soluble layer (11 g of 19 g) was chromatographed on a
silica gel column (EtOAc-MeOH-H O, 10:1:0.1 → 1:1:0.1) to yield 3
fractions (E1–E3). Fraction E1 (5.4 g) was further separated over a silica
gel column (CHCl –MeOH–H O, 6:1:0.1 → 1:1:0.1) to obtain 9 fractions
E1A–E1I). Fraction E1E (117 mg) was subjected to passage over a
Lobar-A RP-C18 (240 × 10 mm) column with 40% aqueous MeOH and
further purified by semipreparative HPLC (20% aqueous CH CN) to
3
4
.3.5. (+)-(8R,7′S,8′R)-6-O-β-D-glucopyranosyl lyoniresinol-9′-O-β-D-
xylopyranoside (20)
2
2
5
Colorless gum; [α] +13.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε)
D
2
76 (3.82), 230 (sh), 208 (4.13) nm; CD (MeOH) λmax (Δε) 287
3
2
−
1
(
−2.75), 272 (+4.39), 241 (+5.52), 222 (−3.25); IR (KBr) νmax cm
:
(
1
3
702, 3670, 3400, 2959, 2868, 2835, 1351, 1054, 1011; H NMR
(
CD
HRESIMS (positive-ion mode) m/z 737.2625 [M + Na] (calcd. for
17Na, 737.2633).
3
OD, 500 MHz) and 13C NMR (CD
3
OD, 125 MHz) data, see Table 2;
3
+
give compound 1 (5 mg). Fraction E1F (1.4 g) was separated over an
RP-C18 silica gel column with 50% aqueous MeOH to give 5 subfrac-
tions (E1F1–E1F5). Compounds 19 (5 mg), 32 (32 mg), and 34 (5 mg)
were purified from fraction E1F3 (949 mg) by semipreparative HPLC
33 46
C H O
4.3.6. Orobol-7-O-α-L-rhamnopyranosyl-(1 → 6)-O-β-D-glucopyranoside
(28)
(
30% aqueous CH
obtained by purifying fraction E1G (260 mg) through semipreparative
HPLC (12% aqueous CH CN) after separation over an RP-C18 silica gel
3
CN). Compounds 36 (18 mg) and 37 (4 mg) were
2
5
Colorless gum; [α] –61.1 (c 0.10, MeOH); UV (MeOH) λ
max
(log ε)
D
−
1
3
290 (sh), 262 (3.66), 205 (3.91) nm; IR (KBr) νmax cm : 3702, 3670,
3492, 3396, 2970, 2946, 2868, 2835, 1351, 1054, 1022; H NMR
(CD OD, 500 MHz) and C NMR (CD OD, 125 MHz) data, see Table 2;
HRESIMS (positive-ion mode) m/z 617.1479 [M + Na] (calcd. for
1
column with 40% aqueous MeOH. Fraction E1H (734 mg) was fractio-
nated over an RP-C18 silica gel column with 35% aqueous MeOH to give
13
3
3
+
3
subfractions (E1H1–E1H3) and further purified by semipreparative
HPLC (17% aqueous CH CN) to yield compound 38 (12 mg). Fraction
3
C27H30O15Na, 617.1482).
E2 (0.7 g) was separated on an RP-C18 silica gel column with 40%
aqueous MeOH resulting in 7 subfractions (E2A–E2G). Fraction E2E
4
.4. Acid hydrolysis of compounds 1–4, 20, and 28
(
(
101 mg) was subjected to passage over
a Lobar-A Si gel 60
240 × 10 mm) column (CHCl –MeOH–H O, 5:1:0.1) to obtain 4 sub-
3
2
Compounds 1–4, 20, and 28 (each 1.0 mg) were hydrolyzed with
N HCl (1 mL) under reflux for 2 h at 90 °C. The hydrolysate was di-
fractions (E2E1–E2E4). Compounds 30 (4 mg) and 31 (8 mg) were
isolated by purifying subfraction B2E2 (20 mg) through semi-
preparative HPLC (28% aqueous CH CN). Fraction E3 (1.8 g) was
3
chromatographed on an RP-C18 silica gel column with 40% aqueous
1
2
luted with H O and extracted with EtOAc. The organic layer was re-
moved under reduced pressure to give aglycone (2a–4a, 20a, and 28a),
respectively.
MeOH to give 6 subfractions (E3A–E3F). The subfraction E3B (1.2 g)
was purified by semipreparative HPLC (17% aqueous CH
compound 24 (8 mg).
3
CN) to yield
4
.4.1. (7R,8R)-1-(4-hydroxy-3-methoxyphenyl)propan-7,8-diol (2a)
25
1
Brownish gum; [α] –47.4 (c 0.05, MeOH); H NMR (CD
3
OD,
D
7
00 MHz) δ 6.78 (1H, d, J = 1.9 Hz), 6.75 (1H, d, J = 8.0 Hz), 6.65
(
1H, dd, J = 8.0, 1.9 Hz), 3.91 (1H, d, J = 6.8 Hz), 3.65 (1H, overlap),
4
.3.1. 3,5-Dihydroxyphenyl-(6′-O-benzoyl)-1-O-β-D-glucopyranoside (1)
25
0.90 (3H, d, J = 7.2 Hz).
Colorless gum; [α] –56.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε)
D
−1
2
3
5
72 (2.23), 226 (3.13), 205 (3.54) nm; IR (KBr) νmax cm : 3702, 3670,
1
394, 2970, 2868, 2835, 1351, 1054, 1022; H NMR (CD
3
OD,
4.4.2. 4-Hydroxy-1-(2-hydroxyethyl)phenol (3a)
1
3
1
00 MHz) and C NMR (CD
3
OD, 125 MHz) data, see Table 1; HRESIMS
Brownish gum;
H NMR (CD OD, 700 MHz) δ 6.69 (1H, d,
3
+
(
4
positive-ion mode) m/z 415.1004 [M + Na] (calcd. for C19
15.1005).
H
20
O
9
Na,
J = 8.0 Hz), 6.67 (1H, d, J = 2.0 Hz), 6.54 (1H, dd, J = 8.0, 2.0 Hz),
3.69 (2H, t, J = 7.3 Hz), 2.68 (2H, t, J = 7.3 Hz).
6

K.J. Park, et al.
Phytochemistry 167 (2019) 112085
4
.4.3. 3,5-Dihydroxy-1-(hydroxymethyl)phenol (4a)
DMSO, the blue stained live cells were converted to purple color for-
mazan and this color change was quantified by measuring the absor-
bance at 570 nm in a microplate reader. Cell viability was measured as
1
Brownish gum; H NMR (CD
2H, s).
3
OD, 700 MHz) δ 6.45 (2H, s), 4.49
(
%
of only LPS treated group where LPS treated group were set as 100%.
G
4
.4.4. (+)-lyoniresinol (20a = 23)
The NO synthase inhibitor N -monomethyl-L-arginine (L-NMMA) was
Brownish gum; [α] +58.2 (c 0.03, MeOH); 1H NMR (CD
2
5
OD,
00 MHz) δ 6.59 (1H, s), 6.38 (2H, s), 4.31 (1H, d, J = 5.6 Hz), 3.86
D
3
used as a positive control.
7
(
3H, s), 3.85 (1H, overlap), 3.74 (6H, s), 3.60 (1H, dd, J = 10.9,
Acknowledgements
5
4
.1 Hz), 3.49 (2H, overlap), 3.38 (3H, s), 2.70 (1H, dd, J = 15.4,
.7 Hz), 2.58 (1H, dd, J = 15.4, 11.6 Hz), 1.97 (1H, m), 1.63 (1H, m).
This research was supported by the Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by
4
.4.5. Orobol (28a)
the
Ministry
of
Education,
Science
and
Technology
Brownish gum; 1H NMR (CD
OD, 700 MHz) δ 8.06 (1H, s), 7.04
1H, d, J = 2.0 Hz), 6.87 (1H, dd, J = 8.1, 2.0 Hz), 6.84 (1H, d,
J = 8.1 Hz), 6.36 (1H, d, J = 2.2 Hz), 6.24 (1H, d, J = 2.2 Hz).
3
(2016R1A2B2008380). We thank the Korea Basic Science Institute for
the MS spectrometric analysis.
(
Appendix A. Supplementary data
4.5. Determination of absolute configuration of sugar moieties of
compounds 1–4, 20, and 28
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.112085.
The aqueous layer, obtained after the separation of organic layer,
was neutralized through an Amberlite IRA-67 column and repeatedly
evaporated to obtain each sugar fraction. The sugar residue was dis-
solved in anhydrous pyridine (0.5 mL) followed by adding 2.0 mg of L-
cysteine methyl ester hydrochloride. The mixture was stirred at 60 °C
for 1.5 h. Then, 0.1 mL of 1-trimethylsilylimidazole was added and the
mixture was stirred at 60 °C for another 2 h. Finally, the mixture was
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