Bioorganic Chemistry 66 (2016) 97–101

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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Bioactivity-guided isolation of antioxidant triterpenoids from Betula

platyphylla var. japonica bark
Hee Jeong Eom a, Hee Rae Kang a, Ho Kyong Kim a, Eun Bee Jung b, Hyun Bong Park c, Ki Sung Kang b,⇑,
Ki Hyun Kim a,⇑
a
School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
b
College of Korean Medicine, Gachon University, Seongnam 461-701, Republic of Korea
c
Department of Chemistry, Yale University, New Haven, CT 06520, United States

a r t i c l e i n f o a b s t r a c t

Article history: The bark of Betula platyphylla var. japonica (Betulaceae) has been used to treat pneumonia, choloplania,
Received 9 January 2016 nephritis, and chronic bronchitis. This study aimed to investigate the bioactive chemical constituents
Revised 17 March 2016 of the bark of B. platyphylla var. japonica. A bioassay-guided fractionation and chemical investigation of
Accepted 1 April 2016
the bark of B. platyphylla var. japonica resulted in the isolation and identification of a new lupane-type
Available online 1 April 2016
triterpene, 27-hydroxybetunolic acid (1), along with 18 known triterpenoids (2–19). The structure of
the new compound (1) was elucidated on the basis of 1D and 2D NMR spectroscopic data analysis as well
Keywords:
as HR-ESIMS. Among the known compounds, chilianthin B (17), chilianthin C (18), and chilianthin A (19)
Betula platyphylla var. japonica
Betulaceae
were triterpene-lignan esters, which are rarely found in nature. Compounds 4, 6, 7, 17, 18, and 19 showed
Triterpenoids significant antioxidant activities with IC50 values in the range 4.48–43.02 lM in a DPPH radical-
Antioxidant scavenging assay. However, no compound showed significant inhibition of acetylcholine esterase
DPPH radical scavenging (AChE). Unfortunately, the new compound (1) exhibited no significance in both biological activities.
This study strongly suggests that B. platyphylla var. japonica bark is a potential source of natural antiox-
idants for use in pharmaceuticals and functional foods.
Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction model, and the major diarylheptanoids were identified as the

Betula platyphylla var. japonica (Miquel) Hara (Betulaceae), well-
known as ‘‘Asian white birch”, is widely distributed in Japan, main-
land China, Korea, and eastern Siberia, and its bark has been used
in Chinese traditional medicine for the treatment of various
inflammatory diseases, including pneumonia, choloplania, nephri-
tis, and chronic bronchitis [1]. Regarding phytochemical studies of
this plant, it is a rich source of triterpenes, including betulin, which
are reported to display significant activity against multidrug-
resistant cancer cells (KB-C2 or K562/Adr) [2,3]. The presence of
betulin and related triterpenes in the outer bark of B. platyphylla
var. japonica has been determined, while isolation of diarylhep-
tanoids and arylbutanoids from its inner bark and dammarane-
type triterpenes from its leaves, root bark, and pollen has been
reported [2–9]. A recent study showed that B. platyphylla bark
exhibited cognitive-enhancing activity in a scopolamine-induced
active compounds [10].
As part of a continuing search for bioactive constituents from
Korean medicinal plant sources, an EtOH extract of the bark of B.
platyphylla var. japonica was found to exhibit antioxidant activity
in a DPPH radical-scavenging assay, with an IC50 value of
9.85 lg/mL, which was also identified by other groups [11,12].
Bioassay-guided fractionation and repeated chromatography of
the EtOH extract resulted in isolation of a new lupane-type triter-
pene (1), together with 18 known triterpenoids (2–19) from the
active fractions, the EtOAc-soluble and n-BuOH-soluble fractions
(Fig. 1). To our knowledge, the triterpene-lignan esters, compounds
(17–19) are uncommon natural products, and this is the first report
of their isolation from the family Betulaceae. We report herein the
isolation, structural characterization, and biological activities of
constituents 1–19 (Fig. 1).
2. Experimental

2.1. General experimental procedures

⇑ Corresponding authors.
E-mail addresses: kkang@gachon.ac.kr (K.S. Kang), khkim83@skku.edu Optical rotations were measured on a Jasco P-1020 polarimeter
(K.H. Kim). (Jasco, Easton, MD, USA). IR spectra were recorded on a Bruker

http://dx.doi.org/10.1016/j.bioorg.2016.04.001
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.
98 H.J. Eom et al. / Bioorganic Chemistry 66 (2016) 97–101

IFS-66/S FT-IR spectrometer (Bruker, Karlsruhe, Germany). UV Spots were detected on TLC under UV light or by heating after
spectra were acquired on an Agilent 8453 UV–visible spectropho- spraying with anisaldehyde–sulfuric acid.
tometer (Agilent Technologies, Santa Clara, CA, USA). HR-ESI mass
spectra were recorded on a Waters UPLC-QTOF Xevo G2-S mass 2.2. Plant material
spectrometer (Waters Corporation, Milford, CT, USA). NMR spectra
were recorded on a Bruker AVANCE III 700 NMR spectrometer The bark of B. platyphylla var. japonica were collected from
operating at 700 MHz (1H) and 175 MHz (13C), with chemical shifts Danyang, Chungcheongbuk-do, Korea, in October 2014. The mate-
given in ppm (d) (Bruker). Semi-preparative HPLC used a Shimadzu rial was identified by one of the authors (K.R. Lee). A voucher spec-
Prominence HPLC System with SPD-20A/20AV Series Prominence imen (NM-14-063) was deposited in the herbarium of the Natural
HPLC UV–Vis Detectors (Shimadzu, Tokyo, Japan). Column chro- Medicine Research Center of Richwood Pharmaceutical Company,
matography was performed with a silica gel 60 (Merck, Darmstadt, Ltd., Seoul, Korea.
Germany; 70–230 mesh and 230–400 mesh) and RP-C18 silica gel
(Merck, 230–400 mesh). The packing material for molecular sieve 2.3. Extraction and isolation
column chromatography was Sephadex LH-20 (Pharmacia, Upp-
sala, Sweden). Merck precoated silica gel F254 plates and Dried bark of B. platyphylla var. japonica (4.1 kg) was extracted
reversed-phase (RP)-18 F254s plates (Merck) were used for TLC. with 80% EtOH (18 L
1 day
three times) at room temperature

29

30 19 21
20
12 H 18 22 H H
17
11
25 26 13
1 9 14H COOH H COOH H COOR1
2 16 28
OH
10 8
3 5
H 27
15 H H
4
7
R R O
O H HO HO R2
6 H H
24 23 O
1 R = OH 2 R = OH 3 R 1 = CH 3 R 2 = OH
14 R = H 16 R = H
4 R 1 = H R 2 = OH
13 R1 = H R2 = OCH 3

H COOH H COOH H COOH

OH
H H H
R O O
HO HO H R HO
H H
O O
5 R = OH 8
10 R = H 6R =H
7 R = OH

OH

H
H COOR H COOH OH
H
O
H H
H
O H O H HO H
9R =H 11 15
12 R = CH 3

H COOH H COOH

H H
H COOH HO H COOH
HO H H
O O
H H
HO R1
O HO O HO H
H
HO O HO O
O R2 O

OH OH
OH OH 18 R1 = H R2 = OH
17 19 R1 = OH R 2 = H

Fig. 1. Chemical structures of compounds 1–19.

 H.J. Eom et al. / Bioorganic Chemistry 66 (2016) 97–101 99

and filtered. The filtrate was evaporated under reduced pressure 2.3.1. 27-Hydroxybetunolic acid (1)
using a rotavapor to obtain the EtOH extract (351 g), which was White powder, ½a25
D +2.86 (c 0.07, MeOH); ½aD +13.33 (c 0.02,
25

suspended in distilled H2O (2 L) and successively solvent- CHCl3); IR (KBr) mmax: 3300, 2939, 2828, 1720, 1454, 1031 cm1;
partitioned with CHCl3, EtOAc, and n-BuOH, yielding 274 g, 25 g, 1
H (700 MHz) and 13C (175 MHz) NMR spectroscopic data, see
and 30 g of residue, respectively. Each fraction was evaluated for Table 1; ESI-MS (positive mode): m/z 471 [M + H]+; HR-ESI-MS
antioxidant activity using DPPH radical-scavenging assay. The (positive mode): m/z 471.3462 [M + H]+ (calculated for C30H47O4,
EtOAc-soluble and n-BuOH-soluble fractions showed significant 471.3474).
radical-scavenging capacities with IC50 values of 10.21 and
8.79 lg/mL, respectively, while the CHCl3 soluble fraction exhib-
2.4. DPPH radical-scavenging assay
ited weak activity, with an IC50 value of 78.26 lg/mL. To identify
potential antioxidant components, the active fractions, the EtOAc
Antioxidant activities of compounds 1–19 were evaluated in
and n-BuOH soluble fractions, were further investigated. The
terms of their free radical-scavenging capacities by DPPH assay
EtOAc-soluble fraction (25 g) was separated by silica gel column
[13]. In microwells, 100 lL of an aqueous solution of completely
chromatography using a solvent system of CH2Cl2-MeOH-H2O
dissolved sample (control: 100 lL of distilled water) were added
(9:3:0.1) to provide seven fractions (I-VII). Fraction II (5.3 g) was
to an ethanolic solution of DPPH (100 lL, 60 lM). The final concen-
fractionated by reversed-phase (RP)-C18 column chromatography
trations of the tested samples in the assayed solutions were 5, 10,
with 70% MeOH and 100% MeOH to give six sub-fractions
25 and 50 lM. Vitamin C was used as the standard for comparison.
(II-1–II-6). Fraction II-2 (0.7 g) was subjected to a silica
The ability to scavenge DPPH radicals was calculated in terms of
column chromatography using a gradient solvent system of
percentage inhibition according to the following equation: % inhi-
CH2Cl2-MeOH (15:1, 3:1, 1:1), and 100% MeOH to obtain 17
bition = [(A0A1)/A0
100], where A0 is the absorbance of the con-
sub-fractions (II-2a–II-2q). Fraction II-2d (15 mg) was purified by
trol (without sample) and A1 is the absorbance in the presence of
semi-preparative RP HPLC with a solvent system of CH3CN-H2O
the sample.
(56:44, flow rate: 2 mL/min) using a Phenomenex Luna C18(2) col-
umn (250 mm
10 mm i.d., 10 lm) to yield compounds 2
(0.5 mg), 3 (0.8 mg), and 5 (1.4 mg). Fraction II-2e (21 mg) was 2.5. Acetylcholine esterase (AChE) inhibition assay
purified by semi-preparative RP HPLC using a solvent system of
CH3CN-H2O (56:44) to afford compounds 4 (0.3 mg), 6 (0.6 mg), Inhibition of AChE activity by compounds 1–19 was evaluated
7 (0.4 mg), and 8 (0.3 mg). The n-BuOH-soluble fraction (30 g) by a spectrophotometric method described previously [14] with
was applied to silica column chromatography using a gradient sol- minor modification. Briefly, in 96-well plates, a reaction mixture
vent system of CH2Cl2-MeOH (10:1), CH2Cl2-MeOH-H2O (7:4:1), of 25 lL of 15 mM acetylthiocholine iodide in water, 125 ll of
and 100% MeOH to give two fractions (B1 – B2). Fraction B1 3 mM 5,50 -dithiobis [2-nitrobenzoic acid] in buffer and 25 lL of
(4.6 g) was separated by silica column chromatography with a
gradient solvent system of CH2Cl2-MeOH-H2O (7:3:0.5 and 7:4:1)
Table 1
to obtain five fractions (B1a – B1e). Fraction B1a (78.7 mg) was 1
H and 13C NMR data of 1 in CDCl3. (d in ppm, 700 MHz for 1H and 175 MHz for 13C).a
purified by semi-preparative RP HPLC with a solvent system of
Position 1
MeOH-H2O (97:3) to obtain compound 9 (2.0 mg). Fraction B1b
(486.3 mg) was separated by preparative RP HPLC with a gradient dH dC
solvent system of MeOH-H2O (50% MeOH - 100% MeOH, flow rate: 1 1.43 m, 1.90 m 40.0 t
5 mL/min) using an Agilent Eclipse XDB-C18 column (250 mm
2 2.44 m, 2.47 m 34.2 t
21.2 mm i.d., 7 lm) to yield eight fractions (B1b1 – B1b8). Fraction 3 218.3 s
4 47.4 s
B1b2 (20.6 mg) was purified by semi-preparative RP HPLC with a 5 1.41 m 55.1 d
solvent system of MeOH-H2O (83:17) to give compound 13 6 1.47 m, 1.75 m 19.8 t
(2.6 mg) and fraction B1b4 (20.6 mg) was purified by semi- 7 1.42 m, 1.56 m 35.3 t
preparative RP HPLC with a gradient solvent system of MeOH- 8 41.5 s
9 1.44 m 51.5 d
H2O (93% MeOH - 85% MeOH in 30 min) to obtain compounds 1
10 37.3 s
(0.8 mg), 10 (0.8 mg), 15 (0.6 mg), and 16 (1.8 mg). Fraction B1b6 11 1.24 m, 1.44 m 21.8 t
(31.2 mg) was purified by semi-preparative RP HPLC with 85% 12 0.80 m, 1.72 m 25.3 t
MeOH to afford compounds 11 (7.0 mg), and 14 (3.7 mg). Fraction 13 2.35 m 39.3 d
14 46.9 s
B1e (646.2 mg) was separated over preparative RP HPLC with a sol-
15 1.37 m, 1.86 m 23.4 t
vent system of MeOH-H2O (88:12) to yield six fractions (B1e1 – 16 1.35 m, 2.34 m 33.2 t
B1e6). Fraction B1e3 (10.3 mg) was purified by semi-preparative 17 56.2 s
RP HPLC with a solvent system of MeOH-H2O (83:17) to furnish 18 1.77 m 19.7 d
compound 19 (1.3 mg). Fractions B1e4 (4.4 mg) and B1e6 19 3.01 m 46.5 d
20 150.3 s
(9.4 mg) were purified by semi-preparative RP HPLC with a solvent
21 1.41 m, 1.99 m 30.6 t
system of MeOH-H2O (78:22) to give compound 18 (0.8 mg) and 22 1.43 m, 2.00 m 37.0 t
compound 17 (2.7 mg), respectively. Fraction B2 (7.87 g) was sub- 23 1.07 s 27.0 d
jected to silica column chromatography using a gradient solvent 24 1.02 s 21.1 d
25 0.94 s 16.7 d
system of CH2Cl2-MeOH (5:1), CH2Cl2-MeOH-H2O (7:3:0.1, 7:4:1),
26 0.99 s 16.2 d
and 80% MeOH to give four fractions (B2a – B2d). Fraction B2b 27 3.83 d (12.5); 4.24 d (12.5) 61.1 t
(210 mg) was subjected to preparative RP HPLC with a solvent sys- 28 180.1 s
tem of MeOH-H2O (9:1) to obtain nine sub-fractions (B2b1–B2b9). 29 4.62 s, 4.74 s 110.1 t
Fraction B2b8 (9.7 mg) was purified by semi-preparative RP HPLC 30 1.69 s 19.7 d

with a solvent system of MeOH-H2O (88:12) to furnish compound a

J values are in parentheses and reported in Hz; the assignments were based on
1
12 (0.9 mg). H-1H COSY, HSQC, and HMBC experiments.
100 H.J. Eom et al. / Bioorganic Chemistry 66 (2016) 97–101
the compounds (25, 50, 100, and 200 lM) were added, and the
absorbance at 405 nm was measured. Thereafter, 25 lL of AChE
solution (0.22 U/mL) were added to the wells and the microplate
was read again at the same wavelength 10 times at 1 min intervals.
The percentage inhibition of each test solution was then calculated
using the following equation: % inhibition = 1  (Asample/Acontrol)

100, where Acontrol is the absorbance of the control (without sam-
ple) and Asample is the absorbance in the presence of the sample.
3. Results and discussion
3.1. Bioactivity-guided isolation of isolated compounds
The EtOH extract of B. platyphylla var. japonica barks displayed
significant antioxidant activity in the DPPH radical-scavenging
assay, with an IC50 value of 9.85 lg/mL. Based on the bioactivity-
guided isolation principle, the EtOH extract was fractionated to
yield CHCl3, EtOAc, and n-BuOH soluble fractions. Among the
derived soluble fractions, the EtOAc and n-BuOH soluble fractions
showed potent radical-scavenging capacities, with IC50 values of
10.21 and 8.79 lg/mL, respectively, while the CHCl3 soluble frac-
tion exhibited weak radical scavenging capacity, with an IC50 value
of 78.26 lg/mL. These results led us to investigate the EtOAc and n-
BuOH soluble fractions for antioxidant compounds. Further frac-
tionation of the EtOAc soluble fraction using repeated column
chromatography afforded seven known triterpenoids (2–8). Simi-
larly, chemical investigation of the active n-BuOH soluble fraction
resulted in the isolation and identification of a new triterpene (1),
together with 11 known triterpenoids (9–19).
3.2. Structure elucidation of isolated compounds
Compound (1) was isolated as a white powder. The molecular
formula was determined to be C30H46O4 from the molecular ion
peak [M + H]+ at m/z 471.3462 (calculated for C30H47O4,
471.3474) in the positive mode HR-ESIMS (Fig. S1, Sup. material)
and NMR spectroscopic data (Table 1). The IR spectrum exhibited
absorptions of hydroxy (3300 cm1) and carbonyl (1720 cm1)
groups. The 1H NMR spectrum (Table 1) showed the presence of
signals due to five tertiary methyls at dH 0.94 (s), 0.99 (s), 1.02
(s), 1.07 (s), and 1.69 (s), two oxygenated proton signals at dH
3.83 (d, J = 12.5 Hz) and dH 4.24 (d, J = 12.5 Hz), and two olefinic
proton signals at dH 4.62 (s) and 4.74 (s) (Fig. S2, Sup. material).
The characteristic NMR data of vinylic methyl at dH 1.69 (s) and
olefinic proton signals at dH 4.62 (s) and 4.74 (s) suggested the
presence of an isopropenyl group in compound 1. The 13C NMR
spectrum (Table 1) showed 30 carbon signals (Fig. S3, Sup. mate-
rial), which were attributed to 5 methyl, 12 methylene, and 5
methine groups, as well as 8 quaternary carbons, including two
olefinic carbons [dC 150.3 and 110.1], an oxygenated carbon [dC
61.1], and two carbonyl groups [dC 218.3 and 180.1] deduced by
analysis of HSQC (Fig. S4, Sup. material) and HMBC spectra
(Fig. S5, Sup. material). These data suggested that compound 1 is
a lupane-type triterpenoid, which was also implied by comparison
of its data with those of betunolic acid (14) [15]. The 1H and 13C
NMR spectra of 1 were similar to those of 14, with an apparent dif-
ference that a tertiary methyl group in 14 was replaced by an oxy-
genated methylene group [dH 3.83 (d, J = 12.5 Hz) and dH 4.24 (d,
J = 12.5 Hz); dC 61.1] in 1. The location of this oxygenated methy-
lene group was confirmed to be C-27 by the HMBC correlations
of H-26/C-8, H-26/C-14, H-27/C-8, H-27/C-13, H-27/C-14, and H-
27/C-15 (Fig. 2), suggesting that compound 1 was a C-27-
hydroxylated product of 14. The gross structure of 1 was supported
by the cross peaks in the 1H-1H COSY (Fig. S6, Sup. material) and
HMBC spectra (Fig. 2). The absolute stereochemistry of 1 was
established to be identical to 14 by analysis of the NOESY data
(Fig. S7, Sup. material), and by comparing the coupling constants,
chemical shifts, and specific rotation value with those of betunolic
acid (14). In the NOESY spectrum of 1, key NOESY correlations of H-
5/H-9, H-9/H-27, H-13/H-26, H-19/H-13, H-18/H-27, H-23/H-5, H-
23/H-9, H-24/H-25, and H-25/H-26 were observed, and the specific
rotation value of 1 was positive, ½a25
D +13.33 in CHCl3, which was
comparable to the published specific rotation data of 14 (½a25 D
+45.0 in CHCl3, positive) [15]. Thus, the structure of 1 was eluci-
dated to be 27-hydroxybetunolic acid.
The known compounds were identified as cylicodiscic acid (2)
[16], methyl 27-O-trans-caffeoylcylicodiscate (3) [17], 27-O-trans-
caffeoylcylicodiscic acid (4) [18], myricerol (5) [19], uncarinic acid
E (6) [20], myriceric acid B (7) [21], obtusilinin (8) [22], 3b-
acetyloleanolic acid (9) [23], oleanolic acid (10) [24], oleanonic acid
(11) [25], oleanolic acid acetate methyl ester (12) [26], winchic
acid (13) [27], betunolic acid (14) [15], betulin (15) [28], betulinic
acid (16) [28], chilianthin B (17) [29], chilianthin C (18) [29], and
chilianthin A (19) [29] by comparing their obtained spectroscopic
data with values reported previously. To our knowledge, com-
pounds (17–19) are triterpene-lignan esters, which are uncommon
types of natural product, and this is the first report of their isola-
tion from the family Betulaceae.
3.3. DPPH radical-scavenging assay
The DPPH-scavenging activities of isolated compounds 1–19 are
shown in Table 2. Of these compounds, 4, 6, 7, 17, 18, and 19
showed high radical-scavenging activities with IC50 values in the
range 4.48–43.02 lM (Table 2). Particularly, compounds 4
(IC50 = 6.23 lM), 7 (IC50 = 4.48 lM), and 19 (IC50 = 6.54 lM) were
strong antioxidants when compared to the reference radical scav-
enger, ascorbic acid (IC50 = 3.02 lM). The other compounds
showed no radical-scavenging activities within the concentration
range tested (IC50 > 50 lM).
Interestingly, the active compounds 4, 6, 7, 17, 18, and 19 have a
phenylpropanoid moiety, in particular compounds 17, 18, and 19
are triterpene-lignan esters which have a lignan unit, a phenyl-
propanoid dimer. This suggested that the phenylpropanoid moiety
is an essential functional group for the antioxidant activity of the
isolated triterpene. Phenylpropanoids are secondary metabolites
widely distributed in plants and that have therapeutic activity
against hypertension, viral infections, fungal infections, tumors
and cancer, as well as an immunomodulatory effect [30–33]. These
effects are associated with the antioxidant and free radical-
scavenging capacities of their structural components, such as the
number and location of hydroxyl groups on the aromatic ring
and the side-chain structure [34]. Indeed, many studies have
demonstrated that phenylpropanoids are potent antioxidants and
29
30
H
25 26
H COOH
28
3 5
H
27 OH
O H
24 23
1 1
Fig. 2. H- H COSY ( ) and key HMBC ( ) correlations of 1.
 H.J. Eom et al. / Bioorganic Chemistry 66 (2016) 97–101
Table 2
Antioxidant activities of compounds 1–19 in a DPPH radical-scavenging assay.
Compound IC50 (lM)a Compound
1 >50 11
2 >50 12
3 >50 13
4 6.23 14
5 >50 15
6 18.72 16
7 4.48 17
8 >50 18
9 >50 19
10 >50 Ascorbic acidb
a
IC50 value of each compound is presented as mean of three replicates.
b
Ascorbic acid as a positive control.
the presence of phenylpropanoid and/or phenylethanoid groups in
the structure is related to the antioxidant activity by inhibiting the
oxidation of low-density lipoproteins through mechanisms such as
free radical scavenging and metal ion chelation [31–34]. Regarding
the structure–activity relationship (SAR), it appears that the
methyl ester group at C-28 in the isolated triterpene skeleton
decreases its antioxidant activity, since compound 3, which has
an almost identical structure to compound 4, did not exhibit an
antioxidant effect. The presence of a methoxy group on aromatic
ring seems to have a negative effect on antioxidant activity, as
compound 13 was less active than compound 4. In addition,
antioxidant activity may be more negatively influenced by a
cis-type than a trans-type phenylpropanoid group on the basis of
comparison with the activities of compounds 6 and 8. This SAR
information will facilitate future synthetic and pharmacological
studies.
3.4. Acetylcholine esterase (AChE) inhibition assay
Because antioxidants and/or radical scavengers play a crucial
role in the treatment or prevention of neurodegenerative disorders
such as Alzheimer’s disease and Parkinson’s disease [33], the inhi-
bitory effects of isolated compounds 1–19 on AChE activity was
screened using the microwell plate AChE inhibition assay. How-
ever, no compound showed significant inhibition of AChE at con-
centrations up to 200 lM. Recent studies reported tetracyclic
triterpenes to be potent AChE inhibitors; however, these differed
from the isolated compounds, which were pentacyclic triterpenes
[35,36].
4. Conclusions
In the present study, bioassay-guided fractionation and chemi-
cal investigation of the bark of B. platyphylla var. japonica led to the
isolation of a new lupane-type triterpene, 27-hydroxybetunolic
acid (1), along with 18 known triterpenoids (2–19) from the active
EtOAc-soluble and n-BuOH-soluble fractions. In particular, the iso-
lated triterpene-lignan esters (17–19) are uncommon types of nat-
ural product, and this is the first report of their isolation from the
family Betulaceae. This study also suggests that triterpenoids are
the main active constituents of B. platyphylla var. japonica bark
responsible for its antioxidant activity. The phenylpropanoid moi-
ety in the triterpene skeleton is essential for its antioxidant activ-
ity. Therefore, B. platyphylla var. japonica bark could represent an
accessible source of natural antioxidants for use in pharmaceuti-
cals and functional foods.
101
Acknowledgments
IC50 (lM)a Following are results of a study on the ‘‘Leaders in INdustry-
>50 university Cooperation” Project, supported by the Ministry of
>50 Education. This research was also supported by the Basic Science
>50 Research Program through the National Research Foundation of
>50
Korea (NRF) funded by the Ministry of Science, ICT & Future Plan-
>50
>50 ning (2015R1C1A1A02037383).
11.33
43.02 Appendix A. Supplementary material
6.54
3.02
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.04.
001. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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