ejbps, 2015, Volume 2, Issue 3, 38-51.
Research Article
SJIF Impact Factor 2.062
ISSN 2349-8870
European
Journal
of Biomedical
European
Journal of Biomedical
and Pharmaceutical Sciences
Volume: 2
AND
Issue: 3
38-51
Pharmaceutical sciences
Chen et al.
Year: 2015
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SECONDARY METABOLITES FROM THE LEAVES OF
CINNAMOMUM MACROSTEMON HAYATA
Cheng-Ta Li1, Chiu-Li Kao2, Hsing-Tan Li1, Shu-Ting Huang1 and Chung-Yi Chen1*
1
School of Medical and Health Sciences, Fooyin University, Daliao District, Kaohsiung City
83102, Taiwan,
2
Tzu Hui Institute of Technology, Pingtung County, Taiwan
Article Received on 03/04/2015
Article Revised on 24/04/2015
Article Accepted on 15/05/2015
ABSTRACT
*Correspondence for
Author
A chemical investigation of the leaves of Cinnamomum macrostemon
Dr. Chung-Yi Chen
Hayata (Lauraceae) afforded fifteen phytochemicals including three
School of Medical and
butanolides, obtusilactone A (1), isoobtusilactone A (2), and
Health Sciences, Fooyin
tenuifolide B (3); three coumarins, coumarin (4), isoscopoletin (5), and
University, Daliao District,
scopoletin (6); two steroids, β-sitostenone (7), and β-sitosterol (8); two
Kaohsiung City 83102,
benzenoids, cinnamic acid (9), and eugenol (10); two lignans, (+)-
Taiwan.
yangambin (11), and (+)-syringaresinol (12); three dibenzocycloheptenes, tenuifolin (13), reticuol (14), and subamol (15). The structural elucidation was
performed mainly by MS, 1D and 2D NMR spectrum data. Compounds 1-3, 5, 6, 11, 13, and
15 were found for the first time from this plant.
KEYWORDS:
Cinnamomum
macrostemon
Hayata,
Lauraceae,
butanolides,
dibenzocycloheptenes.
INTRODUCTION
Cinnamomum macrostemon Hayata is a medium-sized evergreen tree, and it’s endemic in
Taiwan, distributed at medium altitudes throughout the island (Liao, 1996). The chemical and
cytotoxic activities of several species of Cinnamomum have been evaluated in previously
studies, e.g., C. camphora (Hsieh et al., 2006), C. osmophloeum (Chen et al., 2006; Hsieh et
al., 2005), C. insulari-montanum (Hsieh et al., 2010), C. kotoense (Chen, 2006; Chen et al.,
2008a; Chen & Hong, 2011; Cheng et al., 2010a), C. subavenium (Chen et al., 2007a; Chen
& Wang, 2011a; Chen & Wang, 2011b; Chen et al., 2010a; Kuo et al., 2008b), C. reticulatum
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European Journal of Biomedical and Pharmaceutical Sciences
(Chen, 2011; Chen & Yeh, 2011; Cheng et al., 2010b; Cheng et al., 2009; Chia et al., 2011),
C. tenuifolium (Cheng et al., 2011; Lin et al., 2009), C. burmannii (Chen et al., 2012a; Hong
et al., 2011; Li et al., 2012) and C. philippinense (Chen et al., 2010b; Chen et al., 2011;
Cheng et al., 2012). Butanolide (Chen et al., 2007b; Chen et al., 2008b; Chen et al., 2007c;
Chen et al., 2012c; Kuo et al., 2007; Kuo et al., 2008a; Liu et al., 2011; Liu et al., 2008;
Wang et al., 2011b), lignanoid (Wu et al., 1994) and dibenzocycloheptenoid (Chen et al.,
2010a; Cheng et al., 2010b; Chen et al., 2012a; Lin et al., 2009) compounds are three of the
major classes of bioactive compounds used for their anti-tumor properties. In the course of
screening for biologically and chemically novel agents from Formosan Lauraceous plants
(Chen et al., 2010c; Lin et al., 2010; Lin et al., 2008; Lin et al., 2011; Shen et al., 2011; Wang
et al., 2011a), C. macrostemon Hayata was chosen for further phytochemical investigation.
Its branchlets appear to be erect, slender and glabrous; and it buds with 9 imbricate seales,
scales with brown hairs in winter. Leaves appear to be chartaceous, somewhat polished
above, dull beneath, ovate-lanceolate to oblong-lanceolate, 10-15 cm long, 3-4 cm wide,
acuminate to obtuse apex, acute or suddenly acute at base, with 3 main veins, nerves finely
prominent on both sides, not fragrant when crushed; petioles appear to be 1 cm long and are
widely furrowed above (Liao, 1996). Previously, we isolated 12 compounds, including two
fatty acids, one coumarin, four benzenoids, two steroids, one triterpenoid, one lignin and one
dibenzocycloheptene from the twigs of this plant (Yang et al., 2011; Chen et al., 2012b). This
is also being studied and published the leaves of this plant for the first time. The MeOH
extract of its leaves was subjected to solvent partitioning and chromatographic separation to
afford 15 pure substances. Fifteen compounds including three butanolides, obtusilactone A
(1), isoobtusilactone A (2), and tenuifolide B (3); three coumarins, coumarin (4),
isoscopoletin (5), and scopoletin (6); two steroids, β-sitostenone (7), and β-sitosterol (8); two
benzenoids, cinnamic acid (9), and eugenol (10); two lignans, (+)-yangambin (11), and (+)syringaresinol (12); three dibenzocycloheptenes, tenuifolin (13), reticuol (14), and subamol
(15), were isolated from the leaves of C. macrostemon. Compounds 1-3, 5, 6, 11, 13, and 15
were found for the first time from this plant.
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HO
O
O
1
HO
O
O
2
OCH3
O
O
3
HO
O
O
CH3O
CH3O
O
4
O
HO
O
5
O
O
6
HO
7
8
HO
COOH
CH3O
9
10
OCH3
OCH3
OCH3
OH
O
O
OCH3
H
CH 3O
O
CH3O
H
CH 3O
H
O
HO
OCH3
OCH3
11
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OCH3
H
12
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European Journal of Biomedical and Pharmaceutical Sciences
CH3O
OH
O
13
HO
OH
O
O
14
O
CH3O
OH
HO
OH
15
Figure 1: Chemical structures of compounds 115.
EXPERIMENTAL
General: UV spectra were obtained on a Jasco UV-240 spectrophotometer in MeCN. IR
spectra were measured on a Hitachi 260-30 spectrophotometer (Hitachi, Tokyo, JP). 1H-NMR
(400/500 MHz) and 13C-NMR (100 MHz), HSQC, HMBC, COSY and NOESY spectra were
obtained on a Varian (Unity Plus) NMR spectrometer (Varian, CA, USA). For each sample,
128 scans were recorded with the following settings: 0.187 Hz/point; spectra width, 14400
Hz; pulse width, 4.0 μs; relaxation delay, 2s. Low-resolution ESI-MS spectra were obtained
on an API 3000 (Applied Biosystems, CA, USA) and high-resolution ESI-MS spectra on a
Bruker Daltonics APEX II 30e spectrometer (Bruker, Bremen, Germany). Silica gel 60
(Merck, 70~230 mesh, 230~400 mesh) was used for column chromatography. Precoated
silica gel plates (Merck, Kieselgel 60 F-254), 0.20 mm and 0.50 mm, were used for analytical
TLC and preparative TLC, respectively, and visualized with 10% H2SO4.
Plant material: The leaves of C. macrostemon Hayata were collected from Pinglin Hsiang,
Taipei County, Taiwan, and November 2009. Plant material was identified by Dr. Fu-Yuan
Lu (Department of Forestry and Natural Resources College of Agriculture, National Chiayi
University). A voucher specimen (Cinnamo. 9) was deposited in the Department of Nutrition
and Health Science, School of Medical and Health Sciences, Fooyin University, Kaohsiung
City, Taiwan.
Extraction and isolation: The air-dried leaves of C. macrostemon (3.5 kg) were extracted
with MeOH (10 L×5) at room temperature and a MeOH extract (112.6 g) was obtained upon
concentration under reduced pressure. The residue was placed on a silica gel column and
eluted with CHCl3 gradually enriched with MeOH to afford 4 fractions. Fraction 1 (4.62 g)
eluted with n-hexane–Acetone (30:1) was further purified by silica gel column
chromatography using the same solvent system to obtain obtusilactone A (1) (Chen et al.,
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2010a) (13 mg), isoobtusilactone A (2) (Chen et al., 2010a) (28 mg), tenuifolide B (3) (Lin et
al., 2009) (4 mg), coumarin (4) (Liu et al., 2002) (8 mg), isoscopoletin (5) (Lee et al., 2001)
(5 mg), and scopoletin (6) (Tsukamoto et al., 1984) (10 mg). Fraction 2 (14.38 g) eluted with
n-hexane–Acetone (20:1) was further separated using silica gel column chromatography and
purified by preparative TLC (thin layer chromatography) to yield β-sitostenone (7) (Chen et
al., 1997) (16 mg), β-sitosterol (8) (Chen et al., 1997) (75 mg), cinnamic acid (9) (Chen et al.,
2009) (6 mg), and eugenol (10) (Kuo, et al., 2008b) (34 mg). Fraction 3 (7.43 g) was purified
by silica gel chromatography (CH2Cl2–MeOH, 15:1) to give (+)-yangambin (11) (Kim, et al.,
2010) (6 mg) and (+)-syringaresinol (12) (Chen et al., 1998) (8 mg). Fraction 4 (15.23 g)
eluted with CH2Cl2–MeOH (10:1) was further purified by silica gel column chromatography
using the same solvent system to obtain three dibenzocycloheptenes, tenuifolin (13) (Lin et
al., 2009) (3 mg), reticuol (14) (Chia et al., 2011) (6 mg), and subamol (15) (Chen et al.,
2010c) (9 mg). These compounds were obtained and characterized by comparison of their
physical and spectral data with values obtained in the literature. In addition to 4, 7-10, 12 and
14, all of these compounds were found for the first time from this plant (Yang et al., 2011).
Obtusilactone A (1): Pale yellowish liquid; []25D -11.3 (c 0.05, CHCl3); UV λmax (MeCN)
(log ε) 225 (4.11) nm; IR (neat) max 3400 (br, OH), 1770, 1670 (α,β-unsaturated γ-lactone),
1465, 1365, 1090 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.88 (3H, t, J = 7.2 Hz, H-19), 1.25
(20H, br s, H-9~18), 1.48 (2H, m, H-8), 2.77 (2H, m, H-7), 4.67 (1H, dd, J = 2.8, 1.6 Hz, H5a), 4.89, (1H, dd, J = 2.8, 1.6 Hz, H-5b), 5.11 (1H, br s, H-3), 6.68 (1H, td, J = 7.6, 2.0 Hz,
H-6).
Isoobtusilactone A (2): Pale yellowish liquid; []25D -24.6 (c 0.05, CHCl3); UV λmax
(MeCN) (log ε) 225 (4.12) nm; IR (neat) max 3450 (br, OH), 1770, 1670 (α,β-unsaturated γlactone), 1465, 1360, 1090 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.88 (3H, t, J = 6.8 Hz, H19), 1.25 (20H, br s, H-9~18), 1.52 (2H, m, H-8), 2.45 (2H, m, H-7), 4.72 (1H, dd, J = 2.8,
1.2 Hz, H-5a), 4.94, (1H, dd, J = 2.8, 1.2 Hz, H-5b), 5.24 (1H, br s, H-3), 7.07 (1H, td, J =
7.6, 2.4 Hz, H-6).
Tenuifolide B (3): Colourless oil; []25D -28.9 (c 0.05, CHCl3); UV λmax (MeCN, log ε) 265
(4.05) nm; IR (neat) max 3455 (br, OH), 1780, 1680 (α,β-unsaturated γ-lactone), 1290 cm-1;
1
H NMR (400 MHz, CDCl3) δ 0.87 (3H, t, J = 6.8 Hz, H-20), 1.26 (32H, br s, H-4~19),
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1.43-1.68 (4H, m, H-2, 3), 3.35 (3H, s, OMe-1), 4.12 (1H, dd, J = 7.4, 4.8 Hz, H-1), 4.88,
5.20 (each 1H, d, J = 2.6 Hz, H-6a, b), 7.22 (1H, br s, H-4).
Coumarin (4): Colourless oil; UV λmax (MeCN) 260, 330 nm; IR (neat) max 1730, 1630 cm1 1
; H NMR (400 MHz, CDCl3) δ 6.44 (1H, d, J = 9.5 Hz), 7.31 (1H, d, J = 7.5 Hz), 7.35 (1H,
d, J = 7.5 Hz), 7.50 (1H, d, J = 7.5 Hz), 7.53 (1H, d, J = 7.5 Hz), 7.72 (1H, d, J = 9.5 Hz).
Isoscopoletin (5): White needles (EtOAc), mp 184-186 ℃; UV λmax (MeCN) 260, 315 nm;
IR (neat) max 3400 (OH), 1720 (C=O), 1630 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.90 (3H,
s, OCH3-7), 6.21 (1H, d, J = 9.6 Hz, H-3), 6.80 (1H, s, H-8), 7.12 (1H, s, H-5), 7.82 (1H, d, J
= 9.6 Hz, H-4).
Scopoletin (6): Yellowish needles (CH2Cl2), mp 208-210 ℃; UV λmax (MeCN) 260, 315 nm;
IR (neat) max 3400 (OH), 1720 (C=O), 1630 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.95 (3H,
s, OCH3-6), 6.27 (1H, d, J = 9.6 Hz, H-3), 6.85 (1H, s, H-8), 6.92 (1H, s, H-5), 7.62 (1H, d, J
= 9.6 Hz, H-4).
β-Sitostenone (7): White needles (CH2Cl2), mp 85-86 ℃, IR νmax 1675, 1620, 1450, 1375
cm-1, 1H NMR (400 MHz, CDCl3) : δ 0.68 (3H, s, H-18), 0.81 (3H, d, J = 6.7 Hz, H-26), 0.84
(3H, s, H-27), 0.86 (3H, d, J = 7.0 Hz, H-29), 0.94 (3H, d, J = 6.0 Hz, H-21), 1.02 (3H, s, H19), 5.72 (1H, d, J = 1.4 Hz, H-3).
β-Sitosterol (8): White needles (MeOH), mp 138-140 ℃, IR νmax 3400, 2900, 1625, 1450 cm1 1
, H NMR (400 MHz, CDCl3) : δ 0.68 (3H, s, H-18), 0.81 (3H, d, J = 6.8 Hz, H-26), 0.84
(3H, d, J = 6.8 Hz, H-27), 0.86 (3H, t, J = 7.0 Hz, H-29), 0.92 (3H, d, J = 6.4 Hz, H-21), 1.01
(3H, s, H-19), 3.53 (1H, m, H-3), 5.35 (1H, br s, H-6).
Cinnamic acid (9): Yellowish needles (CH2Cl2), mp 208-210 ℃; UV λmax (MeCN) 260, 315
nm; IR (neat) max 1700, 1680, 1650, 980, 770 cm-1; 1H NMR (400 MHz, CDCl3) δ 6.63 (1H,
d, J = 16.0 Hz, H-2), 7.38 (3H, m, H-6, 7, 8), 7.56 (1H, d, J = 16.0 Hz, H-3), 7.58 (2H, m, H5, 9).
Eugenol (10): Colorless oil; UV λmax (MeCN) 230, 285 nm; IR (neat) max 3500 (OH), 1630,
1500, 1430 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.38 (2H, d, J = 6.8 Hz, H-1), 3.89 (3H, s,
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OCH3), 5.13 (2H, m, H-3), 5.88 (1H, s, OH), 6.03 (1H, m, H-2), 6.75 (1H, dd, J = 8.8, 2.0
Hz, H-5), 6.76 (1H, d, J = 2.0 Hz, H-3), 6.93 (1H, d, J = 8.8 Hz, H-6).
(+)-Yangambin (11): White powder; []25D +32.5 (c 0.50, CHCl3); UV λmax (MeCN) 235,
275 nm; IR (neat) max 1610, 1500 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.11 (2H, m, H-1 and
H-5), 3.84 (6H, s, 2 x OCH3), 3.89 (12H, s, 4 x OCH3), 3.93 (2H, dd, J = 9.6, 3.6 Hz, H4axia. and H-8axia.), 4.30 (2H, dd, J = 9.6, 6.8 Hz, H-4equ. and H-8equ.), 4.75 (2H, d, J =
4.3 Hz, H-2 and H-6), 6.59 (4H, s, H-2, H-2, H-6 and H-6).
(+)-Syringaresinol (12): White powder; []25D +45.3 (c 0.50, CHCl3); UV λmax (MeCN) 235,
270 nm; IR (neat) max 3400 (br, OH), 1610, 1500 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.11
(2H, m, H-1 and H-5), 3.91 (2H, dd, J = 9.2, 3.6 Hz, H-4axia. and H-8axia.), 3.91 (12H, s, 4 x
OCH3), 4.29 (2H, dd, J = 9.2, 6.8 Hz, H-4equ. and H-8equ.), 4.74 (2H, d, J = 4.3 Hz, H-2 and
H-6), 5.50 (2H, s, OH), 6.59 (4H, s, H-2, H-2, H-6 and H-6).
Tenuifolin (13): White amorphous powder; UV λmax (MeCN, log ε) 235 (3.23), 255 (2.65),
290 (2.11) nm; IR (neat) max 3300 (br, OH), 3000, 1700, 1250, 1070, 920 (methylenedioxy)
cm-1; 1H NMR (400 MHz, CDCl3) δ 2.77 (1H, dd, J = 12.8, 6.2 Hz, H-5a), 3.08 (1H, dd, J =
12.8, 8.0 Hz, H-5b), 3.84 (3H, s, OMe-3), 4.32 (1H, d, J = 13.0 Hz, H-13a), 4.49 (1H, dt, J =
13.0, 1.4 Hz, H-13b), 6.02 (each 1H, d, J = 1.6 Hz, H-10), 6.16 (1H, tq, J = 8.0, 1.4 Hz, H-6),
6.76 (1H, d, J = 2.8 Hz, H-4), 6.84 (1H, dd, J = 8.4, 2.8 Hz, H-2), 7.08 (1H, s, H-12), 7.14
(1H, s, H-8), 7.38 (1H, d, J = 8.4 Hz, H-1).
Reticuol (14): White amorphous powder; UV λmax (MeCN, log ε) 235 (3.23), 255 (2.65), 290
(2.11) nm; IR (neat) max 3300 (br, OH), 3000, 1700, 1250, 1070, 920 (methylenedioxy) cm-1;
1
H NMR (400 MHz, CDCl3) δ 2.74 (1H, dd, J = 13.0, 6.5 Hz, H-5a), 3.04 (1H, dd, J = 13.0,
8.5 Hz, H-5b), 3.88 (3H, s, OMe-3), 4.33 (1H, d, J = 13.0 Hz, H-13a), 4.49 (1H, d, J = 13.0
Hz, H-13b), 6.02 (each 1H, d, J = 1.0 Hz, H-10), 6.15 (1H, t, J = 7.5 Hz, H-6), 6.69 (1H, d, J
= 2.5 Hz, H-4), 6.75 (1H, dd, J = 8.5, 2.5 Hz, H-2), 7.06 (1H, s, H-12), 7.12 (1H, s, H-8),
7.32 (1H, d, J = 8.5 Hz, H-1).
Subamol (15): White amorphous powder; UV λmax (MeCN, log ε) 235 (3.23), 255 (2.65),
290 (2.10) nm; IR (neat) max 3400 (br, OH), 3000, 1700, 1250 cm-1; 1H NMR (400 MHz,
CDCl3) δ 2.78 (1H, dd, J = 12.8, 6.2 Hz, H-5a), 3.06 (1H, dd, J = 12.8, 8.0 Hz, H-5b), 3.97
(3H, s, OMe-3), 4.36 (1H, d, J = 13.0 Hz, H-12a), 4.53 (1H, dt, J = 13.0, 1.4 Hz, H-12b),
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6.16 (1H, tq, J = 8.0, 1.4 Hz, H-6), 6.70 (1H, d, J = 2.8 Hz, H-4), 6.76 (1H, dd, J = 8.4, 2.8
Hz, H-2), 7.16 (1H, s, H-11), 7.17 (1H, s, H-8), 7.36 (1H, d, J = 8.4 Hz, H-1).
RESULTS AND DISCUSSION
Compounds 1-3, 5, 6, 11, 13, and 15 were found for the first time from this plant.
Obtusilactone A (1), a pale yellowish liquid, also had molecular formula C19H32O3, as
deduced from FABMS. The UV spectrum of 1 showed and intense absorption band at λ 225
nm, which is typical of β-hydroxy-γ-methylene-α,β-unsaturated-γ-lactone ring. The IR
spectrum showed absorption bands for a hydroxyl group at 3400 cm-1 and an α,β-unsaturatedγ-lactone ring at 1770 and 1670 cm-1. The 1H NMR spectra of 1 presented the β-hydroxy-γmethylene-α,β-unsaturated-γ- lactone skeleton and the Z geometry of the trisubstituted
double bond [ 6.68 (1H, td, J = 7.6, 2.0 Hz, H-6)]. The presence of a broad singlet  1.25
(20H, br s, H-9-18) was attributed to protons in a long methylene chain in 1. The exocyclic
olefinic protons appeared at  4.67, 4.89 (each 1H, dd, J = 2.8, 1.6 Hz, H-5a, b) and one
hydroxymethine proton was located at  5.11 (1H, br s, H-3). Compound 1 showed an []25D
-11.3 (c 0.05, CHCl3), indicating the S configuration at C-3.
Isoobtusilactone A (2), a pale yellowish liquid, also had molecular formula C19H32O3, as
deduced from FABMS. Its spectral data (IR, UV, 1H and 13C NMR) were also very similar to
those of 1. The large difference between them appeared for H-1,  7.07 (td, J = 7.6, 2.4 Hz)
in 2 versus  6.68 in 1, in their 1H NMR spectra, suggesting an E-configuration for Δ2(6) in 2.
The 1H NMR spectra of 2 was similar to that of obtusilactone A, indicating that 2 has the
same β-hydroxy-γ-methylene-α,β-unsaturated- γ-lactone skeleton and the same E geometry
of the trisubstituted double bond [ 7.07 (1H, td, J = 7.6, 2.4 Hz, H-6)]. The presence of a
broad singlet  1.25 (20H, br s, H-9-18) was attributed to protons in a long methylene chain
in 2. The exocyclic olefinic protons appeared at  4.72, 4.94 (each 1H, dd, J = 2.8, 1.2 Hz, H5a, b) and one hydroxymethine proton was located at  5.24 (1H, br s, H-3). Compound 2
showed an []25D -24.6 (c 0.05, CHCl3), indicating the S configuration at C-3. Thus, the
structure of isoobtusilactone A was represented as 2, and elucidated as (3S, 2E)-3-hydroxy-4methylene-2-tetradecylidene-dihydrofuran-1-one, which was further confirmed by COSY and
NOESY experiments. The 1H and
13
C NMR data of 2 were assigned by comparison with
those of 1.
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Tenuifolide B (3) was isolated as a colourless oil. The molecular formula was determined a
C26H46O3 by EIMS ([M]+, m/z 406) and HREIMS. The presence of an α, β-unsaturated γlactone moiety was apparent from UV absorption at 265 nm. The IR peaks at 1780 and 1680
cm-1 supported the presence of the α,β-unsaturated γ-lactone. The 1H NMR spectrum of 3
indicated the presence of an exo methylene group and another downfield alkene proton,
which is located on the β-carbon of the unsaturated lactone from its chemical shift. In
addition, it showed the signals corresponding to a methoxy functionality at  3.35, an
oxymethine proton at  4.12, and long chain aliphatic protons at  1.26 (32H, br s), and 1.421.68 (4H, m). The structure of 3 is similar to that of the known butanolide, 3-(1methoxyoctadecyl)-5-methylene-5H-furan-2-one. Thus, the structure of 3 was elucidated as
3-(1-methoxy-eicosyl)-5-methylene-5H-furan-2- one. The configuration at C-1 remains
undefined, since neither the negative rotation, nor the specific rotation can be correlated to
known compounds.
Tenuifolin (13) was isolated as a white, amorphous powder with a molecular formula of
C18H16O4, as determined by HREIMS (obsd [M]+ at m/z 296.1048; calcd [M]+ 296.1049). This
formula agrees with deductions from the 1H and
13
C NMR data, and corresponds to 11
degrees of unsaturation. The UV spectrum contained absorption bands typical of 5Hdibenzo[a,c] cycloheptene derivatives. IR absorption peaks at 920, 1070 and 3300 cm-1
indicated the presence of methylenedioxy and hydroxy functionalities, respectively. The 1H
NMR resonances of 13 were well dispersed in CDCl3 and displayed an ABX pattern (H-4 at 
6.76, H-2 at 6.84, and H-1 at 7.38), a singlet at  7.08 for H-12, and a singlet at  7.14 of H-8
in the aromatic region, in addition to the methylenedioxy protons at  6.02, accounting for
seven protons. A three proton singlet at  3.84 indicated the presence of the methoxy group.
The C-6 methine proton ( 6.16, tq, J = 8.0, 1.4 Hz) coupling with the neighbor C-5 and C-13
methylene protons, which appeared coupling constant at  2.77 (dd, J = 12.8, 6.2 Hz, H-5a),
3.08 (dd, J = 12.8, 8.0 Hz, H-5b), 4.32 (d, J = 13.0 Hz, H-13a), and 4.49 (dt, J = 13.0, 1.4 Hz,
H-13b), respectively. The
13
C NMR and DEPT spectra of 13 showed 18 resonances
comprising one methyl, three methylene, six methine, and eight quaternary carbons. Structure
13 was also confirmed by 2D NMR experiments. A COSY correlation was observed between
H-1 and H-2, and between H-5 and H-6. A triplet of quartets at  6.16 was assigned to H-6
and showed coupling to the nearby C-5 and C-13 methylene protons, which appeared at 
2.77 and 3.08, and at  4.49, respectively.
www.ejbps.com
46
Chen et al.
European Journal of Biomedical and Pharmaceutical Sciences
Subamol (15) was obtained as a white amorphous powder from CHCl3. The molecular
formula was determined as a C17H16O4 by EIMS ([M]+, m/z 284) and HREIMS. The UV
spectrum of 15 contained absorption bands typical of the 5H-dibenzo[a,c]cycloheptene
derivatives (Budac and Wan, 1996). The IR spectrum of 15 showed characteristic absorption
bands due to the presence of hydroxyl (3400 cm-1) groups. The 1H NMR spectrum of 15
contained an ABX pattern at  6.70 (1H, d, J = 2.8 Hz), 6.76 (1H, dd, J = 8.4, 2.8 Hz) and
7.36 (1H, d, J = 8.4 Hz) for H-4, H-2 and H-1, a singlet at  7.16 for H-11, and a singlet at 
7.17 for H-8 in the aromatic region accounting for five protons. A singlet at  3.97 (3H, s)
was assigned to 3-OMe. This compound was assigned as the C-6 methine proton ( 6.16 (1H,
tq, J = 8.0, 1.4 Hz)) due to the coupling with the neighbor C-5 and C-12 methylene protons
which appeared coupling constant at  2.78 (1H, dd, J = 12.8, 6.2 Hz, H-5a), 3.06 (1H, dd, J
= 12.8, 8.0 Hz, H-5b), 4.36 (1H, d, J = 13.0 Hz, H-12a) and 4.53 (1H, dt, J = 13.0, 1.4 Hz, H12b), respectively. The 13C NMR and DEPT experiments of 15 showed seventeen resonance
lines consisting of one methyl, two methylenes, six methines, and eight quaternary carbons.
The structure of 15 was also confirmed by 2D NMR experiments.
ACKNOWLEDGMENTS
This investigation was supported by two grants from the Ministry of Science and Technology
(MOST-103-2320-B-242-001), and Tzu Hui Institute of Technology (104004 and 104005).
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