Journal of the Chinese Chemical Society, 2004, 51, 665-670 665

Flavones and Cytotoxic Constituents from the Stem Bark of Muntingia

calabura

Jih-Jung Chena* ( ), Ru-Wei Linb ( ), Chang-Yih Duhc ( ),

a
Hung-Yi Huang ( ) and Ih-Sheng Chenb* ( )
a
Department of Pharmacy, Tajen Institute of Technology, Pingtung, Taiwan 907, R.O.C.
b
Institute of Pharmaceutical Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan 807, R.O.C.
c
Institute of Marine Resources, National Sun Yat-sen University, Kaohsiung, Taiwan 804, R.O.C.

Two new flavones, 8-hydroxy-7,3¢,4¢,5¢-tetramethoxyflavone and 8,4¢-dihydroxy-7,3¢,5¢-trimethoxy-

flavone, together with thirteen known compounds have been isolated from the stem bark of Muntingia
calabura. The structures of two new compounds were determined through spectral analyses. Among the iso-
lates, 8-hydroxy-7,3¢,4¢,5¢-tetramethoxyflavone, 8,4¢-dihydroxy-7,3¢,5¢-trimethoxyflavone, and 3-hydroxy-
1-(3,5-dimethoxy-4-hydroxyphenyl)propan-1-one exhibited effective cytotoxicities (ED50 values = 3.56,
3.71, and 3.27 mg/mL, respectively) against the P-388 cell line in vitro.

Keywords: Muntingia calabura; Tiliaceae; Stem bark; Flavones; 8-Hydroxy-7,3¢,4¢,5¢-tetramethoxy-

flavone; 8,4¢-Dihydroxy-7,3¢,5¢-trimethoxyflavone; Cytotoxicity.

INTRODUCTION of C19H18O7, which was confirmed by the HR-FAB-MS. The

Muntingia calabura L. (Tiliaceae) is a medium-sized
evergreen tree, a single species in Genus Muntingia, distrib-
uted in tropical America. It was introduced and cultivated in
gardens and along roadsides for ornamental and edible pur-
poses in southern Taiwan. 1 This plant is rich in flavonoids
with flavones, flavanones, flavans, and biflavans as the major
constituents, and some flavonoids have demonstrated cyto-
toxic activities.2-4 Investigation of the cytotoxic constituents
from the stem bark of the Formosan species led to the isola-
tion and characterization of two new flavones, 8-hydroxy-
7,3¢,4¢,5¢-tetramethoxyflavone (1) and 8,4¢-dihydroxy-
7,3¢,5¢-trimethoxyflavone (2), together with thirteen known
compounds. The structures of these compounds were eluci-
dated by spectroscopic analysis. This paper deals with the
structural elucidation of 1-2 and the cytotoxic activities of the
isolates.
RESULTS AND DISCUSSION
8-Hydroxy-7,3¢,4¢,5¢-tetramethoxyflavone (1) was iso-
lated as light yellow needles. The FAB-MS afforded the posi-
tive ion [M + H]+ at m/z = 359, implying a molecular formula
UV absorptions at 210, 271 and 312 nm were similar to those
of 8,5¢-dihydroxy-7,3¢,4¢-trimethoxyflavone and were char-
acteristic of a 7,8,3¢,4¢,5¢-pentaoxygenated flavone nucleus.2
The presence of a hydroxyl group in the molecule was re-
vealed by a band at 3300 cm-1 in the IR spectrum, which was
confirmed by a signal at d 5.75 (br s, disappeared on addition
of D 2 O) in the 1 H-NMR spectrum. A conjugated carbonyl
group was revealed by IR absorption at 1626 cm-1, along with
a resonance signal in the 13C-NMR spectrum at d 178.2. The
1
H-NMR spectrum of 1 showed four methoxy groups at d
3.93 (3H), 3.97 (6H), and 4.05 (3H), and five aromatic pro-
tons at d 6.78 (1H, s), 7.22 (2H, s), 7.05, 7.79 (each 1H, d, J =
8.8 Hz). In addition, the presence of a 3,4,5-trimethoxy-
phenyl group was clearly demonstrated from two equivalent
aromatic protons at d 7.22 (2H, s, H-2¢ and H-6¢), and three
methoxy signals at d 3.93 (3H, s, OMe-4¢) and d 3.97 (6H, s,
OMe-3¢ and OMe-5¢). According to the above data, the struc-
ture of 1 was elucidated as 8-hydroxy-7-methoxy-2-(3,4,5-
trimethoxyphenyl)chromen-4-one. This was further con-
firmed by 1H-1H COSY, NOESY experiments (Fig. 1). The
assignment of 13 C-NMR resonances was confirmed by the
HSQC and HMBC techniques (Fig. 1); these also supported
the structure of 1.
8,4¢-Dihydroxy-7,3¢,5¢-trimethoxyflavone (2) was iso-

* Corresponding author. Fax: +886-8-7625308; e-mail: jjchen@ccsun.tajen.edu.tw (J. J. Chen)

Fax: +886-7-3210683; e-mail: m635013@kmu.edu.tw (I. S. Chen)
666 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004
lated as light yellow needles. The HR-EI-MS gave an M+ ion
peak at m/z = 344.0894, consistent with a molecular formula
of C 18 H 16 O 7 . The UV absorptions at 211, 270, and 310 nm
were similar to those of 1 and 8,5¢-dihydroxy-7,3¢,4¢-trimeth-
oxyflavone, and were also characteristic of a 7,8,3¢,4¢,5¢-
pentaoxygenated flavone nucleus.2 The presence of a conju-
gated C=O group was revealed by IR absorption at 1628 cm-1,
along with a resonance signal in the 13C-NMR spectrum at d
178.5. The 1H-NMR spectrum of 2 was similar to that of 1 ex-
cept that the OMe-4¢ of 1 was replaced by a OH-4¢ in 2. Anal-
ysis of the 1 H-NMR spectrum of 2 revealed three methoxy
groups at d 3.96 (s, 6H) and 4.05 (s, 3H), which were assigned
to OMe-3¢, OMe-5¢, and OMe-7, respectively. Five aromatic
protons at d 6.77 (s, 1H), 7.21 (s, 2H), and 7.04, 7.78 (each d,
J = 8.8 Hz, each 1H), were assigned to H-3, H-2¢, H-6¢, H-6,
and H-5, respectively. In addition, two broad singlet signals
at d 5.80 and 5.92 (each exchangeable with D 2 O) were as-
signed to OH-8 and OH-4¢, respectively, which could be sup-
ported by IR absorption at 3320 cm-1. According to the above
data, the structure of 2 was elucidated as 8-hydroxy-2-(4-
hydroxy-3,5-dimethoxyphenyl)-7-methoxy-chromen-4-one.
This was further confirmed by 1H-1H COSY and NOESY ex-
periments (Fig. 2). The assignment of 13C-NMR resonances
were confirmed by the HSQC and HMBC techniques (Fig. 2),
which also supported the structure of 2.
Fig. 1. NOESY contacts (a) and HMBC connectivities (b) of compound 1.
Fig. 2. NOESY contacts (a) and HMBC connectivities (b) of compound 2.
Chen et al.
The known isolates including three flavones [6,7-di-
methoxy-5-hydroxyflavone (3),5 5,7-dimethoxyflavone (4),6
and 3,5-dihydroxy-6,7-dimethoxyflavone (5) 7 ], one flavan
[(2S)-5¢-hydroxy-7,8,3¢,4¢-tetramethoxyflavan (6) 2 ], three
steroids [b-sitostenone (7),8 6b-hydroxystigmast-4-en-3-one
(8), 9 and b-sitosterol (9) 10 ], four benzenoids [syringic acid
(10),11 vanillic acid (11), 11 3-hydroxy-1-(3,5-dimethoxy-4-
hydroxyphenyl)propan-1-one (12),12 and tetracosyl ferulate
(13)13], and a mixture of 1-tetracosanol (14) and 1-hexacos-
anol (15)14 were readily identified by comparison of physical
and spectroscopic data (UV, IR, 1 H-NMR, [a] D , and mass
spectrometry data) with corresponding authentic samples or
literature values.
In the previous investigations, several cytotoxic fla-
vonoids and chalcones were isolated from the roots2 and the
leaves and stems 3 of this plant collected in the Philippines
and in Thailand, respectively. In our study of the stem bark of
the Formosan species, the cytotoxic effects of the isolates
were tested in vitro against P-388, A549, and HT-29 cell
lines. The cytotoxicity data are shown in Table 1, and the
clinically applied anticancer agent, mithramycin, was used as
reference compound. 8-Hydroxy-7,3¢,4¢,5¢-tetramethoxy-
flavone (1), 8,4¢-dihydroxy-7,3¢,5¢-trimethoxyflavone (2),
and 3-hydroxy-1-(3,5-dimethoxy-4-hydroxyphenyl)propan-
1-one (12) exhibited effective cytotoxicities (ED50 values =
Flavones and Cytotoxic Constituents from Muntingia calabura J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 667

OCH3
OR
OH 2' 3' 4'
1' 5'
H3CO O 6'
8 1 OCH3
7 9 2
6 10 3
5 4

O
O 7 R=H
1 R = CH3
2 R=H R 8 R = OH

R4 2' 3' 4'

1' 5'
H3CO O 6'
8 1
7 9 2
6 10 3
5 4
R3 R1
OR2 O

3 R1 = R2 = R4 = H, R3 = OCH3
9
4 R1 = R3 = R4 = H, R2 = CH3 HO
5 R1 = OH, R2 = R4 = H, R3 = OCH3

O R2
OCH3
OCH3
OCH3 2' 3' 4'

H3CO O 1'
6'
5'
R1 OCH3
8 1 OH
7 9 2
6
OH
10 3
5 4

6 10 R1 = OCH3, R2 = OH
11 R1 = H, R2 = OH
12 R1 = OCH3, R2 = (CH2)2OH

O
H3CO (CH2)23CH3 CH3(CH2)nOH
O
14 n = 23
13 15 n = 25
HO

3.56, 3.71, and 3.27 mg/mL, respectively) against the P-388
cell line.
From the results of cytotoxic tests, four conclusions on
these isolates can be drawn as follows: (a) Among the fla-
vones, 1 and 2 (with 3¢,4¢,5¢-trioxygenated B-ring) exhibited
effective cytotoxicities against P-388 cell line, on the con-
trary, 3-5 (with unsubstituted B-ring) didn’t exhibit effective
cytotoxicities against the three cell lines. (b) Among the
7,8,3¢,4¢,5¢-pentaoxygenated flavonoids, flavones 1 and 2 ex-
hibited more potent cytotoxic activity than flavan compound
6 against the P-388 cell line. (c) Steroids (7-9) and long chain
alcohols (14 and 15) didn’t exhibit effective cytotoxicities
against the three cell lines. (d) Among the benzenoids (10-
13), only 12 exhibited effective cytotoxicity (ED50 value =
3.27 mg/mL) against the P-388 cell line.
EXPERIMENTAL SECTION
General Experimental Procedures
All melting points were determined on a Yanaco mi-
cro-melting point apparatus and were uncorrected. IR spectra
668 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004
Table 1. Cytotoxic Effects of Compounds Isolated from Muntingia calabura against P-388,
A549, and HT-29 Cell Lines
Compound
Mithramycin*
8-Hydroxy-7,3¢,4¢,5¢-tetramethoxyflavone (1)
8,4¢-Dihydroxy-7,3¢,5¢-trimethoxyflavone (2)
6,7-Dimethoxy-5-hydroxyflavone (3)
5,7-Dimethoxyflavone (4)
3,5-Dihydroxy-6,7-dimethoxyflavone (5)
(2S)-5¢-Hydroxy-7,8,3¢,4¢-tetramethoxyflavan (6)
b-Sitostenone (7)
6b-Hydroxystigmast-4-en-3-one (8)
b-Sitosterol (9)
Syringic acid (10)
Vanillic acid (11)
3-Hydroxy-1-(3,5-dimethoxy-4-hydroxyphenyl)propan-1-one (12)
Tetracosyl ferulate (13)
Mixture of 1-tetracosanol (14) and 1-hexacosanol (15)
* Mithramycin was used as a positive control.
(KBr or neat) were taken on a Perkin Elmer system 2000
FT-IR spectrometer. UV spectra were obtained on a Shimadzu
UV-160A spectrophotometer in EtOH. EI-mass spectra were
recorded on a VG Biotech Quattro 5022 spectrometer. HR-
EI, FAB, and HR-FAB-mass spectra were recorded on a
JEOL JMX-HX 110 spectrometer. NMR spectra including
COSY, NOESY, HMBC and HSQC experiments were re-
corded on a Varian Unity 400 and Varian Inova 500 spec-
trometer either at 400 or 500 MHz (1H), and chemical shifts
are given in ppm (d) with TMS as internal standard. Silica gel
(60-230, 230-400 mesh) (Merck) was used for CC and silica
gel 60 F-254 (Merck) for TLC and prep. TLC. Optical rota-
tions were measured using a Jasco DIP-370 polarimeter in
CHCl3.
Plant Material
The stem bark of M. calabura was collected from
Kaohsiung City, Taiwan, in June 2001 and identified by Dr. I.
S. Chen. A voucher specimen (Chen 6103) was deposited in
the herbarium of the School of Pharmacy, Kaohsiung Medi-
cal University, Kaohsiung, Taiwan, Republic of China.
Extraction and Isolation
The dried stem bark (9.7 kg) was extracted with MeOH
at room temperature, and the extract concentrated under re-
duced pressure. The MeOH extract (880 g), when partitioned
between H2O-CHCl3 (1:1), afforded a CHCl3-soluble fraction
(fr. A, 310 g). Part (95 g) of fr. A was chromatographed on sil-
Chen et al.
ED50 [mg mL-1]
P-388 A549 HT-29
0.06 0.07 0.08
3.56 > 50 > 50
3.71 > 50 > 50
> 50 > 50 > 50
> 50 > 50 > 50
9.39 > 50 > 50
6.28 16.81 26.60
> 50 > 50 > 50
> 50 > 50 > 50
> 50 > 50 > 50
10.72 > 50 > 50
15.62 > 50 > 50
3.27 > 50 > 50
> 50 > 50 > 50
> 50 > 50 > 50
ica gel (2900 g), eluting with n-hexane, gradually increasing
the polarity with EtOAc to obtain 13 frs: fr. A1 (1200 mL,
n-hexane), fr. A2~A4 (each 1200 mL, n-hexane-EtOAc,
20:1), fr. A5~A7 (each 1200 mL, n-hexane-EtOAc, 10:1), fr.
A8~A10 (each 1200 mL, n-hexane-EtOAc, 5:1), fr. A11
(2400 mL, EtOAc-MeOH, 1:1), fr. A12 (1200 mL, EtOAc),
fr. A13 (3000 mL, MeOH). Fr. A2 (3.4 g) was rechromato-
graphed on silica gel (85 g) eluting with n-hexane-Me 2 CO
(3:1) to obtain 6 frs (each 500 mL, fr. A2-1 – fr. A2-6). Fr.
A2-3 (195 mg) was further purified by preparative TLC
(n-hexane-EtOAc, 10:1) to obtain 7 (3.2 mg), mixture of 14
and 15 (2.7 mg). Fr. A4 (5.71 g) was rechromatographed on
silica gel (84 g) using n-hexane-EtOAc (8:1) as eluent to give
10 frs (each 1200 mL, fr. A4-1 – fr. A4-10). Fr. A4-5 (165
mg) was further purified by preparative TLC (CHCl 3 -
Me 2 CO, 20:1) to obtain 9 (85 mg) and 13 (6.8 mg). Fr. A6
(7.8 g) was rechromatographed on silica gel (82 g) using
CHCl3-Me2CO (10:1) as eluent to give 6 frs (each 1200 mL,
fr. A6-1 – fr. A6-6). Fr. A6-2 (225 mg) was further purified
by preparative TLC (n-hexane-EtOAc, 1:1) to obtain 3 (3.5
mg). Fr. A6-3 (154 mg) was further purified by preparative
TLC (CHCl3-Me2CO, 20:1) to obtain 5 (3.3 mg). Fr. A7 (8.6
g) was rechromatographed on silica gel (195 g) using
CHCl3-Me2CO (10:1) as eluent to give 10 frs (each 1500 mL,
fr. A7-1 – fr. A7-10). Fr. A7-6 (305 mg) was further purified
by preparative TLC (CH2Cl2-MeOH, 20:1) to obtain 6 (2.6
mg) and 8 (3.1 mg). Fr. A9 (6.7 g) was rechromatographed on
silica gel (220 g) using CHCl 3 -Me 2 CO (10:1) as eluent to
Flavones and Cytotoxic Constituents from Muntingia calabura
give 12 frs (each 1200 mL, fr. A9-1 – fr. A9-12). Fr. A9-6
(285 mg) was further purified by preparative TLC (CHCl3-
Me 2 CO, 20:1) to obtain 1 (3.8 mg), 2 (1.9 mg), and 4 (5.5
mg). Fr. A9-8 (317 mg) was further purified by preparative
TLC (CH2Cl2-MeOH, 20:1) to obtain 11 (3.4 mg) and 12 (2.8
mg). Fr. A9-10 (437 mg) was further purified by preparative
TLC (CH2Cl2-MeOH, 20:1) to obtain 10 (3.7 mg).
8-Hydroxy-7,3¢,4¢,5¢-tetramethoxyflavone (1)
Light yellow needles from CHCl 3 -MeOH, m.p. 209-
211 °C. UV: lmax (EtOH) nm (log e) = 210 (4.99), 271 (4.23),
312 (4.23). IR (KBr): u max = 3300 (br, OH), 1626 (C=O),
1596, 1504 cm -1 (aromatic ring C=C stretch). EI-MS: m/z
(rel. int.) = 358 (M+, 100), 343 (41), 313 (6), 287 (6), 192 (7),
177 (13), 167 (32), 95 (10), 81 (11), 69 (11). HR-FAB-MS:
C 19 H 19 O 7 , found: 359.1126 [M + H] + , calcd: 359.1131.
1
H-NMR (CDCl3, 500 MHz): d = 3.93 (3H, s, OMe-4¢), 3.97
(6H, s, OMe-3¢ and OMe-5¢), 4.05 (3H, s, OMe-7), 5.75 (1H,
br s, OH-8, exchangeable with D2O), 6.78 (1H, s, H-3), 7.05
(1H, d, J = 8.8, H-6), 7.22 (2H, s, H-2¢ and H-6¢), 7.79 (1H, d,
J = 8.8, H-5). 13 C-NMR (CDCl 3 , 125 MHz): d = 56.3
(OMe-3¢), 56.3 (OMe-5¢), 56.7 (OMe-7), 61.0 (OMe-4¢),
103.8 (C-2¢), 103.8 (C-6¢), 106.5 (C-3), 108.5 (C-6), 116.4
(C-5), 118.3 (C-10), 127.3 (C-1¢), 134.0 (C-8), 141.2 (C-4¢),
145.2 (C-9), 149.8 (C-7), 153.6 (C-3¢), 153.6 (C-5¢), 163.0
(C-2), 178.2 (C-4).
8,4¢-Dihydroxy-7,3¢,5¢-trimethoxyflavone (2)
Light yellow needles from CHCl 3 -MeOH, m.p. 203-
205 °C. UV: lmax (EtOH) nm (log e) = 211 (4.76), 270 (4.32),
310 (4.32). IR (KBr): u max = 3320 (br, OH), 1628 (C=O),
1598, 1502 cm -1 (aromatic ring C=C stretch). EI-MS: m/z
(rel. int.) = 344 (M + , 100), 329 (41), 299 (6), 273 (6), 181
(11), 178 (7), 163 (13), 153 (32), 81 (10), 67 (11), 55 (11).
HR-EI-MS: C18H16O7, found: 344.0894 [M]+, calcd: 344.0896.
1
H-NMR (CDCl 3 , 500 MHz): d = 3.96 (6H, s, OMe-3¢ and
OMe-5¢), 4.05 (3H, s, OMe-7), 5.80 (1H, br s, OH-8, ex-
changeable with D2O), 5.92 (1H, br s, OH-4¢, exchangeable
with D2O), 6.77 (1H, s, H-3), 7.04 (1H, d, J = 8.8, H-6), 7.21
(2H, s, H-2¢ and H-6¢), 7.78 (1H, d, J = 8.8, H-5). 13C-NMR
(CDCl3, 125 MHz): d = 56.2 (OMe-3¢), 56.2 (OMe-5¢), 56.5
(OMe-7), 104.1 (C-2¢), 104.1 (C-6¢), 106.7 (C-3), 108.3 (C-
6), 116.6 (C-5), 118.8 (C-10), 126.7 (C-1¢), 133.7 (C-8),
145.5 (C-9), 149.1 (C-4¢), 149.7 (C-7), 153.2 (C-3¢), 153.2
(C-5¢), 163.4 (C-2), 178.5 (C-4).
Cytotoxicity Assay
P-388 cells were kindly provided by Prof. J. M.
J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 669
Pezzuto, University of Illinois at Chicago; A549 (Human
lung adenocarcinoma) and HT-29 (human colon carcinoma)
were purchased from American Type Culture Collection.
P-388 cells were cultured in Fisher’s medium supple-
mented with 10% heat-inactivated (56 °C for 30 min) fetal
calf serum (FCS). A549 cells were cultured in Eagle Mini-
mum Essential Medium (EMEM) containing Earle’s salts and
supplemented with 0.1 mM of nonessential amino acids and
10% heat-inactivated FCS. HT-29 cells were maintained in
Rosewell Park Memorial Institute (RPMI) 1640 Medium
containing 10% heat-inactivated FCS. All cell lines were
maintained in an incubator at 37 °C in humidified air contain-
ing 5% CO2.
The cytotoxic activities of compounds against P-388,
A549, and HT-29 were assayed by a modification of the MTT
[3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro-
mide] colorimetric method. 15 For P-388 cells, 200 mL cul-
tures were established at 1500 cells/well in 96-well tissue
culture plates (Falcon). Compounds were dispensed to estab-
lished cultures at eight concentrations in triplicate. After
three days of incubation, P-388 cells were enumerated with
MTT.
To measure the cytotoxic activities of purified com-
pounds against A549 and HT-29 cells, each cell line was ini-
tiated at 1000 cells/well in 96-well microtiter plates. Eight
concentrations (triplicate) of test compounds encompassing a
128 fold range were added to each cell line. A549 and HT-29
cells were enumerated using MTT after exposure to test com-
pounds for 6 days, respectively. Fifty mL of 1 mg/mL MTT
were added to each well, and plates were incubated at 37 °C
for a further 5 h. Formazan crystals were redissolved in
DMSO (E. Merck) for 10 min with shaking, and the plate was
read immediately on a microtiter plate reader (Dynatech) at a
wavelength of 540 nm. The ED50 was defined as the concen-
tration of the test compound resulting in a 50% reduction of
absorbance compared to untreated cells in the MTT assay.
The above assays were repeated three times.
ACKNOWLEDGMENTS
This work was kindly supported by grants from the Na-
tional Science Council of the Republic of China (NSC 91-
2320-B-127-006) and Tajen Institute of Technology (Tajen-
91006).
Received November 10, 2003.
670 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004
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