Constituents of Chinese propolis and their antiproliferative activities

Article abstract of DOI:10.1021/np010486c

Two new flavonoids, 3-O-[(S)-2-methylbutyroyl] pinobanksin (1) and 6-cinnamylchrysin (2), were isolated from the EtOAc-soluble fraction of the MeOH extract of Chinese propolis, along with 12 known compounds (3-14). The structures of the isolated compounds were elucidated on the basis of spectroscopic and chemical analyses. The isolated compounds were tested for their antiproliferative activity toward five different cancer cell lines. Benzyl caffeate (13) and phenethyl caffeate (14) showed potent antiproliferative activity toward tested cell lines with a selective activity toward colon 26-L5 carcinoma cell line (EC₅₀ values: 13, 1.01; 14, 0.30 μM).

Full text of DOI:10.1021/np010486c

J . Nat. Prod. 2002, 65, 673-676
673
Con stitu en ts of Ch in ese P r op olis a n d Th eir An tip r olifer a tive Activities
Tepy Usia, Arjun H. Banskota, Yasuhiro Tezuka, Kiyoshi Midorikawa, Katsumichi Matsushige, and
Shigetoshi Kadota*
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630-Sugitani, Toyama 930-0194, J apan
Received October 3, 2001
Two new flavonoids, 3-O-[(S)-2-methylbutyroyl]pinobanksin (1) and 6-cinnamylchrysin (2), were isolated
from the EtOAc-soluble fraction of the MeOH extract of Chinese propolis, along with 12 known compounds
(3-14). The structures of the isolated compounds were elucidated on the basis of spectroscopic and chemical
analyses. The isolated compounds were tested for their antiproliferative activity toward five different
cancer cell lines. Benzyl caffeate (13) and phenethyl caffeate (14) showed potent antiproliferative activity
toward tested cell lines with a selective activity toward colon 26-L5 carcinoma cell line (EC50 values: 13,
1.01; 14, 0.30 µM).
In tr od u ction
new flavonoids and antiproliferative activities of the iso-
lated compounds toward murine B16-BL6 melanoma, hu-
man HT-1080 fibrosarcoma, human lung A549 adenocar-
cinoma, human cervix HeLa adenocarcinoma, and murine
colon 26-L5 carcinoma cell lines.
Propolis (sometimes referred to “bee glue”), a sticky
material that honeybees collect from buds and exudates of
plants, is used in the construction and adaptation of bee
hives. There is a long history of the use of propolis for
various purposes dating back to at least 300 BC,1 and even
now in the 21st century, it is used in home remedies and
personal products. Propolis has also gained popularity as
a health food in various parts of world, including the United
States, J apan, and the European Union, where it is claimed
to improve human health and prevent diseases such as
inflammation, heart disease, diabetes, and even cancer.
Several biological activities such as anticancer, antioxidant,
antiinflammatory, antiseptic, antimycotic, bacteriostatic,
astringent, choleric, spasmolytic, and anaesthetic proper-
ties have been reported for propolis and its constituents.2-4
Due to these purported beneficial effects, there is a renewal
of interest in the composition and biological activities of
propolis. The composition of propolis depends on the
vegetation of the area in which it is collected. Propolis from
Europe, North and South America, Asia, and Africa differ
in their composition because of the differences in the local
vegetation. More than 300 constituents have been identi-
fied so far from propolis, among which phenolic compounds
such as flavonoids and cinnamic acid derivatives have been
reported as major constituents of propolis from the temper-
ate zone.2,5
In our previous studies, we examined the ability of
Brazilian propolis to protect â-cells against the toxicity of
streptozotocin in rats6 and isolated four potent antihepa-
totoxic dicaffeoylquinic acids,7 a new prenylated chromane
derivative,8 and two novel benzofuran derivatives.9 In other
continuing work on propolis, we evaluated the quality of
propolis from Brazil, Peru, The Netherlands, and China,
based on the hepatoprotective, antiproliferative, and free
radical scavenging activities and LCMS analysis.10,11 In this
study, we observed that an extract of Chinese propolis
possessed potent antiproliferative activity.10 However,
there are only a few reports on the constituents of Chinese
propolis,12-15 and no report has been found on antiprolif-
erative constituents. Thus, we examined the constituents
of Chinese propolis and isolated two new flavonoids (1, 2),
together with 12 known compounds (3-14). In this paper,
we report the isolation and structure elucidation of the two
Resu lts a n d Discu ssion
Chinese propolis was successively extracted with H2O,
MeOH, and CHCl3. The MeOH extract, having the stron-
gest antiproliferative activity (EC50 values: 8.1 and 6.7
µg/mL against murine B16-BL6 melanoma and human
cervix HeLa adenocarcinoma cell lines, respectively), was
partitioned into EtOAc-soluble and EtOAc-insoluble frac-
tions. The EtOAc-soluble fraction (EC50
: 7.9 and 6.5
µg/mL against B16-BL6 and HeLa cell lines, respectively)
showed antiproliferative activity similar to the MeOH
extract and thus was subjected to further chemical analysis
to give two new flavonoids, namely, 3-O-[(S)-2-methylbu-
tyroyl]pinobanksin (1) and 6-cinnamylchrysin (2), together
with 12 known compounds: chrysin (3),16 galangin (4),17
izalpinin (5),18 apigenin (6),16 techtochrysin (7),19 pino-
strobin (8),20 pinocembrin (9),16 isoferulic acid (10),21 3,4-
dimethoxycinnamic acid (11),21 benzyl ferulate (12),2 benzyl
caffeate (13),22 and phenethyl caffeate (14).23 Among these,
compounds 11, 12, and 14 were found for the first time
from Chinese propolis.
3-O-[(S)-2-Methylbutyroyl]pinobanksin (1) was obtained
* To whom correspondence should be addressed. Tel: 81-76-434-7625.
Fax: 81-76-434-5059. E-mail: kadota@ms.toyama-mpu.ac.jp.
as a yellow amorphous solid, having [R]25 +24.5° (c 0.1,
D
10.1021/np010486c CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/18/2002

674 J ournal of Natural Products, 2002, Vol. 65, No. 5
Usia et al.
Ta ble 1. 1H and 13C NMR Data for Compounds 1 (in CDCl3) and 2 (in DMSO)a
1
2
position
δH
δC
HMBCa
3, 2′, 6′
2
2, 3
6, 8, 5-OH
6, 5-OH
8, 5-OH
6, 8
6
δH
δC
HMBCb
2
3
4
4a
5
6
7
8
8a
5-OH
7-OH
1′
2′, 6′
3′, 5′
4′
1′′
2′′
3′′
5.36 d (12.0)
5.85 d (12.0)
81.5
71.9
191.7
101.9
164.2
97.3
165.1
95.8
162.5
162.7
105.0
181.7
103.7
158.6
109.3
162.1
93.2
3, 2′, 6′
6.90 s
6.57 s
3
3, 8, 5-OH
1′′, 5-OH
1′′, 8, 5-OH
8, 1′′
6.04 d (2.2)
6.00 d (2.2)
11.49 br s
8
155.4
8
13.09 s
10.86 s
135.0
127.6
128.7
129.6
175.1
40.7
2, 3′, 5′
2, 3′, 4′, 5′
2′, 4′, 6′
2′, 3′, 5′, 6′
3, 2′′, 3′′, 5′′
3′′, 4′′, 5′′
2′′, 4′′, 5′′
130.7
126.1
128.8
131.5
25.1
3, 3′, 5′
3′, 4′, 5′
2′, 4′, 6′
2′, 3′, 5′, 6′
3′′
7.47 m
7.41 m
7.39 m
8.03 d (6.4)
7.56 m
7.56 m
3.45 d (5.1)
6.31 dt (16.1, 5.1)
6.37 d (16.1)
2.36 qt (7.1, 6.9)
1.29 dqd (7.6, 14.3, 14.3)
1.47 dqd (7.6, 14.3, 14.3)
0.60 t (7.6)
127.5
129.4
1′′, 3′′
1′′, 5′′, 9′′
26.4
4′′
5′′
6′′
7′′
8′′
9′′
10.9
16.4
2′′, 3′′
2′′, 3′′
137.1
125.5
128.2
126.5
128.2
125.5
2′′, 6′′, 8′′
3′′, 7′′
5′′, 7′′
5′′, 9′′
5′′, 7′′
1.05 d (6.8)
7.31 d (7.3)
7.25 dd (7.6, 7.3)
7.15 br t (7.6)
7.25 dd (7.6, 7.3)
7.31 d (7.3)
3′′, 7′′
a
The 1H and 13C NMR spectra were measured at 100 and 400 MHz, respectively, and coupling constants (parentheses) are in Hz. b 1H
correlating with 13C resonance.
Ta ble 2. Antiproliferative Activity of the Isolated Compounds
(EC50 Values in µΜ)
CHCl3) and molecular formula C20H20O6 (HRFABMS). The
IR spectrum of 1 shows absorption bands corresponding
to hydroxyl (3350 cm-1) and ester carbonyl (1735 cm-1
)
colon
compound
B16-BL6 HT-1080
A549
160
>270
233
93.6
>350
101
>370
>370
229
>515
>480
92.8
35.0
27.9
3.61
0.04
HeLa
116
>270
111
79.0
106
92.7
>370
202
108
>515
>480
51.1
2.33
2.36
0.23
0.02
26-L5
groups. The 1H NMR spectrum of 1 (Table 1) displays
signals for a monosubstituted benzene ring, meta-coupled
aromatic protons (J ) 2.2 Hz), vicinally coupled oxygenated
methines (J ) 12.0 Hz), and a 1-methylpropyl group,
together with a signal for a hydrogen-bonded phenolic
proton (δ 11.49, s). The 13C NMR spectrum shows 20 carbon
signals including those corresponding to the above groups,
as well as ketone (δ 191.7) and ester (δ 175.1) carbonyl
carbons (Table 1). These data suggested 1 is a flavonol
having a 2-methylbutyroyl group. The analyses of the
COSY, HMQC, and HMBC spectra (Table 1) indicated the
flavonol to be 3,5,7-trihydroxyflavonol (pinobanksin).24 The
location of the 2-methylbutyroyl group was determined to
be at C-3, on the basis of a low-field shift of H-3 (1, δ 5.85;
pinobanksin,24 δ 4.54) and the HMBC correlation between
the ester carbonyl carbon (δ 175.1) and the protons H-3,
H-2′′, H-3′′, and H-5′′. Alkaline hydrolysis of 1 with 1 N
KOH gave 2-methylbutyric acid and pinobanksin. The
absolute configuration of pinobanksin was determined to
1
2
3
4
5
6
7
8
185
143
78.9
>270
109
19.2
30.3
25.0
273
>270
>270
74.4
26.8
>350
31.6
226
111
68.0
94.9
35.7
>350
36.6
358
156
92.4
128
9
75.6
>515
>480
46.4
10
11
12
13
14
>515
>480
>515
>480
63.7
9.78
46.7
9.74
1.01
6.79
1.23
0.11
9.50
1.38
0.02
0.30
0.31
0.02
5-fluorouracil
doxorubicin HCl
analyses of the COSY, HMQC, and HMBC spectra indi-
cated 2 to be a chrysin derivative having a cinnamyl group
at C-6 or C-8. The location of the cinnamyl group was
determined to be at C-6 from the HMBC correlation of H-1′′
with C-5, C-6, and C-7. Thus, compound 2 was determined
to be 6-cinnamylchrysin. Cinnamic acid and its derivatives
are widely distributed in the plant kingdom, and there are
several reports of the attachment of the cinnamyl group
with other classes of compounds.5,25,26 However, this is the
first report of a flavone with a cinnamyl moiety from
propolis.
The isolated compounds were tested for their antipro-
liferative activity toward murine B16-BL6 melanoma,
human HT-1080 fibrosarcoma, human lung A549 adeno-
carcinoma, human cervix HeLa adenocarcinoma, and mu-
rine colon 26-L5 carcinoma cell lines (Table 2). Benzyl
caffeate (13) and phenethyl caffeate (14) showed potent
antiproliferative activity toward all cell lines, especially
toward colon 26-L5 carcinoma cell line (EC50 values: 13,
1.01; 14, 0.30 µM), with EC50 values identical to that of
5-fluorouracil. Isoferulic acid (10) and 3,4-dimethoxycin-
be 2R,3R on the basis of the [R]25 value (+16.5°, MeOH;
D
lit.24 [R]D +15°, MeOH). On the other hand, the absolute
configuration of 2-methylbutyric acid was determined to
be S by GC analysis of its methyl ester with a chiral
column. Thus, compound 1 was identified as 3-O-[(S)-2-
methylbutyroyl]pinobanksin. To the best of our knowledge,
this is the first report of the presence of a (S)-2-methylbu-
tyroyl group in flavonoids.
6-Cinnamylchrysin (2) was isolated as a yellow amor-
phous solid with the molecular formula C24H18O4, and its
IR spectrum shows the presence of hydroxyl (3125 cm-1
)
and carbonyl (1640 cm-1) groups. The H NMR spectrum
of 2 shows signals for two phenyl groups, two isolated
olefins, a trans-olefin (J ) 16.1 Hz) coupled with a
methylene, and two phenolic protons (Table 1). The 13C
NMR spectrum shows 24 carbon signals including a car-
bonyl carbon signal (δ 181.7) (Table 1). These data and
1

Constituents of Chinese Propolis
J ournal of Natural Products, 2002, Vol. 65, No. 5 675
F igu r e 1. DPPH free radical scavenging activity of isolated compounds.
portion was separated by filtration. The filtrate, on cooling,
gave a fatty substance (145 g), and the remaining filtrate was
lyophilized to give a H2O extract (12.5 g). On the other hand,
the residue was further extracted with MeOH (3 L, reflux, 2
h, ×2) and then by CHCl3 (2 L, room temperature, ×2) to yield
a MeOH extract (220 g) and a CHCl3 extract (123 g), respec-
tively. The MeOH extract (120 g) was further fractionated into
EtOAc-soluble (115 g) and EtOAc-insoluble fractions (3.2 g).
The EtOAc-soluble fraction (90.0 g) was subjected to silica gel
column chromatography with a CHCl3-MeOH gradient sys-
tem to afford eight fractions: fraction 1: CHCl3 eluate, 4.4 g;
fraction 2: 1% MeOH-CHCl3 eluate, 4.5 g; fraction 3: 2%
MeOH-CHCl3 eluate, 7.8 g; fraction 4: 3% MeOH-CHCl3
eluate, 10.8 g; fraction 5: 5% MeOH-CHCl3 eluate, 3.7 g;
fraction 6: 10% MeOH-CHCl3 eluate, 11.5 g; fraction 7: 20%
MeOH-CHCl3 eluate, 22.3 g; fraction 8: 30% MeOH-CHCl3
eluate, 25.5 g. Repeated column chromatography of these
fractions over silica gel, followed by preparative TLC, yielded
the following compounds: fraction 1: 5 (33.0 mg), 7 (37.0 mg),
and 8 (614 mg); fraction 2: 7 (168 mg) and 12 (740 mg);
fraction 3: 1 (8.8 mg), 2 (30.5 mg), and 9 (57.8 mg); fraction 4:
3 (110 mg), 4 (333 mg), 11 (306 mg), and a mixture of 4 and 9
(550 mg); fraction 5: 3 (3.5 g); fraction 6: 3 (65.0 mg), 13 (9.9
mg), and 14 (27.3 mg); fraction 7: 6 (15.6 mg) and 10 (33.4
mg); fraction 8: 3 (120 mg) and 6 (12.5 mg).
namic acid (11), on the other hand, had no antiproliferative
effect, indicating that the ester moiety is important for
antiproliferative activity. The hydroxyl groups at the caffeic
acid moiety might also contribute to the antiproliferative
activity, since it was observed that benzyl ferulate (12),
which has a methoxy group at C-3, showed weaker anti-
proliferative activity. Among the flavonoids, galangin (4)
and apigenin (6) showed moderate antiproliferative activi-
ties toward B16-BL6, HT-1080, and colon 26-L5 cell lines,
with EC50 values less than 40 µM. The remaining com-
pounds, including both of the new flavonoids, possessed
only weak or no antiproliferative activities. Phenethyl
caffeate (14), also called CAPE, has been reported to have
strong antitumor activity both in vitro and in vivo.23
Moreover, CAPE (14) has been reported to have strong
antioxidative activities,27,28 which seem to correspond to
its antiproliferative activity. Thus, as a measure of anti-
oxidative effect, we also examined the 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging activities of 4,
6, 12, 13, and 14 (Figure 1), which showed a range of
antiproliferative activities from mild to potent. Interest-
ingly, 13 and 14, both of which show potent antiprolifera-
tive activities, also strongly scavenge DPPH free radical
(Figure 1). Thus, it appears that the antiproliferative
activities of these caffeic acid derivatives may also be
associated with their antioxidative properties.
3-O-[(S)-2-Meth ylbu tyr oyl]pin oban ksin (1): yellow amor-
phous solid; [R]25D +24.5° (c 0.1, CHCl3); IR (CHCl3) νmax 3350,
1735, 1635, 1460, 1145, 1085 cm-1; HRFABMS m/z 357.1328
(calcd for C20H21O6 [M + H]+, 357.1341); 1H and 13C NMR, see
Table 1.
Exp er im en ta l Section
6-Cin n a m ylch r ysin (2): yellow amorphous solid; IR (KBr)
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured on a J ASCO DIP-140 digital polarimeter. IR
spectra were measured with a Shimadzu IR-408 spectropho-
tometer in a KBr disk or in CHCl3 solution. HRFABMS
measurements were performed on a J EOL J MS-700T spec-
trometer, and glycerol was used as a matrix. Gas chromatog-
raphy was done on a Shimadzu GC-14AH gas chromatograph
using a Chiraldex G-TA column with nitrogen as carrier gas.
UV spectra were recorded on a Shimadzu UV-160A UV-visible
spectrophotometer. The 1H, 13C, and 2D NMR spectra were
taken on a J EOL J NM LA-400 spectrometer with tetrameth-
ylsilane (TMS) as an internal standard. Column chromatog-
raphy was performed with silica gel 60 (Nacalai tesque, Inc.,
Kyoto, J apan). Analytical and preparative TLC was conducted
on precoated Merck Kieselgel 60F254 and RP-18F254 plates (0.25
or 0.50 mm thickness).
Biologica l Ma ter ia l. Propolis used in this study was a gift
from Saitama Bee-Keeping Co., Ltd. A voucher sample (TMPW
20925) is preserved in the Museum for Materia Medica,
Analytical Research Center for Ethnomedicines, Institute of
Natural Medicine, Toyama Medical and Pharmaceutical Uni-
versity, Toyama, J apan.
Extr a ction a n d Isola tion . Chinese propolis (834 g) was
extracted with H2O (3 L, 2 h, 80 °C, ×2), and the insoluble
ν
max 3125, 1640, 1610, 1580, 1350, 1300, 1240, 1175, 1090 cm-1
;
HRFABMS m/z 371.1326 (calcd for
C
24H19O4 [M + H]+,
371.1343); H and 13C NMR, see Table 1.
1
Alk a lin e Hyd r olysis of Com p ou n d 1. A solution of 1 (2.0
mg) in MeOH (1.0 mL) and 1 N KOH (1.0 mL) was stirred
overnight at room temperature. The reaction mixture was
neutralized with 1 N HCl and extracted with EtOAc (10 mL).
The EtOAc layer was evaporated, and the residue was dis-
solved in MeOH (1.0 mL) and treated with excess CH2N2. After
evaporation, the residue was dissolved in CHCl3 (5.0 mL) and
filtrated. An aliquot of the filtrate was analyzed by GC
(column, Astec Chiraldex G-TA G0012-08, 30 m × 0.25 mm;
column temperature, 50 °C; detector temperature, 250 °C;
injection temperature, 250 °C), to give a peak at tR 11.05.
Standard (R)- and (S)-2-methylbutyrate gave one peak at tR
9.99 and 10.92 min, respectively. The remaining filtrate was
subjected to preparative TLC with 1% MeOH-CHCl3 to give
pinobanksin (0.2 mg) ([R]25 +16.5°, c 0.01, MeOH).
D
An tip r olifer a tive Assa y. All the cancer cell lines were
maintained in Eagle’s minimum essential medium (Nissui
Pharmaceutical Co., Ltd., Tokyo, J apan), except for murine
colon 26-L5 carcinoma cell line, which was maintained in
RPMI (Nissui Pharmaceutical Co., Ltd., Tokyo, J apan). Both
of these media were supplemented with 10% fetal calf serum

676 J ournal of Natural Products, 2002, Vol. 65, No. 5
Usia et al.
(7) Basnet, P.; Matsushige, K.; Hase, K.; Kadota, S.; Namba, T. Biol.
Pharm. Bull. 1996, 19, 1479-1484.
(Gibco BRL Products, Gaithersburg, MD), 0.1% sodium bicar-
bonate (Nacalai tesque Inc., Kyoto, Japan), and 2 mM glutamine
(Wako Pure Chemicals Ind., Ltd., Osaka, J apan).
(8) Banskota, A. H.; Tezuka, Y.; Prasain, J . K.; Matsushige, K.; Saiki,
I.; Kadota, S. J . Nat. Prod. 1998, 61, 896-900.
Cellular viability in the presence and absence of experi-
mental agents was determined using the standard 3-(4,5-
dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT;
Sigma, St. Louis, MO) assays as described previously.8,29-31
Briefly, exponentially growing cells were harvested, and a 100
µL suspension containing 2500 cells was plated in 96-well
microtiter plates (Corning Incorporated, Costar, 3595). After
24 h of incubation at 37 °C under 5% CO2 to allow cell
attachment, the cells were treated with varying concentrations
of test specimens in their respective medium (100 µL) and
incubated for 72 h under the same conditions as above. After
adding a solution of MTT for 2 h, the amount of formazan
formed was measured spectrophotometrically at 550 nm using
a Perkin-Elmer HTS-7000 Bio Assay Reader. Test specimens
were dissolved in DMSO and then diluted by medium. DMSO
less than 0.1% in the test solution had no effect. 5-Fluorouracil
(Tokyo Kasei Kogyo Co., Ltd., Tokyo, J apan) and doxorubicin
HCl (Kyowa Hakko Co., Ltd., Tokyo, J apan) were used as
positive controls, and EC50 values were calculated from the
mean values of data from four wells.
DP P H Ra d ica l Sca ven gin g Assa y. DPPH radical scav-
enging activity was measured according to the procedure
described previously.31 Briefly, samples dissolved in EtOH (500
µL) were mixed with an equal volume of DPPH solution (60
mM). The resulting solution was thoroughly mixed by vortex,
and absorbance was measured at 520 nm after 30 min. The
scavenging activity was determined by comparing the absor-
bance with that of the blank (100%) containing only DPPH
and solvent.
(9) Banskota, A. H.; Tezuka, Y.; Midorikawa, K.; Matsushige, K.; Kadota,
S. J . Nat. Prod. 2000, 63, 1277-1279.
(10) Banskota, A. H.; Tezuka, Y.; Adnyana, I. K.; Midorikawa, K.;
Matsushige, K.; Message, D.; Huertas, A. A. G.; Kadota, S. J .
Ethnopharmacol. 2000, 72, 239-246.
(11) Midorikawa, K.; Banskota, A. H.; Tezuka, Y.; Nagaoka, T.; Mat-
sushige, K.; Message, D.; Huertas, A. A. G.; Kadota, S. Phytochem.
Anal. 2001, 12, 366-373.
(12) Chi, J . P.; Chen, H. S.; Xue, B. W. Acta Pharm. Sinica 1996, 31, 558-
560.
(13) Qiao, Z.; Chen, R. Zhongguo Zhongyao Zazhi 1991, 16, 481-482.
(14) Zhou, L. D.; Guo, J .; Yu, J . G. Zhongguo Zhongyao Zazhi 1999, 24,
162-164.
(15) Chi, J . P.; Xue, B. W.; Chen, H. S. Chin. Pharm. J . 1996, 31, 264-
266.
(16) Wagner, H.; Chari, V. M. Tetrahedron Lett. 1976, 21, 1799-1802.
(17) Wenkert, E.; Gottlieb, H. E. Phytochemistry 1977, 16, 1811-1816.
(18) Garcia-Viguera, C.; Greenaway, W.; Whatley, F. R. Z. Naturforsch.
1992, 47c, 634-637.
(19) Gaydou, E. M.; Bianchini, J . P. Bull. Soc. Chim. Fr. 1978, II, 43-47.
(20) Gonzalez, A. G.; Aguiar, Z. E.; Luis, J . G.; Ravelo, A. G.; Vazquez, J .
T.; Dominguez, X. A. Phytochemistry 1989, 28, 2871-2872.
(21) Kelley, C. J .; Harruff, R. C.; Carmack, M. J . Org. Chem. 1976, 41,
449-455.
(22) Yamauchi, R.; Kato, K.; Oida, S.; Kanaeda, J .; Ueno, Y. Biosci. Biotech.
Biochem. 1992, 56, 1321-1322.
(23) Grunberger, D.; Banerjee, R.; Eisinger, K.; Oltz, E. M.; Efros, L.;
Caldwell, M.; Estevez, V.; Nakanishi, K. Experientia 1988, 44, 230-
232.
(24) Buckingham, J . Dictionary of Natural Products; Chapman & Hall:
London, 1994, Vol. 5, p 5764.
(25) Gregson, M.; Ollis, W. D.; Sutherland, I. O.; Gottlieb, O. R.; Magal-
haes, M. T. Phytochemistry 1978, 17, 1375-1377.
(26) Carlson, R. E.; Dolphin, D. H. Phytochemistry 1982, 21, 1733-1736.
(27) Natarajan, K.; Singh, S.; Burke, T. R.; Grunberger, D.; Aggarwal, B.
B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 9090-9095.
(28) Mirzoeva, O. K.; Grishanin, R. N.; Calder, P. C. Microbiol. Res. 1997,
152, 239-246.
Refer en ces a n d Notes
(29) Goren, N.; Woerdenbag, H. J .; Bozok-J ohansson, C. Planta Med. 1996,
62, 419-422.
(1) Ghisalberti, E. L. Bee World 1979, 60, 59-84.
(2) Marcucci, M. C. Apidologie 1995, 26, 83-99.
(3) Burdock, G. A. Food Chem. Toxicol. 1998, 36, 347-363.
(4) Banskota, A. H.; Tezuka, Y.; Kadota, S. Phytother. Res. 2001, 15, 561-
571.
(5) Greenaway, W.; May, J .; Scaysbrook, T.; Whatley, F. R. Z. Naturfor-
sch. 1991, 46c, 111-121.
(6) Matsushige, K.; Basnet, P.; Hase, K.; Kadota, S.; Tanaka, S.; Namba,
T. Phytomedicine 1996, 3, 203-209.
(30) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.; Tosini,
S.; Skehan, P.; Scudiero, D. A.; Monks, A.; Boyd, M. R. J . Natl. Cancer
Inst. 1990, 82, 1113-1118.
(31) Hatano, T.; Edamatsu, R.; Hiramatsu, M.; Mori, A.; Fujita, Y.;
Yasuhara, T.; Yoshida, T.; Okuda, T. Chem. Pharm. Bull. 1989, 37,
2016-2021.
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