Chemical constituents of Brazilian propolis and their cytotoxic activities

Article abstract of DOI:10.1021/np980028c

The EtOAc-soluble fraction of the MeOH extract of propalia afforded a new prenylated chromane derivative, 3-hydroxy-2,2-dimethyl-8-prenylchromane- 6-propenoic acid (1), along with 22 known compounds, 2-23. Of the known compounds, 4, 7, 12-19, and 22 were isolated for the first time from propolis, and the absolute configuration of 23 was established as (2S,3R). Investigation suggested that Baccharis spp. are a significant source of tropical Brazilian propalia, in addition to Clusia minor, Clusia major, and Araucaria heterophylla. All the compounds were tested for their cytotoxicity toward human HT-1080 fibrosarcoma and murine colon 26-L5 carcinoma cells. Among these compounds, 9 and 19-21 showed potent cytotoxicity, having ED₅₀ values equal to or less than 10 μg/mL.

Full text of DOI:10.1021/np980028c

8
96
J . Nat. Prod. 1998, 61, 896-900
Ch em ica l Con stitu en ts of Br a zilia n P r op olis a n d Th eir Cytotoxic Activities
†
†
†
†
‡
Arjun H. Banskota, Yasuhiro Tezuka, J eevan K. Prasain, Katsumichi Matsushige, Ikuo Saiki, and
Shigetoshi Kadota*
,
†
Department of Natural Products Chemistry and Department of Pathogenic Biochemistry, Research Institute for
Wakan-Yaku (Traditional Sino-J apanese Medicines), Toyama Medical and Pharmaceutical University,
2
630-Sugitani, Toyama 930-01, J apan
Received J anuary 29, 1998
The EtOAc-soluble fraction of the MeOH extract of propolis afforded a new prenylated chromane
derivative, 3-hydroxy-2,2-dimethyl-8-prenylchromane-6-propenoic acid (1), along with 22 known
compounds, 2-23. Of the known compounds, 4, 7, 12-19, and 22 were isolated for the first
time from propolis, and the absolute configuration of 23 was established as (2S,3R).
Investigation suggested that Baccharis spp. are a significant source of tropical Brazilian
propolis, in addition to Clusia minor, Clusia major, and Araucaria heterophylla. All the
compounds were tested for their cytotoxicity toward human HT-1080 fibrosarcoma and murine
colon 26-L5 carcinoma cells. Among these compounds, 9 and 19-21 showed potent cytotoxicity,
having ED50 values equal to or less than 10 µg/mL.
Propolis is a resinous hive product collected by
honeybees from parts of plants, buds, and exudates and
has been used as a folk medicine since around 300 BC.
Resu lts a n d Discu ssion
The methanolic extract of propolis showed compara-
tively strong cytotoxicity toward HT-1080 and colon 26-
L5 tumor cells. It was further partitioned into EtOAc-
soluble and -insoluble fractions. The EtOAc-soluble
portion had the strongest cytotoxicity and was thus
subjected to further investigation. After repeated col-
umn chromatography and preparative TLC on Si gel,
the fraction afforded a new prenylated chromane de-
rivative (1) along with 22 known compounds: 2,2-
dimethyl-8-prenylchromene-6-propenoic acid (2); 2,2-
1
Various biological activities, such as anticancer, anti-
oxidant, antiinflammatory, antibiotic, and antifungal
effects, have been reported for propolis and its constitu-
ents.2 Recently, propolis has also been extensively used
in food and beverages to improve health and prevent
diseases such as inflammation, heart disease, diabetes,
3
and even cancer. Because of its wide range of biological
activities and its use as a health food, there is a renewed
interest in the composition of propolis. Propolis pos-
sesses a pleasant aromatic smell and varies in color
depending on its source and age. The composition of
the propolis depends on the place and time of collection.
As a consequence, more than 160 constituents have been
identified so far, among which phenolic compounds,
dimethylchromene-6-propenoic
dimethylchromene-6-carboxylic acid (4); artepillin (5);
acid
(3);7
2,2-
8
9
9
4
-dihydrocinnamoyloxy-3-prenylcinnamic acid (6); 4-hy-
1
0
droxy-3-prenylcinnamic acid (7); vanillin (8); coniferyl
1
1
12
aldehyde (9); isocupressic acid (10); 15-acetoxyiso-
1
2
13
cupressic acid (11); agathic acid (12); agathic acid
1
3
14
1
5-methyl ester (13); agathalic acid (14); cupressic
including flavonoids, are major constituents (more than
1
5
16
acid (15); tremetone (16); viscidone (17); 12-acetox-
3
5
0% of the weight of propolis). We evaluated the
yviscidone (18); betuletol (19); kaempferide (20);19
1
7
18
quality of propolis collected at different places in Brazil
20
20
ermanin (21); 3,5,7-trihydroxy-4′-methoxyflavanol (22);
4
based on free radical scavenging activity, examined the
21
and dimeric coniferyl acetate (23). To the best of our
knowledge, compounds 4, 7, 12-19, and 22 were
isolated for the first time from propolis.
5
â-cell protective effect against streptozotocin in rats,
and isolated four potent antihepatotoxic dicaffeoyl qui-
nic acids.6 In our continuing study on Brazilian pro-
polis, guided by tumor cell growth inhibition assay, we
have now isolated a new prenylated chromane deriva-
tive (1), together with 22 known compounds (2-23). The
structures of these compounds were elucidated by
spectroscopic analysis and chemical transformation. In
this paper, we wish to report the isolation of these
compounds from propolis and their cytotoxic activity
toward human HT-1080 fibrosarcoma and murine colon
Dimeric coniferyl acetate (23), which was previously
21
22
reported from Lasiolaena morii, Baccharis spp., and
23
propolis, is a simple diacetate derivative of dihydrodi-
coniferyl alcohol (DCA), an important intermediate of
lignin formation in woody plants. These reports con-
firmed only the relative configuration (2,3-trans) of 23
on the basis of coupling constant between H-2 and H-3
protons (J ) 7.4 Hz) and of NOE experiments. We
determined the absolute configuration of 23 through
alkaline hydrolysis to DCA. A previous report con-
firmed the absolute configuration of DCA, according to
which the [R]D value of two possible trans isomers, [i.e.,
2
6-L5 carcinoma.
*
To whom correspondence should be addressed. Toyama Medical
(2S,3R)-(+)-DCA and (2R,3S)-(-)-DCA] are +63.3° (c
and Pharmaceutical University, 2630-Sugitani, Toyama 930-01, J apan.
24
2
.1, Me2CO) and -54.1° (c 2.7, Me2CO), respectively.
Tel.: +81-764-34-2281(2825). Fax: +81-764-34-5059.
†
The [R]D value +38.74° (c 0.12, Me2CO) of the hydro-
lyzed product of 23 indicated it should be (2S,3R)-(+)-
Department of Natural Products Chemistry.
Department of Pathogenic Biochemistry.
‡
S0163-3864(98)00028-7 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/12/1998

Cytotoxicity of Brazilian Propolis
J ournal of Natural Products, 1998, Vol. 61, No. 7 897
1
13
were determined by 2D NMR including H- C long-
range COSY spectrum (Figure 1). Based on these
evidences, the structure of 1 was concluded to be
3
-hydroxy-2,2-dimethyl-8-prenylchromane-6-propenoic
acid.
There are many investigations on the origin of pro-
polis from temperate zones, and almost always poplar
buds, especially Populus nigra, appeared to be the
2
5
dominant source of propolis. But little work has been
done in connection with the source of tropical propolis
(including Brazilian), even though it has biological
activities similar to those of propolis found in temperate
zones. There are no poplar buds in tropical areas. The
resins exuded by the flowers of Clusia minor and Clusia
major (Guttiferae) were previously reported as the
dominant sources of tropical Venezuelan propolis, by the
occurrence of polyprenylated benzophenones as major
2
6
components.
Araucaria heterophylla (Compositae),
rich in labdane diterpenes, is also reported as a possible
source of Brazilian propolis on consideration of isolated
3
labdane diterpenes. In the present study, we isolated
prenylated compounds, labdane diterpenes, and others.
Among them, 3, 5, 7, 16-18, 23, and methyl ester of 6
were previously reported from different Baccharis spp.
of Compositae grown in the tropical South American
1
0,27-31
zone.
Further, Baccharis spp. are also a rich
source of various diterpenes, including labdane-type and
1
0,27-31
prenylated compounds.
It thus appears that
probable sources of Brazilian propolis would be Bac-
charis spp., in addition to C. minor, C. major and A.
heterophylla.
All the isolated compounds were tested against hu-
man HT-1080 fibrosarcoma and murine colon 26-L5
carcinoma for their in vitro cytotoxicity, and their ED50
values are given in Table 2 along with those of the
extracts. Only 9 and 19-21, showed potent cytotoxic
activity, having ED50 values equal to or less than 10
µg/mL. Compounds 2, 5, 17, and 22 have ED50 values
less than 50 µg/mL in fibrosarcoma, whereas in carci-
noma they showed less cytotoxic activity. Not only
these, but the other compounds isolated from propolis,
except 10 and 19, were found to be more active toward
human fibrosarcoma than toward murine carcinoma.
Neolignan 23 has moderate activity in both cell lines,
with ED50 values of nearly 40 µg/mL. Compounds 1,
DCA. The above result suggests the absolute configu-
ration of 23 to be (2S,3R).
1
0-13, and 15-16 have ED50 values less than 100 µg/
Compound 1 was obtained as a colorless amorphous
powder having UV maxima at 320 and 245 nm. It was
confirmed to be a racemic mixture because the methyl
ester of 1 gave the same mixture of (R)- and (S)-MTPA
esters. The molecular formula of 1 was found to be
C19H24O4 by HREIMS measurement, and the IR spec-
trum showed the presence of hydroxyl and carboxylic
acid groups (3400 and 1675 cm ). The H and C NMR
spectra of 1 were similar to those of 2 and 3 (Table 1)
and showed the signals of meta-coupled benzene protons
mL. The rest of the compounds showed weak cytotoxic
activity, having ED50 values greater than 100 µg/mL.
According to previous studies, the constituents show-
ing strong anticancer activity from propolis are caffeic
acid derivatives, flavonoids, 2,2-dimethyl-8-prenyl-
chromene-6-propenoic acid (2), artepillin (5), and 17-
2
,32-34
-1
1
13
hydroxycleroda-3,(13Z)-dien-15-oic acid.
Even
though the clerodane diterpene was reported to have
potent cytotoxic activity against human hepatocellular
carcinoma HuH13, lung carcinoma HLC-2, HeLa, KB,
(δ 7.19, 7.11), two tertiary methyls (δ 1.36, 1.32), a
3
4
and rat W3Y cells, the labdane diterpenoids 10-15,
showed weak cytotoxicity against HT-1080 and colon 26-
L5 cells. These results indicate that the anticancer
activity of Brazilian propolis is primarily due to the
phenolic compounds, which are also responsible for
3
-methyl-2-butenyl group, and a propenoic acid group.
Protons of one methylene (δ 3.06, 2.77) and one hydroxy
methine (δ 3.82) group were observed, however, in the
1
H NMR spectrum, instead of the olefinic protons in 2
and 3, suggesting a chromane skeleton. The position
of the 3-methyl-2-butenyl and propenoic acid groups
1
,2
other biological activities.

8
98 J ournal of Natural Products, 1998, Vol. 61, No. 7
Banskota et al.
Ta ble 1. 1H and 13C NMR Data of Compounds 1-3 (CDCl3)a
1
2
3
1H
13C
1H
13C
1H
13C
2
3
4
76.5 s
69.5 d
31.4 t
76.2 s
131.0 d
121.0 d
77.2 s
131.4 d
121.7 d
3.82 dd (5.5, 5)
3.06 dd (17, 5.5)
5.64 d (10)
6.31 d (10)
5.67 d (10)
6.29 d (10)
2
.77 dd (17, 5)
4
5
6
7
8
8
9
0
1
2
3
1
2
3
4
5
a
a
119.0 s
128.6 d
126.3 s
127.8 d
130.7 s
153.2 s
147.2 d
114.2 d
172.4 s
25.1 q
22.1 q
28.4 t
122.0 d
132.8 s
25.8 q
122.1 s
129.8 d
126.4 s
124.4 d
129.9 s
153.3 s
147.1 d
114.8 d
172.8 s
28.2 q
28.2 q
28.1 t
122.1
132.7 s
25.8 q
126.9 s
126.4 d
121.4 s
129.9 d
114.5 d
155.6 s
146.8 d
116.9 d
172.5 s
28.3 q
7.11 d (2)
7.19 d (2)
7.03 d (2)
7.18 d (2)
7.13 d (2)
7.32 dd (9, 2)
6.76 d (9)
7.67 d (16)
6.26 d (16)
7.67 d (16)
6.27 d (16)
7.69 d (16)
6.29 d (16)
1
1
1
1
1.36 s
1.32 s
3.27 br t (6.5)
5.25 tqq (7.5, 1.5)
1.43 s
1.43 s
3.26 br t (6.5)
5.26 tqq (7.5, 1.5)
1.45 s
1.45 s
28.3 q
′
′
′
′
′
1.74 s
1.72 s
1.74 s
1.73 s
17.9 q
17.9 q
a
Chemical shifts (δ) are in ppm with coupling constants (J in Hz) in parentheses.
and IR specta were measured with a Shimadzu IR-408
spectrophotometer in CHCl3 solution. EIMS and HRE-
IMS measurements were performed on J EOL D-300
spectrometer using a direct inlet system at the ioniza-
1
13
tion voltage of 70 eV. H, C, and 2D NMR were
obtained on a J EOL GX-400 spectrometer with tetra-
methylsilane (TMS) as an internal standard.
Biologica l Ma ter ia l. Propolis (Yukari propolis) was
collected in Brazil in 1995, and a voucher sample (P-2)
is preserved in our laboratory.
F igu r e 1. Significant correlations observed in the long-range
H- C COSY spectrum of 1.
1
13
Extr a ction a n d Isola tion . Brazilian propolis (1.8
kg) was treated with distilled H2O (2 L × 2) for 2 h at
Ta ble 2. Cytotoxicity of Propolis Extracts and the Compounds
Isolated from the EtOAc-soluble Portion of MeOH Extract
8
0 °C, and the insoluble portion was separated by
(
ED50 values; µg/mL)a
filtration. The filtrate was partially evaporated and
lyophilized to give a H2O extract (130 g), while the
insoluble portion was extracted with MeOH (2 L, reflux,
ext./compds.
HT-1080
colon L5-26
H2O extr a ct
MeOH extr a ct
CHCl3 extr a ct
EtOAc-solu ble
>100
67.33
73.12
53.15
89.55
71.53
46.86
45.47
25.94
4.05
>100
62.35
>100
50.74
84.59
77.07
50.22
59.32
77.90
10.44
63.54
73.27
95.92
>100
>100
58.09
57.15
4.95
2
×
h × 2) and then with CHCl3 (2 L, room temperature,
2) to yield a MeOH extract (331 g) and a CHCl3
extract (315 g). The MeOH extract was further frac-
tionated into EtOAc-soluble (271 g) and -insoluble
fractions (42 g).
EtOAc-in solu ble
1
2
5
6
9
The EtOAc-soluble fraction, which showed the great-
est cytotoxicity, was subjected to Si gel column chro-
matography with CHCl3-MeOH gradient system to give
seven fractions [fraction 1, 5% MeOH-CHCl3 eluate, 9.6
g; fraction 2, 10% MeOH-CHCl3 eluate, 29.4 g; fraction
3, 10% MeOH-CHCl3 eluate, 8.7 g; fraction 4, 10%
MeOH-CHCl3 eluate, 3.0 g; fraction 5, 20% MeOH-
CHCl3 eluate, 44.8 g; fraction 6, 20% MeOH-CHCl3
eluate, 49.3 g; fraction 7, 30% MeOH-CHCl3 eluate,
1
1
1
1
1
1
1
1
2
2
2
2
0
1
2
3
5
6
7
9
0
1
2
3
72.91
70.10
75.43
72.82
94.86
57.40
45.48
5.83
2.91
2.30
26.97
38.92
0.034
5.95
7.64
70.98
39.86
0.015
4
6.9 g].
Fraction 1 contained fatty material, and further Si
gel column chromatography and preparative TLC of
d oxor u bicin HCl
fractions 2-6 yielded the following compounds: fraction
a
Compounds having ED50 value >100 µg/mL are not included
2
2 (74.2 mg), 3 (41.0 mg), 4 (6.0 mg), 5 (100.1 mg), 6
in table.
(
(
22.8 mg), 9 (14.8 mg), 11 (103.0 mg), 13 (66.8 mg), 14
8.5 mg), 16 (24.4 mg), 18 (12.3 mg), 19 (45.0 mg), and
Exp er im en ta l Section
2
3 (24.0 mg); fraction 3 3 (165.0 mg), 8 (63.8 mg), 9 (90.5
Gen er a l Exp er im en ta l P r oced u r es. Optical rota-
tions were measured on a J ASCO DIP-140 digital
polarometer. UV spectra were taken in CHCl3 solution
on a Shimadzu UV-160A UV-vis spectrophotometer,
mg), and 12 (14.5 mg); fraction 4 3 (10.0 mg); fraction 5
10 (83.0 mg), 15 (28.2 mg), 20 (55.6 mg), 21 (11.0 mg),
and 22 (53.5 mg); fraction 6 1 (53.2 mg), 7 (202.6 mg),
10 (22.2 mg), and 17 (25.9 mg). Compounds 3-23 were

Cytotoxicity of Brazilian Propolis
J ournal of Natural Products, 1998, Vol. 61, No. 7 899
identical in all respects (mp, UV, IR, NMR, MS) to
respective compounds described in its literature. The
detail data of 2 and 16 were not reported yet, so they
were included here.
with EtOAc (5 mL × 3). The EtOAc extract was
evaporated and separated by preparative TLC to give
2
5
DCA (1.8 mg), [R] D +38.74° (c 0.12, Me2CO).
Cytotoxicity Assa y. Human HT-1080 fibrosarcoma
and murine colon 26-L5 carcinoma cells were main-
tained in Eagle’s minimum essential medium and RPMI
(both Nissui Pharm. Co., Ltd., Tokyo, J apan), respec-
tively. These media were supplemented with 10% fetal
calf serum (Gibco BRL Products, Gaithersburg, MD),
0.1% sodium bicarbonate, and 2 mM glutamine (Wako
Pure Chemicals Ind., Ltd., Kyoto, J apan).
3
-Hydr oxy-2,2-dim eth yl-8-pr en ylch r om an e-6-pr o-
p en oic a cid (1): colorless amorphous powder; UV
CHCl3) λmax (log ꢀ) 320 (3.5), 245 (3.19) nm; IR (CHCl3)
(
-
1
νmax 3400, 1675, 1625, 1440, 1260, 1140 cm ; EIMS m/z
3
1
+
16 [M] (100), 283 (20), 246 (20), 245 (30), 189 (20),
05 (40), 44 (58); HREIMS m/z 316.1712 (calcd for
1
13
C19H24O4, 316.1712); H and C NMR, Table 1.
P r ep a r a tion of (S)-(-)-MTP A a n d (R)-(+)-MTP A
ester s of 1. To a solution of 1 (10 mg) in MeOH (0.5
mL), excess CH2N2 was added under stirring. After 12
h, the mixture was evaporated under reduced pressure
to obtain methyl ester of 1 (11.2 mg). (S)-(-)-MTPA-
Cl (10 µL) was added to a solution of methyl ester of 1
Cellular viability in the presence and absence of
experimental agents was determined using the standard
3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bro-
mide (MTT, Sigma, St. Louis, MO) assays as described
3
5,36
previously.
In brief, exponentially growing cells
were harvested, and a 50-µL suspension containing 2500
cells was plated in 96-well microtiter plates (Falcon,
Becton Dickinson, NJ ). After 24 h of incubation at 37
(5 mg) in CHCl3 (0.5 mL) and pyridine (0.5 mL), and
the mixture was stirred for 12 h at room temperature.
The reaction mixture was purified by preparative TLC
with MeOH-CHCl3 (1:19) to give a mixture of (R)- and
°C under 5% CO to allow cell attachment, the cells were
2
treated with varying concentrations of test specimens
(S)-MTPA esters (3.1 mg). The same product (4.3 mg)
in their respective medium (100 µL) and incubated for
4 days under the same conditions as above. After
adding a solution of MTT for 4 h, the amount of
formazan formed was measured spectrophotometrically
at 590 nm using Immuno Mini NJ -2300 plate reader.
was obtained when (R)-(+)-MTPA-Cl was reacted with
methyl ester of 1 instead of (S)-(-)-MTPA-Cl in the
1
above reaction. The H NMR (CDCl3) of the mixture
was as follows: δ 7.60, 7.53 (each 1H, d, J ) 16.0 Hz,
H-9), 7.50-7.31 (m, aromatic proton of MTPA moiety),
Test specimens were dissolved in DMSO and then
diluted by medium. DMSO less than 0.1% in the test
solution had no effect on the all. Doxorubicin HCl
7
1
.20, 7.13 (each 1H, d, J ) 2.0 Hz, H-7), 7.03, 7.01 (each
H, d, J ) 2.0 Hz, H-5), 6.28, 6.25 (each 1H, d, J ) 16.0
Hz, H-10), 5.20, 5.15 (each 1H, tqq, J ) 7.5, 1.5 Hz,
H-2′), 3.78, 3.79 (each 6H, -OMe), 3.45 (each 2H, br d,
J ) 8.0 Hz, H-3), 3.24, 3.20 (each 2H, br t, J ) 6.0 Hz,
H2-1′), 2.92 (2H, dd J ) 17.0, 5.5 Hz, H-4), 2.80 (2H, dd
J ) 17.0, 5.0 Hz, H-4), 1.72, 1.69 (each 3H, H3-4′), 1.69,
(Kyowa Hakko Co., Ltd., Tokyo, J apan) was used as a
positive control, and ED50 values were calculated from
the mean values of data from six wells.
Ack n ow led gm en t. We thank Nihon Propolis Co.,
Ltd., Tokyo, J apan, for the kind gift of the research
material, propolis.
1
1
.68 (each 3H, H3-5′), 1.31, 1.28 (each 3H, H3-12), 1.26,
.25 (each 3H, H3-13).
2
,2-Dim eth yl-8-pr en ylch r om en e-6-pr open oic acid
(
ꢀ
2): colorless amorphous powder; UV (CHCl3) λmax (log
Refer en ces a n d Notes
) 323 (4.16), 279 (4.29), 248 (4.24) nm; IR (CHCl3) νmax
(
1) Ghisalberti, E. L. Bee World 1979, 60, 59-84.
2) Marcucci, M. C. Apidologie 1995, 26, 83-99.
-
1
1
675, 1625, 1595, 1375, 1270, 1145, 1120, 980 cm
;
(
+
EIMS m/z 298 [M] (27), 283 (100), 275 (9), 256 (18),
1
(
1
Tr em eton e (16): yellow oil; [R]25D -60.33° (c 0.46,
EtOH); UV (EtOH) λmax (log ꢀ) 279 (3.9), 227 (3.8), and
2
1
(3) Bankova, V.; Marcucci, M. C.; Simova, S.; Nikolova, N.; Ku-
jumgiev, A.; Popov, S. Z. Naturforsch. 1996, 51c, 277-280.
4) Matsushige, K.; Kusumoto, I. T.; Yamamoto, Y.; Kadota, S.;
49 (36), 121 (18), 95 (45), 81 (63); HREIMS 298.1558
(
1
13
calcd for C19H22O3, 298.1559); H and C NMR, Table
Namba, T. J . Trad. Med. 1995, 12, 45-53.
(5) Matsushige, K.; Basnet, P.; Hasa, K.; Kadota, S.; Tanaka, K.;
.
Namba, T. Phytomedicine 1996, 3, 203-209.
(
6) Basnet, P.; Matsushige, K.; Hase, K.; Kadota, S.; Namba, T. Biol.
Pharm. Bull. 1996, 19, 1479-1484.
(
7) Labbe, C.; Rovirosa, J .; Faini, F.; Mahu, M.; San-Martin, A.;
Castillo, M. J . Nat. Prod. 1986, 49, 517-518.
07 (3.7) nm; IR (CHCl3) νmax 1620, 1600, 1580, 1485,
-
1
355, 1280, 1260, 1120, 910, 810 cm ; EIMS m/z 202
(8) Pedro, P. Diaz, D.; Tiberio, A. C.; Pedro, J . N. Phytochemistry
1987, 26, 809-811.
+
[
M] (93), 187 (100), 159 (43), 141 (20), 131 (16), 115
(
9) Aga, H.; Shibuya, T.; Sugimoto, T.; Kurimoto, M.; Nakajima, S.
1
(
12); H NMR (CDCl3) δ 7.80 (2H, br d, J ) 8.0 Hz, H-4
Biosci. Biotech. Biochem. 1994, 58, 945-946.
(10) Bohlmann, F.; Knauf, W.; King, R. M.; Robinson, H. Phytochem-
istry 1979, 18, 1011-1014.
and H-6), 6.81(1H, d, J ) 8.0 Hz, H-7), 5.26 (1H, dd, J
)
8.0, 9.0 Hz, H-2),5.08 (1H, br s, H-11), 4.93 (1H, br s,
(
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