Bioactive oleanolic acid saponins and other constituents from the roots of Viguiera decurrens

Article abstract of DOI:10.1016/S0031-9422(00)00283-1

The bisdesmoside oleanolic acid saponin, 3-O-(methyl-β-D-glucuronopyranosiduronoate)-28-O-β-D-glucopyranosyl -oleanoate, along with nine known compounds (two diterpenic acids, one chromene, three triterpenes, one steroidal glycoside, and two monodesmoside oleanolic acid saponins), were obtained from Viguiera decurrens roots. The chemical structure of the bisdesmoside oleanolic saponin was determined by chemical and NMR spectral evidence. A mixture of monodesmoside saponins displayed cytotoxic activity against P388 and COLON cell lines (ED₅₀ = 2.3 and 3.6 μg/ml, respectively). Two of the known compounds showed insecticidal activity against the Mexican bean beetle larvae (Epilachna varivestis).

Full text of DOI:10.1016/S0031-9422(00)00283-1

Phytochemistry 56 (2001) 93±97
www.elsevier.com/locate/phytochem
Bioactive oleanolic acid saponins and other constituents
from the roots of Viguiera decurrens
a
a
Ä
Silvia Marquina , Nora Maldonado , Marõa Luisa Garduno-Ramõ
 Â
rez a,
b
b
c
d
Eduardo Aranda , Marõa Luisa Villarreal , Võctor Navarro , Robert Bye ,
 Â
e
a,
Guillermo Delgado , Laura Alvarez *
aCentro de Investigaciones QuõÂmicas de la Universidad Aut o noma del Estado de Morelos, Avenida Universidad 1001,
6
2210 Cuernavaca, Morelos, Mexico
bCentro de Investigaci o n en BiotecnologõÂa de la Universidad Aut o noma del Estado de Morelos, Av. Universidad 1001,
Chamilpa, 62210, Cuernavaca, Morelos, Mexico
cCentro de Investigaci o n Biom e dica del Sur, Instituto Mexicano del Seguro Social, Argentina 1, Xochitepec, Morelos, Mexico
dInstituto de BiologõÂa, de la Universidad Nacional Aut o noma de M e xico, Circuito Exterior, Ciudad Universitaria, Coyoacan 04510, Mexico
eInstituto de QuõÂmica de la Universidad Nacional Aut o noma de M e xico, Circuito Exterior, Ciudad Universitaria, Coyoac a n 04510,
M e xico, D. F., Mexico
Received 2 June 1999; received in revised form 7 July 2000
Abstract
The bisdesmoside oleanolic acid saponin, 3-O-(methyl-b-d-glucuronopyranosiduronoate)-28-O-b-d-glucopyranosyl-oleanolate,
along with nine known compounds (two diterpenic acids, one chromene, three triterpenes, one steroidal glycoside, and two mono-
desmoside oleanolic acid saponins), were obtained from Viguiera decurrens roots. The chemical structure of the bisdesmoside
oleanolic saponin was determined by chemical and NMR spectral evidence. A mixture of monodesmoside saponins displayed
cytotoxic activity against P388 and COLON cell lines (ED50=2.3 and 3.6 mg/ml, respectively). Two of the known compounds
showed insecticidal activity against the Mexican bean beetle larvae (Epilachna varivestis). # 2001 Elsevier Science Ltd. All rights
reserved.
Keywords: Viguiera decurrens; Asteraceae; Root; Oleanolic acid; Bisdesmoside; Saponin; Chikusetsusaponin methyl ester; Cytotoxic and insecticidal
activity
1. Introduction
Â
to the Tarahumara Indians as ``nakarori'' (`naka'=ear,
rori'=movement) are used medicinally and in ®shing
`
Chemical investigation of several species belonging to
Viguiera (Asteraceae) have yielded diterpenes (Delgado
et al., 1984a), sesquiterpene lactones (Alvarez et al.,
1985) and ¯avonols (Delgado et al., 1984b) as the major
constituents of their aerial parts. The following con-
tribution is based upon our continuing study of the
genus Viguiera and our search for bioactive natural
products. Viguiera decurrens (Asteraceae) is a perennial
herb endemic to the upper desert grasslands and lower
oak-pine forests of the eastern Sierra Madre Occidental
of western Chihuahua. The thick, elongated roots known
(Brambila, 1976). The distinctive decurrent leaves and
winged stems (over one meter tall with yellow terminal
¯owers) appear as ears when the plant is blown by the
wind. Upon placing the crushed roots (fresh or dried) in
stream pools, the ®sh rise to the surface and are col-
lected by the Indians as food. A poultice from the pun-
gent root is also used to treat infections, wounds and
boils, and an infusion of the root or leaves is drunk to
alleviate gastric ulcers (Pennington, 1963; Bye, 1985).
This paper describes the bioactivity-isolation of olea-
nolic acid saponins from the roots of V. decurrens, the
structure elucidation of the new saponin, and the cyto-
toxic and insecticidal evaluations of some of the isolated
compounds.
* Corresponding author. Fax: +52-73-29-70000-3501.
E-mail address: lalvarez@servm.fc.uaem.mx (L. Alvarez).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00283-1

9
4
S. Marquina et al. / Phytochemistry 56 (2001) 93±97
2. Results and discussion
and 6-methylglucuronosiduronoate. The 1H NMR
CDCl -DMSO-d ) (see experimental) and 13C NMR (see
(
3
6
Using a bioassay-directed fractionation of the roots of
Table 1) spectra of (7) showed signals assignable to an
oleanolic acid moiety [ꢀ 5.26 (1H, br t, J=3.5 Hz, H-12),
3.34 (1H, m, H-3), 2.83 (1H, dd, J=13.5, 4.0 Hz, H-18], a
V. decurrens, three cytotoxic fractions and three non-
cytotoxic fractions were obtained. Chromatographic
puri®cation of each cytotoxic fraction led to the isola-
tion of two kaurenoic diterpenes, namely ent-kaur-16-
en-19-oic acid (1) and 15a-angeloyloxy-ent-kaur-16-en-
b-d-methylglucuronopyranosiduronoate moiety [ꢀ 4.35
0
(1H, d, J=7.5 Hz, H-1 ), 3.74 (3H, s, OCH ], and a b-d-
3
00
glucopyranosyl moiety [ꢀ 5.43 (1H, d, J=8.0 Hz, H-1 ].
In order to clarify the type of cyclization of the sugar
moieties, H and 13C NMR spectroscopic analysis was
undertaken on the heptaacetyl derivative 8 by means of
HMBC and HMQC experiments, and all the proton
signals due to the sugar moieties were assigned. The
pro®les of these signals indicated that both methyl glu-
curonosiduronoate and glucose were b-pyranoses.
The bisdesmoside nature of 8 was demonstrated by
the HMBC experiment which showed cross peaks
between H-1 of the methyl glucuronopyranosidur-
onoate moiety and C-3 of the oleanolic acid moiety, and
between H-1 of the b-d-glucopyranosyl moiety and C-
28 of the genin. Similarly, the detection of all possible
two- and three-bond correlations of the anomeric pro-
1
9-oic acid (2); three saponins: b-d-glucopyranosyl-
1
oleanolate (3) (Yoshikawa et al., 1996a), b-sitosteryl-3-
O-b-d-glucopyranoside, and oleanolic acid-3-O-methyl-
b-d-glucuronopyranosiduronoate (4) (Sakai et al.,
1
994), and one chromene, 8-hydroxy-6-acetyl-2,2-dime-
thylchromene (5) (Bohlmann and Jakupovic, 1978).
From the non-active fractions, the triterpenes friedelin,
friedelan-3b-ol and oleanolic acid (6) as well as the
oleanolic acid bisdesmoside saponin 3-O-(methyl-b-d-
glucuronopyranosiduronoate)-28-O-b-d-glucopyranosyl
oleanolate (7) were also obtained.
0
00
Compound 7 was obtained as a colorless powder (mp
ꢀ
+
216±218 C). In the positive mode FABMS, the quasi
molecular ion-peak was observed at m/z 809 M+H ,
indicating a molecular formula of C H O . On acid
4
3
68 14
hydrolysis compound 7 a€orded oleanolic acid, glucose
Table 1
13C NMR spectral data for 7 (CDCl
3
-DMSO-d
6
3
) and 8 (CDCl ) at 125
MHz, ꢀ values
7
8
7
8
Sapogenol moiety
Sugar moieties
0
1
38.6
38.9
6 Methyl-
glucuronosiduronoate
moiety
1
104.8
102.9
0
0
0
0
0
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
27.1
88.8
39.6
54.6
17.3
32.9
39.6
46.7
35.8
24.9
27.7
90.6
39.4
55.5
18.1
33.8
41.0
47.6
36.7
23.4
122.9
142.9
41.7
29.7
22.9
45.8
41.7
46.8
31.8
33.8
32.9
27.7
16.3
15.3
16.9
25.6
175.6
32.9
25.5
2
3
4
5
6
73.0
71.8
71.9
72.9
68.1
72.5
167.2
52.7
70.8
76.3
168.9
3
51.41
OCH
0
0
0
0
0
0
0
0
0
0
0
0
Glcp moiety
1
2
3
4
5
6
93.3
74.4
76.3
69.4
75.9
62.9
91.6
71.6
72.0
69.6
70.0
61.6
0
1
2 121.5
3 142.7
4
5
6
7
8
9
0
1
2
3
4
5
6
7
39.1
29.8
22.5
45.7
40.8
45.7
31.1
32.9
32.2
27.1
14.6
15.8
16.1
26.6
8 175.4
9
0
32.2
24.9

S. Marquina et al. / Phytochemistry 56 (2001) 93±97
95
tons con®rmed the glycosidic linkages and the assign-
ments for proton and carbon resonances.
Table 2
Evaluation of cytotoxic activity
Based on the above mentioned evidence and compar-
ison of the 13C NMR (Table 1) data for 7 and 8 with
those for oleanolic acid 3, 28-O-bisdesmosides (Yoshi-
kawa et al., 1995), the structure of this compound was
con®rmed as 3-O-(methyl-b-d-glucuronopyranosidur-
onoate)-28-O-b-d-glucopyranosyl oleanolate (7).
The demethyl derivative of 7 has been reported as a
natural product of Aralia elata (Lin et al., 1976), Talinum
triangulare (Kohda et al., 1992), Aralia elata SEEM.
ED50a
Fractions
KBb
P388c OVCARd COLONe UISOf
I
>20
>20
>20
>20
>20
>20
7.24
3.3
9.1
12
±
±
±
II
III
±
±
±
21.4
25.1
8.9
47.9
±
>20
>20
>20
>20
3.6
22.4
>20
3.02
>20
±
IV
V
VI
2.3
5.7
Mixture of 3, 4
and b-sitosteryl-
>4.0 2.3
(
Yoshikawa et al., 1993), Panax pseudoginseng (Ida et
al., 1994), and Dolichos lablab L. (Yoshikawa et al.,
998) and named chikusetsusaponin IVa; therefore,
3-O-b-d-Glcp
3
4
>4.0 >4.0
>4.0 >4.0
±
±
±
±
±
±
1
chikusetsusaponin IVa methyl ester (7) is a natural
saponin from V. decurrens. The NMR spectral data of
its heptaacetyl derivative (8) have not previously been
recorded in the literature (see Table 1).
a
(
KB, human epidermal carcinoma.
ED50 mg/ml).
b
c
d
e
f
P388, murine leukemia.
OVCAR, ovarian cancer.
COLON, colon carcinoma.
UISO, uterine-cervix cancer.
Although 7 could be considered an artifact due to the
fact that MeOH was originally used for the isolation
procedure, this possibility was ruled out from the fol-
lowing observations: (a) the corresponding acid was not
isolated, and it is well known that the use of MeOH
does not allow per se the formation of esters; (b) com-
pound 7 was isolated from an EtOH extract of addi-
tional material of Viguiera decurrens roots (3.4 mg of
compound) obtained from big root tissue (data not
shown) and (c) the presence of methyl b-d-glucur-
onopyranosiduronoate triterpenoidal saponins as nat-
ural products, has been well documented recently in the
literature (Sang et al., 1998; Zhang et al., 1999).
1996b), as well as hypoglycemic activity (Yoshikawa et
al., 1996c), along with other oleanolic acid glycosides,
isolated from Aralia elata and Beta vulgaris.
This is the ®rst report of oleanolic acid saponins from
the roots of a Viguiera species.
3. Experimental
None of the six main fractions displayed anti-
microbial activity against Staphylococcus aureus, Sal-
monella typhimurium, Escherichia coli, Pseudomonas
aeruginosa, Candida albicans and Trycophyton menta-
grophytes, at concentrations up to 400 mg/ml.
Mps are uncorr. NMR instrumentation: Varian
VXR-200 and Varian Unity Plus-500. MS: JEOL JMS-
AX 505 HA.
3.1. Plant material
The mixture of compounds 3, 4 and b-sitosteryl-3-O-
b-d-glucopyranoside obtained from fraction V (see
Table 2) showed cytotoxic activity against the P388 cell
line (ED =2.3 mg/ml); however, the pure compounds
were not toxic (ED50 > 4 mg/ml) (Geran et al., 1972).
Compounds 1, 3, 5, 7 and friedelin were also evaluated
for their insecticidal activity at di€erent concentrations
against Epilachna varivestis larvae, the Mexican bean
beetle (Coleoptera: Coccinellidae), an important pest in
The roots of Viguiera decurrens A. Gray were obtained
in the eastern Sierra Tarahumara, Chihuahua, Mexico,
on 12 October 1991 and identi®ed by R. Bye. The her-
barium voucher specimen (R. Bye 18338) is deposited in
the National Herbarium of Mexico (MEXU).
5
0
3.2. Bioactivity-directed isolation of compounds
Mexico (MacGregor and Gutie
Â
these experiments indicated that friedelin and com-
rrez, 1983). The results of
Dried and powdered roots (1.07 kg) of V. decurrens
were extracted by percolation using a gradient of n-
hexane±EtOAc±MeOH, yielding six fractions: Fr. I
(100:0:0, 18.0 g); Fr. II (9:1:0, 1.6 g); Fr. III (7:3:0, 4 g);
Fr. IV (1:1:0, 2.8 g), Fr. V (0:100:0, 12.0 g); Fr. VI
(0:3:7, 30.6 g). These fractions were evaluated for their
antimicrobial and cytotoxic activities (Table 2). Frac-
tions I, II and V showed cytotoxic activity. Fr. I after
®ltration a€orded 2.23 g of ent-kaur-16-en-19-oic acid
pounds 1 and 5 were not toxic (LC >5000 mg/ml),
50
while saponins 3 and 7 displayed toxicity (LC =1380
and 80 mg/ml (calculated), respectively).
50
The presence of oleanolic acid mono- and bidesmoside
saponins in the roots of Viguiera decurrens is noteworthy,
and chemically validates the Tarahumara people's use of
the roots to stupefy ®sh (Bye, 1985).
Recently, 4 has been reported to have in vivo inhibi-
tory e€ects on ethanol absorption (Yoshikawa et al.,
ꢀ
(1, 0.1151%, mp 170±174 C). Fr. II was ground with n-
hexane to yield 68 mg of 15a-angeloyloxy-ent-kaur-16-

9
6
S. Marquina et al. / Phytochemistry 56 (2001) 93±97
en-19-oic acid (2, 0.104%). Fr. III was methylated with
diazomethane and the residue was chromatographed on
a silica gel column using mixtures of n-hexane±EtOAc;
fractions 1±31 eluted with 95:5 (n-hexane±EtOAc) yiel-
ded 105 mg of friedelin (0.0098%, mp 242±245 C), from
fractions 15±46 eluted with n-hexane±EtOAc 9:1 crys-
29), 0.90 (3H, s, H-27), 0.89 (3H, s, H-24), 0.81 (3H, s,
H-25), 0.71 (3H, s, H-26); 13C NMR spectral data (125
MHz, CDCl -DMSO-d ): see Table 1.
3
6
ꢀ
3.2.2. Compound (8)
Obtained by usual acetylation of 7: powder, mp 99±
ꢀ
+
tallized 50 mg of friedelan-3b-ol (0.0046%, mp 249±
101 C; positive FABMS m/z: 1125 [M+Na] . High-
resolution positive FABMS m/z: calcd for C H
82
ꢀ
2
53 C), fractions 55±60 (8:2 n-hexane±EtOAc) were
5
7
+
combined and the residue (700 mg) was subjected to
silica gel column chromatography eluted with (CH Cl ±
MeOH 9:1) to yield 60 mg of oleanolic acid methyl ester
NaO : 1125.5246; found: 1125.5318 [M+Na] , 769
21
+
+
[M- (Glu-Me)Ac +H] , 741 [M- (Glu-Me)Ac +K] ,
3
331 [(Glc)Ac ] ; H NMR spectral data (500 MHz,
4
CDCl ): ꢀ 5.58 (1H, d, J=8.0 Hz, H-1 ), 5.31 (1H, br t,
2
2
3
+
1
ꢀ
00
(
0.0056%, mp 198±200 C). Fr. IV was applied to a silica
gel column using a gradient system of CH Cl ±MeOH
3
0
J=3.5 Hz, H-12), 5.24 (1H, t, J=9.5 Hz, H-3 ), 5.22
0
2
2
00
to yield 35.8 mg of oleanolic acid (6, 0.0033%, mp 196±
ꢀ
(1H, t, J=9.5 Hz, H-4 ), 5.21 (1H, t, J=9.5 Hz, H-3 ),
00
198 C). Fr. V was subjected to chromatography with
mixtures of CH Cl ±MeOH, to obtain a cytotoxic frac-
5.19 (1H, t, J=9.0 Hz, H-2 ), 5.12 (1H, t, J=8.5 Hz, H-
00 0
4 ), 5.04 (1H, t, J=9.0 Hz, H-2 ), 4.58 (1H, d, J=8.0
0 00
Hz, H-1 ), 4.27 (1H, dd, J=12.5, 4.0, Hz, H-6a ), 4.04
2
2
tion (see Table 2) which was composed of a mixture of
saponins. The mixture was further separated by repe-
ated column chromatography to yield 47 mg of b-d-
00
(1H, dd, J=12.5, 2.0 Hz, H-6b ), 3.98 (1H, d, J=9.5
0
00
Hz, H-5 ), 3.78 (1H, ddd, J=10.5, 4.5, 2.5 Hz, H-5 ),
3.77 (3H, s, OCH ), 3.10 (1H, dd, J=8.0, 4.5 Hz, H-3),
glucopyranosyl olean-12-en-28-oate (3, 0.0043%, mp
3
ꢀ
2
35±237 C), 60 mg of b-sitosteryl-3-O-b-d-glucopyrano-
2.81 (1H, dd, J=16.0, 4.5 Hz, H-18), 1.10 (3H, s, H-27),
0.90 (6H, s, H-23, H-30), 0.89 (6H, s, H-24, H-29), 0.73
(3H, s, H-25), 0.72 (3H, s, H-26); 13C NMR spectral
ꢀ
side (0.0056%, mp 267 C), 125 mg of oleanolic acid 3-O-
methyl-b-d-glucuronopyranosiduronoate (4, 0.011%,
amorphous powder), and 22 mg of 8-hydroxy-6-acetyl-
data (125 MHz, CDCl ): see Table 1.
3
ꢀ
2,2-dimethyl chromene (5, 0.002%, mp 135±137 C) (lit.
mp 132 C, Bohlmann and Jakupovic, 1978). Fr. VI
ꢀ
3.2.3. Acid hydrolysis of 7
(
ing with EtOAc±MeOH (7:3), CH Cl ±MeOH (95:5) and
30.6 g) was subjected to repeated CC on silica gel, elut-
Compound 7 (25 mg) was re¯uxed with 10% HCl-
methanol (5 ml) for 2 h. The aglycone was extracted with
EtOAc and identi®ed by comparison with an authentic
sample. The aqueous layer was adjusted to pH 6 with
2
2
CH Cl ±MeOH (85:15) respectively to a€ord 80 mg of 3-
2
2
O-(methyl-b-d-glucuronopyranosiduronoate)-28-O-b-d-
glucopyranosyl oleanolate (7, 0.0074%).
NaHCO . After evaporation to dryness, the sugars were
3
Compounds 1, 2, 6, b-sitosteryl-3-O-b-d-glucopyr-
anoside, friedelin, friedelan-3-b-ol, were identi®ed by
direct comparison (IR, TLC) with authentic samples,
while compounds 3±5 and 7 were characterized by
means of physicochemical evidence.
extracted with pyridine and analyzed by TLC on silica
gel with EtOAc±MeOH±H O±AcOH (11:2:2:2); detec-
tion was accomplished with p-anisidine phthalate.
2
3.3. Biological activity
3
2
.2.1. 3-O-(Methyl-ꢁ-d-glucuronopyranosiduronoate)-
8-O-ꢁ-d-glucopyranosyl oleanolate (7)
3.3.1. Antimicrobial activity
Evaluations were performed with cultures of Staphylo-
coccus aureus (ATCC 6538), Salmonella typhimurium
(ATCC 06539), Escherichia coli (ATCC 8937), Pseudo-
monas aeruginosa (ATCC 9027); Candida albicans
(ATCC 10231) and Trycophyton mentagrophytes (NRRL
1942), using the conventional disc assay procedure (Van-
den Berghe and Vlietinck, 1991); nystatin (5±160 mg/ml)
and gentamicin (2±128 mg/ml) were included as controls.
ꢀ
Colorless powder (MeOH, mp 216±218 C) (lit. mp
ꢀ
209±212 C, Sakai et al., 1994), a +5.7 (CHCl , c 0.28);
D
3
�
1
IR nmax cm : 3420, 1720, 1700; positive FABMS m/z:
831 [M+Na] ; high-resolution positive FABMS m/z:
Calcd for C H NaO : 831.4506; found: 831.4478
+
4
3
68
14
+
+
+
(
(
M+Na) , 809 [M+H ], 663 [M- Glc+Na ], 475 [M-
+
+
+ 1
Glu-Me)-Glc+Na] , 207 [Glu] , 179 [Glc] ; H NMR
spectral data (500 MHz, CDCl -DMSO-d ): ꢀ 5.43 (1H,
3
6
d, J=8.0 Hz, H-1''), 5.26 (1H, br t, J=3.5 Hz, H-12),
0
3.3.2. Cytotoxic activity
4
.35 (1H, d, J=7.5 Hz, H-1 ), 3.78 (1H, t, J=9.0 Hz, H-
0
These studies were performed using the cell cultures
listed in Table 2 according to Geran and Greenberg's
screening protocols (Geran et al., 1972).
0
2
H-6a ), 3.63 (1H, t, J=8.5 Hz, H-4 ), 3.56 (1H, dd,
), 3.74 (3H, s, OCH ), 3.72 (1H, dd, J=11.0, 5.0 Hz,
0
3
0
0
00
00
J=11.0, 5.5 Hz, H-6b ), 3.48 (1H, t, J=8.5 Hz H-3 ),
.46 (1H, d, J=9.0 Hz, H-5 ), 3.44 (1H, t, J=9.0 Hz, H-
0
0
3
4
3
1
3.3.3. Insecticidal activity
0
0
00
), 3.40 (1H, t, J=8.5 Hz, H-3 ), 3.36 (1H, m, H-5 ),
0
This assay was carried out following the published
protocol (Kubo, 1991), using ®rst instar larvae of Epi-
lachna varivestis (Coleoptera: Coccinellidae) which were
.34 (1H, m, H-2 ), 2.83 (1H, dd, J=13.5, 4.0 Hz, H-18),
.11 (3H, s, H-30), 1.02 (3H, s, H-23), 0.92 (3H, s, H-

S. Marquina et al. / Phytochemistry 56 (2001) 93±97
97
fed with di€erent concentrations of compounds 1, 3, 5,
and friedelin in contaminated leaf-discs. Leaves were
Delgado, G., Alvarez, L., Romo de Vivar, A., 1984a. Phytochemistry
3, 2674.
Delgado, G., Alvarez, L., Romo de Vivar, A., 1984b. Phytochemistry
3, 675.
2
7
obtained from greenhouse bean plants (criollo variety)
free of pests and chemical insecticides. Concentrations
ranged from 100 to 6000 mg/ml. The toxicity data were
analyzed by the Probit method (Finney, 1962) and
expressed as the LC .
2
Finney, D.J., 1962. Probit analysis. Cambridge University Press,
Cambridge, UK.
Geran, R.I., Greenberg, N.H., MacDonald, M.N., Schumacher, A.M.,
Abbott, B.J., 1972. Cancer Chemother. Rep. 3, 1.
50
Ida, Y., Katsumata, M., Satoh, Y., Shoji, J., 1994. Planta Med. 60, 286.
Kohda, H., Yamaoka, Y., Morinaga, S., Ishak, M., Darise, M., 1992.
Chem. Pharm. Bull. 40, 2557.
Acknowledgements
Kubo, I., 1991. In: Dey, P.M., Harborne, M.J.B., Hostettmann, K.
(Eds.), Methods in Plant Biochemistry, vol. 6. Academic Press, New
York, p. 33.
We thank M. Sc. Marõ
Quõmica de la UNAM) for her assistance in obtaining
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