1310 HELVETICA ACTA- Vol.

67 (1984)
CHIMICA

147. New Saponins from Phytolacca dodecandra HERIT IT

by Bruno Domon and Kurt Hostettmann*
Institut de Pharmacognosie et Phytochimie, Ecole de Pharmacie, UniversitC de Lausanne,
2, rue Vuillermet, CH-1005 Lausanne

(9.V.84)

Summary
Three bidesmosidic saponins (1-3) have been isolated from the methanolic extract
of the berries of Phytolacca dodecandra HERIT IT (Phytolaccaceae) by a combination of
rotation locular counter-current chromatography and column chromatography on
reversed phase (RP-8) with MeOH/H,O. The structures have been established by
'H-NMR, I3C-NMR, FAB-MS, and D/CI-MS, as well as on the basis of acidic and
basic hydrolyses. The monodesmosidic saponins la-3a obtained after partial hydro-
lysis with a base exhibit strong molluscicidal activity against the schistosomiasis-
transmitting snail Biomphalaria glabrata. Saponins 1-3, la, and 3a are reported for
the first time, whereas 2a has been identified previously in the aqueous extract of
P. dodecandra berries.

Introduction. - The rising costs of chemotherapy and of synthetic molluscicides

have led to an increasing interest in plants and plant-derived compounds which are
lethal to the intermediate host of schistosomiasis (bilharzia). The use of plants with
molluscicidal properties appears to be a simple and inexpensive technology for the
control of the snail vector. Phytolacca dodecandra HERIT IT (Phytolaccaceae) is one of
the most promising plants. Water extracts of the berries have been evaluated in both
laboratory and field trials [ 1-31. The discovery of the remarkable molluscicidal proper-
ties of P.dodecandra, used in Ethiopia as a soap substitute, was made in 1965 by
Lemma [4]. He observed dead snails in rivers where people washed their clothes with
dried fruits. Subsequently, the water extract was studied by Parkhurst et al. [5] who
isolated very potent monodesmosidic saponins formed of oleanolic acid and of various
glycosidic chains.
In the course of our systematic screening studies on compounds with molluscicidal
activity from medicinal plants [6] [7], we undertook the reinvestigation of P. dodecandra
in order to improve the extraction procedure and to develop a method for the quantita-
tive determination of the saponins. The dried berries were extracted by the classical
method using solvents of increasing polarity, namely petroleum ether, CHCl,, MeOH,
and H,O. The extracts obtained showed no molluscicidal activity against Biomphalaria
glubruta snails, whereas direct water extraction of the fruits yielded a highly potent
 H~LVETICA
CHIMICA
ACTA- Vol. 67 (1984) 1311

solution. The MeOH extract, composed of numerous bidesmosidic saponins, was in-
vestigated. In the present paper, we report on the isolation and structure elucidation of
three new saponins.
Results. - Dried berries (48 g) obtained from plants cultivated in Ethiopia (strain
1733) were extracted successively with petroleum ether, CHCl,, MeOH, and H,O. A
part of the MeOH extract (8 g) was submitted in two portions to rotation locular
counter-current chromatography (RLCC) [8] with the solvent system AcOEt/EtOH/
H,O 40: 20: 40 in the ascending mode. The eluate was monitored by TLC and collected
in six fractions: Z (200 mg), ZZ (400 mg), ZZZ (440 mg), ZV (470 mg), V (1370 mg), and
VZ (1520 mg). These fractions were further separated by reversed-phase chromato-
graphy (see Exper. Part). Pure saponin 1 (50 mg) was obtained from Fraction ZZZ, 2
(540 mg) from Fraction ZV and V , and 3 (45 mg) from Fraction V.
Acidic hydrolysis of saponins 1-3 afforded glucose and as aglycones bayogenin,
oleanolic acid, and hederagenin, respectively, identified by MS and 'H-NMR and by
comparison with authentic samples. Basic hydrolysis of 1-3 yielded glucose and the
monodesmosidic saponins la, 2a, and 3a, respectively. The mol.wt. of the saponins

Compound R' (C(23)) R2 R3 R4

x OH Glc

Hoao<o,
,OH
1 CH20H H
la CH,OH OH H H
HO
2 CH, H Glc Glc
2a CH3 H Glc H
3 CH,OH H Glc Glc
k3 3a CH,OH H Glc H

was established by the combination of desorption/chemical-ionization (D/CI) MS [9]

and fast atom bombardment (FAB) MS [lo]. The MS (D/CI) of 1 (reactant gas NH,,
negative-ion mode) showed a quasi-molecular ion ( [ M - HI-) at m/z 973 and a signal
at m/z 955 after loss of H,O. Additional signals were observed at m/z 81 1 ( [ ( M - H)-
162]-) and m/z 649 ( [ ( M - H)-324]-), corresponding to the loss of one and two glucosyl
moieties, respectively.
The quasi-molecular ion of 1 was not obtained in the positive-ion spectrum, but the
following signals were observed: m/z 830 ( [ ( M+ NH,)-I62]+), m/z 668 ([(M + NHJ-
3241') and m/z 506 ([(M+ NHJ-486]+), corresponding to the loss of one, two, and
three glucosyl units. These results suggest that one sugar moiety is attached at C(28)
and is very easily eliminated. This is confirmed by the spectrum of l a where identical
+
signals are observed, the quasi-molecular ion appearing at m/z 830 ( [ M NH,]').
Thus, 1 is a bidesmosidic saponin with three hexose units, one glucose being attached
to the aglycone by an ester linkage. Compound l a is a monodesmosidic bayogenin-
diglucoside.
The MS (FAB) of 2 (matrix thioglycerol, negative-ion mode) shows quasi-molec-
ular ions at m/z 1139 ( [ M + Cll-) and m/z 1103 ( [ M - HI-). Signals at m/z 941
( [ ( M- H)-162]-), 779 ([(M - H)-324]-), 617 ( [ ( M - H)-486]-), and 455 ( [ ( M - H)-
648]-) correspond to the subsequent loss of four glucosyl moieties. The partially hydro-
lyzed saponin 2a shows a similar spectrum with one hexose unit less: quasi-molecular
1312 HELVETICA ACTA- Vo1.67 (1984)
CHIMICA

+
ions are observed at m/z 977 ( [ M a ] - ) and m/z 941 ( [ M - HI-); other signals are
present at m/z 779, 617, and 455. Compound 2 is a bidesmosidic oleanolic acid-tetra-
glucoside with one sugar unit linked to the aglycone at C(28), whereas 2a is the corre-
sponding prosapogenin with three glucosyl moieties attached at C(3). Compounds 3
and 3a show similar fragmentation patterns to 2 and 2a, respectively, with a shift of
16 u resulting from the substitution of a CH,-group attached on C(4) of the aglycone
by CH,OH.
The interglycosidic linkages as well as the position of attachment of the sugar
chains to the aglycones were established by I3C-NMR spectroscopy. In all the saponins,
a sugar chain is linked to the aglycone at C(3), the 13C-NMRsignal of which appears at
83.2 ppm for 1 and la, at 89.1 ppm for 2 and 2a, and at 82.1 ppm for 3 and 3a. These
chemical shifts are in agreement with values reported from the literature for olean-12-
ene-derived triterpene-3-0-glycosides [6] [ 111. The free COOH-group is observed at 180
ppm (monodesmodic saponins la-3a) whereas, when esterified with a glucosyl moiety,
the chemical shift is 176 ppm (bidesmosidic saponins 1-3). The /3-D-pyranosyl configu-
ration of glucose was established by 'H- and 13C-NMR.

Table. I3C-NMR Chemical Shifts of Saponins l a and 2a (sugar moieties). Pertinent shifts discussed in the text
are shown in italics.

Saponin C(1) C(2) C(3) Ci4) C(5) C(6)

l a inner sugar moiety 104.8") 74.8 76.8 81.3 76.2 62.1')
terminal sugar moiety 105.2a) 75.0 78.2 71.6 78.4 62.5')
2a inner sugar moiety 104.8') 81.6 74.8 81.2 77.1 62Xd)
terminal sugar moiety 104.9') 76.2 77.8 71.5 78.0 62.2d)
terminal suga'r moiety lO5.Sc) 76.6 78.2 71.8 78.4 62.3d)
a)h)c)d) Signals may be interchangeable.

The I3C-NMR data of saponins l a and 2a, which allow the determination of the interglycosidic linkages,
are summarized in the Table. The chemical shifts of the C-atoms of the inner 8-o-glucopyranosyl moiety of
compound l a clearly indicate that the terminal sugar unit is attached at position 4. The C(4)-signal is shifted
downfield by 9.7 to 81.3 ppm; the C(3) and C(S)-signals are shifted upfield by 1.4 and 2.2 ppm, respectively,
whereas the other C-atoms remain almost unaffected. These results are in agreement with the glycosylation rule
previously established by Konishi et al. [12]. Namely, when a OH-group of a sugar is glycosylated, the a-C-
atoms are shifted downfield by 6 to 9 ppm, while p-C-atoms are observed at higher field. The spectrum of
saponin 1 shows six additional signals for the P-D-glucopyranosyl unit esterified on the COOH-group. Com-
pound 2a is also a triglucoside, but with all three sugars branched at position 3 of oleanolic acid. From the
13C-NMR data (Table), it can be seen that two of the P-D-glUCOpyranOSe moieties are terminal with shifts
similar to those of the C-atoms of the terminal sugar of l a . The third p-D-glucopyranose unit (inner moiety)
however, has chemical shifts for C(2) and C(4) at 81.6 and 81.2 ppm, respectively, these downfield shifts indicat-
ing that it is substituted by terminal glucose units at positions 2 and 4. The "C-NMR signals arising from the
sugar moiety of saponin 3a have shifts very similar to those of the sugar moiety of 2a, indicating the same
branched sugar unit.

Thus, the structure of l a is established as 3 - 0 -[0-P-D-glUCOpyranOSyl-(1+~)-P-D-

glucopyranosyl]bayogenin and saponin 2a is identical to 3 - 0 -[2',4'-di-O -@- D-gluco-
pyranosy~)-~-D-g~ucopyranosyl]o~eanolic acid or oleanoglycotoxin-A, previously iso-
lated from the aqueous extract of P. dodecandra by Parkhurst et al. [5]. Compounds 2a
and 3a are the prosapogenins of 2 and 3, respectively tetraglucosides of oleanolic acid
and hederagenin, with one glucosyl unit linked to the aglycone at C(28).
 CHIMICAACTA Val. 67 (1984)
HELVETICA - 1313

Discussion. - Although several phytochemical studies of P. dodecandra berries have

been published [5] [14], it has been possible to isolate the three previously unreported
bidesmosidic saponins 1-3 from the MeOH extract. These glycosides showed no mol-
luscicidal activity, but after treatment with a base afforded the monodesmosidic
saponins la-3a which killed Biomphalaria glabrata snails within 24 h at concentrations
of 12, 6, and 12 ppm, respectively. Compound 2a was previously identified in the
aqueous extract of P. dodecandra berries among other highly active monodesmosidic
saponins [ 5 ] . Direct water extraction afforded la-3a, and only traces of 1-3 could be
detected. The genuine bidesmosidic saponins are easily hydrolyzed during the aqueous
extraction procedure. The monodesmosidic saponins so formed are artefacts since they
are absent in the MeOH extract.
The saponin composition of P. dodecandra berries appears to be very complex and
is formed of numerous glycosides of oleanolic acid, hederagenin, and bayogenin.
Several other bidesmosidic saponins could be isolated. Their structure elucidation is
currently in progress. We undertook also a systematic phytochemical investigation of
berries originating from different cultivated strains which may vary in their saponin
content.
Financial support has been provided by the Swiss National Seience Foundation, the Parasitic Diseasrs
Programme of the World Health Organisation and the UNDPI World Bank1 WHO Special Programme for
Research and Training in Tropical Diseases. Prof. Tekleniariam Ayele, Addis Ababa, Prof. H. Klous and
Dr. Ch.B. Lugt, are gratefully acknowledged for providing plant material. Thanks are also due to Dr. K.B.
Mott, WHO, for helpful advice and to the following who carried out spectral measurements: Prof. W . J . Richter.
Ciba-Geigy, Basel (FAB-MS), Mr. D. Chollet, Zyma, Nyon (D/CI-MS) and Prof. J . Lauterwein, University of
Lausanne (NMR).

Experimental Part
General Remarks. Melting points (m.p.) were determined on a Kojler block and are uncorrected. TLC were
carried out on silica gel precoated A1 sheets (Merck) with CHCl,/MeOH/H,O 58:37:5. Detection was with
Godin reagent [15]. For column chromatography, a Lobar-Liehroprep-RP-8 column (40-63 pin; i.d. 2.5 x 27 cm;
Merck) equipped with a Duramat-80 pump (Chemie und Filter, Regensdorf) and a Sephadex-LH-20 column (i.d.
2.5 x 25 cm; Pharmacia Fine Chemicals) were used. Rotation locular counter-current chromatography (RLCC)
was achieved on a Tokyo Rikakikai apparatus (Tokyo, Japan) with 16 tubes, each divided by a centrally perfo-
rated PTFE disc into 37 loculi. The flow rate was 45 ml/h, the rotation speed 60-70 rpm and the slope 20”.
‘H-NMR and ”C-NMR spectra were recorded on a Bruker- WP-360 apparatus at 360 MHz and 90.52 MHz,
resp., in (DJpyridine as solvent and TMS as internal standard. Desorption/chemical ionization (D/CI) MS were
rccorded on a Riberniag-RIO-IOB quadrupole. Fast-aton-bombardment (FAB) MS were obtained on a Z A B - I S
spectrometer. The target was bombarded with 5-keV Xe-atoms; samples were suspended in thioglycerol. GC
analyses were carried out on a Hewletf-Paekard-5790chromatograph using a 12.5 x 0.2 mm capillary column of
cross-linked dimethylsilicone; the temp. required for the analysis of per(trimethylsily1)-sugar derivatives was
165” for 2 min, then increased at 2”/min.
Acidic Hydrolysis. The saponin ( 2 mg) in MeOH (1 ml) was refluxed in 4 N (10 ml) HCI for 4 h. The
aglycone was extracted with AcOEt and identified by TLC on silica gel with (i-Pr),O/acetone 7 5 : 3 0 . Thc aq.
layer was adjusted to pH 6 with NaHC03. After evaporation to dryness, the sugars were extracted with pyridine
from the residue and analyzed by TLC on silica gel with AcOEt/MeOH/H20/AcOH 95: 15:15:20; detection
with p-anisidine phthalate, and by GC as per(trimethylsily1) derivatives, prepared by adding 60 p1 of hexa-
methyldisilazane/trimethylchlorosilane 2: 1 to 100 11 of the pyridine solution.
Basic Hydrolysis. The saponin (30 mg) in MeOH (5 ml) was refluxed in 0 . 5 aq.
~ KOH (20 ml) for I h. The
mixture was adjusted to pH 6 with aq. HC1 and then extracted with 2 x 25 ml of BuOH; the org. phase was
washed with 20 rnl of H 2 0 , and evaporated to dryness. The partially hydrolyzed saponin was purified on a
Sephadex-LH-20 column eluted with MeOH.
1314 HELVETICA ACTA- Vol. 67 (1984)
CHIMICA

Isolation. Dried berries (48 g) of Phytolacca dodecundra I'HERITcollected in Ethiopia (strain 1733) were
extracted successively with petroleum ether (2 x 500 ml), CHCI, (2 500 ml), MeOH (3 x 500 ml), and finally
H,O (2 x 300 ml). The MeOH extract (13 g) was further studied. A first separation was carried out by RLCC
with AcOEt/EtOH/H,O 40:20:40 in the ascending mode with two portions of 4 g of extract. The eluate was
collected into six fractions: I(200 mg), 11 (400 mg), 111 (440 mg), 1V (470 mg), V (1370 mg), and Vl(1520 mg).
Chromatography of Fraction 111on a Lobar-RP-8 column with MeOH/H,O 65: 35 yielded compound 1, purified
by a second chromatographic separation under the same conditions and then on a Sephadex-LH-20 column
with MeOH: 50 mg of pure 1. Fraction 1V was submitted to reversed-phase chromatography on a Lobar-RP-8
column with MeOH/H,O 65:35 to give 2 (240 mg; further purified on a Sephadex-LH-20 column with MeOH).
Four separations of Fraction V (each time 300 mg) on a Lobar-RP-8 column afforded 2 (300 mg; further
purified on a Sephadrx-LH-20 column with MeOH) and a mixture of 2 other saponins. Chromatography of this
mixture on a Scphadex LH-20 column with MeOH and then on a Lobar-RP-8 column with MeOH/H,O 55:45
yielded 45 mg of pure 3.
28-0-jp- o-Glucopyrunosyl)-3-0-[ O-/i-u-gl~tcopyrano.syf-(I +4)-8- u-gfucopyranosyllbayogrnin ( = p-D-
Glucopyranosyl-38-f ( 0-p- D-glucopyranosyl-( I +4 ) -~-D-glucopyranc~syl) oxy]-2p. 23-dihydroxyolean-12-m-
28-oate; 1; C48H78020): white powder, m.p. 240-250" (dec.). I3C-NMR (90.52 MHz; (D5)pyridine): F's of the
aglycone and the diglucosyl moiety at C(3) identical to those of l a ; 6's of the a-o-glucopyranosyl ester unit:
95.7 (C(l)), 74.1 (C(2)), 79.1 (C(3)), 71.2 (C(4)), 78.9 (C(5)), 62.0 (C(6)). MS (D/CI; NH,, negative ions): 973
( [ M - HI-), 955 ([(M- H)-18]-), 81 I ( [ ( M- H)-162]-), 649 ([(M-H)-324]-). MS (DjCI; NH,, positive ions):
830 ( [ ( M + NH4)-162]+),668 ( [ ( M+ NH4)-324]+), 506 ( [ ( M + NH4)-486]+).
Acidic hydrolysis of 1 afforded D-ghCose and an aglycone identified as bayogenin ( = 2p,3b.23-trihydroxyy-
olean-12-en-28-oic acid): 'H-NMR (360 MHz, (D,)acetone): 0.93 (s, CH,); 1.04 (s, CH,); 1.06 (s, CH,); 1.07 (s,
CH,); 1.28 (s, CH,); 1.42 (s,CH,); 3.66 ( A B , J A 8 = 9.5, CH2OH); 3.71 (d, J = 3.6, H-C(3)); 4.17 (m, H-C(2));
5.38 ( t , J = 3, H-C(I2)). MS (DjCl; CH4. positive ions): 489 ( [ M + HI'), 487, 471, 453, 435, 391, 248.
3-0-1 0-&D-Ghfcopyranosyl-( I -t4)-/3-~-gluco~~yranosyl]bayogenin ( = 3/r\-[0-p- 1~-Glucopyranosyl-(1 44)-
~-~-~luco~pyrunosyl)o,~yJ-2~.23-dihydroxyolean-I2-en-28-oic Acid; l a ; C42H68015) was obtained after basic hy-
drolysis of I : white powder, m.p. 250-260" (dec.). "C-NMR (90.52 MHz, (D,)pyridine): aglycone: 15.0 (C(24)),
17.3 (C(25)), 17.6 (C(26)), 18.1 (C(6)), 23.8 (C(11)), 23.8 (C(30)), 24.1 (C(16)), 26.3 (C(27)), 28.4 (C(I5)), 31.0
(C(20)), 33.4 (C(22)), 33.1 (C(7)), 33.3 (C(29)), 34.3 (C(21)), 37.1 (C(10)), 40.0 (C(8)), 42.1 (C(14)), 42.4 (C(18)),
42.8 (C(4)), 44.1 (C(19)), 46.5 (C(17)), 46.7 (C(I)), 47.9 (C(9)), 48.6 (C(5)), 65.6 (C(2)), 70.6 (C(23)), 83.2 (C(3)),
122.9 (C(12)), 144.9 (C(13)), 180.1 (C(28)); sugar moieties: see Table. MS (D/CI; NH,, positive ions): 830
( [ M + NH4]+), 813 ( [ M +HI+), 668 ([(MNH4)-162]+). 651 ( [ ( M+ H)-162]+), 506 ( [ ( M + NH4)-324]+), 489
+
([(M H)-324]').
p- D-Glucopyrano.Yy1 3- 0-[2',4'-Di- O-(p-u-Glucopyranosylj-b-~-glucopyranosyl]oleanoIate ( = p- 0-Gluco-
-a-
pyranosyl 3p-[ (2'.4'-Pi- 0-(p-o-Glucopyranosyl) D-g~ucopyranosy~)oxy]olean-l2-en-28-oate: 2; C54H8R023):
white powder, m.p. 248-252" (dec.). I3C-NMR (90.52 MKz; (D,)pyridine): 6's of the aglycone and the 3 sugar
units at C(3) identical with those of 2a; 6's of the 8-o-glucopyranosyl ester moiety: 95.8 (C(l)), 74.1 (C(2)), 79.3
(C(3)), 71.2 (C(4)), 78.9 (C(5)), 62.2 (C(6)). MS (FAB; thioglycerol, negative ions): 1139 ( [ M + Cll-), 1103
( [ M + Cll-), 941 ([(M- H)-162]-), 779 ( ( ( M - H)-324]-), 617 ([(M- H)-486]-), 455 ( [ ( M- H)-648]-).
Acidic hydrolysis of 2 afforded oleanolic acid and D-glucose.
3-0-[2'.4'-Di-0-(p-D-Glucopyranosyl)-P-~-gIucopyranosyl]oleanolic Acid ( = 3p-[(2',4'-Pi- O-(B- D-G~UCO-
pyrano.~yl)-~-o-glucopyranosyl)oxyJolean-12-en-28-oic Acid; 2a; C4,H,,OI8) was obtained after partial (basic)
hydrolysis of 2; white powder, m.p. 230-240" (dec.). 13C-NMR (90.52 MHz, (D,)pyridine): 6's of the aglycone
correspond to those previously described for oleanolic acid [I I]; sugar signals: see Table. MS (FAB; thioglyce-
rol, negative ions): 977 ( [ M + Cll-), 941 ( [ M - HI-), 579 ( [ ( M- H)-162]-), 617 ( [ ( M - H)-324]-).
Acefylation 4 2 a : Overnight, 2a (12 mg) was stirred with 2 ml of anh. pyridine/Ac20 1: I. Addition of H,O
induced the precipitation of the peracetate, m.p. 156-158".
3- 0-[2'.4'-Di-0-(p- D-Glucop.vranosy1) -8-D-glucopyranosy~]-28-o-(p-D-glucopyranosylj hederagenin ( = I(-D-
Glucopyrunosyl3~-[(2',4'-Di- O-(/l-Glucopyranosylj -b-o-glucopyranosyl) oxy /-23-hydroxyolean- 12-en-28-oate; 3;
C,4H,8024): white powder, m.p. 25Ck255" (dec.). 13C-NMR (90.52 MHz, (Ds)pyridine): 6's of the aglycone and
the 3 sugar units at C(3) identical with those of 3a; 6's of the p-D-glucopyranosyl ester moiety: 95.7 (C(l)), 74.1
(C(2)), 79.1 (C(3)), 71.4 (C(4)), 78.9 (C(5)), 62.0 (C(6)). MS (FAB; thioglycerol, negative ions): 1155
( [ M + a]-), 1 I19 ([M-HI-), 957 ([(M- H)-l62]-), 795 ([(M- H)-324]-), 633 ( [ ( M- H)-486]-), 471
([(M - H)-648]-).
3- 0-12' ,4'-Di- O-(a- D-GIucopyranosylJ-P-D-glucopyranosyl]hederagenin ( = 3P-[ (2',4'-Di- 0-($ ~-G[ucopy-
ranosyl)-p- D-g~ucopyranosy~)oxyJ-23-hydroxyolean-12-en-28-oic Acid; 3a; C4,H,,0,,) was obtained after basic
 HELVE'rICA CHIMICAACTA vol. 67 (1984)
- 1315

hydrolysis of 3: white powder, m.p. 245-250" (dec.). I3C-NMR (90.52 MHz, (DJpyridine): 6's of the aglycone
correspond to those previously described [Ill; inner sugar moiety: 103.6 (C(l')), 82.3 (C(2')), 74.7 (C(3')). 80.5
(C(4')), 76.7 (C(5')), 62.8 (C(6')); terminal sugar moieties: 104.7 (C(l")), 76.0 (C(2")), 77.9 (C(3")), 71.7 (C(4")),
78.1 (C(5")), 62.2 (C(6")), 105.3 (C(l"')), 76.7 (C(2"')), 78.2 (C(3"')), 71.6 (C(4"')), 78.3 (C(5"')), 62.4 (C(6)).

REFERENCES

[I] H . Kloos & F . S . McCullough. Planta Med. 46, 195 (1982).

[2] C. 5. Lugt, 'Phytolacca dodecandru Berries as a Means of Controlling Bilharzia Transmitting Snails', Litho
Printers, Addis Ahaba, 1981.
[3] P.H . Goll, A . Lemma, J . Duncan & B. Mazengia, Tropenmed. Parasit. 34, 177 (1983).
[4] A . Lemma, Ethiopian Med. J. 3, 84 (1965); idem, ibid. 3, 187 (1965).
[S] R. M . Parkhurst, D. W. Thomas, W.A. Skinner & L. W. Cary, Phytochemistry 12, 1437 (1973); iidem, Indian
J. Chem. 11, 1192 (1973); iidem, Can. J. Chem. 52, 702 (1974).
[6] 5.Domon & K . Hostettmann, Helv. Chim. Actd 66, 422 (1983).
171 K . Hostettmann, Naturwissenschaften 71, 247 (1984).
[8] J. K . Snyder, K. Nakunishi, K . Hostettmann & M . Hostettmann, J. Liquid Chromatogr. 7, 243 (1984).
[9] K. Hostettmann, J. Doumus & M . Hardy, Helv. Chim. Acta 64, 297 (1981).
101 M . Barber, R.S. 5ordoli. G . J . Elliott, R. D. Sedgwick & A. N . Tyler, Anal. Chem. 54, 645A (1982).
.I11 K . Tori, S . Seo, A . Shimuoka & Y. Tomita, Tetrahedron Lett. 1974, 4227.
121 T. Konishi, A . Tada, J. Shoji. R. Kasai & 0 . Tanaka, Chem. Pharm. Bull, 26, 668 (1978).
131 J . W. Powell & W.B. Whalley, Phytochemistry X, 2105 (1969).
141 P. Godin, Nature 174, 134 (1954).