Molluscicidal saponins from Phytolacca icosandra

Article abstract of DOI:10.1016/S0031-9422(00)00233-8

Five monodesmosidic serjanic acid saponins and a monodesmosidic spergulagenic acid saponin were isolated from an aqueous extract of the berries of Phytolacca icosandra. A methanol extract of the berries furnished three bidesmosidic serjanic acid glycosides. Their structures were established by spectroscopic (ES-MS, ¹H NMR, COSY, HSQC, HMBC, ¹³C NMR) and chemical methods. The molluscicidal, spermicidal and haemolytic properties of the saponins were investigated. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00233-8

Phytochemistry 55 (2000) 603±609
www.elsevier.com/locate/phytochem
Molluscicidal saponins from Phytolacca icosandra
a
a
b
a,
Virginie Treyvaud , Andrew Marston , Wahyo Dyatmiko , Kurt Hostettmann *
aInstitut de Pharmacognosie et Phytochimie, Universit e de Lausanne, BEP, CH-1015 Lausanne, Switzerland
bFakultas Pascasarjana, Universitas Airlangga, Surabaya-60286, Indonesia
Received 28 December 1999; received in revised form 5 April 2000
Abstract
Five monodesmosidic serjanic acid saponins and a monodesmosidic spergulagenic acid saponin were isolated from an aqueous
extract of the berries of Phytolacca icosandra. A methanol extract of the berries furnished three bidesmosidic serjanic acid glyco-
sides. Their structures were established by spectroscopic (ES±MS, H NMR, COSY, HSQC, HMBC, 13C NMR) and chemical
methods. The molluscicidal, spermicidal and haemolytic properties of the saponins were investigated. # 2000 Elsevier Science Ltd.
All rights reserved.
1
Keywords: Phytolacca icosandra; Phytolaccaceae; Triterpene saponins; Molluscicidal activity; Haemolytic activity; Spermicidal activity
1
. Introduction
islands) but which is now naturalized in the hilly and
mountainous regions of central and east Java. It is a
noxious weed in Australia, New Zealand and South
Africa.
The discovery of the potent molluscicidal properties
of the berries of Phytolacca dodecandra L'Herit (Phyto-
Â
laccaceae) from Ethiopia (Lemma, 1965) has prompted
a large amount of work on plant-derived compounds
which show toxicity to schistosomiasis-transmitting
snails (Marston and Hostettmann, 1985). Known as
In the present paper, we report the isolation of eight
glycosides of serjanic acid (1±5, 7±9) from the fruits of
P. icosandra, together with a glycoside of spergulagenic
acid (6).
`
2
`endod'' or soapberry, P. dodecandra contains up to
5% (w/w) saponins in the dried berries. Both mollus-
cicidal monodesmosidic oleanolic acid saponins (Par-
khurst et al., 1974) and inactive bidesmosidic glycosides
of oleanolic acid, hederagenin and bayogenin (Domon
and Hostettmann, 1984; Dorsaz and Hostettmann,
2. Results and discussion
The berries of P. icosandra were extracted sequentially
with dichloromethane and methanol. A second batch
was extracted with water alone, since it is known in the
case of P. dodecandra that aqueous extraction converts
bidesmosidic saponins into monodesmosidic saponins,
with a consequent increase in molluscicidal activity
(Domon and Hostettmann, 1984).
1986) have been identi®ed in the berries. Several ®eld
trials with aqueous extracts of the berries of P. dode-
candra have been performed in Ethiopia in order to
control Schistosoma mansoni by killing the intermediate
snail host of this parasite (see, for example, Goll et al.,
1
983).
In an attempt to evaluate the molluscicidal potential
The methanol and water extracts showed molluscici-
dal activity at 200 mg/ml and 25 mg/ml, respectively,
after 24 h, against the snail Biomphalaria glabrata.
The aqueous extract was partitioned between water
and n-butanol. The molluscicidal n-butanol fraction was
separated on a silica gel column using a chloroform-
methanol-water gradient, to give 14 fractions (I±XIV).
Further chromatography of these fractions on silica gel
and Sephadex LH-20, together with low-pressure LC
(LPLC) on Lobar RP-18 columns, gave saponins 1±6.
of other species of the genus Phytolacca, the berries
from P. icosandra L., found in Indonesia, were investi-
gated for their saponin content.
P. icosandra is a shrub native to tropical America
from Mexico to Peru and found also on the Caribbean
(
* Corresponding author. Tel.: +41-692-4561; fax: +41-692-4565.
E-mail address: kurt.hostettmann@ipp.unil.ch (K. Hostettmann).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00233-8

6
04
V. Treyvaud et al. / Phytochemistry 55 (2000) 603±609
After partition of the methanol extract of the berries
between n-butanol and water, the n-butanol fraction
Signals at ꢀ 106.2 and 106.4 for the anomeric carbons
of the sugar moieties in the 13C NMR spectrum of 2
suggested the presence of a disaccharide moiety
attached at C-3. Comparison of spectral data with lit-
erature values (Haraguchi et al., 1988) proved the
saponin to be 3-O-(b-d-galactopyranosyl-(1!3)-b-d-
glucopyranosyl)serjanic acid.
was separated by centrifugal partition chromatography
CPC) into eight fractions (I±VIII). Gel ®ltration on
(
Sephadex LH-20 and silica gel column chromatography
yielded saponins 7±9.
The aglycone part of compounds 1±5 and 7±9 gave
signals in the 13C NMR spectrum which corresponded
to serjanic acid (methyl spergulagenate) (Kohda et al.,
The ES mass spectrum of 3 gave a pseudomolecular
�
ion at m/z 823 [M� H] , indicating the presence of two
1992) (Table 1). The signal for C-30 in all cases was at ꢀ
176.9±177.1, con®rming the presence of a methyl ester
hexosyl units attached to the aglycone (serjanic acid).
2
An MS experiment on this ion gave two fragments at
at this position. In 1±5, C-28 appeared at ꢀ 179.7±180.0,
suggesting a free acid function, while C-28 appeared at ꢀ
m/z 661 and 499, corresponding to the sequential loss of
the two sugars. Acid hydrolysis of the glycoside gave
glucose as the only sugar. Comparison of the 13C NMR
spectrum of 3 with that of 1 showed a down®eld shift of
C-2 of the inner hexosyl moiety from ꢀ 75.8 to 83.4. The
part of the 13C NMR spectrum corresponding to the
sugar moiety was virtually identical to that of other
diglucosides substituted at position C-2, as found for
example in Astragalus sieberi (Verotta et al., 1998).
Consequently, the structure of 3 is 3-O-(b-d-glucopyrano-
syl-(1!2)-b-d-glucopyranosyl)serjanic acid.
1
76.0±176.1 in 7±9, implying esteri®cation at this posi-
tion.
For saponin 6, the signals for the aglycone in the 13C
NMR spectrum corresponded to spergulagenic acid
Haraguchi et al., 1988) (Table 1).
(
�
Saponin 1 gave an ion at m/z 661 [M� H] in the
2
electrospray (ES) mass spectrum. MS fragmentation of
this ion gave the deprotonated aglycone at m/z 499
�
[
A� H] , corresponding to the loss of a hexosyl unit.
1
3
Signals of the monosaccharide moiety in the C NMR
spectrum were attributed to glucose. The anomeric sig-
nal at ꢀ 106.9 showed the glucose to be attached at C-3
and not at C-28. Thus the structure of 1 was established
as 3-O-b-d-glucopyranosylserjanic acid. Saponin 1 has
previously been obtained as a partial hydrolysis product
of a saponin from P. thyrsi¯ora (Haraguchi et al., 1988).
Table 1
13C NMR spectral data for the aglycone parts of saponin 3 (serjanic
5
acid) and saponin 6 (spergulagenic acid) (125 MHz in pyridine-d )
Carbon
3
6
1
2
3
4
5
6
7
8
9
38.6
26.6
89.1
39.4
56.0
18.5
33.2
39.7
47.9
36.9
23.7
38.6
26.4
89.6
39.7
55.9
18.5
33.2
39.6
47.9
36.9
23.7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
±
0
1
2
123.1
144.2
42.0
28.2
23.9
46.2
42.6
43.3
44.2
30.7
34.5
28.4
17.5
15.4
16.9
26.0
179.7
28.5
177.1
51.7
123.3
144.3
42.0
28.4
23.9
46.3
43.0
43.3
44.0
31.1
34.7
28.4
17.3
15.4
16.8
26.1
180.0
29.0
179.1
±
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OCH
3

V. Treyvaud et al. / Phytochemistry 55 (2000) 603±609
605
A pseudomolecular ion at m/z 985 [M� H]� was
respectively. Thus, saponin 6 is 3-O-(a-l-rhamnopyr-
anosyl-(1!2)-b-d-glucopyranosyl-(1!2)-b-d-glucopyr-
anosyl) spergulagenic acid.
observed in the ES mass spectrum of 4. Fragments at m/z
2
8
23, 661 and 499 in an MS experiment corresponded to
the sequential loss of three hexosyl moieties from the
molecular ion. These were identi®ed as glucose after
acid hydrolysis. The anomeric proton resonances of 4
were at ꢀ 4.88 (d, J=7.8 Hz), 5.27 (d, J=7.3 Hz) and
Saponins 7±9 gave pseudomolecular ions at m/z 985,
1131 and 985, respectively, in their ES mass spectra. All
three were bidesmosidic saponins since basic hydrolysis
with 0.1 M KOH gave the corresponding mono-
desmosidic saponins of spergulagenic acid (the methyl
ester at C-30 was also hydrolysed) and glucose. Their
structures were con®rmed by analysis of the H, 13C and
2D NMR spectra. Saponin 7 was identi®ed as 3-O-(b-d-
galactopyranosyl - (1!3) - b - d - glucopyranosyl)serjanic
acid 28-O-b-d-glucopyranoside and 9 as 3-O-(a-l-
rhamnopyranosyl-(1!2)-b-d-glucopyranosyl-(1!2)-b-
d-glucopyranosyl) serjanic acid 28-O-b-d-glucopyrano-
side, both ®rst isolated from Phytolacca bogotensis
(Nielsen et al., 1995). Saponin 8 was identi®ed as 3-O-
(b-d-glucopyranosyl-(1!2)-b-d-glucopyranosyl) serjanic
acid 28-O-b-d-glucopyranoside, found for the ®rst time
in the fruits of P. thyrsi¯ora (Haraguchi et al., 1988).
Saponins 7, 8 and 9 are the corresponding bidesmosi-
dic glycosides of 2, 3 and 5, respectively. Since all the
major bidesmosides present in the methanol extract
were isolated, the corresponding bidesmosides of 1, 4
and 6 were either absent from this extract or only pre-
sent in trace amounts. At the same time, saponins 1±6
were only found in the aqueous extract and not in the
5.39 (d, J=7.8 Hz). Proof of attachment of the inner
glucose residue to C-3 of the aglycone was shown by the
correlation of the anomeric proton at ꢀ 4.88 with the
signal at ꢀ 89.1 in the HMBC spectrum. The 1!3
interglycosidic linkage with the second glucose moiety
was deduced from the correlation in the HMBC experi-
1
0
0
0
ment of the H-1 signal (ꢀ 5.27) with the C-3 at ꢀ 88.9.
The terminal glucose was attached to the C-3 position
of the middle sugar, as shown by the HMBC correlation
0
00
00
between H-1 (ꢀ 5.39) and C-3 (ꢀ 84.4). Accordingly, 4
is 3-O-(b-d-glucopyranosyl-(1!3)-b-d-glucopyranosyl-
(
1!3)-b-d-glucopyranosyl)serjanic acid.
The ES mass spectrum of 5 showed a pseudomole-
�
2
cular ion at m/z 969 [M� H] , while MS generated
fragments at m/z 823, 661 and 499 which were attrib-
uted to the sequential loss of a deoxy sugar, followed by
two hexosyl units. Acid hydrolysis of 5 gave glucose and
rhamnose. Consequently a linear trisaccharide with a
rhamnosyl terminal moiety is attached to the serjanic
acid aglycone. The interglycosidic linkages and the
linkage position of the sugar chain to the aglycone were
solved by concerted use of 2D NMR spectroscopic
techniques. The H± H COSY experiment allowed
sequential assignment of the proton resonances, while
the HMQC spectrum led to unambiguous assignment of
the carbon shifts (Table 2). In the HMBC spectrum, the
anomeric proton signals at ꢀ 6.42 (s, terminal rhamnose),
1
1
Table 2
13C NMR spectral data for the sugar moieties of saponins 3±6 (125
MHz in pyridine-d5)
Carbon
3
4
5
6
0
0
5.85 (d, J=7.3 Hz, glucose) and 4.96 (d, J=7.3 Hz,
1 (Glc)
2
104.9
83.4
78.4
71.7
78.0
62.7
105.9
74.6
88.9
69.7
77.9
62.6
105.0
78.9
79.4
72.8
77.9
62.6
105.0
79.0
79.5
72.8
77.9
63.4
inner glucose) exhibited correlations with the carbon
signals at ꢀ 78.5 (C-2 of second glucose), 79.0 (C-2 of
inner glucose) and 89.6 (C-3 of aglycone), respectively.
Saponin 5 is thus 3-O-(a-l-rhamnopyranosyl-(!)-b-d-
glucopyranosyl-(1!2)-b-d-glucopyranosyl) serjanic acid.
In the case of 6, the ES mass spectrum gave a pseudo-
0
3
0
4
0
5
0
6
0
0
1
2
3
4
5
6
(Glc)
(Glc)
(Rha)
106.0
77.2
78.1
71.6
78.3
62.8
106.3
72.0
84.4
69.5
77.0
62.0
102.0
78.5
79.4
72.6
77.5
62.7
102.0
78.5
79.4
72.6
77.5
62.8
00
00
00
0
0
�
2
molecular ion at m/z 955 [M� H] , MS fragments of
3
this ion at m/z 809, 647 and MS at m/z 647, 485, cor-
responding to the cleavage of a deoxy sugar, followed
0
0
by two hexosyl moieties. The signal at m/z 485 was due
�
0
0
0
000
000
00
00
00
1
2
3
4
5
6
106.2
75.8
78.3
71.5
78.6
62.5
to the deprotonated [A� H] ion of spergulagenic acid.
Acid hydrolysis revealed the presence of glucose and
1
rhamnose in the sugar portion, as in 5. In fact the H
and 13 C NMR spectra (Tables 2 and 3) of the sugar
moieties of 5 and 6 were virtually identical. The inter-
glycosidic linkages of 6 were con®rmed to be the same
as those found in 5 by an HMBC experiment, in which
the anomeric proton signals at ꢀ 6.41 (s, terminal
rhamnose) and 5.84 (d, J=7.8 Hz, middle glucose)
exhibited correlations with the carbon signals at ꢀ 78.5
0
00
000
1
2
3
4
5
6
101.9
72.4
71.9
74.3
69.5
19.0
101.9
72.4
71.9
74.4
69.5
19.0
0
0
0
0
0
00
00
00
00
00
(
C-2 of middle glucose) and 79.0 (C-2 of inner glucose),

6
06
V. Treyvaud et al. / Phytochemistry 55 (2000) 603±609
methanol extract (TLC and HPLC analysis). The gly-
cosides 3±5 have not previously been reported in the
literature.
Finnigan-MAT LCQ ion trap mass spectrometer in the
negative ion mode. H (500 MHz) and 13C (125 MHz)
NMR spectra were run on a Varian Inova Unity 500
1
1
1
The isolated saponins were tested for their mollusci-
cidal, spermicidal and haemolytic activities (Table 4).
The three bidesmosidic saponins (7±9) from the metha-
nol extract were all inactive against Biomphalaria glab-
rata snails. Saponins with molluscicidal activity were
only isolated from the aqueous extract. All the glyco-
sides from this extract (1±6) were monodesmosidic
saponins. Of these, 1 and 2 were the most active, with
minimum concentrations of 3.1 mg/ml required to kill
the snails. Saponins 3 and 4 were also toxic to snails at
the same concentration as that shown by the reference
compound, 3-O-(b-d-glucopyranosyl-(1!4)-b-d-gluco-
pyranosyl)bayogenin, one of the most active triterpene
glycosides isolated from P. dodecandra (Domon and
Hostettmann, 1984; Dorsaz and Hostettmann, 1986).
Glycoside 5, a trisaccharide with rhamnose as the term-
inal sugar, gave borderline toxicity to the snails. Sapo-
nin 6, di€ering only from 5 in having a free C-30
carboxyl group, was inactive in the bioassay. The hae-
molytic activities of the saponins parallelled very closely
the molluscicidal activities (Table 4). Again glycosides 1
and 2 showed the highest activities. The pattern was not
as clear for the spermicidal activities (Table 4). The dis-
accharide 3 was more active than the monosaccharide
derivative 1. However, saponins 1±4 still remained the
only compounds which gave positive results in this test,
as in the case of haemolytic activity. It is interesting to
note that the spergulagenic acid glycoside 6 was inactive
in all three biological tests.
spectrometer, in C D N. 2D experiments H± H DQF-
5 5
COSY (double quantum ®ltered direct chemical shift
1
13
correlation spectroscopy), inverse detected H± C
HSQC (heteronuclear single quantum coherence) and
HMBC (heteronuclear multiple bond connectivity) were
obtained using Varian VNMR software. LPLC was
performed on Lobar RP-18 size B columns (Merck) and
semi-prep. HPLC on a Waters PREPLC assembly with
2 Nova-Pak columns (PrepHR C18, 6 mm, 100Â40
mm). For gel ®ltration, Sephadex LH-20 (Pharmacia)
was used, with MeOH as eluent.
3.2. Plant material
Berries of P. icosandra were collected near Purwodadi
in Java, Indonesia in October 1992. Voucher specimens
have been deposited at the Herbarium Bogoriensis,
Bogor, Indonesia and at the Institute of Pharmacognosy
and Phytochemistry, University of Lausanne, Switzer-
land.
3.3. Extraction and isolation
Dried berries (50 g) of P. icosandra were ground and
extracted with H O (3Â500 ml). After partition of this
2
extract (15.7 g) between n-BuOH and H O, a part (4.4
2
g) of the n-BuOH fraction (4.5 g) was chromatographed
on silica gel, with a CHCl ±MeOH±H O (40:10:1!
3
2
13:7:1) gradient, to give 14 fractions (I±XIV). LPLC of
The high molluscicidal activity of the aqueous extract
25 mg/ml) can thus be attributed to the presence of
fr. II (MeOH±H O 70:30!75:25) gave saponin 1 (55
2
(
mg), while LPLC of fr. IX under the same condi-
tions gave 5 (102 mg). Gel ®ltration of fr. IV yiel-
ded 2 (164 mg). Saponins 3 (4 mg, from fr. V) and
4 (32 mg, from fr. VII) were puri®ed by a combi-
monodesmosidic saponins of serjanic and spergulagenic
acids. No monodesmosidic glycosides were isolated
from the methanol extract and the corresponding weak
molluscicidal activity (200 mg/ml) re¯ects the very low
concentration of monodesmosidic saponins in this
extract. At least in certain cases (2, 3, 5), the formation
of monodesmosidic glycosides presumably occurs dur-
ing extraction of the berries by water as a result of acti-
vation of enzymes present and cleavage of the glycoside
chains at C-28 (7±9) (Domon and Hostettmann, 1984).
In view of the potent molluscicidal activity of the
aqueous extract of the berries of P. icosandra, this could
be a promising candidate plant for the local control of
schistosomiasis in south-east Asia.
nation of silica gel chromatography (CHCl ±MeOH±
3
H O) and semi-prep HPLC (MeOH±H O 70:30). Fr.
2
2
XIV contained 6 (20 mg), which was further puri®ed by
gel ®ltration (MeOH) and LPLC with MeOH±H O
2
60:40.
A second batch of berries (400 g) was extracted ®rst
with CH Cl (3Â2.5 l) and then with MeOH (3Â2.5 l).
2
2
The MeOH extract (33 g) was partitioned between n-
BuOH and H O, and a portion (3 g) of the n-BuOH
2
fraction (19 g) was chromatographed by CPC on a
Pharma-Tech CCC-1000 instrument (capacity 650 ml).
Elution with the solvent CHCl ±MeOH±i-PrOH±H O
3
2
5
:6:1:4 (lower phase as mobile phase) at a ¯ow-rate of 3
ml/min gave a total of seven fractions (I±VII). Saponin
(236 mg) was obtained from fr. II after gel ®ltration,
3. Experimental
7
3.1. General procedures
saponin 8 (23 mg) from fr. III after silica gel chromato-
graphy (CHCl ±MeOH±H O 65:35:5) and saponin 9
3
2
Optical rotations were measured with a Perkin-Elmer
41 MC polarimeter. ESI±MS were conducted using a
(100 mg) from fr. VI after silica gel chromatography
with the same solvent.
2

V. Treyvaud et al. / Phytochemistry 55 (2000) 603±609
607
3.4. Saponin 1
3.8. Saponin 5
2
3
ꢀ
23
ꢀ
White amorphous powder. [a] +21 C (MeOH, c
d
White amorphous powder. [a] +62 C (MeOH, c
d
�
2
13
1
0
.1). Negative ion mode ES±MS m/z: 661 [M� H] ; MS
0.1). C NMR: Table 2. H NMR: Table 3. Negative
2
�
�
of m/z 661: m/z 499 [A� H] .
ion mode ES±MS m/z: 969 [M� H] ; MS of m/z 969:
�
m/z 823, 661, 499 [A� H] .
3
.5. Saponin 2
3
.9. Saponin 6
2
3
ꢀ
White amorphous powder. [a] +60 C (MeOH, c
d
�
2
23
ꢀ
0
.1). Negative ion mode ES±MS m/z: 823 [M� H] ; MS
White amorphous powder. [a] +22 C (MeOH, c
d
0.1). C NMR: Tables 1 and 2. H NMR: Table 3.
�
13
1
of m/z 823: m/z 661, 499 [A� H] .
�
2
Negative ion mode ES±MS m/z: 955 [M� H] ; MS of
3
�
3
.6. Saponin 3
m/z 955: m/z 809, 647; MS m/z 647, 485 [A� H] .
2
3
ꢀ
White amorphous powder. [a] +49 C (MeOH, c
3.10. Acid hydrolysis of saponins
d
.1). C NMR: Tables 1 and 2. H NMR: Table 3.
1
3
1
0
�
2
Negative ion mode ES±MS m/z: 823 [M� H] ; MS of
Saponin (1 mg) was heated at re¯ux with 5 ml of 2 N
HCl±MeOH (1:1) for 2 h. The MeOH was evaporated
and the aqueous solution was extracted with EtOAc.
The aglycone in the EtOAc layer was analysed by TLC
�
m/z 823: m/z 661, 499 [A� H] .
3
.7. Saponin 4
[(i-Pr) O±acetone 75:30]. The aqueous layer was evapo-
rated to dryness and monosaccharides were identi®ed
2
2
3
ꢀ
White amorphous powder. [a] +47 C (MeOH, c
d
.1). C NMR: Table 2. H NMR: Table 3. Negative
1
3
1
0
by comparison with authentic samples on TLC (i-
PrOH±H O 85:15), after detection with p-anisidine
phthalate.
�
2
ion mode ES±MS m/z: 985 [M� H] ; MS of m/z 985:
2
�
m/z 823, 661, 499 [A� H] .
Table 3
1H NMR spectral data for the sugar moieties of saponins 3±6 (500 MHz in pyridine-d
)a
5
Proton
3
4
5
6
0
1
2
3
4
5
6
6
(Glc)
4.94 d (7.8)
4.37 dd (8.8, 7.8)
4.29 m
4.88 d (7.8)
4.96 d (7.3)
4.95 d (7.3)
0
0
0
0
0
0
4.03 dd (8.9, 7.8)
4.32 dd (8.9, 8.9)
4.04 dd (8.9, 8.9)
4.01 m
4.43 dd (8.9, 7.3)
4.56 dd (8.9, 8.9)
4.10 dd (8.9, 8.9)
3.94 m
4.42 dd (8.8, 7.3)
4.55 dd (8.8, 8.8)
4.09 dd (8.8, 8.8)
3.92 m
4.17 dd (8.8, 8.8)
3.94 m
a
4.57 dd (12.0, 2.5)
4.39 dd (12.0, 5.5)
4.52 dd (11.5, 2.0)
4.31 m
4.51 dd (12.0, 2.5)
4.35 dd (12.0, 6.0)
4.51 dd (12.0, 2.5)
4.34 dd (12.0, 6.0)
b
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
6
(Glc)
5.39 d (7.8)
4.14 dd (8.8, 7.8)
4.20 m
5.27 d (7.3)
4.70 dd (9.0, 7.3)
4.31 m
5.85 d (7.3)
5.84 d (7.8)
4.30 dd (8.9, 7.3)
4.25 dd (8.9, 8.9)
4.08 dd (8.9, 8.9)
3.87 m
4.28 dd (8.9, 7.8)
4.24 dd (8.9, 8.9)
4.07 dd (8.9, 8.9)
3.83 m
4.33 dd (8.8, 8.8)
3.96 m
4.40 dd (9.0, 9.0)
3.93 m
a
4.50 dd (11.5, 2.4)
4.49 dd (11.5, 5.6)
4.52 dd (12.0, 2.2)
4.26 dd (12.0, 6.0)
4.48 dd (12.0, 2.2)
4.32 dd (12.0, 6.0)
4.46 dd (12.0, 2.5)
4.31 dd (12.0, 6.0)
0
b
0
0
0
0
0
0
0
00
00
00
00
00
00
00
1
2
3
4
5
6
6
(Glc)
5.39 d (7.8)
4.09 m
4.26 dd (8.9, 8.9)
4.22 dd (8.9, 8.9)
4.11 m
a
4.70 dd (12.0, 2.5)
4.39 dd (12.0, 6.0)
b
0
0
0
0
0
0
00
00
00
00
00
00
1
2
3
4
5
6
(Rha)
6.42 s
4.76 br s
6.41 s
4.75 br d (3.4)
4.69 dd (9.3, 3.4)
4.36 dd (9.3, 9.3)
5.04 dq (9.3, 6.3)
1.81 d (6.3)
4.71 dd (9.2, 3.1)
4.36 dd (9.2, 9.2)
5.06 dq (9.2, 6.3)
1.82 d (6.3)
a
Assignments based on COSY, HMQC and HMBC spectra.

6
08
V. Treyvaud et al. / Phytochemistry 55 (2000) 603±609
Table 4
Molluscicidal, spermicidal and haemolytic activities of saponins isolated from Phytolacca icosandra
Saponin
1
2
3
4
5
6
7
8
9
Controla
Molluscicidal activityb
Spermicidal activityc
Haemolytic activityd
3.1
700
1.9
3.1
500
3.8
10.0
250
7.5
12.5
1333
7.5
50.0
>50
>2000
>60
>50
>2000
>60
>50
>2000
>60
>50
>2000
>60
12.5
500
7.5
>2000
>60
a
3
-O-(b-d-Glucopyranosyl-(1!4)-b-d-glucopyranosyl)bayogenin.
b
c
Minimum concentration required to kill 100% of snails (Biomphalaria glabrata) after 24 h (mg/ml).
Minimum concentration required to cause 100% inhibition of the motility of human spermatozoids after 3 min (mg/ml).
Minimum concentration for total haemolysis of bovine erythrocytes (mg/ml).
d
3
.11. Alkaline hydrolysis of bidesmosidic saponins
were tested with bovine erythrocytes until haemolysis
was no longer observed. The suspension of erythrocytes
was prepared by washing bovine blood (5 ml) with 0.9%
isotonic solution (5 ml) and centrifuging. The supernatant
was removed and the washing procedure repeated until
the supernatant remained colourless. These erythrocytes
were mixed with an equal volume of 0.9% NaCl and 50
ml of this suspension was added to the solutions under
test. After 1 h, the mixture was centrifuged and the
transmittance of the supernatant was measured by a
spectrophotometer at 540 nm, using the supernatant
resulting after the washing procedure as blank.
Saponin (1 mg) was heated at re¯ux with 10 ml of 0.1
N KOH for 1 hour. The cooled reaction mixture was
then neutralised with 1 N HCl and then extracted with
n-BuOH. The aqueous layer was analysed for sugars (as
above) and the n-BuOH layer was evaporated to dry-
ness and checked by TLC with the solvent CHCl3±
MeOH-H O 65:35:5.
2
3.12. Molluscicidal activity
Molluscicidal activity was determined by the method
of Hostettmann et al. (1982). Extracts were initially tested
at 400 mg/ml and pure compounds at 50 mg/ml in water.
Toxicity to Biomphalaria glabrata snails was established
by observing heart beat under a microscope after 24 h.
Successive dilutions of the test solutions were made in
order to ®nd the minimum concentration required to
kill snails.
Acknowledgements
Financial support has been provided by the Swiss
National Science Foundation. The Herbette Foundation
of the University of Lausanne is gratefully acknowl-
edged for the award of a travel grant.
3.13. Spermicidal activity
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