1
38
V. Lanzotti et al. / Phytochemistry 74 (2012) 133–139
solvent system from H
eluted fraction followed by TLC analyses (SiO
water 60:15:25, v/v/v) revealed that only one fraction contained
saponin compounds. This last fraction (72 mg, eluted with H O–
MeOH (1:1) was further purified by HPLC on a semipreparative
18 column with the mobile phase H O–MeOH (40:60), giving pure
compound 1 (39.1 mg, t = 3.2 min), ascalonicoside A (5.8 mg,
= 4.3 min), and crude compounds 2 (12.1 mg, t = 3.9 min) and
(8.3 mg, t = 2.9 min). These latter compounds have been finally
purified by HPLC on a analytical C18 column with the mobile phase
2
O to MeOH. Preliminary NMR study of the
standard D-galactose and L-rhamnose for the hydrolysate of 3 gave
single peaks.
2
, butanol/acetic acid/
2
3.8. Biological assays
C
2
Antifungal activity of the three ceposides was tested on 10 dif-
ferent fungal species. Three soil-borne pathogens (Fusarium oxy-
sporum f. sp. lycopersici, R. solani and Sclerotium cepivorum), five
air-borne pathogens (Alternaria alternata, Aspergillus niger, B. cine-
rea, Mucor sp., Phomopsis sp.) and two antagonistic fungi (T. atrovi-
ride and Trichoderma harzianum) were selected. S. cepivorum was
used because is a pathogen specific of the Allium genus. All mi-
crobes were obtained from Department of Arboriculture, Botany
and Plant pathology, Agriculture Faculty (NA), Italy. Antifungal
R
t
R
R
3
R
2
H O–MeOH (40:60), giving respectively pure compounds
2
(
10.2 mg) and 3 (6.8 mg).
3.4. Ceposide A
activity was assessed by in vitro test following Barile et al.
3
(
25R)-furost-5(6)-en-1b,3b,22
a
,26-tetraol 1-O-b-
D
-xylopyrano-
(2006). Briefly, a suspension of 10 spores was prepared in 50
ll
syl
(
26-O- -rhamnoyranosyl-(1 ? 2)-O-b-
a
-D
D
-galactopyranoside
of 0.1 strength PDB with 15 ll of 5 mM potassium phosphate buf-
2
d
5
1); Yield: 39.1 mg; colorless amorphous solid;
c = 0.1 MeOH); IR (KBr)
H NMR data, see Tables 1 and 3; C NMR data, see Table 2.
½
aꢃ
ꢀ30.7°
fer (pH 6.7). Saponins were added at three concentrations (200, 50
and 10 p.p.m.) in a 96-well plate and incubated at 25 °C. Fungal hy-
phal length of germinating spore was measured after 14 to 24 h
depending on the fungal species. For Rhizoctonia and S. cepivorum
the antifungal activity was evaluated with a petri dish growth as-
say because such microbes do not produce conidia. Plates of 9 cm
of PDA added with the saponins at three concentrations as above
described, were inoculated with a 5 mm plug containing the fungi.
Plates were incubated at 25 °C and the fungi radial growth was
measured after 24, 48 and 72 h. Finally, antifungal synergistic ef-
fects of the three saponins were assessed on all fungi, by applying
the compounds in even mixtures (33% for each one) at three final
concentrations (200, 50 and 10 p.p.m.). Inhibition of fungal growth
was statistically evaluated by one-way ANOVA by using molecule
concentrations as a factor for each fungus. Significance was evalu-
ated in all cases at P < 0.05 and P < 0.01.
ꢀ
1
(
m
max 3410, 2930, 1150 and 1045 cm
;
1
13
ꢀ
HRFABMS (negative ion): found m/z 887.4632 [MꢀH] ; calculated
for C44
71
H O18 m/z 887.4647.
3
.5. Ceposide B
(
25R)-furost-5(6)-en-1b,3b,22
syl 26-O- -rhamnoyranosyl-(1 ? 2)-O-b-
Yield: 10.2 mg; colorless amorphous solid; ½
MeOH); IR (KBr)
a,26-tetraol 1-O-b-D-xylopyrano-
a-
D
D
-glucopyranoside (2);
25
aꢃ
ꢀ35.2° (c = 0.1
d
ꢀ1 1
m
max 3413, 2932, 1150 and 1043 cm
; H NMR
1
3
data, see Tables 1 and 3; C NMR data, see Table 2. HRFABMS (neg-
ꢀ
ative ion): found m/z 887.4628 [MꢀH] ; calculated for C44
71 18
H O
m/z 887.4647.
3.6. Ceposide C
Acknowledgements
(
25R)-furost-5(6)-en-1b,3b,22
anosyl 26-O- -rhamnoyranosyl-(1 ? 2)-O-b-
side (3); Yield: 6.8 mg, colorless amorphous solid; ½
c = 0.1 MeOH); IR (KBr)
a,26-tetraol 1-O-b-D-galactopyr-
a
-D
D
-galactopyrano-
Mass and NMR spectra were recorded at CSIAS, University of
Naples Federico II. The assistance of the staff is gratefully acknowl-
edged. We also thank. Alessandra Gargiulo and Vincenzo Antignani
for technical assistance in laboratory analyses.
25
aꢃ
ꢀ41.7°
d
ꢀ1
(
m
max 3400, 2934, 1159 and 1048 cm
;
1
13
H NMR data, see Tables 1 and 3; C NMR data, see Table 2.
ꢀ
HRFABMS (negative ion): found m/z 917.4715 [MꢀH] ; calculated
for C45
73
H O19 m/z 917.4726.
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.7. Determination of sugar absolute configurations
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1
L
D
D
L
D-
D
L