Steroidal saponins from the roots of Smilax aspera subsp. mauritanica

Article abstract of DOI:10.1248/cpb.56.1324

Two new steroidal saponins (1, 2) were isolated from the roots of Smilax aspera subsp. mauritanica (POIR.) ARCANG. (Liliaceae), together with the known curillin G (3), asparagoside E (4), asparoside A (5), asparoside B (6) and the phenolic compound resveratrol (7). Their structures were established mainly on the basis of 600 MHz 2D-NMR spectral data. 3 exhibited antifungal activity against the human pathogenic yeasts Candida albicans, C. glabrata and C. tropicalis (minimum inhibitory concentrations of 25, 25 and 50 μg/ml, respectively) whereas the other compounds were inactive.

Full text of DOI:10.1248/cpb.56.1324

1324
Notes
Chem. Pharm. Bull. 56(9) 1324—1327 (2008)
Vol. 56, No. 9
Steroidal Saponins from the Roots of Smilax aspera subsp. mauritanica
Zineddine BELHOUCHET,a Marc SAUTOUR,a Tomofumi MIYAMOTO,b and
Marie-Aleth LACAILLE-DUBOIS*, a
a Laboratoire de Pharmacognosie, Unité UMIB UPRES-EA 3660, Faculté de Pharmacie, Université de Bourgogne; 7 Bd.
Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France: and b Graduate School of Pharmaceutical Sciences, Kyushu University;
Fukuoka, 812–8582, Japan. Received March 15, 2008; accepted June 9, 2008; published online June 11, 2008
Two new steroidal saponins (1, 2) were isolated from the roots of Smilax aspera subsp. mauritanica (POIR.)
ARCANG. (Liliaceae), together with the known curillin G (3), asparagoside E (4), asparoside A (5), asparoside B
(6) and the phenolic compound resveratrol (7). Their structures were established mainly on the basis of 600 MHz
2D-NMR spectral data. 3 exhibited antifungal activity against the human pathogenic yeasts Candida albicans,
C. glabrata and C. tropicalis (minimum inhibitory concentrations of 25, 25 and 50 mg/ml, respectively) whereas
the other compounds were inactive.
Key words Smilax aspera subsp. mauritanica; Liliaceae; steroidal saponin; resveratrol; 2D-NMR; antifungal activity
The genus Smilax contains 350 species, which are distrib- aglycon was identified as sarsapogenin [(25S)-5b-spirostane-
uted widely mainly in tropical regions of east Asia, and 3b-ol]13,14) from the 1D and 2D NMR spectral data of 1 (see
South and North America. Several Smilax species have al- Table 1). The A/B cis-ring fusion was confirmed by observa-
ready been studied chemically and found to contain steroidal tion of the signals at dC 35.3 (C-5), 39.8 (C-9), and 23.6 (C-
saponins.1—6) From a biological point of view, some species 19), indicating that the aglycon is a 5b-steroidal sapogenin.15)
were documented to exhibit antiinflammatory,7) NO-modulat- The 25S stereochemistry of the 27-methyl group was de-
ing8) and antileprosic9) activity and recently, antifungal duced based on the presence of the two proton signals [dH
steroidal saponins were isolated from the rhizome of S. med- 3.34 (1H, d, Jꢂ10.9 Hz) and 4.00 (1H, m)], which corre-
1
ica.5,6) As part of our ongoing search for new antifungal sponded to the H2-26 in the H-NMR16) and the higher field
steroid saponins, 5,6,10—12) we have investigated the roots of resonance of C-27 (dC 15.8) when compared to the 13C-NMR
1
Smilax aspera subsp. mauritanica. The present paper reports shift of 25R-spirostanes (dC 17.1—17.2).17) The H-NMR
the isolation and characterization of two new steroidal spectrum of 1 displayed signals for three anomeric protons at
saponins (1, 2) along with four known saponins (3—6) and dH 4.84, 5.54 (d, Jꢂ7.5 Hz) and 6.0 (s), which gave correla-
the phenolic compound resveratrol (7). Their structures were tions, in the HSQC spectrum, with 13C-NMR signals at dC
determined by spectroscopic methods including 1D and 2D 100.0, 101.2 and 101.9, respectively. Evaluation of chemical
NMR experiments and FAB-MS and HR-ESI-MS. Further- shifts and spin–spin couplings allowed the identification
more, their antifungal activity was tested against three human of two b-glucopyranosyl units (Glc I, Glc II) and one a-
pathogenic yeasts (Candida albicans, C. glabrata, and C. rhamnopyranosyl units (Rha I). The sequence of the oligosac-
tropicalis).
charide chain was determined from the HMBC and NOESY
The n-BuOH-soluble fraction of the MeOH–H2O (7 : 3) spectra. Correlations observed in the HMBC spectrum be-
extract of the roots of S. aspera subsp. mauritanica was puri- tween the 1H-NMR signal at dH 4.84 (Glc I H-1) and the 13C-
fied by precipitation with diethyl ether to give a crude NMR signal at dC 74.7 (Agly C-3), and in the NOESY spec-
saponin mixture. This mixture was submitted to multiple trum between dH 4.84 (Glc I H-1) and dH 4.26 (m) (Agly H-
chromatographic steps involving vacuum-liquid chromatog- 3) proved the Glc I to be linked at C-3 of the aglycon. The
1
raphy (VLC) on reversed-phase C18 silica gel and medium- correlation in the HMBC spectrum between the H-NMR
pressure liquid chromatography (MPLC) on normal silica gel signal at dH 5.54 (d, Jꢂ7.5 Hz) (Glc II I H-1) and dC 78.2
to yield compounds 1—6. The diethyl ether fraction was sub- (Glc I C-2) and a NOESY cross-peak between dH 5.54 (d,
mitted to multiple MPLC to give the pure phenolic com- Jꢂ7.5 Hz) (Glc II H-1) and dH 4.34 (Glc I H-2), revealed a
pound 7.
(1→2) linkage between these two sugars. The linkage of Rha
Compound 1, a white amorphous powder, exhibited in I to the 2-position of Glc II was deduced by the HMBC cor-
FAB-MS (negative-ion mode) a quasimolecular ion peak at relation observed between dH 6.0 (s) (Rha I H-1) and dC 79.4
m/z 885 [MꢀH]ꢀ, indicating a molecular weight of 886. (Glc II C-2) and the NOESY cross-peak between dH 6.0 (s)
Its molecular formula was established as C45H74O17 by the (Rha I H-1) and dH 3.94 (Glc II H-2). On the basis of the
positive ion-mode HR-ESI-MS showing a pseudo-molecular above results, the structure of 1 was established as (25S)-5b-
ion peak at m/z 909.5392 [MꢁNa]ꢁ (Calcd for C43H70O17Na: spirostane-3b-ol 3-O-a-L-rhamnopyranosyl-(1→2)-b-D-glu-
909.5352). Other fragment ion peaks were observed at m/z copyranosyl-(1→2)-b-D-glucopyranoside.
739 [(MꢀH)ꢀ146]ꢀ and 577 [(MꢀH)ꢀ146ꢀ162]ꢀ, corre-
Compound 2 was isolated as a white amorphous powder.
sponding to the successive loss of one deoxy-hexosyl and Its HR-ESI-MS (positive-ion mode) exhibited a pseudo-mo-
one hexosyl moieties. Acid hydrolysis of 1 yielded glucose, lecular ion peak at m/z 1089.5195 [MꢁNa]ꢁ (Calcd for
rhamnose (TLC) and their absolute configuration was deter- C51H86O23Na: 1045.5245), ascribable to a molecular formula
mined to be D and L by GC analysis of chiral derivatives of C51H86O23. Its FAB-MS spectrum (negative-ion mode) showed
the sugars in the acid hydrolysate (see Experimental). The a quasi-molecular ion peak at m/z 1065 [MꢀH]ꢀ indicating a
∗ To whom correspondence should be addressed. e-mail: malacd@u-bourgogne.fr
© 2008 Pharmaceutical Society of Japan

September 2008
1325
Fig. 1. Chemical Structures of 1—3
Table 1. 1H- and 13C-NMR Dataa) of the Aglycone Part of 1—2, d in ppm
(J in Hz)
molecular weight of 1066. A fragment ion peak was ob-
served at m/z 919 [(MꢀH)ꢀ146]ꢀ corresponding to the loss
of one deoxy-hexosyl moiety. The comparison of NMR data
of 2 (Table 1) with literature data allowed the identification
of the aglycon as the previously reported (25S)-3b,5b,22a-
furostane-3,22,26-triol (the aglycon of aspafilioside D).18)
The 25S stereochemistry of the Me-27 group was deduced
from the resonances of protons and carbons at C-25 (dC
33.1), C-26 (dC 74.5), and C-27 (dC 17.0) in comparison
with literature data.19,20) The differences observed in 1H-
NMR chemical shifts of the geminal protons H-26a and H-
26b (daꢀdbꢂ0.58) supported a 25S furostane-type steroid
since this difference is usually more than 0.57 ppm in 25S
compounds and less than 0.48 ppm in 25R compounds.19,20)
The 1H-NMR spectrum of 2 displayed signals for four
anomeric protons at dH 4.90 (d, Jꢂ7.5 Hz), 5.60 (d, Jꢂ7.5
Hz), 6.06 (s) and 4.70 (d, Jꢂ7.6 Hz), which gave correla-
tions, in the HSQC spectrum, with 13C-NMR signals at dC
99.4, 101.6, 101.9 and 104.3, respectively. Evaluation of
chemical shifts and spin–spin couplings allowed the identifi-
cation of three b-glucopyranosyl units (Glc I, Glc II, Glc III)
and one a-rhamnopyranosyl unit (Rha I). The sequence of
the oligosaccharide chain was determined from the HMBC
1
2
b),c)
b),c)
dC
dH
dC
dH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
30.2
26.4
74.7
29.6
35.3
25.6
25.8
35.1
39.8
34.8
20.7
39.9
40.5
56.0
31.6
81.1
62.2
16.1
23.6
42.1
14.4
109.7
31.6
26.3
26.9
64.9
15.8
1.28,b) nd
1.52,b) 1.80b)
4.26 m
29.4
26.5
74.5
29.4
35.2
30.3
26.4
34.9
39.9
34.8
21.6
39.9
40.9
55.9
31.8
81.0
63.2
16.3
23.6
40.2
16.0
110.5
36.4
27.8
33.1
1.20,b) nd
1.10,b) 1.84b)
4.29 m
1.70,b) nd
2.35b)
1.20,b) nd
2.36b)
1.30,b) 1.40b)
nd, nd
1.80,b) nd
1.10,b) nd
1.33 m
1.30 m
1.28b)
1.20b)
1.10,b) nd
nd
1.00,b) 1.60b)
1.20,b) 1.65b)
1.00b)
0.98b)
nd
1.30,b) 1.62b)
4.55 m
4.94 m
1.92b)
0.79 s
0.90 s
2.16 m
1.26b)
1.80 dd (6.7, 8.8)
0.74 s
0.88 s
1.84 m
1.10 d (6.7)
1
and the NOESY spectra. H- and 13C-NMR signals of the
1.25,b) 1.95b)
1.30,b) 2.08b)
1.80
1.96,b) 2.07b)
1.63,b) 1.97b)
1.86 m
oligoside at C-3 were superimposable with those of 1 reveal-
ing the same sequence of sugars as described above. The
linkage of the sugar at the C-26 position was indicated by the
correlation in the NOESY spectrum between the anomeric
proton of Glc III at dH 4.70 (d, Jꢂ7.9 Hz) and dH 3.42 (Agly
C-26). On the basis of the above results, the structure of 2
was established as (25S)-3b,5b,22a-furostane-3,22,26-triol
3-O-a-L-rhamnopyranosyl-(1→2)-b-D-glucopyranosyl-
(1→2)-b-D-glucopyranosyl 26-O-b-D-glucopyranoside.
3.34 d (10.9), 4.00b) 74.5
3.42,b) 4.00b)
0.98 d (6.5)
0.99 d (6.9)
17.0
a) Measured at 600 MHz for 1H and 150 MHz for 13C with reference to pyridine-d5.
b) Overlapping 1H-NMR signals are reported without designated multiplicities. c)
nd: not determined.
NOESY, HSQC and HMBC) as well by comparison with lit-
Compounds 3—7 were identified by interpretation of their erature data as curillin G (3),21) asparagoside E (4),22) asparo-
spectral data, mainly FAB-MS and 2D-NMR (COSY, TOCSY, side A (5),23) asparoside B (6)23) and resveratrol (7),24) re-

1326
Vol. 56, No. 9
Table 2. 1H- and 13C-NMR Data of the Sugar Moieties of 1—2 (in Pyri-
dine-d5),a),b),c) d in ppm (J in Hz)
Table 3. Antifungal Activity of 1—6 and Ketoconazole against Candida
Species Given as MIC (mg/ml)a)
Compounds Candida albicans Candida glabrata Candida tropicalis
1
2
1
2
3
ꢄ200
ꢄ200
25
ꢄ200
1.56
ꢄ200
ꢄ200
25
ꢄ200
1.56
ꢄ200
ꢄ200
50
ꢄ200
3.12
dC
dH
dC
dH
Glc I
1
2
3
4
5
6
100.0
78.2
79.4
71.0
77.6
62.8
4.84b)
4.34b)
4.02
99.4
78.4
79.2
71.1
77.6
62.1
4.90 d (7.5)
4.39b)
4—6
Ketoconazoleb)
4.52
3.94b)
4.04b)
a) Compounds with MIC values ꢄ200 mg/ml are considered not active. b) Posi-
tive control.
3.82 br d (9.3)
3.85b)
4.12,b) 4.50b)
4.20,b) 4.40b)
Glc II
Table 4. Antifungal Activity of 1, 3 and Other Spirostanol Saponins
(SS1—SS3) According to the Osidic Part at the Position C-3
1
2
3
4
5
6
101.2
79.4
78.3
72.1
77.6
61.7
5.54 d (7.5)
3.94b)
101.6
79.4
78.4
72.2
77.6
62.9
5.60 d (7.5)
4.00 dd (9.3, 9.8)
4.08b)
4.02 dd (8.6, 8.8)
3.78 (8.6, 9.8)
3.84 m
MIC values against
3.84b)
Osidic part at the position C-3
C. albicans, C. glabrata Ref.
and C. tropicalis
3.84b)
4.12,b) 4.32b)
4.19,b) 4.52b)
Rha I
3
: b-D-Glc-(1→4)-b-D-Glc
25 to 50 mg/ml
1
2
3
4
5
6
101.9
71.6
71.7
73.5
68.7
18.4
6.0 s
101.9
71.7
71.7
73.1
68.8
18.5
6.06 s
4.57 br s
4.57 dd (7.6, 11.2)
4.26 dd (9.9, 9.1)
4.89b)
SS1 : b-D-Glc-(1→6)-b-D-Glc
6.25 to 25 mg/ml
-Glc 12.5 to 50 mg/ml
-Glc 25 to 50 mg/ml
6
5
5
4.63 br s
SS2 : b-
D
-Glc-(1→6)-[b-
D
-Glc-(1→4)]-b-
-Glc-(1→2)]-b-
D
D
4.52b)
SS3 : b-
D
-Glc-(1→6)-[b-
D
4.22b)
1
: a-L-Rha-(1→2)-b-D-Glc-(1→2)-b-D-Glc
ꢄ200 mg/ml
4.86 dq (5.9, 9.3)
1.70 d (6.2)
1.72 d (4.5)
Glc III
1
2
3
4
5
6
104.3
73.7
77.6
71.1
77.6
62.9
4.70 d (7.6)
3.92b)
trisacharride moiety (Table 4), while the osidic chain of 1 is
linear. The primary mode of action of saponins is believed to
be through interaction with sterols of the plasma mem-
brane,28) and the presence of a linear-chain trisaccharide moi-
ety at the position C-3 seems to reduce the antifungal activ-
ity.
4.18 dd (8.1, 8.3)
3.97 dd (8.6, 8.3)
3.85b)
4.19,b) 4.38b)
a) The assignments were based on the DEPT, HSQC and HMBC experiment
(150 MHz for 13C-NMR, 600 MHz for 1H-NMR). b) Overlapping 1H-NMR signals
are reported without designated multiplicities. c) 1H- and 13C-NMR chemical shifts
of substituted residues are italicized.
Experimental
General Experimental Methods IR spectra (CHCl3) were recorded on
a Perkin-Elmer 881 spectrophotometer. FAB-MS (negative-ion mode, glyc-
erol matrix) was conducted on a JEOL SX 102 spectrometer. HR-ESI-MS
was carried out on a Q-TOF 1 micromass spectrometer. Optical rotations
spectively. The presence of resveratrol in some plants species
was of no interest until 1992 when it was reported the oc-
curence of this compound as a potent antioxidant.25) This were taken with a AA-10R automatic polarimeter. The 1D and 2D NMR
spectra (1H–1H COSY, TOCSY, NOESY, HSQC and HMBC) were per-
formed using a UNITY-600 spectrometer at the operating frequency of
600 MHz on a Varian INOVA 600 instrument equipped with a SUN 4 L-X
compound was previously isolated in some Liliaceae such as
Veratrum sp.26) or Yucca sp.27) but to our knowledge, this is
the first account of the isolation of resveratrol from the Smi-
lax genus.
computer system (600 MHz for 1H and 150 MHz for 13C spectra). All chemi-
cal shifts (d) are given in ppm and the samples were solubilized in pyridine-
d5 (dC 150.3, 135.9, 123.9). GC analysis was carried out on a Thermoquest
gas chromatograph using a DB-1701 capillary column (30 mꢃ0.25 mm, i.d.)
(J & W Scientific), with detection by FID, and the initial temperature main-
tained at 80 °C for 5 min and then raised to 270 °C at the rate of 15 °C/min;
carrier gas: He. Compound isolations were carried out using a medium-pres-
sure liquid chromatography (MPLC) system [Gilson M 305 pump, 25 SC
head pump, M 805 manometric module, Büchi column (460ꢃ25 mm and
460ꢃ15 mm), Büchi precolumn (110ꢃ15 mm)] and silica gel 60 (Merck,
15—40 mm). Vacuum-liquid chromatography (VLC) was performed on a
C18 reversed phase (Merck, 25—40 mm) (12ꢃ3 cm). TLC and HPTLC em-
ployed precoated silica gel 60 F254 plates (Merck). The following TLC sol-
vent systems were used: for saponins (a) CHCl3–MeOH–H2O (13 : 7 : 2,
lower phase); for sapogenins (b) CHCl3–MeOH (9 : 1); for monosaccharides
(c) CHCl3–MeOH–H2O (8 : 5 : 1). Spray reagents for the saponins were:
Komarowsky reagent, a mixture (5 : 1) of p-hydroxybenzaldehyde (2% in
MeOH) and H2SO4 50%; for the sugars: diphenylaminephosphoric acid
reagent.
The antifungal activity of compounds 1—6 was evaluated
at concentrations less than 200 mg/ml against strains of Can-
dida albicans, C. glabrata and C. tropicalis (Table 3). Ac-
cording to our previous observations, saponins having a
furostan skeleton (2, 4, 5, 6) were devoid of activity against
the tested fungi.5,6,10—12) Concerning the spirostanol saponins,
only compound 3, having two sugars for the osidic chain
showed an antifungal activity against the yeast tested with
minimum inhibitory concentration (MIC) values between 25
and 50 mg/ml (Table 4). Antifungal activities were also ob-
served previously with SS1 which differ of 3 by the (1→6)
linkage of the second glucose. Sautour et al. showed that
spirostanol saponins (SS2 and SS3) from Smilax medica hav-
ing three sugars for the osidic chain exhibited antifungal ac-
tivities with MIC values between 12.5 to 50 mg/ml.5) In the
present study, compound 1 was devoid of activity. The differ-
ence between the osidic part of 1 and other saponins having
three sugars (SS2 and SS3) is the presence of a rhamnose
unit, but espacially the number of saccharides connected at
Glc I. Indeed, SS2 and SS3 possess a branched-chain
Plant Material The roots of Smilax aspera subsp. mauritanica (POIR.)
ARCANG. were collected in Mas de Jau from Case de Pènes (Roussillon,
France) and identified by Gerard Ducerf. A voucher specimen (No. 6624) is
deposited in the Herbarium of the Laboratory of Pharmacognosy, Faculty of
Pharmacy, University of Burgundy, France.
Extraction and Separation Dried powdered roots (410 g) of S. aspera
subsp. mauritanica were refluxed three times with MeOH–H2O (7 : 3, 2 l),

September 2008
1327
evaporated to dryness yielding a MeOH–H2O extract. This was partitioned
successively with hexane, CH2Cl2 and n-BuOH (each 3ꢃ200 ml) yielding
after evaporation of the solvents the corresponding hexane (1 g), CH2Cl2
(1.5 g) and n-BuOH (17 g) fractions. Nine grams of the n-BuOH residue was
dissolved in MeOH and purified by precipitation with diethyl ether (2ꢃ250
ml), yielding a crude saponin mixture (5.2 g). This latter was submitted to
the VLC on C18 reversed-phase (12ꢃ3 cm) using H2O (100 ml), MeOH–H2O
mixtures (5 : 5; 4 : 1, each 100 ml) and finally MeOH (100 ml). The fraction
eluted with MeOH–H2O (4 : 1) (435 mg) was submitted to MPLC column
chromatography (Si gel (15—40 mm), system a), yielding 11 fractions, 1—
11. Fraction 1 was rechromatographed in the same conditions to give the
pure compound 6 (5 mg). Fraction 3 was rechromatographed in the same
conditions to give the pure compound 2 (6 mg). Fractions 4, 6 and 9 were
rechromatographed in the same conditions to give the pure compounds 3
(9 mg), 4 (5 mg) and 5 (11 mg), respectively. The fraction eluted with 100%
istry, 31, 2439—2443 (1992).
3) Kubo S., Mimaki Y., Sashida Y., Nikaido T., Ohmoto T., Phytochem-
istry, 31, 2445—2450 (1992).
4) Bernardo R. R., Pinto A. V., Parente J. P., Phytochemistry, 43, 465—
469 (1996).
5) Sautour M., Miyamoto T., Lacaille-Dubois M. A., J. Nat. Prod., 68,
1489—1493 (2005).
6) Sautour M., Miyamoto T., Lacaille-Dubois M. A., Planta Med., 72,
667—670 (2006).
7) Shao B., Guo H., Cui Y., Ye M., Han J., Guo D., Phytochemistry, 68,
623—630 (2007).
8) Chung H. S., Shin C. H., Lee E. J., Hong S. H., Kim H. M., Comp.
Biochem. Physiol. C, 135, 197—203 (2003).
9) Paris R., Vaillant M., Benard M., Ann. Pharm. Fr., 10, 328—335
(1952).
MeOH (200 mg) was submitted to MPLC (system a) to give 10 fractions, 10) Sautour M., Mitaine-Offer A. C., Miyamoto T., Dongmo A., Lacaille-
1—10. Fraction 6 was concentrated to dryness, yielding the pure compound Dubois M. A., Planta Med., 70, 90—92 (2004).
1 (21 mg). The diethyl ether fraction (1.13 g) was submitted to MPLC col- 11) Sautour M., Mitaine-Offer A. C., Miyamoto T., Dongmo A., Lacaille-
umn chromatography (system a) yielding the pure compound 7 (9 mg).
(25S)-5b-Spirostane-3b-ol 3-O-a- -Rhamnopyranosyl-(1→2)-b- -gluco-
pyranosyl-(1→2)-b-D-glucopyranoside (1): White amorphous powder,
Dubois M. A., Chem. Pharm. Bull., 52, 1353—1355 (2004).
12) Sautour M., Miyamoto T., Lacaille-Dubois M. A., Phytochemistry, 68,
2554—2562 (2007).
L
D
HR-ESI-MS (positive ion-mode) m/z: 909.5392 [MꢁNa]ꢁ, (Calcd for 13) Agrawal P. K., Bunsawansong P., Morris G. A., Phytochemistry, 47,
C45H74O17Na: 909.5352). FAB-MS (negative ion mode) m/z: 885 [MꢀH]ꢀ.
255—257 (1998).
[a]D20 ꢀ33.3° (cꢂ0.07, MeOH). IR nmax (CHCl3) cmꢀ1: 3255 (OH), 2980 14) Agrawal P. K., Magn. Reson. Chem., 41, 965—968 (2003).
(CH), 1040 (C–O–C). 1H- and 13C-NMR: see Tables 1 and 2.
15) Yang Q.-X., Xu M., Zhang Y.-J., Li H.-Z., Yang C.-R., Helv. Chim.
Acta, 87, 1248—1253 (2004).
16) Debella A., Haslinger E., Kunert O., Michl G., Abebe D., Phytochem-
istry, 51, 1069—1075 (1999).
(25S)-3b,5b,22a-Furostane-3,22,26-triol 3-O-a-L-Rhamnopyranosyl-
(1→2)-b-D-glucopyranosyl-(1→2)-b-D-glucopyranosyl 26-O-b-D-Glucopy-
ranoside (2): White amorphous powder, HR-ESI-MS (positive ion-mode)
m/z: 1089.5195 [MꢁNa]ꢁ, (Calcd for C51H86O23Na: 1045.5245). FAB-MS 17) Agrawal P. K., Jain D. C., Pathak A. K., Magn. Reson. Chem., 33,
(negative ion mode) m/z: 1065 [MꢀH]ꢀ. [a]D20 ꢀ69.2° (cꢂ0.053, MeOH).
923—953 (1995).
IR nmax (CHCl3) cmꢀ1: 3355 (OH), 2927 (CH), 1067 (C–O–C). 1H- and 13C- 18) Li Y.-F., Hu L.-H., Lou F.-C., Hong J.-R., Li J., Shen Q., J. Asian Nat.
NMR: see Tables 1 and 2.
Prod. Res., 7, 43—47 (2005).
Acid Hydrolysis A solution of each saponin (3 mg) in 2 N aqueous
19) Agrawal P. K., Magn. Reson. Chem., 42, 990—993 (2004).
CF3COOH (5 ml) was refluxed on a water bath for 3 h. After extraction with 20) Agrawal P. K., Steroids, 70, 715—724 (2005).
CH2Cl2 (3ꢃ5 ml), the aqueous layer was repeatedly evaporated to dryness 21) Sharma S. C., Sharma H. C., Phytochemistry, 3, 683—686 (1993).
with MeOH until neutral and glucose was identified by TLC with a standard 22) Goryanu G. M., Nistryan A. K., Nauka-Farm. Prakt., 1984, 38—39
using CHCl3–MeOH–H2O (8 : 5 : 1). Furthermore, a silylated derivated of
the sugar was prepared according to the procedure previously described.29) L-
Cysteine methyl ester hydrochloride (0.06 mol/l) and HMDS–TMCS (hexa-
(1984).
23) Sharma S. C., Chand R., Sati O. P., Phytochemistry, 8, 2075—2078
(1982).
methyldisilazane–trimethylchlorosilane, 3 : 1) were added to the aqueous 24) Bangani V., Crouch N. R., Mulholland D. A., Phytochemistry, 51,
residue. After centrifugation of the precipitate, the supernatant was concen- 947—951 (1999).
trated and partitioned between n-hexane and H2O, and the hexane layer was 25) Siemann, E. H., Creasy L. L., Am. J. Enol. Vitic., 43, 49—52 (1992).
analyzed by GC. D-Glucose and L-rhamnose were detected.
26) Hanawa F., Tahara S., Mizutani J., Phytochemistry, 31, 3005—3007
Antifungal Activity Minimum inhibitory concentrations (MICs) were
(1992).
performed using the broth dilution test.30) For these bioassays three human 27) Oleszek W., Sitek M., Stochmal A., Piacente S., Pizza C., Cheeke P., J.
pathogenic yeasts were used: Candida albicans (IP 1180-79), C. glabrata
Agric. Food Chem., 49, 747—752 (2001).
and C. tropicalis (clinical isolates). The reference compound ketoconazole 28) Bader G., Seibold M., Tintelnot K., Hiller K., Pharmazie, 55, 72—74
(Sigma) was used as positive control.
(2000).
29) Haddad M., Miyamoto T., Laurens V., Lacaille-Dubois M. A., J. Nat.
Prod., 66, 372—377 (2003).
References
1) Ju Y., Jia Z. H., Phytochemistry, 31, 1349—1351 (1992).
2) Sashida Y., Kubo S., Mimaki Y., Nikaido T., Ohmoto T., Phytochem-
30) Quiroga E. N., Sampietro A. R., Vattuone M. A., J. Ethnopharmacol.,
74, 89—96 (2001).