A new steroidal saponin from Allium ampeloprasum var. porrum with antiinflammatory and gastroprotective effects

Article abstract of DOI:10.1016/j.phytol.2011.05.009

A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum var. porrum. On the basis of chemical conversions and detailed analyses of ¹H and ¹³C NMR spectra including 2D NMR spectroscopic techniques, its structure was established as 3-[(O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 2)-O-[O-β-d-glucopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranosyl)oxy] -2,6-dihydroxy-(2α,3β,5α,6β,25R)-spirostane. Results of the present study indicated that the steroidal saponin showed haemolytic effects in the in vitro assays and demonstrated antiinflammatory activity and gastroprotective property using in vivo models.

Full text of DOI:10.1016/j.phytol.2011.05.009

Phytochemistry Letters 4 (2011) 306–310
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
A new steroidal saponin from Allium ampeloprasum var. porrum with
antiinflammatory and gastroprotective effects
*
´
Camila Rodrigues Ada˜o, Bernadete Pereira da Silva, Jose Paz Parente
Laborato´rio de Quı´mica de Plantas Medicinais, Nu´cleo de Pesquisas de Produtos Naturais, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro, P.O. Box 68045,
CEP 21941-971 Rio de Janeiro, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history:
A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum var. porrum. On the basis of
chemical conversions and detailed analyses of 1H and 13C NMR spectra including 2D NMR spectroscopic
Received 23 February 2011
Received in revised form 25 May 2011
Accepted 30 May 2011
techniques, its structure was established as 3-[(O-
(1 ! 2)-O-[O- -glucopyranosyl-(1 ! 3)]-O- -glucopyranosyl-(1 ! 4)-
2,6-dihydroxy-(2 ,3 ,5 ,6 ,25R)-spirostane. Results of the present study indicated that the steroidal
b
-
D
-glucopyranosyl-(1 ! 3)-
b-D-glucopyranosyl-
-galactopyranosyl)oxy]-
b
-
D
b
-
D
b-D
Available online 17 June 2011
a
b a b
saponin showed haemolytic effects in the in vitro assays and demonstrated antiinflammatory activity
and gastroprotective property using in vivo models.
Keywords:
Allium ampeloprasum var. porrum
Alliaceae
ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Steroidal saponin
Antiinflammatory activity
Gastroprotective effect
1. Introduction
positive to Liebermann–Burchard test. It revealed a quasi-
molecular weight ion peak at m/z 1282.3401 [M+Na]+ in the
positive-ion mode MALDI-TOFMS. In the 13C NMR spectrum, fifty-
seven carbon signals observed belong to four methyl groups,
fourteen methylene groups (six of which were oxygenated), thirty-
six methine groups (twenty-nine of which were oxygenated) and
three quaternary carbon atoms (one of which was oxygenated).
The number of hydrogen atoms attached to each individual carbon
atom was determined by DEPT spectrum. On the basis of the above
mentioned MS and 13C NMR spectral data (Table 1), compound 1
was assumed to be a saponin with the molecular formula
Allium ampeloprasum L. var. porrum (Alliaceae), a bulbous
perennial plant, is one of the daily edible green vegetables for
Brazilian people. The bulbs have been used for treating inflamma-
tory symptoms. The crushed bulb is used to treat initial stages of
cough, mucous secretion and sore throat. The fresh juice is taken
orally as a stomachic and antispasmodic and is also reputed to
ˆ
possess digestive properties (Correa, 1926).
The Allium plants have been found to be rich in steroidal
saponins, as well as organosulfur compounds (Kravets et al., 1990;
Matsuura, 2001). As part of our ongoing efforts in discovering
potentially bioactive and/or new steroidal saponins from Brazilian
medicinal plants (Parente and da Silva, 2008), we describe the
isolation, structural elucidation and evaluation of the antiinflam-
matory and antiulcerogenic activities of a new steroidal saponin
from the methanolic extract of A. ampeloprasum var. porrum.
C57H94O30, bearing a chain of five monosaccharide moieties.
In addition to this, the spirostanol glycosidic nature of
compound 1 was indicated by the strong absorption band at
3400 cmꢀ1 for the hydroxyl group and characteristic 25R-
spiroketal absorption bands at 890 and 910 cmꢀ1 (intensity,
910 < 890 cmꢀ1) in the IR spectrum (Wall et al., 1952). The 25R
stereochemistry of the Me-27 group was confirmed by hydrogens
and carbons resonances at positions 25, 26 and 27 in comparison
with data reported in the literature (Carotenuto et al., 1999;
Agrawal, 2003). The A/B trans-ring fusion was deduced from the
2. Results and discussion
The MeOH extract of A. ampeloprasum var. porrum was
suspended in H2O and partitioned with n-BuOH. The n-BuOH
extract was subjected to chromatographic purification steps to
afford compound 1, a colorless amorphous powder, which was
signals at
d
47.32 (CH, C-5), 54.03 (CH, C-9) and 16.72 (CH3, C-19)
H steroidal sapogenin,
H)
indicating that the aglycone of 1 is a 5
confirmed by one hydrogen at
(Carotenuto et al., 1997). On inspection of the 1H and 13C NMR
a
d 1.27 (td, J = 2.8, 2.8, 12.0 Hz, 5a
spectral data of compound 1, the structure of the aglycone
moiety was identical with (2a,3b,5a,6b,25R)-2,6-dihydroxyspiro-
* Corresponding author. Tel.: +55 21 2562 6791; fax: +55 21 2562 6513.
E-mail address: parente@pq.cnpq.br (J.P. Parente).
stan-3-yl.
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2011.05.009

C.R. Ada˜o et al. / Phytochemistry Letters 4 (2011) 306–310
307
Table 1
104.84, 104.33, 103.77 and 103.29, respectively. Evaluation of
chemical shifts and spin–spin couplings allowed the identification
13C NMR data of compound 1 (150 MHz, pyridine-d5).
Position
d
C
DEPTa
Position
d C
DEPTa
of one
units (
b
-galactopyranosyl unit (
b
-Gal) and four
-GlcIV). The attachments of the
b-glucopyranosyl
b
-GlcI, -GlcII, -GlcIII and b
b
b
1
46.7
71.1
84.1
33.8
47.3
70.2
39.6
29.5
54.0
36.6
20.9
40.2
40.4
55.8
31.7
80.7
62.5
16.1
16.7
41.5
14.6
108.8
31.3
29.5
30.1
66.7
16.9
CH2
CH
CH
CH2
CH
CH
CH2
CH
CH
C
b
b
b
b
-
D
-GlcI
sugar moieties to the aglycone moiety were established by 1H–1H-
COSY, HMBC and HMQC experiments. The HMBC and HMQC
2
1
2
3
4
5
6
104.3
80.0
87.4
69.2
77.5
62.5
CH
CH
CH
CH
CH
CH2
3
4
spectra displayed long range couplings between Gal-H-1 at
and aglycone-C-3 at 84.07. In addition to this, long range
couplings were observed between GlcI-H-1 at 5.12 and Gal-C-4 at
79.35, between GlcII-H-1 at 5.56 and GlcI-C-2 at 80.01,
between GlcIII-H-1 at 5.05 and GlcII-C-3 at 87.01, between
GlcIV-H-1 at 5.22 and GlcI-C-3 at 87.39, which accounted for the
saccharide part linkage to the C-3 OH group of agigenin. The NMR
d 4.89
5
d
6
d
7
d
d
d
8
9
d
d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
-
D
1
2
3
4
5
6
-GlcII
-GlcIII
-GlcIV
d
d
CH2
CH2
C
103.3
74.3
87.0
68.8
77.5
61.5
CH
CH
CH
CH
CH
CH2
b
signals of compound 1 were assigned by 2D NMR experiments
including COSY, HSQC, HMQC and HMBC and by comparing the
NMR data of 1 with those reported in the literature (Mimaki et al.,
1995; Carotenuto et al., 1999; Itabashi et al., 2000; Ohtsuki et al.,
2004; da Silva and Parente, 2006).
The sequence of the sugar chain of 1 was confirmed by
methylation analysis (Parente et al., 1985) which furnished 1,5-di-
O-acetyl-2,3,4,6-tetra-O-methyl glucitol, 1,3,5-tri-O-acetyl-2,4,6-
tri-O-methyl glucitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galac-
titol and 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl glucitol. Conse-
quently, on the basis of the results described above, the structure of
CH
CH2
CH
CH
CH3
CH3
CH
CH3
C
-
D
1
2
3
4
5
6
104.8
74.7
77.5
69.5
77.9
62.0
CH
CH
CH
CH
CH
CH2
CH2
CH2
CH
CH2
CH3
compound
(1 ! 3)-
(1 ! 3)]-O-
l)oxy]-2,6-dihydroxy-(2
1
was established as 3-[(O-
-glucopyranosyl-(1 ! 2)-O-[O-
-glucopyranosyl-(1 ! 4)-
,3 ,5 ,6 ,25R)-spirostane (1).
b
b-D
-
D
-glucopyranosyl-
-glucopyranosyl-
b-D-galactopyranosy-
b-
D
b
-D
-Gal
-
D
1
2
3
4
5
6
b
-D
1
2
3
4
5
6
102.5
72.0
75.1
79.3
75.1
60.2
CH
CH
CH
CH
CH
CH2
103.8
74.7
77.9
69.8
77.9
62.0
CH
CH
CH
CH
CH
CH2
a
b a b
According to the literature, steroidal saponins isolated from
medicinal plants have been reported to have important biological
activities, such as stimulation of lymphocyte proliferation (Gautam
et al., 2009) and inhibition of the production of inflammatory
cytokines by activated macrophages (Kim et al., 2009). With the
aim of evaluating the biological properties, we tested the in vivo
antiinflammatory and gastroprotective activities of compound 1
(Fig. 1) in order to explore its contribution to the traditional
medicinal use of this species. On the toxicological side, the in vitro
haemolytic potential was also measured.
Generally, steroidal saponins possess powerful haemolytic
capacity because steroids have higher affinities for cholesterol on
erythrocyte membranes. The ability of compound 1 to induce
haemolysis of human red blood cells was investigated and
compared with the saponin QS-21 isolated from Quillaja saponaria
a
Multiplicities were assigned from DEPT spectrum.
On acid hydrolysis, compound 1 gave galactose, glucose and an
aglycone, which was identical to agigenin (1a) by comparative
analysis of 1H and 13C NMR and EIMS spectra of 1a with those of
known agigenin (Kel’ginbaev et al., 1974; Carotenuto et al., 1997).
Analysis of the sugars by GC/MS indicated the presence of
galactose and glucose in a ratio of 1:4, respectively (Kamerling
et al., 1975). Their absolute configurations were determined by GC
of their trimethylsilylated (ꢀ)-2-butylglycosides (Gerwig et al.,
1978).
spectrum of compound 1 displayed five anomeric hydrogen atoms
at 4.89 (d, J = 7.8 Hz), 5.05 (d, J = 7.5 Hz), 5.12 (d, J = 7.8 Hz), 5.22
(d, J = 7.8 Hz), 5.57 (d, J = 7.5 Hz) which gave correlations in the
HSQC spectrum with five anomeric carbon atoms at 102.54,
D-Galactose and
D
-glucose were detected. The 1H NMR
(HD50 5 mg/mL), a substance commonly used in animal and human
experimental models. According to procedures described in the
literature (Santos et al., 1997), compound 1 was shown to possess
d
potent haemolytic capacity (HD50 20
mg/mL), which can be
d
explained by the amphipathic characteristic of its structure,
Fig. 1. Structure of compound 1.

308
C.R. Ada˜o et al. / Phytochemistry Letters 4 (2011) 306–310
225
200
175
150
125
100
75
in gastric hyperemia and in number and severity of lesions. This
protective action is closely related to the reference compound
cimetidine at the same dosage. The intensity of gastric ulcers was
quantified by the percentage of the injury area in relation to the
control group (Fig. 3). This result suggests that compound 1
probably interfere with the ulcerogenic mechanism, showing a
cytoprotective property.
In conclusion, the investigation of the biological properties of
compound 1 indicated that this substance may be the potential
therapeutic agent involved in the gastroprotective property and
the treatment of inflammatory conditions, justifying the use of this
plant as a food source and in the traditional medicine.
*
*
carrageenan
dexamethasone
compound 1
**
**
**
*
*
**
50
**
**
25
0
0
1
2
3
4
5
Time after injection (h)
Fig. 2. Antiinflammatory activity of compound 1 (100 mg/kg) and the reference
compound dexamethasone (25 mg/kg) against mouse paw oedema induced by
carrageenan. Results are mean ꢂ S.E.M. (n = 5); *p < 0.05, **p < 0.01, significantly
different from the control group.
3. Experimental
3.1. General experimental procedures
containing a hydrophobic steroidal nucleus and two hydrophilic
carbohydrate moieties.
Carbohydrate content was analyzed by gas-chromatography–
electron impact mass spectrometry (GC–EIMS) of the alditol
acetates (Sawardeker et al., 1965). The experimental data were
tested for statistical differences using the Student’s t-test. Melting
points were determined by an Electrothermal 9200 micro-melting
point apparatus and are uncorrected. The optical rotations were
measured on a Perkin Elmer 243B polarimeter. IR spectra were
measured on a Perkin Elmer FT-IR 1600 spectrometer. 1H and 13C
NMR, DEPT, COSY, HMBC, HSQC and HMQC experiments were
performed in deuterated pyridine on a Bruker DRX-600 NMR
In addition to this, the antiinflammatory activity of compound 1
was investigated using an acute inflammation model. The results
were measured by inhibition of carrageenan-induced mouse paw
oedema (Levy, 1969). The carrageenan-induced inflammation is a
biphasic phenomenon. The early phase of oedema is attributed to
the release of histamine, serotonin and similar substances. The
later phase results mainly from the potentiating effects of
prostaglandins on mediator release. Compound 1 showed signifi-
cant antiinflammatory potential, promptly controlling both phases
of inflammation and provoking an inhibition of oedema formation
similar to the reference compound dexamethasone (Fig. 2). In
comparison with the literature reports, compound 1 showed
antiedematous properties with potency similar to other bioactive
compounds, such as anemarsaponin B, a steroidal saponin isolated
from the rhizomes of Anemarrhena asphodeloides, a medicinal plant
used against inflammatory disorders (Kim et al., 2009).
The gastroprotective property of compound 1 was investigated
with the aim to confirm its utilization in the traditional medicine,
since there are popular informations about the use of this plant for
the treatment of digestive disorders. The antiulcerogenic activity
was evaluated by measuring the inhibition of acute gastric lesions
induced by acidified ethanol (Hamauzu et al., 2008). By macro-
scopic observations, in the control animals that received only
water before acidified ethanol administration, intense and
widespread gastric hyperemia and thickened lesions were evident.
In contrast, the stomachs of the animals which received compound
1 showed an aspect close to normality, with significant reduction
spectrometer (600 MHz for dH and 150 MHz for
dC). All chemical
shifts ( ) are given in ppm units with reference to tetramethylsi-
d
lane (TMS) as the internal standard and the coupling constants (J)
are in Hz. Gas chromatography (GC) was carried out with flame
ionization detector (FID), using
a glass capillary column
(0.31 mm ꢁ 25 m) SE-30. GC–EIMS were taken on a VG Auto
SpecQ spectrometer operating at 70 eV. The MALDI-TOFMS was
obtained using a perseptive Voyager RP mass spectrometer. Silica
gel columns (230–400 mesh ASTM, Merck) and Sephadex LH-20
(Pharmacia) were used for column chromatography (CC). Thin-
layer chromatography (TLC) of monosaccharides was performed
on silica gel coated plates (Merck) using the following solvent
systems: (A) CHCl3–MeOH–H2O (65:35:10, v/v/v, lower phase) for
steroidal saponin 1, (B) CHCl3–MeOH (95:5, v/v) for sapogenin 1a,
and (C) n-BuOH–acetone–H2O (4:5:1, v/v/v) for monosaccharides.
Spray reagents were orcinol/H2SO4 for steroidal saponin 1 and
monosaccharides and CeSO4 for sapogenin 1a.
3.2. Plant material
The bulbs of A. ampeloprasum var. porrum were purchased from
a nursery in Rio de Janeiro, Brazil, in September 2007. The bulbs
were cultivated, and a voucher specimen (LQPM 60) of the plant is
on file in our laboratory.
compound 1
**
3.3. Extraction and isolation
cimetidine
*
Fresh bulbs of A. ampeloprasum var. porrum (1.5 kg), previously
cut into small pieces, were extracted with MeOH (6 L) for 72 h at
rm. temp. The extract was concentrated under reduced pressure to
remove most of the MeOH and the resulting aq. phase (500 mL)
was extracted with n-BuOH (500 mL). This procedure was repeated
and the resulting organic phase was evaporated in vacuo to give a
crude material (4.93 g). It was dissolved in MeOH (70 mL) and
roughly chromatographed (0.70 g/10 mL, each time) by column
chromatography over Sephadex LH-20 (3.8 cm ꢁ 65 cm) using
MeOH as eluent to yield fractions containing saponins (143 mg).
The resulting residue containing saponins (1.0 g) was subjected to
column chromatography over silica gel (2.8 cm ꢁ 90 cm), using
control
0
10 20 30 40 50 60 70 80 90 100 110
Area of gastric lesion (percentage of total area)
Fig. 3. Antiulcerogenic activity of compound 1 (100 mg/kg) and the reference
compound cimetidine (100 mg/kg) against acidified ethanol induced gastric lesions
(0.3 M HCl/EtOH). Results are mean ꢂ S.E.M. (n = 5); *p < 0.05, **p < 0.01,
significantly different from the control group.

C.R. Ada˜o et al. / Phytochemistry Letters 4 (2011) 306–310
309
CHCl3–MeOH–H2O (65:35:10, lower phase) as eluent to afford
compound 1 (186 mg).
(100 mg/kg) or the reference compound dexamethasone (25 mg/
kg) dissolved in saline solution as positive control. Acute
inflammation was produced by subplantar injection of 50 (L of
1% freshly prepared colloidal suspension of carrageenan in
physiological saline injected into the subplantar region of the
right hind paw of the mice, 1 h after the oral administration of test
sample as well as the negative and positive controls. The footpad
thickness was measured with a spring-loaded dial gauge (Mitutoyo
Corp., Tokyo, Japan) before and every 1 h during 5 h after induction
of inflammation. Percent inhibition of the inflammation was
determined by applying statistical methods followed by the
calculation of percent inhibition for each group by comparing
with control group.
3.4. 3-[(O-
b
-
D
-Glucopyranosyl-(1 ! 3)-
-glucopyranosyl-(1 ! 3)]-O-
-galactopyranosyl)oxy]-2,6-dihydroxy-
,25R)-spirostane (1)
b
-
D
-glucopyranosyl-
(1 ! 2)-O-[O-
b
-D
b-D-glucopyranosyl-
(1 ! 4)-
b
-
D
(2 ,3 ,5
a
b
a
,6
b
Colorless amorphous powder (186 mg); m.p. 262–264 8C;
25
[a
]
D
ꢀ21 (c 0.1, MeOH); IR (KBr) nmax cmꢀ1: 3400 (OH), 2930
(CH), 1445, 1365, 1235, 1145, 1045, 970, 910, 890, 855; 1H NMR
(C5D5N, 600 MHz)
d 5.57 (1H, d, J = 7.5 Hz, GlcII-H-1), 5.22 (1H, d,
J = 7.8 Hz, GlcIV-H-1), 5.12 (1H, d, J = 7.8 Hz, GlcI-H-1), 5.05 (1H, d,
J = 7.5 Hz, GlcIII-H-1), 4.89 (1H, d, J = 7.8 Hz, Gal-H-1), 4.84 (1H,
ddd, J = 11.2, 8.9, 5.0 Hz, H-3), 4.55 (1H, brq, J = 7.1 Hz, H-16), 4.15
(1H, ddd, J = 11.5, 8.9, 4.7 Hz, H-2), 3.89 (1H, brs, H-6), 3.58 (1H, dd,
J = 10.6, 3.7 Hz, H-26eq), 3.50 (1H, dd, J = 10.6, 10.6 Hz, H-26ax),
3.9. Antiulcerogenic activity
Antiulcerogenic activity was evaluated by measuring acute
gastric lesions induced by acidified ethanol. Male Swiss mice (three
months old, 25–35 g) in groups of five were fasted for 24 h before
the experiment and administered orally with 1 mL of pure water as
the negative control, or compound 1 (100 mg/kg), or the reference
compound cimetidine (100 mg/kg) dissolved in vehicle as positive
control. One hour after the treatments, all animals received orally
200 (L of acidified ethanol solution (0.3 M HCl/EtOH) to induce
gastric lesions. The animals were killed 1 h after treatment with
the ulcerogenic agent and the stomachs removed, opened along
the greater curvature and rinsed with physiological saline to
determine the lesion damage. The degree of gastric mucosal
damage was evaluated from digital pictures using a computerized
image analysis system. The percentage of the total lesion area
(hemorrhagic lesions) to the total surface area of the stomach was
defined as the ulcer index (Hamauzu et al., 2008).
1.27 (1H, td, J = 2.8, 2.8, 12.0 Hz, 5aH), 1.13 (3H, d, J = 6.9 Hz, Me-
21), 0.82 (3H, s, Me-18), 0.77 (3H, s, Me-19), 0.69 (3H, d, J = 5.8 Hz,
Me-27). 13C NMR (C5D5N, 150 MHz): see Table 1. MALDI-TOFMS m/
z: 1282.3401 [M+Na]+ (calcd for C57H94NaO30+, 1282.3564).
3.5. Acid hydrolysis of compound 1
Compound 1 (100 mg) was dissolved in 1 M HCl soln./1,4-
dioxane 1:1 (10 mL) and heated in a sealed tube at 100 8C during
2 h. After cooling, the mixture was neutralized with 1 M NaOH in
MeOH and the salts that deposited on addition of MeOH were
filtered off, and the filtrate was evaporated in vacuo. The residue
was chromatographed on silica gel column (10 mm ꢁ 100 mm)
eluted with CHCl3–MeOH mixtures (19:1 ! 7) to furnish the
aglycone moiety (29 mg) and sugar mixture (47 mg). A sample of
the sugar mixture (1 mg) was dissolved in pyridine (100
mL) and
analyzed by TLC in comparison with -glucose and -galactose.
D
D
Acknowledgements
3.6. Molar carbohydrate composition and D,L configurations
This work was financially supported by FINEP, CAPES and CNPq.
The molar carbohydrate composition of compound 1 (1 mg)
was determined by GC–MS analyses of their monosaccharides as
their trimethylsilylated methylglycosides obtained after metha-
nolysis (0.5 M HCl in MeOH, 24 h, 80 8C) and trimethylsilylation
(Kamerling et al., 1975). The configurations of the glycosides were
established by capillary GC and GC–EIMS of their trimethylsily-
lated (ꢀ)-2-butylglycosides (Gerwig et al., 1978).
References
Agrawal, P.K., 2003. Spectral assignments and reference data—25R/25S stereochem-
istry of spirostane-type steroidal sapogenins and steroidal saponins via chemi-
cal shift of geminal protons of ring-F. Magn. Res. Chem. 41, 965–968.
Carotenuto, A., Fattorusso, E., Lanzotti, V., Magno, S., 1999. Spirostanol saponins of
Allium porrum L. Phytochemistry 51, 1077–1082.
Carotenuto, A., Fattorusso, E., Lanzotti, V., Magno, S., De Feo, V., Carnuccio, R.,
Dacquisto, F., 1997. Porrigenins A and B, novel cytotoxic and antiproliferative
sapogenins isolated from Allium porrum. J. Nat. Prod. 60, 1003–1007.
3.7. Methylation analysis
´
Correˆa, M.P., 1926. Diciona´rio das Plantas Uteis do Brasil e das Exo´ticas Cultivadas.
Imprensa Nacional, Rio de Janeiro.
da Silva, B.P., Parente, J.P., 2006. A new bioactive steroidal saponin from leaves of
Furcraea gigantea. Z. Naturforsch. 61b, 1153–1157.
Compound
(200 L) in a Teflon-lined screw-cap tube. Lithium methylsulfinyl
carbanion (200 L) was added to the solution under an inert
atmosphere and the mixture was sonicated for 60 min. After
cooling to ꢀ4 8C, cold methyl iodide (400 L) was added.
Sonication was conducted in sonication bath (20 8C) for
45 min. The methylation was terminated by addition of water
(4 mL) containing sodium thiosulfate, and the permethylated
product extracted with chloroform (3ꢁ 2 mL) and evaporated
(Parente et al., 1985). The methyl ethers were obtained after
hydrolysis (4 N TFA, 2 h, 100 8C) and analyzed as alditol acetates by
GC–EIMS (Sawardeker et al., 1965).
1 (1 mg) was dissolved in dimethylsulfoxide
m
Gautam, M., Saha, S., Bani, S., Kaul, A., Mishra, S., Patil, D., Satti, N.K., Suri, K.A.,
Gairola, S., Suresh, K., Jadhav, S., Qazi, G.N., Patwardhan, B., 2009. Immuno-
modulatory activity of Asparagus racemosus on systemic Th1/Th2 immunity:
implications for immunoadjuvant potential. J. Ethnopharmacol. 121, 241–247.
Gerwig, G.J., Kamerling, J.P., Vliegenthart, J.F.G., 1978. Determination of the D and L
configuration of neutral monosaccharides by high-resolution capillary G.L.C.
Carbohydr. Res. 62, 349–357.
Hamauzu, Y., Irie, M., Kondo, M., Fujita, T., 2008. Antiulcerative properties of crude
polyphenols and juice of apple, and Chinese quince extracts. Food Chem. 108,
488–495.
Itabashi, M., Segawa, K., Ikeda, Y., Kondo, S., Naganawa, H., Koyano, T., Umezawa, K.,
2000. A new bioactive steroidal saponin, furcreastatin, from the plant Furcraea
foetida. Carbohydr. Res. 323, 57–62.
Kamerling, J.P., Gerwig, G.J., Vliegenthart, J.F.G., Clamp, J.R., 1975. Characterization
by gas-liquid chromatography–mass spectrometry and proton-magnetic-reso-
nance spectroscopy of pertrimethylsilyl methyl glycosides obtained in the
methanolysis of glycoproteins and glycopeptides. Biochem. J. 151, 491–495.
Kel’ginbaev, A.N., Gorovits, M.B., Abubakirov, N.K., 1974. Steroidal saponins and
sapogenins of Allium. 7. Structure of neoagigenin and agigenin. Khim. Prir.
Soedin. 6, 801–802.
m
m
a
3.8. Antiinflammatory activity
Antiinflammatory activity was evaluated by the carrageenan-
induced oedema method (Levy, 1969). Male Swiss mice (three
months old, 25–35 g) in groups of five were administered orally
with 1 mL of saline solution as the negative control or compound 1
Kim, J.-Y., Shin, J.-S., Ryu, J.H., Kim, S.Y., Cho, Y.-W., Choi, J.-H., Lee, K.-T., 2009. Anti-
inflammatory effect of anemarsaponin B isolated from the rhizomes of Anemar-
rhena asphodeloides in LPS-induced RAW 264.7 macrophages is mediated by

310
C.R. Ada˜o et al. / Phytochemistry Letters 4 (2011) 306–310
kB and p38 pathways. Food Chem. Parente, J.P., Cardon, P., Leroy, Y., Montreuil, J., Fournet, B., Ricart, G., 1985. A
negative regulation of the nuclear factor-
Toxicol. 47, 1610–1617.
convenient method for methylation of glycoprotein glycans in small
amounts by using lithium methylsulfinyl carbanion. Carbohydr. Res. 141,
41–47.
Kravets, S.D., Vollerner, Y.S., Gorovits, M.B., Abubakirov, N.K., 1990. Steroids of the
spirostan and furostan series from plants of the genus Allium. Khim. Prir. Soedin.
4, 429–443.
Levy, L., 1969. Carrageenan paw oedema in the mouse. Life Sci. 8, 601–606.
Matsuura, H., 2001. Saponins in garlic as modifiers of the risk of cardiovascular
disease. J. Nutr. 131, 1000S–1005S.
Mimaki, Y., Kanmoto, T., Kuroda, M., Sashida, Y., Nishino, A., Satomi, Y., Nishino, H.,
1995. Steroidal saponins from the underground parts of Hosta longipes and their
inhibitory activity on tumor promoter-induced phospholipid metabolism.
Chem. Pharm. Bull. 43, 1190–1196.
Ohtsuki, T., Koyano, T., Kowithayakorn, T., Sakai, S., Kawahara, N., Goda, Y., Yama-
guchi, N., Ishibashi, M., 2004. New chlorogenin hexasaccharide isolated from
Agave fourcroydes with cytotoxic and cell cycle inhibitory activities. Bioorg.
Med. Chem. 12, 3841–3845.
Parente, J.P., da Silva, B.P., 2008. Bioactive steroidal saponins from Brazilian medic-
inal plants. Curr. Trends Med. Chem. 5, 45–57.
Santos, W.R., Bernardo, R.R., Pec¸anha, L.M.T., Palatnik, M., Parente, J.P., de Sousa,
C.B.P., 1997. Haemolytic activities of plant saponins and adjuvants. Effect of
Periandra mediterranea saponin on the humoral response to the FML antigen of
Leishmania donovani. Vaccine 15, 1024–1029.
Sawardeker, J.S., Sloneker, J.H., Jeanes, A., 1965. Quantitative determination of
monosaccharides as their alditol acetates by gas liquid chromatography. Anal.
Chem. 37, 1602–1604.
Wall, M.E., Eddy, C.R., McClennan, M.L., Klump, M.E., 1952. Detection and
estimation of steroidal sapogenins in plant tissue. Anal. Chem. 24,
1337–1341.