Haemolytic activity and immunological adjuvant effect of a new steroidal saponin from allium ampeloprasum var. porrum

Article abstract of DOI:10.1002/cbdv.201100005

A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum L. var. porrum. On the basis of chemical evidence, comprehensive spectroscopic analyses, and comparison with known compounds, its structure was established as (3β,5α,6β,25R)-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} -6-hydroxyspirostan-2-one (1). Results of the present study indicated that 1 exhibited haemolytic activity in the in vitro assays, and immunological adjuvant activity on the cellular immune response against ovalbumin antigen. Copyright

Full text of DOI:10.1002/cbdv.201100005

5
8
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Haemolytic Activity and Immunological Adjuvant Effect of a New Steroidal
Saponin from Allium ampeloprasum var. porrum
by Camila Rodrigues Ad a˜ o, Bernadete Pereira da Silva, Luzineide Wanderley Tinoco, and
Jos e´ Paz Parente*
Laborat o´ rio de Quꢀmica de Plantas Medicinais, N u´ cleo de Pesquisas de Produtos Naturais, Centro de
Ci eˆ ncias da Sa u´ de, Universidade Federal do Rio de Janeiro, Caixa Postal 68045, CEP 21941-971 Rio de
Janeiro, Brazil
(
phone: þ552125626791; fax: þ552122702683; e-mail: parente@pq.cnpq.br)
A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum L. var. porrum. On the
basis of chemical evidence, comprehensive spectroscopic analyses, and comparison with known
compounds, its structure was established as (3b,5a,6b,25R)-3-{(O-b-d-glucopyranosyl-(1!3)-b-d-
glucopyranosyl-(1!2)-O-[O-b-d-glucopyranosyl-(1!3)]-O-b-d-glucopyranosyl-(1!4)-b-d-galacto-
pyranosyl)oxy}-6-hydroxyspirostan-2-one (1). Results of the present study indicated that 1 exhibited
haemolytic activity in the in vitro assays, and immunological adjuvant activity on the cellular immune
response against ovalbumin antigen.
Introduction. – Plants of the genus Allium are known for their production of
steroidal saponins, as well as organosulfur compounds [1][2]. Steroidal saponins have a
wide spectrum of biological properties, including antifungal, antibacterial, cytotoxic,
haemolytic, hypocholesteremic, anti-inflammatory, and immunomodulatory activities
[
3]. Allium ampeloprasum L. var. porrum (Liliaceae), a bulbous perennial plant, is one
of the daily edible green vegetables for Brazilian people. This plant is used not only as
food but also as medicine. The bulbs have been used for treating inflammatory
symptoms. The crushed bulb is used to treat initial stages of cough, mucous secretion,
and sore throat. The fresh juice is taken orally and is reputed to possess
immunostimulating properties [4]. While there are reports on saponins isolated from
Allium [1–3], no chemical investigation of A. ampeloprasum var. porrum has been
reported as yet. Here, we describe the isolation and structure elucidation of a new
steroidal saponin, 1, from the MeOH extract of A. ampeloprasum var. porrum.
Compound 1 was evaluated for its haemolytic and immunological-adjuvant activity on
the cellular immune response against ovalbumin antigen.
Results and Discussion. – The MeOH extract of A. ampeloprasum var. porrum was
suspended in H O and partitioned with BuOH. The BuOH extract was subjected to
2
chromatography to afford compound 1, a colorless amorphous powder, which was
positive to LiebermannꢀBurchard test. It revealed a pseudomolecular-ion peak at m/z
þ
13
1
280.3365 ([MþNa] ) in the positive-ion mode MALDI-TOF-MS. In the C-NMR
spectrum, of the 57 C-atom signals were observed, attributed to four Me, 14 CH (six of
2
which were O-bearing), 35 CH groups (28 of which were O-bearing), and four
ꢁ
2012 Verlag Helvetica Chimica Acta AG, Zꢂrich

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
59
quaternary C-atoms (two of which were O-bearing). The number of H-atoms attached
to each individual C-atom was determined by the DEPT spectrum. On the basis of the
13
above mentioned MS and C-NMR data (Table), compound 1 was assumed to be a
saponin with the molecular formula C H O , bearing five monosaccharide moieties.
57
92 30
In addition to this, the spirostanol glycosidic nature of compound 1 was indicated by
ꢀ
1
the strong absorption bands at 3400 cm for the OꢀH group and the characteristic
25R)-spiroketal absorption bands at 890 and 910 cm (intensity, 910<890 cm ) in
the IR spectrum [5]. The (R)-configuration at C(25) was confimed by the comparison
ꢀ
1
ꢀ1
(
1
13
of H and C resonances of HꢀC(25), CH (26), and Me(27) with data reported in the
2
literature [6][7]. In the HMBC spectrum (Fig. 1), the Me(18) signal at d(H) 0.85
showed correlations with C-atom signals at d(C) 40.3 (C(13)), 55.7 (C(14)), and 62.3
(C(17)), while the Me(27) signal at d(H) 0.70 showed correlations with C-atom signals
at d(C) 28.7 (C(24)), 30.1 (C(25)), and 66.3 (C(26)). The Me(21) signal at d(H) 1.15
showed correlations with C-atom signals at d(C) 41.5 (C(20)), 62.3 (C(17)), and 108.7
(
C(22)). The Me(19) signal at d(H) 1.19 showed correlations with C-atom signals at
1
d(C) 40.8 (C(10)), 47.3 (C(5)), and 54.0 (C(1)). The H-NMR spectrum of 1 displayed
five anomeric H-atom signals at d(H) 4.97 (d, J¼7.8), 5.10 (d, J¼7.5), 5.18 (d, J¼7.8 ) ,
5
.22 (d, J¼7.8), and 5.58 (d, J¼7.5; see Exper. Part) which showed correlations in the
HSQC spectrum with five anomeric C-atom signals at d(C) 101.2, 103.8, 104.7, 104.4,
and 103.4, respectively. Evaluation of chemical shifts and spinꢀspin couplings allowed
1
the identification of one b-galactopyranosyl unit (b-Gal ) and four b-glucopyranosyl
2
3
4
5
units (b-Glc , b-Glc , b-Glc and b-Glc ). Direct support of the sequence of the sugars
and binding sites was provided by the results of the HMBC experiment (Fig. 1), which
1
showed the following cross-peaks: 4.97 (HꢀC(1) (Gal ))/80.2 (C(3)), 5.22 (HꢀC(1)
2
1
3
2
(
(
Glc ))/79.3 (C(4) (Gal )), 5.58 (HꢀC(1) (Glc ))/80.6 (C(2) (Glc )), 5.18 (HꢀC(1)
4 3 5 2
Glc ))/87.0 (C(3) (Glc )), and 5.10 (HꢀC(1) (Glc ))/88.3 (C(3) (Glc )) (Fig. 2). In the
HMBC spectrum of 1, the C-atom signal at d(C) 207.5 (C(2)) showed correlations with
H-atom signals at d(H) 2.10 (H ꢀC(1)), 2.50 (H ꢀC(1)), and 5.00 (HꢀC(3)) which
b
a
allowed the location of the C(2)¼O group.
The NOESY spectrum (Fig. 3) showed the correlations 4.04 (HꢀC(6))/1.25
H ꢀC(7)), 4.04 (HꢀC(6))/1.60 (H ꢀC(5)), 4.04 (HꢀC(6))/2.06 (H ꢀC(4)), and 4.04
(
(
a
a
a
HꢀC(6))/2.50 (H ꢀC(1)), which established the b-configuration for the OH group at
a
C(6). The NOESY correlations 1.19 (Me(19))/2.10 (H ꢀC(1)), 1.19 (Me(19))/2.06
b

6
0
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table. 13C-NMR Data (75 MHz) for 1 in C D N ). C-Atom numbering as indicated in formula 1.
a
5
5
C-Atom
d(C)
C-Atom
d(C)
1
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
53.96 (t)
207.50 (s)
80.20 (d)
33.68 (t)
47.25 (d)
70.94 (d)
40.01 (t)
30.05 (d)
53.96 (d)
40.76 (s)
20.77 (t)
39.37 (t)
40.25 (s)
55.69 (d)
31.64 (t)
80.55 (d)
62.32 (d)
15.94 (q)
15.70 (q)
41.46 (d)
14.50 (q)
108.70 (s)
31.24 (t)
28.69 (t)
30.05 (d)
66.32 (t)
16.79 (q)
C(1 )
101.18 (d)
71.97 (d)
74.88 (d)
79.26 (d)
74.88 (d)
60.07 (t)
104.37 (d)
80.55 (d)
88.27 (d)
70.01 (d)
77.37 (d)
62.32 (t)
103.36 (d)
74.09 (d)
87.04 (d)
68.65 (d)
77.37 (d)
61.18 (t)
104.72 (d)
74.88 (d)
77.37 (d)
70.94 (d)
77.82 (d)
61.77 (t)
103.81 (d)
74.88 (d)
77.82 (d)
70.94 (d)
77.82 (d)
61.77 (t)
1
C(2 )
1
C(3 )
1
C(4 )
1
C(5 )
1
C(6 )
2
C(1 )
2
C(2 )
2
C(9)
C(3 )
2
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
Me(18)
Me(19)
C(20)
Me(21)
C(22)
C(23)
C(24)
C(25)
C(26)
Me(27)
C(4 )
2
C(5 )
2
C(6 )
3
C(1 )
3
C(2 )
3
C(3 )
3
C(4 )
3
C(5 )
3
C(6 )
4
C(1 )
4
C(2 )
4
C(3 )
4
C(4 )
4
C(5 )
4
C(6 )
5
C(1 )
5
C(2 )
5
C(3 )
5
C(4 )
5
C(5 )
5
C(6 )
a) Chemical shifts are in ppm relative to TMS. Spectra were recorded at 258. C-Atom assignments were
accomplished by HMBC and HSQC experiments.
(
(
(
H ꢀC(4)), 1.19 (Me(19))/2.10 (HꢀC(8)), 0.85 (Me(18))/1.43 (H ꢀC(15)), 0.85
b
b
Me(18))/1.94 (HꢀC(20)), 1.85 (HꢀC(17))/1.15 (HꢀC(14)), and 1.85 (HꢀC(17))/4.55
HꢀC(16)) provided evidence for the trans-A/B, trans-B/C, trans-C/D, and cis-D/E ring
fusions (Fig. 2). The NOESY correlation 4.55 (HꢀC(16))/3.48 (H ꢀC(26)) led to the
a
assignment of the 22a-configuration. The above data indicated that the structure of the
aglycone moiety was identical to (3b,5a,6b,25R)-6-hydroxy-2-oxospirostan-3-yl [8].
The NMR signals of 1 were assigned by 2D-NMR experiments and by comparing the
NMR data of 1 with those reported in the literature [6–14].
On acid hydrolysis, compound 1 gave galactose (Gal), glucose (Glc), and an
1
aglycone, which was identified as porrigenin B by comparative analysis of its H- and
13
C-NMR, and EI mass spectra with those reported in the literature [8]. Analysis of the
sugars by GC/MS indicated the presence of Gal and Glc in a ratio of 1:4, respectively

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
61
Fig. 1. HMBC Spectrum of compound 1
Fig. 2. Selected HMBC (H!C) and NOESY (H$H) correlations exhibited by compound 1
[
15]. Their absolute configurations were determined by GC of their trimethylsilylated
(
ꢀ)-2-butylglycosides [16], in both cases, as d.
The sequence of the sugar chain of 1 was confirmed by methylation analysis [17]
which furnished 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol, 1,3,5-tri-O-acetyl-2,4,6-
tri-O-methylglucitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgalactitol, and 1,2,3,5-tetra-
O-acetyl-4,6-di-O-methylglucitol. Consequently, the structure of compound 1 was

6
2
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 3. NOESY Spectrum of compound 1
established as (3b,5a,6b,25R)-3-{(O-b-d-glucopyranosyl-(1!3)-b-d-glucopyranosyl-
(
1!2)-O-[O-b-d-glucopyranosyl-(1!3)]-O-b-d-glucopyranosyl-(1!4)-b-d-galacto-
pyranosyl)oxy}-6-hydroxyspirostan-2-one (1).
According to the literature, steroidal saponins possess several physiological
properties depending on their chemical structures, such as haemolytic activity and
capacity for alteration of membrane permeability. Additionally, these compounds have
been reported to possess therapeutic potential for immune-system modulation via
different mechanisms [3]. Because the original observation that certain saponins cause
substantial enhancement of immune responses, when administered together with an
antigen in a vaccine, their use as adjuvants received special attention [18–20]. Indeed,
QS-21, a complex saponin isolated from Quillaja saponaria Molina, has been
evaluated in a large number of vaccines in Phase-I and Phase-II human clinical trials,
and it was recently synthesized [21]. To investigate the biological properties of
compound 1, it was evaluated for haemolytic activity in vitro [22], and compared with
adjuvants commonly used in animal and human experimental models (Fig. 4),
exhibiting a significant haemolytic potential similar to other steroidal saponins isolated
from medicinal plants, such as Smilax officinalis and Agave americana [22]. Generally,
steroidal saponins with the spirostane skeleton possess high haemolytic activity,
because steroids have higher affinities for cholesterol on erythrocyte membranes [23].

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
63
Fig. 4. Haemolytic activity [mg/ml] of compound 1 and adjuvants commonly used in animal and human
experimental models. The adjuvant concentration inducing 50% of the maximal haemolysis was
considered the median haemolytic dose (HD ; graphical interpolation). Each experiment included
50
triplicates at each concentration.
This is a common property of steroidal saponins as a consequence of its amphipathic
structure with a hydrophobic steroidal nucleus and a hydrophilic carbohydrate moiety,
and this seems to be the case for compound 1.
The formulation of effective vaccines generally requires an adjuvant to stimulate an
appropriate immune response. Adjuvants are usually defined as compounds that can
increase and/or modulate the intrinsic immunogenicity of an antigen. An efficient
adjuvant should have negligible toxicity and enhance the humoral or cellular immune
response to a specific antigen [24].
Several studies have revealed the immunopotentiating properties of compounds
from plant origin, including adjuvant effects [25]. In the traditional medicine, steroidal
saponins isolated from different sources were shown to possess immunomodulatory
activities, such as stimulation of lymphocyte proliferation [26], promotion of antibody
production [27], and enhancement of phagocytic activity [28].
To investigate the immunoadjuvant property of compound 1, its adjuvant potential
on the cellular immune response against ovalbumin (OVA) antigen was investigated
(
Fig. 5). Delayed-type hypersensitivity (DTH) reaction was evaluated as an in vivo
assay of cellular immune response. This type of immunity is typically elicited by soluble
protein antigens that are introduced with adjuvants. In the classical experimental
model, the animal is first immunized by the administration of the antigen conjugated
with adjuvants, which is called sensitization. Then, in the elicitation stage, the animal is
challenged subcutaneously with the same antigen, and the subsequent reaction is

6
4
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 5. Immunological adjuvant activity of compound 1 and commercial adjuvants on the cellular immune
response against ovalbumin antigen. Delayed-type hypersensitivity responses after two subcutaneous
immunizations with 100 mg of antigen and 100 mg of each adjuvant. Male Swiss mice were subcutaneously
immunized twice at weekly intervals with 100 mg of antigen alone or dissolved in saline solution
containing 100 mg of compound 1, FCA, FIA, or QS-21. Results are meanꢁS.E.M. values (n¼5); *: p<
0
.05, **: p<0.01 significantly different from the saline control. Studentꢃs t-test. Abbreviations: OVA:
ovalbumin antigen; SAL: saline solution; FCA: Freundꢃs Complete Adjuvant; FIA: Freundꢃs
Incomplete Adjuvant; QS-21: purified Quillaja saponaria saponin.
analyzed. In this study, mice immunized with OVA conjugated with compound 1
showed responses greater than those when the antigen was combined with commercial
adjuvants. This response developed rapidly after immunization and persisted at high
levels for at least 3 d [29].
The structural similarities between the steroidal saponin 1 and other bioactive
steroidal saponins isolated from medicinal plants, used against immunological
disorders, may help to explain the adjuvant activity of 1. Many adjuvant compounds
have an amphipathic structure, which lends a wide variety of physicochemical features
and biological activities to them [30]. The adjuvant potentials of steroidal saponins also
depend on their amphipathic structures comprising hydrophilic carbohydrate side
chains and lipophilic aglycone backbones. For example, the adjuvant tomatine, a
steroidal saponin isolated from the leaves of wild tomato Lycopersicon pimpinellifo-

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
65
lium, possesses similar structural features and is capable of stimulating potent antigen-
specific humoral and cellular immune responses [27]. In another compound isolated
from Asparagus racemosus, the stimulation of lymphocyte proliferation and macro-
phage activation is directly linked to this amphipathic characteristic [28]. Probably, the
immunopotentiating activity of compound 1 results from enhanced antigen presenta-
tion, since targeting is the mechanism by which an adjuvantꢀantigen complex is
delivered to antigen-presenting cells for processing [24]. In conclusion, the inves-
tigation of the biological properties of 1 indicated that this compound may be the
therapeutic agent involved in the immunomodulatory activity, justifying the use of
Allium ampeloprasum var. porrum in the traditional medicine.
Experimental Part
General. Column chromatography (CC): silica gel (SiO ; 230–400 mesh, Merck) and Sephadex LH-
2
2
0 (Pharmacia). TLC: Kieselgel 60 F254 aluminum support plates (Merck); spots were detected after
spraying with orcinol/H SO , followed by heating. M.p.: Electrothermal 9200 micro-melting point
2
4
apparatus; uncorrected. Optical rotations: Perkin-Elmer 243B polarimeter. IR Spectra: Perkin-Elmer
99B spectrometer. NMR Spectra: Mercury-300 NMR and Varian VNMR500 spectrometers; chemical
5
shifts in ppm rel. to Me Si and J values in Hz; o, overlapped; 2D experiments (HSQC, HMBC, NOESY)
4
were performed using standard Varian pulse sequences. HR-MALDI-TOF- and EI-MS: Perseptive
Voyager RP and VG AutoSpec spectrometers, resp.; in m/z. GC/MS: VG AutoSpec GC/MS apparatus,
with ZB glass cap. column (50 mꢂ0.25 mm i.d.).
Plant Material. The bulbs of Allium 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
6
0) of the plant is on file in our laboratory.
Extraction and Isolation. The fresh bulbs (1.5 kg) were cut into pieces and extracted with MeOH (6 l)
for 72 h at r.t. The extract was concentrated under reduced pressure to remove most of the MeOH, and
the resulting aq. phase (500 ml) was extracted with BuOH (500 ml). This procedure was repeated, and
the resulting org. phase was evaporated in vacuo to give a crude material (4.63 g). It was dissolved in
MeOH (60 ml) and subjected to CC (0.771 mg/10 ml; Sephadex LH-20 (3.8ꢂ65 cm); MeOH). The
fraction containing saponins (940 mg) was subjected to CC (SiO ; 2.8ꢂ90 cm; CHCl /MeOH/BuOH/
2
3
H O 7:7:7:2), afforded a TLC-homogeneous compound 1 (236 mg; R 0.38).
2
f
(
3b,5a,6b,25R)-3-{(O-b-d-Glucopyranosyl-(1!3)-b-d-glucopyranosyl-(1!2)-O-[O-b-d-glucopyr-
anosyl-(1!3)]-O-b-d-glucopyranosyl-(1!4)-b-d-galactopyranosyl)oxy}-6-hydroxyspirostan-2-one (¼
5a,9b,25R)-6-Hydroxy-2-oxospirostan-3-yl b-d-Glucopyranosyl-(1!3)-[b-d-glucopyranosyl-(1!3)-
(
b-d-glucopyranosyl-(1!2)]-b-d-glucopyranosyl-(1!3)-b-d-galactopyranoside; 1). Colorless amor-
2
5
phous powder. M.p. 252–2548 (dec.). [a] ¼ ꢀ18 (c¼0.1, MeOH). IR (KBr): 3400 (OH), 2930
D
1
(
CH), 1720 C¼O), 1450, 1370, 1145, 1045, 910, 890, 855. H-NMR (C D N, 500 MHz): 0.70 (d, J¼5.8,
5
5
Me(27)); 0.85 (s, Me(18)); 0.90 (overlapped (o), HꢀC(9)); 1.10 (o, HꢀC(12a)); 1.15 (o, HꢀC(14)); 1.15
(
d, J¼6.9, Me(21)); 1.19 (s, Me(19)); 1.20 (o, H ꢀC(7)); 1.40 (o, CH (11)); 1.43 (o, H ꢀC(15)); 1.55 (o,
a
2
b
H ꢀC(24)); 1.56 (dd, J¼3.2, 10.5, HꢀC(25)); 1.60 (o, HꢀC(5)); 1.62 (o, H ꢀC(23)); 1.70 (o, H ꢀC(12));
b
a
b
1
.70 (o, H ꢀC(23)); 1.85 (o, HꢀC(17)); 1.95 (o, HꢀC(20)); 2.05 (o, H ꢀC(7)); 2.06 (o, CH (4)); 2.10 (o,
b
b
2
H ꢀC(1)); 2.10 (o, HꢀC(8)); 2.10 (o, H ꢀC(15)); 2.50 (d, J¼12.5, H ꢀC(1a)); 3.51 (dd, J¼10.5, 10.5,
b a a
H ꢀC(26)); 3.59 (dd, J¼3.2, 10.5, H ꢀC(26)); 4.04 (o, HꢀC(6)); 4.55 (o, HꢀC(16)); 4.97 (d, J¼7.8,
a
b
1
5
4
HꢀC(1 )); 5.00 (dd, J¼3.7, 11.4, HꢀC(3)); 5.10 (d, J¼7.5, HꢀC(1 )); 5.18 (d, J¼7.8, HꢀC(1 )); 5.22 (d,
2
3
13
J¼7.8, HꢀC(1 )); 5.58 (d, J¼7.5, HꢀC(1 )). C-NMR (C D N): see the Table. MALDI-TOFMS:
5
5
þ
þ
30
1
280.3365 ([MþNa] , C H NaO ; calc. 1280.3405).
57
92
Acid Hydrolysis of 1. Saponin 1 (100 mg) was dissolved in 1m HCl soln./1,4-dioxane 1:1 (10 ml) and
heated in a sealed tube at 1008 for 1 h. After cooling, the mixture was neutralized with 1m NaOH in
MeOH, and the salts that deposited on addition of MeOH were filtered, and the filtrate was evaporated
in vacuo. The residue was submitted to CC (SiO (10 g); CHCl /MeOH 19 :1!7:3) to furnish porrigenin
2
3

6
6
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
B (30 mg) [8] and a sugar mixture (48 mg). A sample of the sugar mixture (1 mg) was dissolved in
pyridine (100 ml), applied to a TLC plate, and subjected to TLC (BuOH/acetone/H O 4 :5 :1; detection:
2
orcinol/H SO ) with authentic sugars: glucose (R 0.38) and galactose (R 0.35) were identified.
2
4
f
f
Molar Carbohydrate Composition and Configurations. The molar carbohydrate composition of 1
(
1 mg) was determined by GC/MS analyses of its monosaccharides as their trimethylsilylated
methylglycosides obtained after methanolysis (0.5m HCl, 808, 24 h) and trimethylsilylation [15]. The
configurations of the glycosides were established by cap. GC and GC/MS of their trimethylsilylated (ꢀ)-
2
-butylglycosides [16].
Methylation Analysis. Compound 1 (1 mg) was dissolved in DMSO (200 ml) in a Teflon-lined screw-
cap tube. Lithium methylsulfinyl carbanion (200 ml) was added to the soln. under an inert atmosphere,
and the mixture was sonicated for 60 min. After cooling to ꢀ48, cold MeI (400 ml) was added. Sonication
was conducted in a sonication bath at 208 for 45 min. The methylation was terminated by addition of H O
2
(
4 ml) containing Na S O , and the permethylated product was extracted with CHCl (3ꢂ2 ml) and
2
2
3
3
evaporated in vacuo [17]. The methyl ethers were obtained after hydrolysis (4n CF COOH, 1008, 2 h)
3
and analyzed as alditol acetates by GC/MS [31].
Haemolytic Assay. Normal human red blood cell suspension (0.5 ml of 0.5%) was mixed with 0.5 ml
of diluent containing 5, 10, 20, 30, 40, 50, 100, 250, and 500 mg/ml of compound 1, Al(OH) , purified
3
Quillaja saponin (QS-21), and 5–500 mg/ml of Freundꢃs Complete Adjuvant (FCA) and Freundꢃs
Incomplete Adjuvant (FIA) in saline soln. The mixtures were incubated for 30 min at 378 and centrifuged
at 70g for 10 min. Saline and dist. H O were included as minimal and maximal haemolytic controls, resp.
2
The haemolytic percents developed by the saline control were subtracted from those of all groups. The
adjuvant concentration inducing 50% of the maximal haemolysis was considered the median haemolytic
dose (HD ; graphical interpolation). Each experiment included triplicates at each concentration [22].
50
Immunological-Adjuvant Activity. The adjuvant activities of compound 1 and commercial adjuvants
were evaluated by the enhancement of the cellular immune response produced by the delayed-type
hypersensitivity (DTH) reaction against the ovalbumin (OVA) antigen. In the in vivo assay, male Swiss
mice (three-months-old) were subcutaneously immunized twice at weekly intervals with 100 mg of
ovalbumin antigen (OVA) dissolved in 100 ml of sterile saline (SAL) as the negative control group, or
with 100 mg of OVA mixed with 100 mg of compound 1 or Freundꢃs Complete Adjuvant (FCA) or
Freundꢃs Incomplete Adjuvant (FIA) as the positive control groups, each one dissolved in 100 ml of saline
as vehicle. A reference compound, the commercial purified Quillaja saponaria saponin (QS-21; 100 mg),
was mixed with 100 mg of the antigen (OVA) and dissolved in 100 ml of saline, for comparison. The DTH
responses were assessed by measuring the increment in the right footpad thickness after subcutaneous
challenge with 100 mg of OVA in 100 ml of saline a week after the second immunization. The footpad
thickness was measured with a spring-loaded dial gauge (Mitutoyo Corp., Tokyo, Japan) before, and 24,
4
8, and 72 h after injection. Injecting each animal with 100 ml of saline in the left hind footpad served as
control. The OVA-specific responses were obtained by subtracting the response to OVA challenge in
unimmunized control mice [29].
Animals. Male Swiss mice (three-months-old) weighing 25–35 g were purchased from Health
Sciences Center, Federal University of Rio de Janeiro, Brazil, and acclimatized for 7 d before. Rodent
laboratory chow and tap water were provided ad libitum, and maintained under controlled conditions:
temp., 24ꢁ18; humidity, 50ꢁ10%; 12-h light/12-h dark cycle. All procedures were in strict accordance
with the ꢄPrinciples of Laboratory Animal Careꢃ (National Institute of Health Publication 85-23, revised
1
985).
Statistical Analysis. The experimental data were expressed as meanꢁstandard error (SE) and
examined for their statistical significance of difference P with Studentꢃs t test.
We thank for the financial supports from the Brazilian Funding Agencies CNPq (Conselho Nacional
de Desenvolvimento Cientꢀfico e Tecnol o´ gico), CAPES (CoordenaÅ a˜ o de AperfeiÅoamento de Pessoal
de Nꢀvel Superior), and FINEP (Financiadora de Estudos e Projetos).

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
REFERENCES
67
[
[
[
[
1] S. D. Kravets, Y. S. Vollerner, M. B. Gorovits, N. K. Abubakirov, Khim. Prir. Soedin. 1990, 429.
2] H. Matsuura, J. Nutr. 2001, 131, 1000S.
3] M. A. Lacaille-Dubois, H. Wagner, Phytomedicine 1996, 2, 363.
´
4] M. P. Correa, ꢄDicion a´ rio das Plantas Uteis do Brasil e das Ex o´ ticas Cultivadasꢃ, Imprensa Nacional,
Rio de Janeiro, 1926.
[
[
[
[
5] M. E. Wall, C. R. Eddy, M. L. McClennan, M. E. Klumpp, Anal. Chem. 1952, 24, 1337.
6] A. Carotenuto, E. Fattorusso, V. Lanzotti, S. Magno, Phytochemistry 1999, 51, 1077.
7] P. K. Agrawal, Magn. Reson. Chem. 2003, 41, 965.
8] A. Carotenuto, E. Fattorusso, V. Lanzotti, S. Magno, V. De Feo, R. Carnuccio, F. DꢃAcquisto, J. Nat.
Prod. 1997, 60, 1003.
[
9] E. Fattorusso, V. Lanzotti, S. Magno, O. Taglialatela-Scafati, J. Agric. Food Chem. 1998, 46, 4904.
[
[
[
[
10] E. Fattorusso, V. Lanzotti, O. Taglialatela-Scafati, M. Di Rosa, A. Ianaro, J. Agric. Food Chem. 2000,
48, 3455.
11] Y. Mimaki, T. Kanmoto, M. Kuroda, Y. Sashida, A. Nishino, Y. Satomi, H. Nishino, Chem. Pharm.
Bull. 1995, 43, 1190.
12] M. Itabashi, K. Segawa, Y. Ikeda, S. Kondo, H. Naganawa, T. Koyano, K. Umezawa, Carbohydr.
Res. 2000, 323, 57.
13] T. Ohtsuki, T. Koyano, T. Kowithayakorn, S. Sakai, N. Kawahara, Y. Goda, N. Yamaguchi, M.
Ishibashi, Bioorg. Med. Chem. 2004, 12, 3841.
[
[
[
[
[
[
[
[
14] B. P. da Silva, J. P. Parente, Z. Naturforsch., B 2006, 61, 1153.
15] J. P. Kamerling, G. J. Gerwig, J. F. Vliegenthart, J. R. Clamp, Biochem. J. 1975, 151, 491.
16] G. J. Gerwig, J. P. Kamerling, J. F. G. Vliegenthart, Carbohydr. Res. 1978, 62, 349.
17] J. P. Parente, P. Cardon, Y. Leroy, J. Montreuil, B. Fournet, G. Ricart, Carbohydr. Res. 1985, 141, 41.
18] Z.-G. Yang, H.-X. Sun, W.-H. Fang, Vaccine 2005, 23, 5196.
19] H.-X. Sun, Vaccine 2006, 24, 1324.
20] H.-X. Sun, Vaccine 2006, 24, 3432.
21] Y.-J. Kim, P. Wang, M. Navarro-Villalobos, B. D. Rohde, J. M. DerryBerry, D. Y. Gin, J. Am. Chem.
Soc. 2006, 128, 11906.
[
[
22] W. R. Santos, R. R. Bernardo, L. M. T. PeÅanha, M. Palatnik, J. P. Parente, C. B. P. de Sousa, Vaccine
1
997, 15, 1024.
23] K. Oda, H. Matsuda, T. Murakami, S. Katayama, T. Ohgitani, M. Yoshikawa, Biol. Chem. 2000, 381,
7.
6
[
[
[
[
[
24] D. J. Marciani, Drug Discovery Today 2003, 8, 934.
25] H.-X. Sun, Y. Xie, Y.-P. Ye, Vaccine 2009, 27, 1787.
ˇ
26] M. Coli c´ , D. Vu cˇ evi c´ , V. Kilibarda, N. Radi cˇ evi c´ , M. Savi c´ , Phytomedicine 2002, 9, 117.
27] W. J. W. Morrow, Y.-W. Yang, N. A. Sheikh, Vaccine 2004, 22, 2380.
28] M. Gautam, S. Saha, S. Bani, A. Kaul, S. Mishra, D. Patil, N. K. Satti, K. A. Suri, S. Gairola, K.
Suresh, S. Jadhav, G. N. Qazi, B. Patwardhan, J. Ethnopharmacol. 2009, 121, 241.
29] A. M. Mowat, A. M. Donachie, G. Reid, O. Jarrett, Immunology 1991, 72, 317.
30] K. Oda, H. Matsuda, T. Murakami, S. Katayama, T. Ohgitani, M. Yoshikawa, Vaccine 2003, 21, 2145.
31] J. S. Sawardeker, J. H. Sloneker, A. Jeanes, Anal. Chem. 1965, 37, 1602.
[
[
[
Received January 5, 2011