Spirostane and cholestane glycosides from the bulbs of Allium nigrum L

Article abstract of DOI:10.1016/j.foodchem.2010.09.028

A phytochemical investigation of the fresh bulbs of Allium nigrum L. led to the isolation of new spirostane-type glycosides as two inseparable isomer mixtures, nigrosides A1/A2 (1a/1b) and nigrosides B1/B2 (2a/2b), two new cholestane-type glycosides, nigrosides C and D (3 and 4), together with the known compounds, 25(R,S)-5α-spirostan-2α,3β,6β-trio1-3-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (5a/5b) and 25(R,S)-5α-spirostan-2α,3β,6β-trio1 3-O-β-d-glucopyranosyl-(1→2)-O-[4-O-(3S)-3-hydroxy-3-methylglutaryl-β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (6a/6b), isolated from this plant for the first time. All structures were elucidated mainly by spectroscopic analysis (1D and 2D NMR experiments, FABMS, HRESIMS) and by comparison with literature data. Cytotoxicity of the isolated compounds was assessed against human colon carcinoma (HT-29 and HCT 116) cell lines. Compounds 5a/5b and 6a/6b were found to be the most active with IC₅₀ values 1.09 and 2.82μM against HT-29 and 1.59 and 3.45μM against HCT 116, respectively.

Full text of DOI:10.1016/j.foodchem.2010.09.028

Food Chemistry 125 (2011) 447–455
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Spirostane and cholestane glycosides from the bulbs of Allium nigrum L
Aymen Jabrane a,b, Hichem Ben Jannet c, Tomofumi Miyamoto d, Jean-François Mirjolet e, Olivier Duchamp e,
Féthia Harzallah-Skhiri b, Marie-Aleth Lacaille-Dubois a,
⇑
a Laboratoire de Pharmacognosie, Unité de Molécules d’Intérêt Biologique, UMIB UPRES-EA 3660, Faculté de Pharmacie, Université de Bourgogne, F-21079 Dijon Cedex, France
b Institut Supérieur de Biotechnologie de Monastir, Université de Monastir, 5000, Monastir, Tunisia
c Laboratoire de Chimie des Substances Naturelles et de Synthèse Organique, Faculté des Sciences de Monastir, Université de Monastir, 5000 Monastir, Tunisia
d Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
e Oncodesign, 20 Rue Jean Mazen, BP 27627, 21076 Dijon Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A phytochemical investigation of the fresh bulbs of Allium nigrum L. led to the isolation of new spirostane-
type glycosides as two inseparable isomer mixtures, nigrosides A1/A2 (1a/1b) and nigrosides B1/B2 (2a/
2b), two new cholestane-type glycosides, nigrosides C and D (3 and 4), together with the known
Received 26 February 2010
Received in revised form 23 July 2010
Accepted 7 September 2010
compounds, 25(R,S)-5
(1 ? 3)]-O-b- -glucopyranosyl-(1 ? 4)-b-
6b-trio1 3-O-b- -glucopyranosyl-(1 ? 2)-O-[4-O-(3S)-3-hydroxy-3-methylglutaryl-b-
(1 ? 3)]-O-b- -glucopyranosyl-(1 ? 4)-b- -galactopyranoside (6a/6b), isolated from this plant for the first
a
-spirostan-2
a
,3b,6b-trio1-3-O-b-
D
-glucopyranosyl-(1 ? 2)-O-[b-
-spirostan-2
-xylopyranosyl-
D
-xylopyranosyl-
D
D-galactopyranoside (5a/5b) and 25(R,S)-5
a
a
,3b,
D
D
Keywords:
Allium nigrum
Liliaceae
Cholestane
Steroidal saponins
Nigrosides
NMR
D
D
time. All structures were elucidated mainly by spectroscopic analysis (1D and 2D NMR experiments,
FABMS, HRESIMS) and by comparison with literature data. Cytotoxicity of the isolated compounds was
assessed against human colon carcinoma (HT-29 and HCT 116) cell lines. Compounds 5a/5b and 6a/6b were
found to be the most active with IC50 values 1.09 and 2.82
HCT 116, respectively.
l
M against HT-29 and 1.59 and 3.45
lM against
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, steroidal glycosides have attracted serious attention
within the scientific community owing to the significant cytotoxic
activity against a leukaemia cell line (Yokosuka, Jitsuno, Yui,
Yamazaki, & Mimaki, 2009), breast cancer, HeLa cervical cells
(Kaskiw et al., 2009) and human colon cancer cell lines (Acharya
et al., 2010; Wang et al., 2004).
Allium nigrum L. (black garlic, black onion, broad leaved garlic),
synonym of A. bauerianum Baker., A. multibulbosum Jacq., A. magicum
L., A. monspessulanum Gouan., A. odorum Ten., is an ornamental plant
of the Liliaceae family, first described by Linnaeus in 1762, the name
comes from the greenish-black lobed ovaries in the centre of each
flower. Some are blacker than others. This species has a large bulb
(3–4 cm) with an ovoid-spherical shape and an entire smooth white
tunic. It differs from other Allium species by having relatively broad
leaves (Burnie, 1995). Ethnobotanical reference in Tunisia has
indicated that this species was not used in popular medicine (Le
Floc’H, 1983) but it was reported to be used as a food spice in Sicily
(Lentini & Venza, 2007).
Allium is a large, and the most important, representative genus of
the Liliaceae family, containing about 500 species, widely distrib-
uted in the northern hemisphere (Block, 1985). Since early times,
many Allium species, such as garlic (A. sativum L.), onion (Allium
cepa L.), chives (A. schoenoprasum L.), leeks (A. porrum L.) and
shallots (A. ascalonicum Hort.) have been used as foods, spices,
and herbal remedies (Fattorusso, Lanzotti, Taglialatela-Scafati, Di
Rosa, & Ianaro, 2000). Previous phytochemical investigations of
Allium sp. showed that these plants are a rich source of steroidal
saponins, as well as sulphur-containing compounds (Hostettmann
& Marston, 1995; Iciek, Kwiecien, & Wlodek, 2009; Lanzotti,
2005). The steroidal saponins, an important class of natural prod-
ucts, have been reported to possess various important biological
effects, such as haemolytic (Wang et al., 2007), antifungal (Barile
et al., 2007; Sautour, Miyamoto, & Lacaille-Dubois, 2007), antidia-
betic (Yoshikawa et al., 2007), platelet aggregation inhibitory
(Zhang et al., 1999), anti-inflammatory (Shao et al., 2007) and
immunomodulatory activities (Lacaille-Dubois, 2005; Zhang et al.,
2007).
No previous phytochemical or pharmacological study has been
reported on this plant. As a part of our continuous search for bio-
active saponins from the Liliaceae family (Acharya et al., 2010),
we now examine the bulbs of A. nigrum (L.). In this paper we
describe the isolation and structural elucidation of new steroid
saponins, among them two pairs of inseparable epimer mixtures,
⇑
Corresponding author. Tel.: +33 3 80 39 32 29; fax: +33 3 80 39 33 00.
E-mail address: m-a.lacaille-dubois@u-bourgogne.fr (M.-A. Lacaille-Dubois).
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.09.028

448
A. Jabrane et al. / Food Chemistry 125 (2011) 447–455
nigrosides A1/A2 (1a/1b), B1/B2 (2a/2b) and two cholestane-type
saponins, nigrosides C (3) and D (4), together with the epimers of
known compounds (5a/5b) and (6a/6b) (Fig. 1). Their structures
were determined mainly by spectroscopic methods, including 1D
and 2D NMR experiments, FABMS and HRESIMS, and compared
with literature data for the known compounds. In addition, the
cytotoxicity of all compounds was evaluated against the human
colon cancer (HCT 116 and HT-29) cell lines.
R2
21
O
25
R1
18
22
19
O
H
R2
HO
O
21
2
3
O
25
R1
H
H
18
OH
O
6
22
HO
OH
19
H
O
O
HO
HO
HO
H
OH
O
O
O
OH
2
3
H
H
OH
H3C
HO
OH
O
6
O
H
HO
HO
OH
OH
OH
1a R1= CH3 R2 = H
2a R1= CH3 R2 = H
1b R1=H
R2 = CH3
2b R1=H
R2 = CH3
OH
HO
HO
HO
27
21
18
22
O
20
H3C
25
26
S1 =
19
OR1
H
16
OR2
1
OH
H
H
3
5
O
HO
6
3 R1= S1 R2 = S2
4 R1 = OH R2 = S2
HO
OH
O
HO
O
OH
CH3
S2 =
OH
R2
21
O
25
R1
18
22
19
O
OH
H
O
HO
HO
O
O
2
3
R3O
HO
O
O
H
H
O
OH
O
OH
H
HO
OH
O
HO
OH
OH
HO
HO
OH
O
O
OH
(S)-HMG: (S)-3hydroxy-3-methylglutaric acid
5a
R1 = CH3
R1 = H
R2 = H
R3 = H
R3 = H
5b
6a
6b
R2 = CH3
R2 = H
R1 = CH3
R1 = H
R3 = (S)-HMG
R3 = (S)-HMG
R2 = CH3
Fig. 1. Structures of coupounds 1a/1b, 2a/2b, 3, 4, 5a/5b and 6a/6b.

A. Jabrane et al. / Food Chemistry 125 (2011) 447–455
449
2.2. Plant material
2. Materials and methods
Bulbs of A. nigrum L. were collected in the region of Béja (Tunisia),
in April, 2008 and identified by Professor Féthia HARZALLAH-
SKHIRI, in the Institut Supérieur de Biotechnologie de Monastir,
Tunisia. A voucher specimen (An 30.4.08) was deposited at the
Laboratory of Plant Biology in the above Institute.
2.1. General experimental procedures
Optical rotations were recorded on an AA-OR automatic polar-
imeter. The 1D and 2D NMR spectra (1H and 13C NMR, 1H–1H COSY,
TOCSY, NOESY, HSQC and HMBC) were performed 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
computer system (600 MHz for 1H and 150 MHz for 13C spectra).
Conventional pulse sequences were used for COSY, HSQC, and
HMBC spectra. TOCSY spectra were acquired using the standard
MLEV17 spin-locking sequence and a 90 ms mixing time. The mix-
ing time in the NOESY experiment was set to 500 ms. The carbon
type (CH3, CH2, CH) was determined by DEPT experiments. Chem-
ical shifts are reported as d values (ppm), referenced with respect
to the residual solvent signal of C5D5N, and coupling constants (J)
were measured in Hz. The samples were solubilised in pyridine-
d5. FABMS (negative-ion mode, glycerol matrix) was conducted
on a JEOL-SX-102 mass spectrometer. HRESIMS was carried out
on a Q-TOF 1 micromass spectrometer. Compound isolations were
2.3. Extraction and isolation of compounds
The fresh bulbs of A. nigrum (2.5 kg) were refluxed twice with
MeOH–H2O (7:3, 3 l) for 1 h and evaporated in vacuo yielding an
aqueous extract that was partitioned successively with CHCl3
(3 l) and H2O-satd n-BuOH (2 l), yielding, after evaporation of the
solvents, the corresponding CHCl3 (2.4 g) and n-BuOH (18 g) frac-
tions. A 5.5 g aliquot of the n-BuOH residue was submitted to
VLC on RP-18 silica gel using H2O (500 ml), MeOH–H2O mixtures
(5:5, 2 ꢀ 500 ml), and finally MeOH (2 ꢀ 500 ml) as eluents. After
evaporation of the solvents, five fractions were obtained: AN I–V.
AN-II (1.35 g) was submitted to MPLC [silica gel (15–40 lm), sys-
carried out using
a medium-pressure liquid chromatography
tem a: CHCl3-MeOH–H2O (7/3/1) lower phase)] to give eight frac-
tions (Fr1–Fr8). Fr3 (120 mg), rechromatographed under the
same conditions by MPLC (system a), afforded compound 5a/5b
(14 mg, 0.0025% w/w of the fresh bulbs). Fr7 was concentrated to
dryness, to give compound 6a/6b (29 mg, 0.0052% w/w of the fresh
bulbs). AN-IV (3 g) was submitted to VLC using silica gel 60 (15–
(MPLC) system [Gilson M 305 pump, 25 SC head pump, M 805
manometric module, Büchi glass column (460 ꢀ 25 mm and
460 ꢀ 15 mm), Büchi precolumn (110 ꢀ 15 mm)] on silica gel 60
(Merck, 15–40
75–200
lm), and reversed-phase RP-18 silica gel (Silicycle
m), and with a Gilson Pump Manager C-605, having
l
two pumps (2 ꢀ Büchi pump modul C-601) and one fraction collec-
40 lm), eluted with a gradient of CHCl3–MeOH (9/1–2/8), to give
tor (Büchi C-660). Vacuum-liquid chromatography (VLC) was per-
four sub-fractions, sF1–sF4. An aliquot of 250 mg of sF3 (1.24 g)
was rechromatographed successively by MPLC on RP-18 silica gel
MeOH–H2O (7/3), using a Gilson Pump Manager, affording the pure
compounds 3 (11 mg, 0.0019% w/w of the fresh bulbs) and 4
(13 mg, 0.0023% w/w of the fresh bulbs). Fraction sF4 was submit-
ted to successive chromatographies (MPLC on silica gel, system a)
to give compounds 1a/1b (12 mg, 0.0021% w/w of the fresh bulbs)
and 2a/2b (15 mg, 0.0027% w/w of the fresh bulbs).
formed on a reversed-phase RP-18 silica gel (Silicycle, 75–200 lm)
(12 ꢀ 6 cm). Thin-layer chromatography (TLC, Silicycle) and
high-performance thin-layer chromatography (HPTLC, Merck)
were performed on precoated silica gel plates 60 F254. The spray re-
agent for saponins was the Komarowsky reagent, which is a mix-
ture (5:1) of p-hydroxybenzaldehyde (2% in MeOH) and ethanolic
H2SO4 (50%).
Table 1
a,b
1H NMR spectroscopic data of the aglycone moieties of 1a/1b and 2a/2b. (Pyridine-d5, d in ppm, J in Hz)
.
Pos.
1a/1b
2a/2b
dH
dH
a
1
2
3
4
1.19 (1H, t, 11.9), 2.12b (1H, m)
1.13, 2.35 (1H, m)
4.31 (1H, m)
4.25 (1H, m)
2.22, 2.28 (1H, q, 11.9)
1.01
4.08 (1H, m)
3.98 (1H, m)
2.08 (1H, m), 2.36 (1H, q, 12.8)
5
1.06
6
7
8
9
11
12
14
15
16
17
18
19
3.94
1.06, 1.96
2.04
3.90 (1H, m)
1.03, 1.95
2.00 (1H, m)
0.56 (1H, m)
1.28, 1.48
0.62
1.31, 1.45
0.99, 1.58
1.03
1.38 (1H, m), 1.98
4.45
1.74 (1H, m)
0.73 (3H, s)
1.25 (3H, s)
0.92, 1.55
1.00
1.38 (1H, m), 1.95
4.44 (1H, m)
1.73 (1H, m)
0.70 (3H, s)
1.15 (3H, s)
20
21
23
24
25
26
1.85 (1H, dq, 6.6, 6.9)
1.06 (3H, d, 6.9)
1.63, 1.52
1.45, 1.48
1.48
3.42 (1H, t, 10.5)
3.53 (1H, br d, 9.2)
0.62 (3H, d, 4.3)
1.80 (1H, m)
1.06 (3H, d, 6.9)
1.32, 1.79
1.27, 2.04
1.53
3.33 (1H, d, 10.5)
3.97 (1H, m)
0.98 (3H, d, 6.9)
1.84 (1H, m)
1.06 (3H, d, 6.9)
1.64, 1.58
1.46, 1.48
1.49
3.39 (1H, t, 10.5)
3.53 (1H, br d, 9.2)
0.62 (3H, d, 4.3)
1.81 (1H, m)
1.06 (3H, d, 6.9)
1.35 (1H, m), 1.80
1.28 (1H, m), 2.03 (1H, m)
1.52
3.33 (1H, d, 10.5)
3.97 (1H, m)
27
0.98 (3H, d, 6.9)
a
Overlapped signals are reported without designated multiplicity.
b
a
axial proton, bequatorial proton.

450
A. Jabrane et al. / Food Chemistry 125 (2011) 447–455
779.4194). 1H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyri-
dine-d5, 150 MHz) data: see Tables 1, 2 and 4.
Table 2
13C NMR spectroscopic data of the aglycone moieties of 1a/1b and 2a/2b. (Pyridine-d5,
d in ppm).
Nigrosides B1/B2 (compound 2a/2b): white amorphous powder:
a 2D1
ꢁ
: ꢂ28.5 (MeOH, c 0.30). Negative FABMS: m/z 771 [M–H]ꢂ,
Pos.
Dept
1a/1b
2a/2b
½
HRESIMS (positive-ion mode): m/z 795.4147 [M + Na]+ (calcd. for
795.4143). 1H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyri-
dine-d5, 150 MHz) data: see Tables 1, 2 and 4.
dC
dC
1
2
3
4
5
6
7
8
(CH2)
(CH)
(CH)
(CH2)
(CH)
(CH)
(CH2)
(CH)
(CH)
(C)
46.7
70.2
84.5
30.8
47.1
69.7
39.7
29.5
54.1
36.6
21.0
39.9
40.5
55.8
31.4
80.9
62.3
16.2
16.7
41.6
14.6
109.2
31.4
28.7
30.2
66.5
16.9
44.5
76.4
78.4
31.4
46.9
69.4
39.6
29.4
53.9
36.5
20.9
39.8
40.4
55.7
31.6
80.9
62.2
16.1
16.3
41.6
14.5
109.1
31.3
28.7
30.0
66.5
16.8
Nigroside C (compound 3): yellow amorphous powder: ½a D21
ꢁ
:
ꢂ60.6 (MeOH, c 0.20). Negative FABMS: m/z 887 [M–H]ꢂ, HRESIMS
(positive-ion mode): m/z 911.4986 [M + Na]+ (calcd. for 911.4980).
1H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5,
150 MHz) data: see Tables 3 and 4.
9
Nigroside D (compound 4): yellow amorphous powder: ½a D21
ꢁ
:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
ꢂ58.2 (MeOH, c 0.18). Negative FABMS: m/z 741 [M–H]ꢂ, HRESIMS
(positive-ion mode): m/z 765.4409 [M + Na]+ (calcd. for 765.4401).
1H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5,
150 MHz) data: see Tables 3 and 4.
(CH2)
(CH2)
(C)
(CH)
(CH)
(CH2)
(CH)
(CH3)
(CH3)
(CH)
(CH3)
(C)
(CH2)
(CH2)
(CH)
(CH2)
(CH3)
Compounds 5a/5b: white amorphous powder: ½a D21
ꢁ : ꢂ59.8
(MeOH, c 0.50). Negative FABMS: m/z 1065 [M–H]ꢂ, m/z 933 [(M–
H)–132]ꢂ, 771 [(M–H)–132–162]ꢂ, 609 [(M–H)–132–162–162]ꢂ,
HRESIMS (positive-ion mode): m/z 1089.5203 [M + Na]+ (calcd. for
1089.5198). 1H NMR and 13C NMR data of aglycone moiety ap-
peared similar to those obtained for 1a/1b: 1H NMR (pyridine-d5,
42.1
14.4
109.6
25.9
25.7
27.0
65.0
15.9
42.0
14.3
109.6
25.8
25.6
27.0
65.1
15.8
600 MHz) dH 2.08 (1H, m, Ha-4), dH 2.28 (1H, q, 12.4, Hb-4), dH
0.73 (3H, s, Me-18), dH 1.14 (3H, s, Me-19), dH 1.06 (3H, d, 6.9,
Me-21), dH 1.48 (1H, m, H-25) of (25R)-isomer, dH 1.52 (1H, m, H-
25) of (25S)-isomer, dH 3.40 (1H, t, 11.0, H
a-26) and dH 3.52 (1H,
m, Hb-26) of (25R)-isomer, dH 3.31 (1H, d, 11.0, H
a-26) and dH
3.96 (1H, m, Hb-26) of (25S)-isomer, dH 0.62 (3H, d, 4.3, Me-27) of
(25R)-isomer and dH 0.98 (3H, d, 7.1, Me-27) of (25S)-isomer. 13C
NMR (pyridine-d5, 600 MHz) dC 46.5 (C-1), dC 70.2 (C-2), dC 84.0
(C-3), dC 31.8 (C-4), dC 47.3 (C-5), dC 69.6 (C-6), dC 39.7 (C-7), dC
29.6 (C-8), dC 54.0 (C-9), dC 36.6 (C-10), dC 21.0 (C-11), dC 40.5
(C-12), dC 40.0 (C-13), dC 55.8 (C-14), dC 31.2 (C-15), dC 81.0
(C-16), dC 62.3 (C-17), dC 16.2 (C-18), dC 16.7 (C-19), dC 42.1
2.4. Characterisation of isolated compounds
Nigrosides A1/A2 (compound 1a/1b): white amorphous powder:
½
a 2D1
ꢁ
: ꢂ39.5 (MeOH, c 0.50). Negative FABMS: m/z 755 [M–H]ꢂ,
HRESIMS (positive-ion mode): m/z 779.4202 [M + Na]+ (calcd. for
Table 3
1H and 13C NMR spectroscopic data of the aglycone moieties of 3 and 4. (Pyridine-d5, d in ppm, J in Hz)a,b
.
Pos.
Dept
3
4
dC
dH
dC
dH
1
2
3
4
5
6
7
8
(CH)
(CH2)
(CH)
(CH2)
(C)
(CH)
(CH2)
(CH)
(CH)
(C)
81.4
3.60 (1H, br d, 11)
77.7
3.65 (1H, dd, 11.7, 4.0)
2.20, 2.56
3.83 (1H, m)
2.53, 2.58
a
35.2
67.6
42.8
138.6
125.1
31.1
33.1
50.4
42.6
24.4
40.1
41.8
54.8
36.1
82.0
58.0
13.4
14.1
35.3
12.1
73.0
33.2
36.7
28.3
22.6
22.7
1.80 , 2.68b (1H, br d, 11)
42.1
67.7
43.0
139.6
124.3
31.6
32.6
50.8
43.0
23.8
40.1
41.7
54.8
36.8
82.0
57.6
13.2
13.5
35.4
12.1
73.5
33.1
36.1
28.4
22.6
22.7
3.75 (1H, m)
2.50, 2.58 (1H, t, 12.3)
5.42 (1H, br d, 5.0)
1.32, 173
1.24
5.44 (1H, br d, 5.0)
1.35, 1.78
1.28
9
1.18
1.19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
(CH2)
(CH2)
(C)
1.51, 2.47
1.42, 2.02 (1H, br d, 6.7)
1.60, 2.61
1.20, 1.91 (1H, m)
(CH)
(CH2)
(CH)
(CH)
(CH3)
(CH3)
(CH)
(CH3)
(CH)
(CH2)
(CH2)
(CH)
(CH3)
(CH3)
0.81
0.80
1.69, 2.22 (1H, m)
4.50
1.91 (1H, t, 8.8)
0.91 (3H, s)
1.07 (3H, s)
2.42
1.08 (1H, d, 6.9)
4.22
1.64, 1.75
1.55, 1.78
1.50
1.67, 2.25 (1H, m)
4.51
1.87 (1H, br d, 10.9)
0.92 (3H, s)
1.21 (3H, s)
2.40
1.08 (1H, d, 6.9)
4.19
1.63, 1.75
1.54, 1.78
1.49
0.80 (3H, d, 6.2)
0.83 (3H, d, 6.4)
0.79 (3H, d, 6.2)
0.81 (3H, d, 6.4)
a
Overlapped signals are reported without designated multiplicity.
b
a
axial proton, bequatorial proton.

A. Jabrane et al. / Food Chemistry 125 (2011) 447–455
451
Table 4
1H and 13C NMR spectroscopic data of the sugar moieties of 1a/1b, 2a/2b, 3 and 4. (Pyridine-d5, d in ppm, J in Hz)a.
1a/1b
2a/2b
3
4
dC
dH
dC
dH
dC
dH
dC
dH
Glc
Glc
1
2
3
4
5
6a
6b
100.3
78.3
78.6
71.4
77.7
62.1
4.90 (1H, d, 7.8)
4.06 (1H, t, 8.1)
4.15 (1H, t, 9.0)
3.89 (1H, t, 9.5)
3.91 (1H, m)
4.47 (1H, dd, 12.3, 3.4)
4.10
101.3
73.8
77.2
70.6
77.9
61.9
4.99 (1H, d, 7.6)
4.50 (1H, t, 9.0)
4.07
4.36
3.98 (1H, m)
4.34
4.19 (1H, dd, 10.9, 5.1)
Rha
Rha I
Rha
1
2
3
4
5
6
101.9
71.7
71.9
73.5
69.1
18.1
6.11 (1H, br s)
4.70 (1H, br s)
4.51 (1H, dd, 3.0, 9.0)
4.21 (1H, t, 9.5)
4.81 (1H, dq, 6.4, 6.1)
1.63 (3H, d, 5.9)
103.2
71.8
71.9
72.9
69.4
18.1
5.86 (1H, br s)
4.63 (1H, br s)
4.52 (1H, dd, 3.1, 9.5)
4.21
4.51
1.50 (3H, d, 6.1)
103.5
71.5
71.9
72.8
69.5
18.1
5.86 (1H, br s)
4.63 (1H, br s)
4.52 (1H, dd, 3.1, 9.5)
4.21
4.51
1.50 (3H, d, 6.1)
Gal
Gal
Gal
1
2
3
4
5
6a
6b
101.8
71.1
74.2
69.4
76.5
61.7
4.97 (1H, d, 7.6)
4.03 (1H, t, 8.1)
4.16 (1H, dd, 10.2, 5.9)
4.05
3.89 (1H, m)
4.37 (1H, br d, 10.0)
4.14
106.5
71.5
81.5
69.0
75.9
61.3
4.62 (1H, d, 6.7)
4.38 (1H, t, 8.6)
4.06 (1H, dd, 9.7, 2.6)
4.60
3.88 (1H, m)
4.33 (1 h, br d, 9.8)
4.24
106.7
71.5
81.5
69.1
76.0
61.2
4.62 (1H, d, 6.7)
4.38 (1H, t, 8.6)
4.06 (1H, dd, 9.5, 2.6)
4.61
3.88 (1H, m)
4.35 (1H, br d, 9.8)
4.25
Rha II
1
2
3
4
5
6
97.4
72.2
72.3
73.5
70.2
18.20
5.48 (1H, br s)
4.41 (1H, br s)
4.35 (1H, t, 6.6)
4.18
4.10
1.55 (3H, d, 5.9)
a
Overlapped signals are reported without designated multiplicity.
(C-20), dC 14.1 (C-21), dC 109.6 (C-22), dC 26.0 (C-23), dC 25.7 (C-24),
[dC 28.8 (C-25), dC 66.6 (C-26), dC 16.8 (C-27) of (25R)-isomer], [dC
27.0 (C-25), dC 64.8 (C-26), dC 15.9 (C-27) of (25S)-isomer], 1H
NMR and 13C NMR data of sugar moieties are shown in Table 5.
man colon cancer cells (HT-29 and HCT 116). Isolated compounds
and Paclitaxel (positive control) were dissolved in DMSO to make
stock solutions, which were diluted to a working solution before
use (the final DMSO concentration was 0.1% v/v). HT-29 and HCT-
116 cells were seeded at an initial density of 5000 or 10,000 cells/
well in 96-well plates and treated with medium having various con-
centrations of the test compounds. DMSO controls (0.1%) did not af-
fect cell proliferation. After 96 h, 20 ll of MTT solution (5 mg/ml in
PBS) were added to the culture medium and the reaction mixture
was incubated at 37 °C in a 5% CO2 atmosphere for 4 h. The MTT solu-
tion was aspired and 200 ll of DMSO were added. The optical den-
Compounds 6a/6b: white amorphous powder: ½a D21
ꢁ : ꢂ61.6
(MeOH, c 0.50). Negative FABMS: m/z 1209 [M–H]ꢂ, m/z 1065
[(M–H)–144]ꢂ, 933 [(M–H)–144–132]ꢂ, 771 [(M–H)–144–132–
162]ꢂ, 609 [(M–H)–144–132–162–162]ꢂ. HRESIMS (positive-ion
mode): m/z 1233.5621 [M + Na]+ (calcd. for 1233.5617). 1H NMR
and 13C NMR data of aglycone moiety appeared similar to those ob-
tained for 5a/5b. 1H NMR and 13C NMR data of sugars moieties and
acyl group are shown in Table 5.
sity (OD) was measured spectrophotometrically at 570 nm. The
results were expressed as concentrations of compound producing
50% toxicity (IC50 value). The experiments were repeated twice.
2.5. Acid hydrolysis
A solution of each saponin (2 mg) in H2O (2 ml) was treated with
2 N aq. CF3COOH (5 ml) and refluxed for 3 h at 95 °C. After extrac-
tion with CH2Cl2 (3 ꢀ 5 ml), the aq. layer was repeatedly concen-
trated with MeOH until neutral, and then analysed by TLC (SiO2,
CHCl3/MeOH/H2O 8:5:1) with authentic samples. Furthermore, a
silylated derivative of the sugars was prepared according to the pre-
3. Results and discussion
3.1. Phytochemical investigation
The n-BuOH fraction from the MeOH:H2O (7:3) extract of the A.
nigrum bulbs was submitted to several chromatographic steps (VLC
and MPLC on normal and reversed RP-18 silica gel), yielding new
saponins, two of them as inseparable mixtures named nigrosides
A1/A2 (1a/1b), B1/B2 (2a/2b), and the pure compounds nigrosides
C (3) and D (4), together with the known saponins, also as insepa-
rable mixtures (5a/5b) and (6a/6b). The structures of the new
saponins were elucidated by 600 MHz NMR analyses, including
1D and 2D NMR experiments, and compared with literature data
for the known compounds. These latter were identified as
viously described procedure (Haddad, Miyamoto, Laurens,
Lacaille-Dubois, 2003) and analysed by GC. The following sugars
were characterised: -glucose, -rhamnose for (1a/1b), -galactose,
-glucose for (2a/2b), -rhamnose, -galactose for (3 and 4),
-xylose for (5a/5b) and (6a/6b).
&
D
L
D
D
L
D
D-
glucose, D-galactose and D
2.6. MTT cytotoxicity assay
The bioassay was carried out according to the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method
(Carmichael, Degraff, Gazdar, Minna, & Mitchell, 1987) with two hu-
25(R,S)-5
O- [b- -xylopyranosyl-(1 ? 3)]-O-b-
b- -galactopyranoside (aginoside/turoside A) (5a/5b) and 25
a
-spirostan-2
a
,3b,6b-trio1-3-O-b-
D-glucopyranosyl-(1 ? 2)-
D
D
-glucopyranosyl-(1 ? 4)-
D

452
A. Jabrane et al. / Food Chemistry 125 (2011) 447–455
Table 5
far as we know, the isolation of a steroidal mixture of C-25 R and
S epimers is sometimes difficult (Zheng, Zhang, Li, & Yang, 2004).
Thus, their structures were elucidated as C-25 epimeric mixtures.
Compound 1a/1b, in high-resolution electrospray ionisation
mass spectroscopy (HR-ESIMS) (positive-ion mode), exhibited a
pseudo-molecular ion peak at m/z = 779.4202 [M + Na]+, consistent
with a molecular formula C39H64O14. Its fast-atom bombardment
mass spectrum (FABMS) (negative-ion mode) displayed a pseu-
do-molecular ion peak at m/z 755 [M–H]ꢂ, indicating a molecular
weight of 756. A fragment-ion peak was observed at m/z 609
([M–H–146]ꢂ), corresponding to the loss of one deoxyhexosyl unit.
The 1H and 13C NMR spectra, in combination with DEPT and HSQC
spectra of 1a/1b, displayed the presence of two angular methyl
proton signals at dY 0.73 (3H, s), 1.25 (3H, s), three secondary
methyl proton signals at dY 1.06 (3H, d, J = 7.0 Hz), 0.62 (3H, d,
J = 4.3 Hz, (R)-form), 0.9 8 (3H, d, J = 7 Hz, (S)-form) and character-
istic acetal carbon signal at dC 109.6 (S)-form and at dC 109.2 (R)-
form, indicating the presence of a steroidal spirostanol skeleton
(Agrawal, Jain, & Pathak, 1995). Analysis of NMR data showed 13C
NMR signals at dC 47.1 (C-5), 54.1 (C-9), and 16.7 (C-19), character-
1H and 13C NMR spectroscopic data of the sugar moieties of 5a/5b and 6a/6b
(Pyridine-d5, d in ppm, J in Hz)a.
5a/5b
6a/6b
dC
dH
dC
dH
Gal
Gal
1
2
3
4
5
6a
6b
102.4
72.0
75.0
79.1
75.3
60.6
4.89 (1H, d, 7.9)
102.4
72.0
75.0
79.1
75.3
60.6
4.89 (1H, d, 7.9)
4.44
4.08
4.48
4.01
4.20
4.44
4.44
4.07
4.45
4.01
4.20
4.44
Glc I
Glc I
1
2
3
4
5
6a
6b
103.8
80.4
86.7
69.6
76.9
62.1
5.06 (1H, d, 7.9)
103.8
80.4
86.7
69.6
76.9
62.1
5.06 (1H, d, 7.9)
4.18
3.98
3.65
3.70
3.94
4.35
4.18
3.97
3.65
3.69
3.93
4.35
Xyl
Xyl
1
2
3
4
5
104.3
74.8
77.7
70.8
66.6
5.10 (1H, d ,7.9)
104.3
74.8
77.7
72.9
66.6
5.15 (1H, d,7.9)
istic of A/B trans-ring fusion, indicating that 1a/1b is a 5a-steroidal
3.85
4.01
3.97
3.56
4.12
3.85
4.01
4.00
3.56
4.12
spirostanol derivative. This was confirmed by the NOESY correla-
tions observed between dH 3.98 (Agly H-3) and 1.06 (Agly H-5).
The HMBC correlations between dY 4.08 (Agly H-3), and dC 70.2
(Agly C-2) and between dY 1.06 (Agly H-5) and dC 69.7 (Agly C-
6), indicated a secondary alcoholic function at positions C-2 and
Glc II
Glc II
C-6, respectively. The 2a orientation of the hydroxyl unit at (C-2)
1
2
3
4
5
6a
6b
103.8
75.4
77.4
71.0
77.7
62.1
5.50 (1H, d, 7.6)
103.8
75.4
77.4
71.0
77.7
62.1
5.50 (1H, d, 7/6)
3.94
4.11
4.00
3.85
4.30
4.40
3.94
4.11
4.00
3.85
4.30
4.40
was proved by the NOESY cross-peak between dH 1.25 (s, ax H3-
19) and dH 4.08 (m, H-2), proving the axial orientation of this pro-
ton, while the NOESY cross-peak between dH 1.06 (s, H-5) and dH
3.94 (m, H-6) indicated the 6b orientation of the hydroxyl at (C-
6). The above aglycone was identified as 25(R,S)-5
a-spirostan-
2a
,3b,6b-triol (agigenin/neoagigenin) by comparison of 13C and
Acyl
1H NMR chemical shifts of 1a/1b obtained from the 2D NMR data
(Tables 1 and 2) with those reported in the literature (Carotenuto
et al., 1997; Sata et al., 1998). The 25 (R,S) stereochemistry of the
Me-27 group was deduced from the resonance of protons and car-
bons from the ring F portion (C-22–C-27) (Tables 1 and 2) (Agra-
wal, 2003). The NOESY correlations between H3-19/H-2, H3-19/
H-8, H3-18/H-8, H3-18/H-20, H-5/H-9, H-9/H-14, H-14/H-17 and
H-16/H-17 confirmed the conventional A/B trans, B/C trans, C/D
1
2
3
4
5
6
171.2
48.3
70.2
48.7
173.4
28.4
–
2.68, 2.91
–
2.74, 3.02
–
1.48
a
Overlapped signals are reported without designated multiplicity.
trans, D/E cis, and C-20a stereochemistry of the aglycone of 1a/1b.
The 1H NMR spectrum showed the presence of two anomeric
proton signals at dY 4.90 (1H, d, J = 7.2 Hz) and 6.11 (1H, br s), giv-
ing correlation of the HSQC spectrum with two anomeric carbons
at dC 100.3 and 101.9, respectively. The evaluation of chemical
shifts and spin–spin couplings allowed the identification of one
(R,S)-5
O-[4-O-(3S)-3hydroxy-3-methylglutaroyl-b-
O-b- -glucopyranosyl-(1 ? 4)-b- -galactopyranoside (6a/6b),
previously isolated in Allium ssp (Kawashima, Mimaki, & Sashida,
1991; Kawashima, Mimaki, & Sashida, 1993), but reported for the
first time in A. nigrum.
a
-spirostan-2
a
,3b,6b-trio1–3-O-b-
D
-glucopyranosyl-(1 ? 2)-
D-xylopyranosyl-(1 ? 3)]-
D
D
b-glucopyranosyl unit (Glc) and one
(Rha). The absolute configuration of Glc was determined as
that of Rha as , as described above. COSY and TOCSY experiments
a
-rhamnopyranosyl unit
D
and
After acid hydrolysis of each compound, the sugar moieties
were characterised by TLC with authentic samples as: rhamnose
and glucose (in the case of 1a/1b), galactose and glucose (in the
case of 2a/2b), rhamnose and galactose (in the case of 3, 4), and
galactose, glucose and xylose for 5a/5b, 6a/6b. The absolute config-
uration was determined by GC of the chiral derivatives of the
hydrolysate of each compound (see expr part) as D for glucose,
xylose and galactose and L for rhamnose. In the 1H NMR spectra
L
allowed the sequential assignments of all of the proton resonances
to the individual monosaccharides, as shown in (Table 4). The se-
quence of the disaccharide chain of 1a/1b was determined by
HMBC and NOESY experiments. The correlations observed in the
HMBC spectrum between the 1H NMR signals at dY 4.90 (d,
J = 7.7 Hz, Glc H-1) and the 13C NMR signal at dC 84.5 (Agly C-3),
and in the NOESY spectrum between the signal at dY 4.90 (Glc
H-1) and 3.98 (Agly H-3), suggested that the glucose was linked
at C-3 of the aglycone. The correlation in the HMBC spectrum,
between the 1H NMR signal at dY 6.11 (Rha H-1) and the 13C
NMR signal at dC 78.3 (Glc C-2), revealed a (1 ? 2) linkage between
these two sugars, which was confirmed by the cross-peak in the
NOESY spectrum between dY 6.11 (Rha H-1) and dY 4.06 (Glc
H-2), On the basis of the above data, compound 1a/1b was eluci-
3
of the compounds, the relatively large JH-1,H-2 values of the
glucose, xylose, and galactose (between 7.0 and 8.0 Hz) moieties
indicated a b anomeric proton. The broad singlet of the anomeric
proton of the rhamnose unit indicated an
et al., 2008).
a-orientation (Avunduk
Saponins 1a/1b and 2a/2b were isolated as amorphous white
powders. The 1H and 13C NMR data obtained from 1D and 2D spec-
troscopic experiments revealed that 1a/1b and 2a/2b were spirost-
anol derivatives, existing as mixtures of C-25 R and S epimers. As
dated as 25 (R,S)-5a-spirostan-2a,3b,6b-triol 3-O-a-L-rhamnopyr-
anosyl-(1 ? 2)-b- -glucopyranoside, called nigrosides A1/A2.
D

A. Jabrane et al. / Food Chemistry 125 (2011) 447–455
453
Compound 2a/2b, in high-resolution electrospray ionisation
mass spectroscopy (HR-ESIMS) (positive-ion mode), exhibited a
pseudo-molecular ion peak at m/z = 795.4147 [M + Na]+, consistent
with a molecular formula C39H64O15. Its fast-atom bombardment
mass spectrum (FABMS) (negative-ion mode) showed a pseudo-
molecular ion peak at m/z 771 [M–H]ꢂ, indicating a molecular
weight of 772. Significant fragment-ion peaks were observed at
m/z 609 ([M–H–162]ꢂ), suggesting the elimination of a one hexosyl
moiety. A detailed comparison of the 1H, 13C NMR chemical shift
(Tables 1 and 2), and the 2D NMR spectral analysis of the aglycone
part of 1a/1b and 2a/2b, revealed that all signals were superimpos-
able, except those of the carbon signals C-2 and C-3 in 2a/2b. The
downfield shift of C-2 by 6.2 ppm at dC 76.4 and the upfield shift of
C-3 by 6.1 ppm at dC 78.4 indicated that the aglycone of 2a/2b (agi-
genin/neoagigenin) was substituted at those two positions (Mima-
ki, Kuroda, Fukasawa, & Sashida, 1999). Full assignment of the 1H
and 13C NMR signals of the aglycone part of 2a/2b showed glyco-
sylation shifts for C-2 and C-3, suggesting a bidesmosidic spiros-
tane-type saponin. The 1H NMR spectrum showed the presence
of two anomeric proton signals at dH 4.97 (1H, d, J = 7.6 Hz) and
4.99 (1H, d, J = 7.6 Hz), giving correlations in the HSQC spectrum
with two anomeric carbon signals at dC 101.8 and 101.3, respec-
tively. The complete assignment of the glycosidic NMR signals
was achieved by analysis of COSY, TOCSY, NOESY, HSQC, and HMBC
experiments (Table 4). Evaluation of the spin–spin couplings and
chemical shift from the 2D NMR spectra of 2a/2b allowed the
identification of one b-glucopyranosyl (Glc) and one b-galactopyr-
anosyl (Gal) unit. The site of attachment of the sugar was deter-
mined by combination of the HMBC and NOESY experiments. In
the HMBC spectrum, the anomeric proton signals at dH 4.99 (Glc
H-1) and 4.97 (Gal H-1) showed correlation with the carbon reso-
nances at dC 76.3 (C-2) and 78.4 (C-3), respectively. On the other
hand, the NOESY cross-peaks, dH 4.99 (Glc H-1)/dH 4.31 (Agly
H-2), dH 4.97 (Gal H-1)/dH 4.25 (Agly H-3), dH 4.99 (Glc H-1)/dH
The 1H NMR spectrum of 3 displayed three anomeric proton sig-
nals at dH 5.86 (1H, br s), 5.48 (1H, br s) and 4.62 (1H, d, J = 6.7),
which gave correlation with three anomeric carbon signals at dC
103.2, 97.4 and 106.5, respectively, in the HSQC spectrum. Com-
plete assignments of each glycosidic proton system were achieved
by 2D NMR spectroscopic experiments allowing the identification
of two rhamnopyranosyl units (Rha I and Rha II) and one b-galac-
topyranosyl (Gal) unit (Table 4).
The site of attachment of sugar moieties on the aglycone was
determined by HMBC and NOESY experiments. The cross-peak in
the HMBC spectrum between dH 4.62 (Gal I H-1) and dC 82.0 (Agly
C-16), between dH 5.48 (Rha II H-1) and dC 81.4 (Agly C-1), together
the cross-peak observed in NOESY spectrum between the proton
signal at dH 4.62 (Gal H-1) and dH 4.50 (Agly H-16) and between
the proton signal at dH 5.48 (Rha II H-1) and dH 3.60 (Agly H-1),
confirmed this glycosylation at positions C-1 and C-16 by Rha II
and Gal, respectively. The deshielded value at dC 81.5 (Gal C-3) sug-
gested the point of linkage of Rha I. The correlation in the HMBC
spectrum between the signals at dH 5.86 (Rha I H-1) and dC 81.5
(Gal C-3) and a NOESY cross-peak between a signal at dH 5.86
(Rha I H-1) and dH 4.06 (Gal H-3), revealed a (1 ? 3) glycosidic
linkage between Rha I and Gal. Therefore, the structure of 3
was determined as (22S)-cholest-5-ene-1b, 3b, 16b, 22-tetraol
1-O-[
b- -galactopyranoside, named nigroside C.
Compound 4, was obtained as an amorphous yellow powder; in
a-L-rhamnopyranosyl] 16-O-a-L-rhamnopyranosyl-(1 ? 3)-
D
high-resolution electrospray ionisation mass spectroscopy (HR-
ESIMS) (positive-ion mode), it showed a pseudo-molecular ion
peak at m/z = 765.4409 [M + Na]+, consistent with the molecular
formula
C39H66O13. The negative-ion mode FABMS spectrum
showed an [M–H]ꢂ ion at m/z 741, corresponding to a molecular
weight of 742. Another fragment-ion peak was observed at m/z
595 ([M–H–146]ꢂ), suggesting the elimination of a terminal des-
oxyhexosyl unit. The NMR data of this compound revealed that
its aglycone differs from that of compound 3 in the NMR signals
of ring A (Table 3). The main difference was the observation of
an upfield 13C NMR chemical shift at dC 77.7 (C-1), showing a sec-
ondary alcoholic function at this position and the downfield 13C
NMR chemical shift of C-2 at dC 42.1, instead of 35.2 as in com-
pound 3. The molecular weight of 4 was 146 mass units smaller
than that of 3, indicating that 4 had two sugar units. This was
ascertained by observation of two anomeric proton signals at dH
4.62 (1H, d, J = 6.7) and 5.86 (1H, br s), giving correlation in the
HSQC spectrum with two anomeric carbons at dC 106.7 and
1.13 (Agly H-1
firmed Glc and Gal to be located at C-2 and C-3, respectively. Thus,
compound 2a/2b was determined to be 25(R,S)-5 -spirostan-
,3b,6b-trio1-2-O-[b- -glucopyranosyl]-3-O-b- -galactopyrano-
side, called nigrosides B1/B2.
a) and dH 4.97 (Gal H-1)/dH 2.22 (Agly H-4b) con-
a
2
a
D
D
Compound 3, was obtained as an amorphous yellow powder,
and showed, in high-resolution electrospray ionisation mass spec-
troscopy (HR-ESIMS) (positive-ion mode), a pseudo-molecular ion
peak at m/z = 911.4986 [M + Na]+, consistent with the molecular
formula C45H76O17. The fast-atom bombardment mass spectrum
(FABMS) (negative-ion mode) exhibited a quasi-molecular ion peak
at m/z 887 [M–H]ꢂ indicating a molecular weight of 888. A frag-
ment-ion peak was observed at m/z 741 ([M–H–146]ꢂ), corre-
sponding to the elimination of one desoxyhexosyl moiety. The 1H
NMR spectrum of 3 showed signals of the aglycone part and an oli-
gosaccharidic part. The 1H and 13C NMR spectral data for the agly-
cone moiety of 3 (Table 3) showed five methyl groups at dH 0.91
(3H, s), dH 1.07 (3H, s), dH 1.08 (3H, d, J = 6.9), dH 0.79 (3H, d,
J = 6.2 Hz), dH 0.81 (3H, d, J = 6.4 Hz) and an olefinic proton at dH
5.42 (1H, br d, J = 5.0), which gave correlations, in the HSQC spec-
trum, with five methyl carbons at dC 13.4, 14.1, 12.1, 22.6, 22.7 and
one olefinic carbon signal at dC 125.1, respectively. Four secondary
alcoholic carbons at dC 82.0 (C-16), 81.4 (C-1), 73.0 (C-22), 67.6 (C-
3), and three quaternary carbon signals at dC 43.0 (C-10), 41.7 (C-
13), 139.6 (C-5), were also observed. These signals suggested that
103.5, respectively. The identification of one
a-rhamnopyranosyl
(Rha) and one b-galactopyranosyl (Gal) unit was achieved as de-
scribed above (Table 4).
In the HMBC spectrum, the cross-peaks between dH 4.62 (Gal H-
1) and dC 82.0 (Agly C-16) and between dH 5.86 (Rha H-1) and dC
81.5 (Gal C-3) provided the sequence of the oligosaccharide chain
at C-16 to be Rha (1 ? 3) Gal. This sequence was confirmed by
cross-peaks in the NOESY spectrum between dH 4.62 (Gal H-1)
and dH 4.51 (Agly H-16), and between dH 5.86 (Rha H-1) and
dH 4.06 (Gal H-3). Accordingly, the structure of 4 was determined
as (22S)-cholest-5-ene-1b,3b,16b,22-tetraol 16-O-
a-L-rhamnopyr-
anosyl-(1 ? 3)-b- -galactopyranoside, called nigroside D.
D
3.2. Cytotoxic activities
the aglycone of 3 was of a
D
5,6-cholestane type (Inoue et al.,
The cytotoxicity of the isolated compounds was evaluated
against the HT-29 and HCT 116 human colon cancer cell lines in
comparison with Paclitaxel, using the MTT assay (Carmichael
et al., 1987). According to the results (Table 6), the most active
compounds were the spirostanol glycosides 5a/5b (aginoside/turo-
side A) and 6a/6b having four sugar units with IC50 values of 1.59
1995; Mimaki, Kawashima, Kanmoto, & Sashida, 1993;). The agly-
cone stereochemistry of 3 was proposed by analysis of the NOESY
data. Comparison of NMR data of 3 (Table 3) with literature values
allowed the identification of the aglycone as the previously re-
ported (22S)-cholest-5-ene-1b, 3b, 16b, 22-tetrol, the aglycone of
schubertoside
D
, isolated from some Allium sp (Mimaki et al., 1993).
and 3.45 lM, respectively against HCT 116 and 1.09 and 2.82 lM,

454
A. Jabrane et al. / Food Chemistry 125 (2011) 447–455
Agrawal, P. K., Jain, D. C., & Pathak, A. K. (1995). NMR spectral investigation. Part 37.
Table 6
NMR spectroscopy of steroidal sapogenins and steroidal saponins: an update.
Magnetic Resonance in Chemistry, 33(12), 923–953.
Avunduk, S., Mitaine-Offer, A. C., Alankus-Caliskan, O., Miyamoto, T., Senol, S. G., &
Lacaille-Dubois, M. A. (2008). Triterpene glycosides from the roots of Astragalus
flavescens. Journal of Natural Products, 71(1), 141–145.
IC50 (lM) values of isolated saponins towards HCT-116 and HT-29 cell lines.
Compounds
Cell lines
HCT-116
HT-29
Barile, E., Bonanomi, G., Antignani, V., Zolfaghari, B., Sajjadi, S. E., Scala, F., et al. (2007).
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Block, E. (1985). The chemistry of garlic and onions. Science American, 252,
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Burnie, D. (1995). Wild flowers of the Mediterranean. London: Dorling Kindersley
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Carmichael, J., Degraff, W. G., Gazdar, A. F., Minna, J. D., & Mitchell, J. B. (1987).
1a/1b
2a/2b
3
47.8
70.8
>100
>100
>100
1.59
3.45
0.00321
>100
>100
>100
1.09
2.82
0.00140
4
5a/5b
6a/6b
Paclitaxel
Evaluation of
a
tetrazolium-based semiautomated colorimetric assay:
assessment of radiosensitivity. Cancer research, 47(4), 943–946.
Carotenuto, A., Fattorusso, E., Lanzotti, V., Magno, S., De Feo, V., Carnuccio, R.,
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sapogenins isolated from Allium porrum. Journal of Natural Products, 60(10),
1003–1007.
A and B, novel cytotoxic and antiproliferative
respectively, against HT 29. Compounds 2a/2b, 3 and 4 were
considered inactive on both cell lines with IC50 > 100 M and
compound 1a/1b showed moderate cytotoxicity with IC50 47.8–
70.5 M on both cell lines. However, the above mentioned activity
is three times less active than Paclitaxel (IC50 0.00321 and
0.00140 M against HCT 116 and HT-29, respectively). These re-
sults are in good agreement with reported literature data. Namely,
the cytotoxicity of aginoside (5a) was previously reported in the
literature against P388 (murine leukaemia), A549 (lung cancer),
DLD-1(colon cancer) and WS1 (normal skin fibroblast) human cell
l
Fattorusso, E., Lanzotti, V., Taglialatela-Scafati, O., Di Rosa, M., & Ianaro, A. (2000).
Cytotoxic saponins from bulbs of Allium porrum L. Journal of Agricultural and
Food Chemistry, 48(8), 3455–3462.
l
Haddad, M., Miyamoto, T., Laurens, V.,
& Lacaille-Dubois, M. A. (2003). New
biologically active triterpenoidal saponins acylated with salicylic acid from
Albizia adianthifolia. Journal of Natural Products, 66(3), 372–377.
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Steroidal glycosides from allium macleanii and a steroidal glycosides from
Allium macleanii and A. senescens, and their inhibitory activity on tumour
promoter-induced phospholipid metabolism of hela cells. Phytochemistry, 40(2),
521–525.
Kaskiw, M. J., Tassotto, M. L., Moka, M., Tokar, S. L., Pycko, R., Th’ng, J., et al. (2009).
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l
lines with IC50 values of 1.9, 5.8, 7.9, and 3.6
(Mskhiladze et al., 2008; Sata et al., 1998). The same compound
exhibited considerable activity at a concentration of 50
gmlꢂ1
lM, respectively
l
,
against a Hela cell line (Inoue et al., 1995). The remaining com-
pounds (1–4 and 6a/6b) were tested for the first time against
HCT 116 and HT–29 cell lines and their cytotoxic activity against
other cells has not been previously mentioned. Since few com-
pounds have been isolated and tested in this study, it is difficult
to derive structure–activity relationships. However, these results
suggest that special consideration should be given to the steroid
saponins 5a/5b and 6a/6b in biological and pharmacological stud-
ies of garlic and its preparations.
Lanzotti, V. (2005). Bioactive saponins from Allium and Aster plants. Phytochemistry
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4. Conclusions
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(2008). Cytotoxic steroidal saponins from the flowers of Allium leucanthum.
Molecules, 13, 2925–2934.
The phytochemical study of A. nigrum L. has resulted in the iso-
lation and characterisation of new saponins 1a/1b, 2a/2b, 3 and 4,
together with the two known ones 5a/5b and 6a/6b. Their struc-
tures were elucidated by chemical and spectroscopic means,
including NMR and HRESIMS. To the best of our knowledge, this
is the first phytochemical analysis of this plant. The cytotoxicities
of all isolated compounds were evaluated with Paclitaxel as posi-
tive control against HT-29 and HCT 116 human colon cancer cells,
for the first time, by using the MTT assay. Among them, com-
pounds 5a/5b and 6a/6b were found to be most active on both cell
lines. The information presented here could be used as preliminary
data. Moreover, further biological experiments are needed in order
to examine the therapeutic potential of A. nigrum.
Sata, N., Matsunaga, S., Fusetani, N., Nishikawa, H., Takamura, S.,
& Saito, T.
(1998). New antifungal and cytotoxic steroidal saponins from the bulbs of an
elephant garlic mutant. Bioscience, Biotechnology and Biochemistry, 62(10),
1904–1911.
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Asparagus acutifolius. Phytochemistry, 68(20), 2554–2562.
Shao, B., Guo, H., Cui, Y., Ye, M., Han, J., & Guo, D. (2007). Steroidal saponins from
Smilax china and their anti-inflammatory activities. Phytochemistry, 68(5),
623–630.
Acknowledgement
Wang, S. L., Bing, C., Cui, C. B., Liu, H. W., Wu, C. F., & Yao, X. S. (2004). Diosgenin-3-
O-
a
-L-rhamnopyranosyl-(1 ? 4)-b-
D
-glucopyranoside obtained as
a
new
The authors are thankful to the IFC-Tunisie (Institut Français de
Coopération en Tunisie) for a scholarship to A.J.
anticancer agent from Dioscorea futschauensis induces apoptosis on human
colon carcinoma HCT-15 cells via mitochondria-controlled apoptotic pathway.
Journal of Asian Natural Products Research, 6(2), 115–125.
Wang, Y., Zhang, Y., Zhu, Z., Zhu, S., Li, Y., Li, M., et al. (2007). Exploration of
the correlation between the structure, hemolytic activity, and cyto-
toxicity of steroid saponins. Bioorganic and Medicinal Chemistry, 15(7),
2528–2532.
Yokosuka, A., Jitsuno, M., Yui, S., Yamazaki, M., & Mimaki, Y. (2009). Steroidal
glycosides from Agave utahensis and their cytotoxic activity. Journal of Natural
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