Cytotoxic and antioxidant triterpene saponins from Butyrospermum parkii (Sapotaceae)

Article abstract of DOI:10.1016/j.carres.2011.09.014

Three new triterpenoid saponins, elucidated as 3-O-β-d- glucopyranosyloleanolic acid 28-O-β-d-xylopyranosyl-(1→4)-α-l- rhamnopyranosyl-(1→2)-β-d-xylopyranoside (parkioside A, 1), 3-O-[β-d-apifuranosyl-(1→3)-β-d-glucopyranosyl]oleanolic acid 28-O-[β-d-apifuranosyl-(1→3)-β-d-xylopyranosyl-(1→4) -[α-l-rhamnopyranosyl-(1→3)]-α-l-rhamnopyranosyl-(1→2) β-d-xylopyranoside (parkioside B, 2) and 3-O-β-d-glucuronopyranosyl- 16α-hydroxyprotobassic acid 28-O-α-l-rhamnopyranosyl-(1→3)- β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2) -β-d-xylopyranoside (parkioside C, 3), were isolated from the n-BuOH extract of the root bark of Butyrospermum parkii, along with the known 3-O-β-d-glucopyranosyloleanolic acid (androseptoside A). The structures of the isolated compounds were established on the basis of chemical and spectroscopic methods, mainly 1D and 2D NMR data and mass spectrometry. The new compounds were tested for both radical scavenging and cytotoxic activities. Compound 2 showed cytotoxic activity against A375 and T98G cell lines, with IC₅₀ values of 2.74 and 2.93 μM, respectively. Furthermore, it showed an antioxidant activity comparable to that of Trolox or butylated hydroxytoluene (BHT), used as controls, against 2,2-diphenyl-1-picryl hydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), oxygen and nitric oxide radicals.

Full text of DOI:10.1016/j.carres.2011.09.014

Carbohydrate Research 346 (2011) 2699–2704
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Cytotoxic and antioxidant triterpene saponins from Butyrospermum
parkii (Sapotaceae)
a
a
a
b
c
Léon A. Tapondjou , Laurentine B.T. Nyaa , Pierre Tane , Massimo Ricciutelli , Luana Quassinti ,
c
c
d
d,
⇑
Massimo Bramucci , Giulio Lupidi , Beaudelaire K. Ponou , Luciano Barboni
a
Department of Chemistry, Faculty of Science, University of Dschang, Box 183, Dschang, Cameroon
Laboratory of HPLC–MS, University of Camerino, Via S. Agostino 1, I-62032 Camerino, Italy
School of Pharmacy, Drugs and Health Products, University of Camerino, Via Gentile III da Varano, I-62032 Camerino, Italy
School of Science and Technology, Chemistry Division, University of Camerino, Via S. Agostino 1, I-62032 Camerino, Italy
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2011
Received in revised form 14 September
Three new triterpenoid saponins, elucidated as 3-O-b-
D
-glucopyranosyloleanolic acid 28-O-b-
-apifuranosyl-
-xylopyranosyl-(1?4)-
-xylopyranoside (parkioside B, 2) and
-hydroxyprotobassic acid 28-O- -rhamnopyranosyl-(1?3)-b- -xylo-
-rhamnopyranosyl-(1?2)-b- -xylopyranoside (parkioside C, 3), were isolated from
the n-BuOH extract of the root bark of Butyrospermum parkii, along with the known 3-O-b- -glucopyrano-
D-xylopyr-
anosyl-(1?4)-
a
-
L
-rhamnopyranosyl-(1?2)-b-
-glucopyranosyl]oleanolic acid
-rhamnopyranosyl-(1?3)]- -rhamnopyranosyl-(1?2)b-
-O-b- -glucuronopyranosyl-16
pyranosyl-(1?4)-
D
-xylopyranoside (parkioside A, 1), 3-O-[b-
D
(
[
1?3)-b-
D
28-O-[b- -apifuranosyl-(1?3)-b-
D
D
2
011
a
-L
a
a
-
L
D
Accepted 17 September 2011
Available online 24 September 2011
3
D
a
-
L
D
a
-
L
D
D
Keywords:
syloleanolic acid (androseptoside A). The structures of the isolated compounds were established on the
basis of chemical and spectroscopic methods, mainly 1D and 2D NMR data and mass spectrometry.
The new compounds were tested for both radical scavenging and cytotoxic activities. Compound 2
Butyrospermun parkii
Butyrospermun paradoxum
Sapotaceae
Saponin
showed cytotoxic activity against A375 and T98G cell lines, with IC50 values of 2.74 and 2.93
lM,
Cytotoxic activity
Antioxidant activity
respectively. Furthermore, it showed an antioxidant activity comparable to that of Trolox or butylated
0
hydroxytoluene (BHT), used as controls, against 2,2-diphenyl-1-picryl hydrazyl (DPPH), 2,2
bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), oxygen and nitric oxide radicals.
-azino-
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
C (1–3, Fig. 1), from the root bark of this plant. Together with the
new compounds, the known 3-O-b-D-glucopyranosyloleanolic acid
Butyrospermun parkii Kotschy (Sapotaceae), also known as
(androseptoside A) was also isolated.
Butyrospermun paradoxum (Don) Hepper, is a small tree that grows
up to 14 m high. The seeds of this plant contain an edible fat that is
used in traditional medicine to treat scabies, ulcers, wounds and
2
. Results and discussion
The dried and pulverized root barks of B. parkii (3 kg) were ex-
1
,2
nasal stiffness. Previous work on the seeds of this plant collected
in the northern part of Cameroon revealed the presence of fatty
tracted three times (each for 24 h) with MeOH (95%) at room tem-
perature. The filtrate obtained was concentrated under reduced
pressure to yield a dark residue (276 g). This extract was sus-
pended in water and successively partitioned against EtOAc and
n-BuOH, yielding, after evaporation to dryness, 56 and 110 g of
EtOAc and n-BuOH extracts, respectively. One part of the n-BuOH
extract was subjected to Sephadex LH-20 column chromatography
using MeOH as the eluant to separate saponins from polyphenolic
compounds and sugars. The saponin part consisted by two main
fractions (fraction A and B). Fraction A was repeatedly purified
by column chromatography on silica gel to afford the known
1
acids and triglycerides. The antibacterial and antifungal properties
of extracts from leaves, stem bark, root bark, fruit and seed kernels
2
of B. parkii have also been evaluated, and the characterization of
some bioactive metabolites from the seeds of this plant were
3
reported in 2009 by some of us. Because plants belonging to the
Sapotaceae family have been known as rich source of saponins,
4
–7
we examined the n-BuOH extract from the root bark of B. parkii
as part of our research work on bioactive saponins from
Cameroonian medicinal plants.8
–13
In this paper we report the
isolation, structure elucidation, antioxidant and cytotoxic activities
of three new triterpene saponins, designated as parkioside A, B and
3
-O-b-D-glucopyranosyloleanolic acid (androseptoside A) by
1
4
comparison with data reported in the literature. Fraction B affor-
ded three new saponins, parkioside A (1), B (2) and C (3) (Fig. 1),
the structures of which were elucidated through the analysis of
⇑
Corresponding author. Tel: +39 0737402240; fax: +39 0737637345.
E-mail address: luciano.barboni@unicam.it (L. Barboni).
0
008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.09.014

2
700
L. A. Tapondjou et al. / Carbohydrate Research 346 (2011) 2699–2704
2
9
30
1D-TOCSY and HSQC data allowed the complete assignment of the
glycosidic protons and carbons (Table 2), and the identification of
one b-glucopyranosyl (Glc), one a-rhamnopyranosyl (Rha) and
two b-xylopyranosyl (Xyl I and Xyl II) units. Sequencing and points
of attachment in the glycosidic chains were established by analysis
of HMBC and ROESY experiments. The glycosidic side chain at
1
9
2
0
H
18
1
2
2
2
2
5
9
2
6
13
O
OH
2
16 28
1
0
H
6
4
27
HO
RO
O
C-3 of the aglycone was identified as b-D-glucose, with its anomeric
O
3
H
proton at d 4.31 (d, J = 8.2 Hz) and its anomeric carbon at d 105.3
Xyl I
2
4
23
O
OH
O
O
(Table 2). The b-anomeric configuration of this sugar moiety was
Glc
HO
HO
3
based on the observation of the large
JH-1,H-2 coupling constant.
The location of this glucose unit at C-3 was confirmed by the HMBC
correlation between the anomeric proton (d 4.31) and the C-3 of
the aglycone (d 89.4). For the trisaccharide side chain at C-28 posi-
tion of the aglycone, the HMBC spectrum showed correlation be-
tween H-1 of Xyl II (d 4.44) and C-4 of Rha (d 83.1), between H-1
of Rha (d 5.31) and C-2 of Xyl I (d 74.8), between H-1 of Xyl I (d
O
HO
O
H C
3
2
O
R O
O
OH
Rha I
OH
OH
1
R O
Xyl II
HO
OH
5
.46) and C-28 of the aglycone (d 176.4). The tandem mass spec-
β-apiose
n
trometry (MS ), which utilizes the collision-induced dissociation
(CID) of target ions, has been used to confirm the sequences of
: R = H, R1 = H, R2 = H
1
2
2
ꢀ
: R = β-api (Api I), R = α−L-rha (Rha II), R2 = β-api (Api II)
1
the sugar chains. The MS of m/z = 1027.4 [MꢀH] gave fragments
ꢀ
ꢀ
at m/z = 937.3 [MꢀHꢀ90] , m/z = 895.4 [MꢀHꢀ132] correspond-
ing to the cross-link cleavage of the terminal xylose unit1
8,19
and
the loss of the terminal xylose, respectively. Another important
ꢀ
fragment ion was found at m/z = 617.2 [MꢀHꢀ2 ꢁ 132ꢀ146] ,
2
0
attributed to the loss of a triglycosidid chain made of two pentoses
1
2 H
3
(
xyloses) and a deoxyhexose, (rhamnose). The MS of m/z = 617.4
1
3
O
2
ꢀ
gave a fragment at m/z = 411.0 [617ꢀ162ꢀ44] , corresponding to
HO
O
2
8
the loss of the CO
of the above analysis, the structure of 1 was thus elucidated as 3-O-
b- -glucopyranosyloleanolic acid 28-O-b- -xylopyranosyl-(1?4)-
-rhamnopyranosyl-(1?2)-b- -xylopyranoside (parkioside A).
Compound 2 was obtained as a white amorphous powder. Its
ESIMS (negative-ion mode) showed quasi-molecular ion peaks at
2
moiety at C-28 and a glucose unit. On the basis
1
6
HOOC
HO
HO
OH
3
H
4
O
6
D
D
H
OH
a
-L
D
Xyl I
OH
O
HO
GlcA
HO
HO
O
O
ꢀ
ꢀ
m/z = 1473.6 [M+Cl] and m/z = 1437.7 [MꢀH] , indicating a
O
H C
HO
3
O
molecular weight of 1438 amu consistent with a molecular for-
O
O
Rha I
OH
110
mula of C68H O32. Comparison of the NMR data of 2 and 1 (Tables
OH
HO
H C
Xyl II
1 and 2) revealed that the two compounds have the same aglycone
(oleanolic acid) and different side chains at C-3 and C-28, com-
pound 2 containing three more sugar units (two pentosyls and
3
O
HO
HO
Rha II
OH
3
one deoxyhexosyl moieties). The supplementary signals were as-
-apiofuranosyl moieties (Api I and Api II)2
0,21
with
signed to two b-
D
Figure 1. Structures of compounds 1–3.
their characteristic anomeric carbon atoms at d 109.9 and 108.8
and anomeric protons at d 5.21 (d, J = 2.8 Hz) and 5.16 (d,
J = 3.6 Hz), respectively, and one terminal rhamnose moiety (Rha
II) [anomeric carbon at d 100.1, anomeric proton at d 5.44 (br s),
1
ESI-MS, tandem MS–MS and 1D and 2D NMR data, including H,
C, COSY, TOCSY, ROESY, HMBC and HSQC.
1
3
3
CH -6 at d 1.22 (d, J = 6.6 Hz)]. The positions of these three supple-
Compound 1 was obtained as a white amorphous powder. Its
mentary sugar units were evidenced by the HMBC correlations and
C chemical shifts. The anomeric proton of Api I (d 5.21) was found
1
3
ESI-MS (negative-ion mode) spectrum showed quasi-molecular
ꢀ
ꢀ
ion-peaks at m/z = 1063.4 [M+Cl] and m/z = 1027.4 [MꢀH] , indi-
to be connected to C-3 (d 85.0) of the glucopyranosyl moiety in the
HMBC spectrum; this connection is confirmed by the chemical
shift of the C-3 of the glucopyranosyl moiety. The second apifur-
anosyl unit was located at the C-3 of Xyl II due to the deshielded
value observed for the signal of that carbon (d 84.4) compared to
the value observed for compound 1 (d 76.8) (Table 2) and to the
HMBC cross peak correlations between the anomeric proton at d
5.16 (H-1 of Api II) and the carbon at d 84.4, and between the pro-
ton at d 3.39 (H-3 of Xyl II) and the anomeric carbon of Api II at d
108.8. Finally, the terminal rhamnopyranosyl moiety was deduced
to be located at the C-3 position of the inner rhamnosyl moiety
(Rha I) because of its deshielded value observed (d 83.7) compared
to that observed in compound 1 (d 70.9) and its HMBC correlation
cating the molecular weight of 1028 amu, consistent with the
84
molecular formula of C52H O20. Upon acid hydrolysis with 2 N
TFA at 120 °C, compound 1 afforded an aglycone that was identi-
fied as oleanolic acid by co-TLC comparison with an authentic sam-
ple, and confirmed by the 2D NMR spectra of 1 (Table 1) compared
1
5–17
with literature values.
The sugars obtained from the saponin
hydrolysate were identified as glucose, xylose and rhamnose by
TLC comparison with authentic samples, and confirmed by 1D-
1
TOCSY, HSQC and HMBC data of 1, as detailed below. The
H
NMR spectrum of 1 displayed four sugar anomeric protons at d
.31 (d, J = 8.2 Hz), 4.44 (d, J = 7.8 Hz), 5.31 (br s) and 5.46 (d,
4
J = 6.6 Hz), giving HSQC correlations with four anomeric carbons
at d 105.3, 105.9, 99.8 and 94.0, respectively (Table 2), confirming
2
with the Rha II anomeric proton (d 5.44). The MS experiment of
1
ꢀ
ꢀ
that compound 1 contains four sugar units. The H NMR spectrum
1437.7 [MꢀH] gave a fragment at m/z = 1291 [MꢀHꢀ146] , cor-
3
also revealed the presence of one rhamnose moiety, identified by
responding to the loss of one terminal deoxyhexose. The MS
ꢀ
the observation of a methyl doublet at d 1.29 (J = 6.3 Hz) coupled
yielded fragments at m/z = 749 [MꢀHꢀ2ꢁ146ꢀ3ꢁ132] , assigned
1
1
with a glycosidic proton at 3.51 ppm. The analysis of H– H COSY,
to the loss of a pentaglycosidic ester-linked chain at C-28, and

L. A. Tapondjou et al. / Carbohydrate Research 346 (2011) 2699–2704
2701
Table 1
NMR spectroscopic data for the aglycone moieties of compounds 1–3 (400 MHz, CD
3
OD)
Position
Parkioside A (1)
(J in Hz)
1.00, m
.60, m
1.91, m
.66, m
Parkioside B (2)
(J in Hz)
Parkioside C (3)
(J in Hz)
1.18, br d (12.5)
d
C
d
H
d
C
d
H
d
C
d
H
1
2
38.4
25.6
38.5
23.2
0.98, m
1.57, m
1.68, m
1.91, m
45.5
69.7
1
2.04, dd (12.5, 4.5)
4.33, br d (4.5)
1
3
4
5
6
89.4
38.7
55.7
18.0
3.15, dd (11.2, 5.6)
89.6
38.8
55.7
18.0
3.15, dd (11.0, 5.2)
81.7
44.0
47.4
67.5
3.61, br d (3.8)
0.78, m
1.40, m
0.78, m
1.38, m
1.33, br s
4.48, br s
1
.54, m
1.40, m
.47, m
1.58, m
1.41, m 1.47, m
7
32.7
32.6
40.0
1.57, br d (13.2)
1.83, br d (13.2)
1
8
9
39.3
47.6
36.4
23.1
122.3
143.4
41.6
27.8
39.3
47.5
36.5
23.1,
122.3
143.4
41.7
38.5
47.3
35.8
23.2
122.6
142.6
42.0
35.1
1.56, dd (11.0, 4.6)
1.57, m
1.67, dd (11.8, 6.0)
1
1
1
1
1
1
0
1
2
3
4
5
1.88, m
5.26, br t (3.6)
1.89, m
5.26 br, t (3.4)
2.05, m
5.38 br, t (3.5)
1.19, m
27.9
1.19, m
1.56, m
1.61, m
2.05, m
1.46 br, d (14.7)
1.73 br, d (14.7)
4.48 br, s
1
.58, m
1.60, m
.04, m
1
6
22.4
22.3
73.1
2
1
1
1
7
8
9
46.8
41.4
45.9
46.9
41.3
45.9
48.8
40.9
46.4
2.83, dd (13.6, 4.2)
1.12, dd (13.8, 4.2)
2.83, dd (13.4, 4.3)
1.13, dd (13.5, 4.8)
1.71 br, t (13.5)
2.99, dd (13.6, 4.0)
1.07, dd (13.2, 4.0)
2.29 br, t (13.2)
1
.70 br t (13.8)
2
2
0
1
30.2
33.5
30.1
33.6
29.9
35.0
1.22, m
.39, m
1.56, m
.72, m
1.21, m
1.38, m
1.58, m
1.70, m
1.06 s
1.16, m
1.92, m
1.74, m
1.91, m
1
2
2
2
3
31.8
27.2
31.8
27.3
30.6
64.1
1
1.06, s
3.43, d (10.0)
3.75, d (10.0)
2
2
2
2
2
2
3
4
5
6
7
8
9
0
15.6
14.7
16.4
24.7
176.4
32.0
22.6
0.85, s
0.96, s
0.78, s
1.15, s
15.7
14.6
16.3
24.7
176.5
32.0
22.6
0.86 s
0.95 s
0.77 s
1.15 s
14.9
17.5
17.4
25.9
175.6
31.9
23.5
0.88, s
1.60, s
1.05, s
1.35, s
0.90, s
0.92, s
0.90 s
0.92 s
0.95, s
0.88, s
ꢀ
m/z = 455 [749ꢀ132ꢀ162] , suggesting the supplementary loss of
one diglycosidic ether-linked chain, composed by one pentose
and one hexose moieties. This confirms that compound 2 contains
seven sugar units: four pentosyls, two deoxyhexosyls and one
hexosyl moiety. Thus, the structure of 2 was established as 3-O-
between the protons at d 3.43, d 3.64 (H-4 and H-5 of GlcA) and
the carbonyl at d 175.4. The sugars composition was thus identified
as one b-glucuronopyranosyl (GlcA), two a-rhamnopyranosyl (Rha
I and Rha II) and two b-xylopyranosyl (Xyl I and Xyl II) units. The
complete assignments of the glycosidic protons and carbons (Table
1
1
[
2
b-
8-O-[b-
D
-apifuranosyl-(1?3)-b-
D
-glucopyranosyl]-oleanolic acid
-xylopyranosyl-(1?4)-[
-rhamnopyranosyl-(1?2)b- -xylopy-
2) were possible through the analysis of H- H COSY, TOCSY-1D
and HSQC data. The glycosidic side chain at C-3 of the aglycone
was identified as b-glucuronic acid, with its anomeric proton at d
4.49 (d, J = 7.3 Hz) and its anomeric carbon at d 103.1 (Table 2).
The location of glucuronic acid at C-3 was confirmed by the HMBC
correlation between the anomeric proton (d 4.49) and C-3 (d 81.7).
The NMR data of the C-28 tetraglycosidic chain evidenced a close
similarity with compound 1, with the exception of an extra rham-
nose unit (Rha II) present in 3 and the deshielded value observed
for the C-3 carbon atom of Xyl II (d 82.7) in 3 compared to the same
carbon value in compound 1 (d 76.8) (Table 2). Furthermore, the
HMBC correlation observed between the anomeric proton of Rha
II (d 5.15) and the carbon at d 82.7 (C-3 of Xyl II), indicated that
Rha II is connected to C-3 of Xyl II, as further confirmed by the
HMBC correlation between H-3 of Xyl II (d 3.47) and the anomeric
D
-apifuranosyl-(1?3)-b-
D
a-L-
rhamnopyranosyl-(1?3)]
a
-
L
D
ranoside (parkioside B).
Compound 3 was obtained as a white amorphous powder; its
ESI-MS (negative-ion mode) spectrum gave a quasi-molecular ion
ꢀ
peak at m/z = 1251.57 [MꢀH] , consistent with a molecular for-
1
mula of C58H
92
O
29. The H NMR spectrum (Tables 1 and 2) showed
the characteristic signals of a triterpenoidic saponin, the aglycone
of which was identified as 16 -hydroxyprotobassic acid
2b,3b,6b,16 ,23-pentahydroxyolean-12-en-28-oic acid) by NMR
data, which were in good agreement with those reported in the lit-
erature. With respect to compound 1, the H NMR spectra of 3
showed the presence of an extra anomeric proton at d 5.15 (br s),
giving an HSQC correlation with the anomeric carbon at d 101.3,
and an additional methyl doublet at d 1.25 (d, J = 6.2 Hz), indicating
the presence of a second rhamnopyranosyl unit in compound 3.
Moreover, the presence of an additional carbonyl signal (d 175.4)
a
(
a
5
,6
1
2
ꢀ
carbon of Rha II (d 101.3). The MS of m/z = 1251.4 [MꢀH] gave
ꢀ
fragments at m/z = 1105.3 [MꢀHꢀ146]
and m/z = 695.2
ꢀ
[MꢀHꢀ2ꢁ132–2ꢁ146] , attributed to the loss of a terminal deox-
1
3
in the C NMR spectrum of 3 and the loss of a hydroxymethylene
group (d 61.4 in compound 1) suggested the presence in compound
yhexose and a tetraglycosidic chain made of two pentoses and two
3
deoxyhexose, respectively. The MS of m/z = 695.2 gave an ion peak
3
of a glucuronic acid unit (GlcA) instead of a glucopyranosyl
at m/z = 519 [695–176], indicating the loss of one glucuronic acid
moiety. This was confirmed by the HMBC correlations observed
unit. Thus, compound 2 was elucidated as 3-O-b-D-glucuronopyr-

2
702
L. A. Tapondjou et al. / Carbohydrate Research 346 (2011) 2699–2704
Table 2
NMR spectroscopic data for the sugar moieties of compounds 1–3 (400 MHz, CD
3
OD)
Position
Parkioside A (1)
(J in Hz)
Parkioside B (2)
(J in Hz)
Parkioside C (3)
(J in Hz)
d
C
, type
d
H
d
C
, type
d
H
d
C
, type
d
H
C-3: 1
Glc: 105.3
74.3
76.2
70.2
76.8
4.31, d (8.2)
Glc: 105.3
74.1
85.0
69.6
76.1
4.32, d (7.9)
GlcA: 103.1
73.5
82.7
72.2
74.7
4.49, d (7.3)
2
3
4
5
6
3.17 br, t (8.2)
3.27 br, t (8.8)
3.32 br, t (8.3)
3.23, m
3.37 br, t (8.2)
3.40 br, t (8.6)
3.50 br, t (8.8)
3.30, m
3.52, dd (11.6, 5.1)
3.84 br, d (11.6)
5.21, d (2.8)
3.34 br, t (8.5)
3.48 br, t (8.8)
3.43 br, t (9.0)
3.64, d (10.0)
61.4
3.65, dd (11.8, 5.3)
60.1
175.4
3
.83 br, d (11.8)
Api I 1
109.9
77.1
79.2
73.4
2
3
4
3.98, d (2.8)
3.78, d (9.6)
4.29, d (9.6)
5
63.6
94.1
75.0
76.2
68.6
65.4
3.58 br, s
5.43 d (6.1)
C-28: Xyl I 1
94.0
74.8
75.4
69.4
65.2
5.46, d (6.6)
94.0
74.8
76.1
69.6
65.3
5.42, d (6.4)
3.61, dd (9.0, 6.4)
3.56 br, t (9.0)
3.53, m
3.28, dd (11.4, 7.6)
3.91 br, d (11.4)
5.36 br, s
2
3
4
5
3.57, dd (9.1, 6.6)
3.56 br, t (9.0)
3.51, m
3.59 dd (9.0, 6.1)
3.56 br t (9.0)
3.51 m
3.29, dd (11.2, 7.7)
3.89 br, d (11.2)
5.35 br, s
3.29 dd (11.6,7.8)
3
.90 br d (11.6)
Rha I 1
2
3
4
5
6
99.8
70.4
70.9
83.1
67.4
16.8
105.9
74.7
76.8
69.6
65.8
5.31 br, s
3.92 br, s
100.3
70.4
83.7
77.1
67.3
16.8
106.1
74.1
84.4
68.5
65.4
99.9
70.9
71.0
82.5
67.4
16.9
105.5
74.9
82.7
68.4
65.7
3.94 br, s
3.94 br, s
3.85, dd (9.1, 3.4)
3.51 br, t (9.4)
3.75, m
1.29, d (6.3)
4.44, d (7.8)
3.19 br, t (8.3)
3.30 br, t (8.9)
3.45, m
3.82, dd (9.1, 3.3)
3.51 br, t (9.2)
3.75, m
1.29, d (6.4)
4.47, d (7.6)
3.36 br, t (8.0)
3.39 br, t (8.5)
3.51, m
3.20 br, t (11.2)
3.87, dd (11.2, 6.0)
5.44 br, s
3.91, dd (3.4, 1.8)
3.71, (9.3, 3.4)
3.38 br, t (9.3)
4.21, m
3.86, dd (8.9, 3.1)
3.58 br, t (9.0)
3.79, m
1.32, d (6.2)
4.53, d (7.7)
3.40 br, t (8.2)
3.47, t (8.7)
Xyl II 1
2
3
4
5
3.56, m
3.17 br, t (11.1)
3.23 br, t (11.2)
3.90, dd (11.2, 6.1)
5.15 br, s
3.98, dd (3.2, 2.0)
3.72, dd (9.5, 3.2)
3.40 br, t (9.5)
4.01, m
3
.83, dd (11.1, 5.2)
Rha II 1
2
3
4
100.1
71.0
70.5
73.9
68.0
101.3
71.0
71.0
74.0
68.6
16.5
5
6
16.6
108.8
76.9
78.7, C
73.7
1.22, d (6.4)
5.16, d (3.6)
3.83, d (3.6)
1.25, d (6.2)
Api II 1
2
3
4
3.72, d (9.6)
4.13, d (9.6)
5
65.4
3.51 br s
anosyl-16
a
-hydroxyprotobassic acid 28-O-
-xylopyranosyl-(1?4)- -rhamnopyranosyl-(1?2)-b-
-xylopyranoside (parkioside C).
It is worth noting that saponins having oleanolic acid deriva-
tives, and more precisely 16 -hydroxyprotobassic acid, as agly-
a
-
L
-rhamnopyranosyl-
Compound 2 resulted to be the more active against superoxide rad-
ical, also with respect to Trolox and butylated hydroxytoluene
(BHT) used as control and tested at the same concentration.
Furthermore, it also showed lower activity with respect to com-
pounds 1 and 3 towards the hydroxyl radical (Fig. 2). Finally, the
(
1?3)-b-
D
a-L
D
a
cone, with similar sugar chains at C-3 and C-28 positions, as in
compounds 1, 2 and 3 have been frequently encountered in various
plants belonging to Sapotaceae family.5
–7,22
The radical scavenging activity of compounds 1–3 was screened
0
against 2,2-diphenyl-1-picryl hydrazyl (DPPH), 2,2 -azino-bis
(
3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), nitric oxide (NO)
and oxygen radicals. Compound 2 showed a dose dependent DPPH
and ABTS radial scavenging comparable with Trolox used as con-
trol (Table 3). The activity of the new isolated saponins was also
evaluated against oxygen radical scavengers as superoxide,
hydrogen peroxide, hydroxyl radicals and nitric oxide (Fig. 2).
Table 3
DPPH, ABTS radical scavenging activity of compounds 1–3
Compounds
DPPH IC50
lM
ABTS IC50 lM
Parkioside A (1)
Parkioside B (2)
Parkioside C (3)
Trolox
>>1000
16.2(±2)
>>1000
111.0(±4)
25(±2)
70(±2)
Figure 2. Radical scavenging activity of compounds 1–3 towards different radical
species. For all compounds the radical scavenging activity was evaluated with the
different assays as described in experimental and all the derivatives were used at a
18.8(±1.5)
12.5(±0.3)
concentration of 20 lM.

L. A. Tapondjou et al. / Carbohydrate Research 346 (2011) 2699–2704
2703
Table 4
collection were deposited at the Cameroon National Herbarium
in Yaoundé (Ref.: No. 58927/HNC).
In vitro cytotoxic activity of compounds 1–3
Compounds
Cell line
IC50
M)a T98G
MDA-MB 231 A375
HCT116
55.44
(
l
3.3. Extraction and isolation
Parkioside A (1) 23.68
5% CI 20.25–27.68
35.97
27.32
9
32.97–39.23 26.05–28.66 49.46–62.14
The dried and pulverized root barks of Butyrospermun parkii
3 kg) were extracted three times (each for 24 h) with 9 L MeOH
95%) at room temperature. The filtrate obtained was concentrated
under reduced pressure to yield a dark residue (276 g). This extract
was suspended in water and successively partitioned against
EtOAc and n-BuOH, yielding after evaporation to dryness 56 and
Parkioside B (2) 2.93
Parkioside C (3) >100
9.62
>100
7.65–12.10
6.89
5.65–7.39
2.74
>100
2.42–3.11
0.50
0.37–0.68
14.12
>100
12.36–16.13
8.75
8.03–9.54
(
(
9
5% CI
Cisplatin
5% CI
2.61–3.29
7.79
6.83–8.52
9
a
The IC50 value is the concentration of compound that affords a 50% reduction in
1
10 g of EtOAc and n-BuOH extracts, respectively. 100 g of the n-
cell growth (after 72 h of incubation). T98G = human glioblastoma multiforme cell
line. MDA-MB 231 = human breast adenocarcinoma cell line. A375 = human
malignant melanoma cell line. HCT116 = human colon carcinoma cell line.
CI = confidence interval.
BuOH extract was subjected to Sephadex LH-20 column chroma-
tography using MeOH as the eluent to separate the saponins from
polyphenolic compounds and sugars. The saponins part was con-
sisted by two main fractions: fraction A (16 g) and fraction B
(
17 g). Fraction A was repeatedly purified by column chromatogra-
phy over silica gel (32–63 m) with EtOAc–MeOH–H O (95:5:2) as
the eluent and mainly afforded 3-O-b- -glucopyranosyloleanolic
acid (72 mg). Purification of fraction B on silica gel (32–63 m)
with EtOAc–MeOH–H O (85:15:5) and (80:20:10) led to the isola-
tion of compounds 1 (85 mg), 2 (128 mg) and 3 (62 mg).
three new compounds did not show any scavenging activity to-
wards hydrogen peroxide and nitric oxide.
l
2
D
The antiproliferative activity of compounds 1–3 was also evalu-
ated against human breast adenocarcinoma (MDA-MB-231),
malignant melanoma (A375), colon carcinoma (HCT116) and glio-
blastoma multiforme (T98G) cell lines. Compound 3 was not active
in this assay. Compound 1 and 2 showed in vitro cytotoxic activity
against all the cell lines tested (Table 4). Compound 2 was the most
active, with IC50 values of 2.74 and 2.93 lM against A375 and T98G
cell lines, respectively. It also showed a moderate activity against
MDA-MB 231 and HCT116 cell lines (IC50 values of 9.62 and
l
2
3
3
.4. Identification
.4.1. Parkioside A (1)
White amorphous powder; ½
2
D
5
aꢂ
ꢀ7.6 (c 0.25, MeOH); IR (KBr)
ꢀ1 1 13
mmax 3404, 2938, 1735, 1655, 1382, 1047 cm ; H and C NMR
1
4.12
lM, respectively). With respect to compound 2, compound
data, see Tables 1 and 2; ESIMS (negative-ion mode), m/z = 1063.4
ꢀ
ꢀ
2
1
was less active by about nine-fold against T98G and A375 cell
[
M+Cl] and m/z = 1027.4 [MꢀH] ; ESIMS-MS MS (1027.4) m/
ꢀ
ꢀ
lines and about four-fold against MDA-MB 231 and HCT116 cell
lines. The higher cytotoxicity observed for compound 2 compared
to compound 1 could be correlated with the presence of the apiose
z = 937.3 [MꢀHꢀ90] , m/z = 895.4 [MꢀHꢀ132] and m/z = 617.2
ꢀ
3
[
MꢀHꢀ2ꢁ132–146] , MS (617.4) m/z 411.0 [617–162–44].
units, as already suggested for the triterpenoid saponins isolated
3
.4.2. Parkioside B (2)
White amorphous powder; ½
from the aerial parts of Conyza blinii23 and the pectic polysaccha-
25
D
aꢂ
ꢀ51.8 (c 0.13, MeOH); IR (KBr)
ꢀ1 1 13
ride obtained from the cell wall of the marine phanerogam Zostera
mmax 3425, 2933, 1728, 1657,1386,1045 cm ; H and C NMR
2
4
marina. The inactivity of compound 3 in this assay could be
attributed to its aglycone (16 -hydroxyprotobassic acid), which
data, see Tables 1 and 2; ESIMS (negative-ion mode) m/z = 1473.6
ꢀ
ꢀ
2
a
[
[
M+Cl] and 1437.7 [MꢀH] ; ESIMS MS (1437.7) m/z = 1291
ꢀ
3
ꢀ
has been established to be not a structural determinant of
MꢀHꢀ146] , MS (1291) m/z = 749 [MꢀHꢀ2ꢁ146–3ꢁ132] and
6
cytotoxicity.
m/z = 455 [749ꢀ132ꢀ162].
3
3
. Experimental
3.4.3. Parkioside C (3)
2
D
5
White amorphous powder; ½
aꢂ
ꢀ39.9 (c 0.7, MeOH); IR (KBr)
ꢀ1 1 13
.1. General methods
mmax 3417, 2930, 1740, 1637, 1383, 1047 cm ; H and C NMR data,
ꢀ
see Tables 1 and 2; ESIMS (negative-ion mode) m/z 1251.5 [MꢀH] ;
2
ꢀ
Optical rotations were measured on PerkinElmer 241 MC polar-
ESIMS MS (1251.5) m/z = 1105.3 [MꢀHꢀ146] and m/z = 695.2
ꢀ 3
imeter. IR spectra were measured as a film on a KBr pellet using a
FTIR-8400S Shimadzu spectrometer. H and C NMR, DEPT, COSY,
HSQC, HMBC and ROESY experiments were performed on a Varian
Mercury Plus Spectrometer (400 MHz for H and 100 MHz for C).
All chemical shifts (d) are given in ppm units with reference to the
residual solvent signals and the coupling constants (J) are in Hz. ESI
mass spectra were carried out on an Agilent Technologies LC/MSD
[MꢀHꢀ2ꢁ132–2ꢁ146] , MS (695.2) m/z = 519 [695ꢀ176].
3.5. Acid hydrolysis of compounds 1–3
1
13
1
13
A solution of each compound (3 mg) in water (1 mL) and 2 N
aqueous CF COOH (10 mL) was heated at reflux at 100 °C on water
3
bath for 2 h. The mixture was then diluted in water (10 mL) and ex-
n
Trap SL (G2445D SL). ESIMS was performed in FIA (Flow Injection
Analysis) using MeOH as solvent (5 mL/min, nebulizer pressure
tracted with EtOAc (3 ꢁ 3 mL). The combined EtOAc layers were
2
washed with H O and evaporated to dryness to afford the aglycons.
1
5 psi), drying gas flow 4 L/min, drying gas temperature 325 °C.
Column chromatography was performed using Silica Gel 60 Merck
63–200 m and 32–63 m). TLC was carried out on precoated Kie-
The aqueous residue was concentrated to dryness by adding
repeatedly MeOH to remove acid and analysed by TLC (silica gel)
in comparison with standard sugars by using a mixture of
(
l
l
selgel 60 F254 (Merck) plates visualised by spraying with 50% H
2
SO
4
3 2
CHCl –MeOH–AcOH–H O (60:32:12:8) as the eluant. The absolute
and heating for 10 min at 110 °C.
configuration of sugar residues was determined by GC analysis of
their chiral derivatives.
2
5
3
.2. Plant material
3
.6. Radical scavenging activity tests
The root barks of Butyrospermum parkii (Sapotaceae) were
collected in Bangwa village, near the city of Bangangte (West Re-
gion of Cameroon) in February 2009. Specimens documenting the
In order to measure antioxidant activity, the DPPH free radical
scavenging assays were carried out according to the previously de-

2
704
L. A. Tapondjou et al. / Carbohydrate Research 346 (2011) 2699–2704
scribed procedures.26 ABTS radical cation decolorization assay,
which relies on the decolorization of the blue-green ABTS cation
radical caused by the antioxidant, was performed with a micro-
plate assay, as previously described.27 Scavenging activities of
superoxide radicals and hydroxyl radicals were evaluated follow-
ing the methods described by Kanga et al.28 Scavenging of hydro-
gen peroxide was evaluated following the method described by
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The authors are grateful to the IFS (International Foundation for
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.09.014.
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