A triterpenoid saponin possessing antileishmanial activity from the leaves of Careya arborea

Article abstract of DOI:10.1016/j.phytochem.2005.10.028

Bioguided-fractionation of the methanol extract of the leaves of Careya arborea led to isolation of a triterpenoid saponin, designated arborenin, and characterized as 3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl- 2α,3β-dihydroxy-taraxast-20-en-28-oic acid (1), together with desacylescin III (2). The structures were determined on the basis of extensive 2D NMR spectroscopic analysis. The saponin showed in vitro antileishmanial activity against Leishmania donovani (strain AG 83).

Full text of DOI:10.1016/j.phytochem.2005.10.028

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 183–190
www.elsevier.com/locate/phytochem
A triterpenoid saponin possessing antileishmanial activity from
the leaves of Careya arborea
Debayan Mandal, Nilendu Panda, Shrabanti Kumar, Sukdeb Banerjee,
*
Nirup B. Mandal, Niranjan P. Sahu
Indian Institute of Chemical Biology, Department of Steroids and Terpenoids Chemistry, 4 Raja S C Mullick Road, Jadavpur, Kolkata 700 032, India
Received 22 December 2004; received in revised form 25 October 2005
Available online 15 December 2005
Abstract
Bioguided-fractionation of the methanol extract of the leaves of Careya arborea led to isolation of a triterpenoid saponin, designated
arborenin, and characterized as 3-O-b-D-glucopyranosyl(1!2)-b-D-glucopyranosyl-2a,3b-dihydroxy-taraxast-20-en-28-oic acid (1),
together with desacylescin III (2). The structures were determined on the basis of extensive 2D NMR spectroscopic analysis. The saponin
showed in vitro antileishmanial activity against Leishmania donovani (strain AG 83).
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Careya arborea; Triterpenoid saponin; Arborenin; Desacylescin III barringtogenol C; Barringtogenol D; 2D NMR
1. Introduction
2. Results and discussion
Careya arborea is a handsome deciduous tree, widely
distributed throughout the greater part of India. The dif-
ferent parts of the plant enjoy considerable reputation in
Indian medicine as astringent and tonic, and are used as
antipyretic and antipruritic in eruptive fevers (Phondke,
2000). Previous phytochemical investigations on the leaves
and seeds of the plant were limited to the isolation of few
sterols and rearranged triterpenes (Row and Sastry, 1964;
Das and Mahato, 1982). In continuation of our work on
chemical studies on naturally occurring bioactive saponins
(Sahu et al., 2002) we report herein the isolation and
characterization of a novel triterpenoid saponin, desig-
nated as arborenin, with antileishmanial activity, from
the methanolic extract of the leaves of this medicinal
plant, along with a known triterpenoid glycoside, desacy-
lescin III.
The n-BuOH soluble fraction obtained from defatted
MeOH extract of C. arborea leaves showed moderate
in vitro antileishmanial activity against Leishmania dono-
vani (strain AG 83). Bioguided fractionation of the active
fractions eluted with MeOH–H2O mixture from the Diaion
HP-20 chromatography led to isolation of two triterpenoid
saponins.
Arborenin (1) has the molecular formula C42H68O14 as
determined from its high resolution positive ion FABMS
(m/z 819.4457 [M + Na]+). Of the 42 carbon signals dis-
played in the 13C NMR spectrum, 12 could be assigned
to the carbohydrate moiety (10 methine and 2 methylene
signals), whereas the aglycone accounted for seven singlets,
1
eight doublets, eight triplets and seven quartets. The H
NMR spectrum displayed seven methyl signals, of which
six were singlets (d 0.87, 1.03, 1.04, 1.18, 1.31, 1.73) and
one doublet (d 1.11, J = 6.6 Hz). Additional signals
observed include those ascribed to an olefinic proton (d
5.48, t, J = 7.8 Hz), two oxy methine protons (d 3.24, d,
J = 9.4 Hz; 4.04, m) and two anomeric protons (d 4.93, d,
J = 7.6 Hz; 5.49, d, J = 7.6 Hz). These data suggested that
*
Corresponding author. Tel.: +91 33 24733491; fax: +91 33 2473 5197.
E-mail address: npsahu@iicb.res.in (N.P. Sahu).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.10.028

184
D. Mandal et al. / Phytochemistry 67 (2006) 183–190
the aglycone part of 1 was likely to be a pentacyclic triter-
pene with two hydroxyl groups and a trisubstituted double
bond. Comparison of 13C NMR data (Table 1) with those
of known triterpenes (Dai et al., 2001; Reynolds et al.,
1986) suggested that 1 belongs to the taraxastane series.
1
The H-1H COSY spectrum of 1 showed a coupling inter-
action between the signals of d 3.24 and d 4.04, which were
assigned to two hydroxyl groups at C-3 and C-2 positions.
The HETCOR spectrum showed that the corresponding
carbon signals were at d 95.8 and 66.9, respectively. The
assignment of hydroxyl groups to C-2 and C-3 was further
confirmed from the HMBC correlations (Fig. 1) between
the signal for C-2 and those of H-1, H-3 and H-23; simi-
larly, the signal for C-3 showed correlation with those for
H-1, H-2, H-23 and H-24. Both the hydroxyl groups at
C-2 and C-3 must be equatorial as evident from the large
coupling constant (J = 9.4 Hz) for H-3. Furthermore, the
H-2 signal showed cross peaks in the NOESY spectrum
with H-24 (d 1.18), H-25 (d 0.87), and H-1b (d 2.37) signals,
while that of H-3 was correlated with signals for H-1a (d
1.12) and H-5 (d 0.82). The 13C signal at d 178.2, assigned
to a carboxyl group, showed HMBC cross peaks with
H-18, H-16 and H-22 signals, justifying its assignment to
C-28. The HMBC correlations of the signals for C-20 with
H-19, H-22, H-29 and H-30 signals, and C-21 with H-19,
H-22 and H-30 signals suggested the position of the double
bond in the triterpene core (Fig. 1). The relative orienta-
tions of the remaining protons of 1 were established from
coupling patterns and cross peaks generated from the
phase sensitive NOESY spectrum. For example, the
NOESY connectivities for H-5a with H-9 and H-1a, and
for H-27 with H-18 and H-15a showed the spatial proxim-
ity between these protons. Using COSY, HOHAHA,
HMBC and NOESY information, assignments for a and
b protons of other carbons were also made. From the fore-
going evidences it was concluded that the triterpene core of
1 was 2a,3b-dihydroxy-taraxast-20-en-28-oic acid.
Table 1
13C NMR chemical shiftsa of 1, 3 and 5 in pyridine-d5
Carbon no.
1
3
5
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
a
47.8 t
66.9 d
95.8 d
41.2 s
55.8 d
18.5 t
34.6 t
41.0 s
51.0 d
38.0 s
22.1 t
29.6 t
39.4 d
42.3 s
27.9 t
33.7 t
49.1 s
49.4 d
37.9 d
48.2 t
68.9 d
83.8 d
39.9 s
56.1 d
18.7 t
34.3 t
40.9 s
50.9 d
38.7 s
22.3 t
25.4 t
43.2 d
41.4 s
27.7 t
28.1 t
42.2 s
48.4 d
42.4 d
83.9 s
27.3 t
32.3 t
29.2 q
17.5 q
17.8 q
15.9 q
14.4 q
176.7 s
18.6 q
24.1 q
39.1 t
28.1 t
78.1 d
39.4 s
55.7 d
18.8 t
32.5 t
40.3 s
47.3 d
37.3 s
23.9 t
122.6 d
144.4 s
41.1 s
30.8 t
76.6 d
49.7 s
41.1 d
41.0 t
38.1 s
90.7 d
76.4 d
28.8 q
16.6 q
15.5 q
17.2 q
29.0 q
61.0 t
29.8 q
28.8 q
Glucose-1
10
104.6 d
82.5 d
78.6 d
71.3 d
78.4 d
62.4 t
20
30
40
50
60
Glucose-2
100
200
300
400
500
600
105.7 d
76.7 d
78.2 d
72.0 d
78.3 d
63.0 t
143.1 s
117.9 d
38.5 t
28.3 q
17.7 q
17.6 q
16.4 q
15.0 q
178.2 s
23.7 q
22.2 q
Assignments based on 13C, DEPT, HSQC, HMBC experiments and
multiplicities as determined by DEPT.
H
H
COOH
HO
OH
H
H
O
HO
HO
O
H
OH
O
H
O
HO
HO
1
OH
H
Fig. 1. COSY ( ) HMBC (!) NOESY (-----) correlations of 1.

D. Mandal et al. / Phytochemistry 67 (2006) 183–190
185
Attempted enzymatic hydrolysis of arborenin (1) was
precluded due to solubility problem. When hydrolysis
was carried out with mineral acid it afforded an aglycone
(3) and only D-glucose as sugar component identified by
GLC analysis of the hydrolysate after derivatization.
Although most of the 13C NMR signals of 3 showed simi-
larity with that of 1, differences were noticed for signals
attributed to ring E. The presence of a d-lactone ring was
suggested by the appearance of an absorption peak at
1742 cmꢀ1 in its IR spectrum and a signal at d 176.7 in
the 13C NMR spectrum. The HMBC spectrum displayed
cross peaks between C-28 and H-18 (d 1.04), H-16 (d
1.22) and H-22 (d 1.50) signals, as also between C-20 and
H-29 (d 0.88), H-30 (d 1.27) and H-19 (d 1.57) signals, pro-
viding confirmation for a d-lactone ring between C-20 and
C-28. It is presumed that the aglycone was rearranged dur-
ing acid hydrolysis to a six membered lactone. Thus the
structure of 3 was deduced as 2a,3b-dihydroxy-taraxa-
stan-28,20b-olide. It is worthy of mention that Das and
Mahato (1982) isolated the compound (careyagenolide)
from the acid hydrolysis of the ethanolic extract of the
leaves of the plant. The NMR spectral data of 3 is pre-
sented in Tables 1 and 2.
HMBC connectivities of the signal for H-100 (d 5.49) of
the terminal glucose with that of C-20 (d 82.5) of the inner
glucose, and of the signal for H-10 (d 4.93) of the inner glu-
cose with that of C-3 (d 95.8) of the aglycone moiety estab-
lished the sugar–sugar and sugar–aglycone linkages in 1.
The results of NOESY experiment also supported the pro-
posed linkages. The absolute configurations of the mono-
saccharides were chosen in keeping with those mostly
encountered among the plant glycosides. From the forego-
ing evidences, the structure of arborenin was elucidated as
3-O-b-D-glucopyranosyl(1!2)-b-D-glucopyranosyl-2a,3b-
dihydroxy-taraxast-20-en-28-oic acid (1).
Compound 2 showed in its MALDI-TOF MS the
[M + Na]+ ion at m/z 1013 indicating the molecular weight
to be 990. Of the 48 carbon signals displayed in the 13C
NMR, 30 were assigned to the aglycone part and 18 to
the oligosaccharide moiety. The presence of two sp2 carbon
signals at d 122.4 (d) and 143.7 (s), coupled with the infor-
mation from 1H NMR (six methyl singlets and a vinyl pro-
ton triplet) indicated that the aglycone is likely to possess
an olean-12-ene skeleton. Acid hydrolysis of 2 furnished
two aglycones, one major and one minor, together with
glucuronic acid, glucose and galactose identified by GLC.
The major aglycone was identified as barringtogenol C
(4) by comparing its 13C NMR chemical shifts with those
reported in the literature (Pal et al., 1994). The minor prod-
uct (5) had a molecular formula C30H48O4 as determined
from 13C NMR data and the observation of a sodiated-
molecular ion peak [M + Na]+ at m/z 495.07 in the positive
The interglycosidic and sugar–aglycone linkages as well
as signal and structural assignments of the sugars in 1 were
established on the basis of the following arguments. The
anomeric proton signals (H-10 and H-100) appeared as dou-
blets (J = 7.6 Hz,) indicating b-configuration (4C1 confor-
mation) for both the glucopyranosyl units. The observed
Table 2
1H NMR spectral data of 1 (aglycone part), 3 and 5 (pyridine-d5)a, with J value in Hertz
Carbon no.
1
1
3
5
1.12 (+),
1.25 (+),
1.02 (m), 1.59 (+)
2.37 (dd, 4.6, 12.8)
4.04 (m)
3.24 (d, 9.4)
2.36 (dd, 4.4, 12.4)
4.13 (m)
3.41 (d, 9.4)
0.96 (m)
2
3
5
6
7
1.87 (+), 1.87 (+)
3.46 (dd, 5.5, 10.5)
0.88 (br d, 11.9)
1.41 (dq, 3.2, 12.4), 1.59 (+)
1.27 (m), 161 (+)
1.73 (+)
1.87 (+), 1.90 (m)
5.35 (t-like)
–
1.78 (m), 1.87 (+)
4.92 (br d, 3.2)
2.98 (dd, 6.4, 11.9)
1.73 (+), 1.82 (m)
3.92 (s)
4.70 (s)
1.24 (s)
1.06 (s)
0.94 (s)
0.90 (s)
1.71 (s)
3.88, 4.22 (both d, 10.5)
1.0 (s)
1.18 (s)
0.82 (d, 11.7)
1.45 (+), 1.29 (+)
1.33 (+), 1.33 (+)
1.45 (+)
1.29 (+), 1.62 (br d, 10.5)
1.18 (+), 1.79 (+)
2.83 (t, 12.5)
1.18 (+), 1.79 (+)
1.53 (dt, 2.8, 13.4), 2.32 (m)
1.29 (+)
2.44 (t, 6.6)
5.48 (d, 7.8)
2.0 (br d, 14.0), 2.64 (dd, 7.1, 15.4)
1.31 (s)
1.18 (s)
1.36 (+), 1.54 (+)
1.36(+), 1.36 (+)
1.44 (dd, 3.0, 12.8)
1.17 (m), 1.57 (+)
1.54 (+), 1.54 (+)
1.25 (+)
1.10 (m), 2.29 (dt, 4.3, 13.3)
1.22 (m), 2.02 (m)
1.04 (dd, 5.5, 11.7)
1.57 (+)
1.51 (+), 1.80 (m)
1.50 (+), 1.50 (+)
1.26 (s)
1.08 (s)
0.92 (s)
9
11
12
13
15
16
18
19
21
22
23
24
25
26
27
28
29
30
0.87 (s)
1.03 (s)
1.04 (s)
–
1.11 (d, 6.6)
1.73 (s)
0.91 (s)
0.89 (s)
–
0.88 (d, 6.9)
1.25 (s)
(+, overlapped with other signals).
a
Assignments based upon COSY, TOCSY, HETCOR, NOESY, DEPT and HMBC.

186
D. Mandal et al. / Phytochemistry 67 (2006) 183–190
ion MALDITOF mass spectrum. Comparison of the 1D
and 2D NMR spectra of 5 with those of 4 revealed that
5 possesses a similar structure except for the rings D and
cross peaks observed between the 21-H (d 4.70) and C-16
(76.6) signals. Dreiding model studies revealed that such
a ring formation is likely between C-16a and C-21a bonds.
The model studies also suggested H-22 to be b-oriented,
since the 21-H-22-H dihedral angle is then nearly 90ꢁ,
which explains the singlet nature of the two proton signals.
The a-orientation of 22-OH group is also supported by the
NOESY relationship observed between H-22 and H-30 sig-
nals. Thus the structure of 5 was established as 16a,21a-
epoxy-olean-12-en-3b,22a,28-triol (barringtogenol D). It
is noteworthy that barringtogenol D was obtained when
barringtogenol C was refluxed with hydrochloric acid
(Chakraborti and Barua, 1963; Nakano et al., 1969). It is
presumed that barringtogenol D was obtained during acid
hydrolysis of 2. These evidences as well as the 13C NMR
chemical shifts (Table 1) of compound 2 suggested its iden-
tity with desacylescin III (Yoshikawa et al., 1996).
1
E. The H NMR spectrum showed the signals for seven
methyls (d 0.90, 0.94, 1.00, 1.06, 1.18, 1.24 and 1.71, all
s), one olefinic proton (d 5.35, t-like), two hydroxy/oxy
methylene protons (d 3.88 and 4.22, 2 · d, J = 10.5 Hz),
four hydroxy/oxy methine protons (d 3.46, dd, J = 5.5,
10.5 Hz; 3.92, s; 4.70, s; 4.92, br d, J = 3.2 Hz). One of
the hydroxyl groups could be assigned to C-3 on biogenetic
grounds as well as from the HMBC correlation of the pro-
ton signal (d 3.46) with C-4, C-23 and C-24 signals. The
presence of another hydroxyl group at C-22 was estab-
lished from the HMBC correlations between the H-22 sig-
nal (d 4.70) and those of C-21, C-20, C-18, C-17 and C-16.
A hydroxy methyl nature of C-28 could be inferred from
the observed HMBC cross peaks between signals of 28-
O
H
H
COOH
O
C
H
H
HO
O
HO
HO
H
OH
O
H
HO
HO
H
H
O
OH
O
HO
HO
OH
3
1
OH
OH
CH2OH
OH
OH
CH2OH
OH
OH
H
OH
O
HOOC
HO
H
O
O
HO
O
O
HO
HO
H
H
OH
OH
OH
O
HO
4
2
OH
O
OH
CH2OH
HO
H
5
H2(d 3.88, 4.22) and those of C-18, C-16 and C-22. Thus
only one more oxygen atom remained to be assigned.
The downfield chemical shift attributed to H-16 (d 4.92)
and H-21 (d 3.92) as well as to the corresponding carbons
(Table 1) suggested that both these carbons are oxygen-
ated. Since the molecular formula allowed only one oxygen
atom, the presence of an ether linkage between C-16 and C-
21 appeared likely. This was confirmed from the HMBC
3. Antileishmanial activity
To determine whether any of the compounds (1–5) had
any effect on the growth of L. donovani AG 83, promastig-
otes (2.5 · 106 cells mlꢀ1) were exposed to various concen-
trations of the compounds for 7 days. The numbers of
live promastigotes were counted by trypan blue exclusion.
Compound 1 was found to inhibit the growth in a dose

D. Mandal et al. / Phytochemistry 67 (2006) 183–190
187
dependent manner. At a concentration of 15 lg mlꢀ1
1
4. Experimental
inhibited the growth by 55% on the 2nd day, 70% growth
was inhibited on day 4 and 75% on day 7. The other com-
pounds 2–5 virtually had no effect on the survival of L.
donovani AG 83 promastigotes up to a concentration of
30 lg mlꢀ1. The IC50 values for 1 and amphotericin B
(positive control) were calculated to be 15 and 0.22 lg mlꢀ1
which varied inversely with incubation time.
The protozoan parasite L. donovani survives and multi-
plies within mammalian macrophages. It is, therefore, of
interest to test the efficacy of the compounds on intracellu-
lar amastigotes. Peritoneal macrophages of BALB/c mice
were infected with L. donovani AG 83 promastigotes
in vitro. Infected macrophages after subsequent washings
were incubated with different concentrations of the com-
pounds (1–5). Similar to its effect on promastigotes, com-
pound 1, at a concentration of 12.5 lg mlꢀ1, reduced the
parasite burden by 57% on day 1 and 77% on day 2. The
compounds (2–5) reduced intracellular parasite burden
only marginally, compared with medium or DMSO (Table
3). The IC50 of inhibition was calculated to be 12.5 and
0.10 lg mlꢀ1 for arborenin (1) and amphotericin B,
respectively.
Cytotoxicity of the compounds was examined by 18 h
51Cr-release assays with radiolabelled promastigotes and
amastigotes of L. donovani in the presence of varying con-
centrations of these compounds. The antileishmanial drug
sodium antimony gluconate (SAG) was used for compari-
son and normal human peripheral blood mononuclear cells
(PBMC) were used as a control for nonspecific toxicity. Up
to a concentration of 0.1% (v/v), DMSO was found to be
nontoxic to human PBMC or L. donovani promastigotes
and amastigotes. None of the compounds (1–5) and SAG
was found to be toxic to human PBMC up to a concentra-
tion of 20 mg lꢀ1. However, compound 1 was found to
have significant toxicity on both promastigotes and intra-
cellular amastigotes.
4.1. General procedures
All melting points were measured on a Yanagimoto
micromelting point apparatus and are uncorrected. IR
spectra were determined using JASCO 7300FTIR spec-
trometer. Optical rotations were measured using
a
JASCO DIP-370 digital polarimeter. 1H and 13C NMR
spectra were recorded at 500 and 125 MHz, respectively
using a Jeol ECP-500 spectrometer in C5D5N with
TMS as internal standard. HRFABMS (positive) was
performed on
a JEOL MS-700 mass spectrometer.
MALDI-TOF MS (positive) was conducted using Persep-
tive Biosystems Voyager DESTR mass spectrometer.
TLC was carried out on silica gel 60 F254, and spots were
visualized by spraying with 10% H2SO4 solution followed
by heating. Diaion HP-20 (Mitsubishi Chemicals) and sil-
ica gel (silica gel 60, Merck) were used for column chro-
matography. GLC was performed on a Hewlett–Packard
model 5730A instrument using 2% SE-30 on Chromosorb
W (60–80 mesh), 3 mm i.d. · 1.5 m, 150 ꢁC column tem-
perature, nitrogen as carrier gas with a flow rate of
15 ml/min.
4.2. Plant material
The plant material was collected from Indian Botanic
Garden, Howrah, and identified by Dr. (Ms.) Debjani
Basu, Botanist, at Indian Botanic Garden, Howrah, West
Bengal, India. A voucher specimen was deposited at the
Steroids and Terpenoids Chemistry Department, Indian
Institute of Chemical Biology, Kolkata.
4.3. Extraction and isolation
The defatted, air dried powdered leaves of C. arborea
(1 kg) were extracted successively with MeOH (3x5lt) at
ambient temperature. The combined MeOH extract was
concentrated, macerated with water, and then partitioned
with n-BuOH (2 · 1 l). The n-BuOH soluble part (20 g)
was applied to a column of Diaion HP-20 (400 g) and
washed with water followed by 30%, 50%, 80% and
100% of MeOH. Fraction eluted with 80% methanol
(2.5 g) was chromatographed over silica gel (70 g).
Graded elution was carried out with chloroform followed
by various mixtures of CHCl3–MeOH (9:1, 4:1, 7:3, and
3:2). A total of 30 fractions (each 75 ml) were collected
and fractions giving similar spots on TLC were combined.
Fractions eluted with CHCl3–MeOH (7:3) were combined
together and subjected to rechromatography over silica
gel (15 g). Fractions (collected in 15 ml lots) eluted with
CHCl3–MeOH mixture (3:1) furnished arborenin
(65 mg). Further purification of the fraction eluted with
CHCl3–MeOH mixture (3:2) over silica gel (10 g) with
CHCl3–MeOH mixture (13:7) gave desacylescin III
(62 mg).
Table 3
In vitro antileishmanial activity of compounds (1–5) on intracellular
amastigotes of L. donovani at day 1 of drug treatmenta
Treatment
Dose
Amastigotes/100 nuclei (mean SD)
Medium
DMSO
Amphotericin B
1
2
3
4
5
–
527.33 31.34
520.33 10.50
256.04 14.50b
272.00 17.88b
493.52 10.20
501.32 14.60
488.35 19.60
498.78 25.30
0.20%
0.10 lg mlꢀ1
12.5 lg mlꢀ1
30.0 lg mlꢀ1
30.0 lg mlꢀ1
30.0 lg mlꢀ1
30.0 lg mlꢀ1
Statistical analyses were performed by one way ANOVA followed by
DunnettÕs post-hoc test.
a
Thioglycolate elicited peritoneal macrophages of BALB/c mice were
infected in vitro with L. donovani amastigotes and incubated with graded
concentrations 0.1–30 lg mlꢀ1 of 1–5 (dissolved in DMSO) for 1 day at
37 ꢁC for the determination of antileishmanial activity on intracellular
amastigotes.
b
P < 0.01 verses medium control; mean SD of triplicate
determination.

188
D. Mandal et al. / Phytochemistry 67 (2006) 183–190
4.4. Arborenin (1)
followed by chromatographic purification on a silica gel
column furnished two compounds (4, 16 mg) and (5,
9 mg). Compound 4 was identified as barringtogenol C
on the basis of spectral comparison (Pal et al., 1994). Com-
pound 5 was crystallized from MeOH as fine needles, m.p.
Colourless needles from MeOH, m.p. 310–312 ꢁC (dec);
21
½aꢁD ꢀ22.3ꢁ (c 0.583, MeOH); IR: mmax cmꢀ1 (KBr): 3414,
2370, 1628, 1515, 1455, 1377, 1161, 1064 and 660;
HRFAB MS: m/z observed 819.4457, calc. for
C42H68O14Na, 819.4507; MALDI-TOF-MS (positive): m/
z 819.17 [M + Na]+; 1H NMR (C5D5N): aglycone part
(Table 1); sugar moiety: inner glucose: 4.93 (d,
J = 7.6 Hz, H-10), 4.29+ (H-20), 4.26+ (H-30), 4.14 (t,
J = 7.8 Hz, H-40), 4.04 (m, H-50), 4.58 (dd, J = 2.0,
11.6 Hz, H-60a), 4.29+ ðH-60bÞ; Outer glucose: 5.49 (d,
J = 7.6 Hz, H-100), 4.14 (t, J = 7.8 Hz, H-200), 4.31+ (H-
300), 4.30+ (H-400), 3.95 (m, H-500), 4.50 (dd, J = 2.9,
11.5 Hz, H-60a0) and 4.44 (dd, J = 4.4, 11.4 Hz, H-60b0),
(+overlapped with other proton signals); 13C NMR
(C5D5N): (Table 2). (Found: C, 63.37; H, 8.54;
C42H68O14 requires: C, 63.30; H, 8.60%.)
21
299–302 ꢁC; ½aꢁD +10.93ꢁ (c 1.50, MeOH); IR: mmax cmꢀ1
(KBr): 3389, 2370, 2336, 1645, 1462, 1378, 1101, 1039,
784 and 647; MALDI-TOF-MS (positive) m/z 495.0778
[M + Na]+; 1H NMR (Table 1); 13C NMR (Table 2).
(Found: C, 76.32; H, 10.21; C30H48O4 requires: C, 76.23;
H, 10.24%.)
The acid hydrolyzate was worked up following the
same procedure as described for 1. The monosaccharides
were identified as D-glucuronic acid, D-glucose and D-
galactose by GLC analysis on comparison with authentic
samples.
4.8. Parasite culture and growth conditions
4.5. Acid hydrolysis of 1
L. donovani strain AG 83 was originally obtained from
an Indian kala-azar patient (Ghosh et al., 1983) and
maintained in golden hamsters. Amastigotes were isolated
from spleens of L. donovani infected golden hamsters as
described (Jaffe et al., 1984). The spleen was rinsed in
ice-cold phosphate buffered saline (PBS), glucose
(55 mM)/EDTA (2 mM), lightly homogenized, macro-
scopic particles were allowed to settle. The turbid suspen-
sion was decanted, centrifuged at 100g for 10 min at 4 ꢁC.
The amastigote enriched suspension was centrifuged at
800g for 10 min. The pellet was suspended in 45% Percoll
(8.0 ml), and finally 25% Percoll (4.0 ml) was layered over
the amastigote suspension and further centrifuged at
5000g for 1 h. The band containing amastigotes was taken
and washed with PBS (3·) and finally resuspended in
Medium-199 (Gibco Laboratories, New York, NY,
USA), supplemented with 20% FBS. Promastigotes were
obtained by transforming amastigotes and were main-
tained in vitro in Medium-199 supplemented with 8%
FBS.
Arborenin (1) (25 mg) was hydrolyzed with 2 M HCl in
aq. MeOH (10 ml) at 95 ꢁC for 2.5 h on a boiling water-
bath, cooled, and 5 ml water was added. Methanol was
removed, and the solution was extracted with EtOAc
(5 ml · 3). The solvent was washed with water and dried
to give a white powder. Purification of the product over sil-
ica gel column and subsequent crystallization from MeOH
21
afforded the aglycone (3, 11 mg), m.p. 304 ꢁC, ½aꢁD +4.76ꢁ
(c, 0.42, MeOH); IR: mmax cmꢀ1 (KBr): 3430, 2372, 2336,
1742, 1628, 1552, 1518, 1455, 1379, 1232, 1047, 959 and
614.
MALDI-TOF-MS
(positive)
m/z
495.1615
[M + Na]+; 1H NMR (Table 1); 13C NMR (Table 2).
(Found: C, 76.29; H, 10.19; C30H48O4 requires: C, 76.23;
H, 10.24%.)
The aqueous part of the acid hydrolyzate was neutralized
by passing through an anion exchange resin (Amberlite
MB-3) column, concentrated and then treated with 1-(trim-
ethylsilyl) imidazole at room temperature for 2.5 h. After
excess reagent was decomposed with water, the reaction
product was extracted with hexane (3 ml · 3). The TMSi
derivative of the monosaccharide obtained after removal
of the solvent was identified to be that of D-glucose from
GLC analysis.
4.9. Isolation of normal human peripheral blood mononuclear
cells
PBMC were separated from heparinized whole blood of
normal donors by Ficoll–Hypaque density gradient centri-
fugation as described (Boyum, 1968).
4.6. Desacylescin III (2)
Colourless crystals from CH3CN–MeOH–water, m.p.
21
228-230 ꢁC, ½aꢁD +14.8ꢁ (c 0.615, MeOH); MALDI-TOF-
4.10. In vitro growth of L. donovani promastigotes in the
presence of compounds (1–5)
MS (positive): m/z 1013.29 [M + Na]+. Identical with an
authentic sample on the basis of 1H and 13C NMR
(C5D5N) comparison (Yoshikawa et al., 1996).
Promastigotes (1 · 106) were incubated with or without
various concentrations of the compounds or standard
antileishmanial drugs in Medium-199 (1.0 ml) supple-
mented with 8% FBS at 22 ꢁC. Growth of promastigotes
was monitored by counting the number of motile prom-
astigotes microscopically.
4.7. Acid hydrolysis of 2
A solution of 2 (40 mg) was subjected to acid hydrolysis
by the same procedure as described for 1. Usual work up

D. Mandal et al. / Phytochemistry 67 (2006) 183–190
189
4.11. In vitro infection of BALB/c mice peritonial
macrophages
experimental release was that obtained from these cul-
tures incubated with compounds (1–5) or sodium anti-
mony gluconate, and the total release was that
obtained from these cultures incubated with 1 N HCl.
In 18 h assays, the spontaneous release never exceeded
30% of the total release from either form of the parasite
or normal human PBMC.
Thioglycolate-elicited peritoneal exudate was used as the
source of macrophages for better recovery and easier isola-
tion. Approximately 2.5 · 105 macrophages were allowed
to adhere to glass coverslips (20 · 25 mm) in Rose-Well
Park Memorial Institute (RPMI)–1640 medium (Gibco
Laboratories) supplemented with 10% FBS, and cultured
for 5–7 days at 37 ꢁC in 5% CO2 before in vitro infection
with L. donovani. Stationary phase L. donovani promastig-
otes (5.0 · 106) were added to each coverslip and incubated
for 6 h at 37 ꢁC in CO2.
Acknowledgements
We thank Dr. Debjani Basu, Scientist, Botanical Sur-
vey of India, Howrah, West Bengal for supplying and
identifying the plant material, and Dr. Kazuo Koike,
Department of Pharmaceutical Sciences, Toho Univer-
sity, Japan for HRFABMS, MALDI-TOF-MS, 2D
NMR spectra of the compounds. Dr. N.P. Sahu is in-
debted to CSIR for award of Emeritus Scientist and
Mr. D. Mandal is thankful to CSIR for financial assis-
tance as SRF.
4.12. Antileishmanial activity of compounds (1–5) on
L. donovani infected BALB/c mice macrophages in vitro
Cover slips were washed (3·) with 10% FBS-supple-
mented RPMI-1640 to remove uningested parasites and
incubated for 2 days in the presence or absence of graded
concentrations of compounds and amphotericin B (Sigma
Chemical Co., USA). Infected macrophage cultures were
washed with FBS, fixed with pre-chilled methanol, stained
with Giemsa, and examined microscopically under oil
immersion. At least 400 target macrophages were examined
for each coverslip. Antileishmanial activity was determined
by calculating the number of amastigotes per 100
macrophages.
References
Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from
human blood. Isolation of mononuclear cells by one centrifugation,
and granulytes by combining centrifugation and sedimentation at 1g.
Scandinavian Journal of Clinical and Laboratory Investigation Sup-
plement, 77–89.
Chakraborti, S.K., Barua, A.K., 1963. Triterpenoids-XVI, The constitu-
tion of barringtogenol D – a new triterpenoid sapogenin from
Barringtonia acutangula Gaertn. Tetrahedron 19, 1727–1732.
Dai, J., Zhao, C., Zhang, Q., Liu, Z., Zheng, R., Yang, L., 2001.
Taraxastane-type triterpenoids from Saussurea petrovii. Phytochemis-
try 58, 1107–1111.
Das, M.C., Mahato, S.B., 1982. Triterpenoid sapogenols from the leaves
of Careya arborea: Structure of careyagenolide. Phytochemistry 21,
2069–2073.
Ghosh, A.K., Rakshit, M.M., Ghosh, D.K., 1983. Effect of berberine
chloride on Leishmania donovani. Indian Journal of Medical Research
78, 407–416.
Jaffe, C.L., Grimaldi, G., McMohan-Pratt, D., 1984. In: Morel, C.M.
(Ed.), Genes and Antigens of Parasites: A Laboratory Manual, second
ed. Fundacao Oswaldo Cruz, Rio de Janiero, p. 47.
Nakano, T., Hasegawa, M., Fukumaru, T., Durham, L.J., Budzikiewicz,
H., Djerassi, C., 1969. The structure of jegosapogenol (barringtogenol
C, Aescindin) and the configuration at C-21 and C-22 in barringtog-
enol D, Aescigenin, protoaescigenin, iso-aescigenin. Journal of Organic
Chemistry 34, 3135–3146.
Pal, B.C., Chaudhury, T., Yoshikawa, K., Arihara, S., 1994. Saponins
from Barringtonia acutangula. Phytochemistry 35, 1315–1318.
Phondke, G.P., 2000. The Wealth of India, Raw Materials, vol. 1. National
Institute of Science Communication (CSIR), New Delhi, p. 216 (1st
Supplement).
Reynolds, W.F., McLean, S., Poplawski, J., Enriquez, R.G., Escobar,
L.I., Leon, I., 1986. Total assignment of 13C and 1H spectra of three
isomeric triterpenol derivatives by 2D NMR; an investigation of
complex natural products. Tetrahedron 42, 3419–3428.
Row, L.R., Sastry, C.S.P., 1964. Chemical examination of Careya arborea
Roxb. Indian Journal of Chemistry 2, 510.
Sahu, N.P., Panda, N., Mandal, N.B., Banerjee, S., Koike, K., Nikaido,
T., 2002. Polyoxypregnane glycosides from the flowers of Dregea
volubilis. Phytochemistry 61, 383–388.
4.13. Cytotoxicity assays
In vitro grown promastigotes, freshly purified amastig-
otes, or normal human PBMC (2 · 106 of each) were
washed and resuspended in Medium-199 (1.0 ml) contain-
ing 8% FBS. Fifty microcuries of sodium chromate-51Cr
(Na251CrO4, BARC, Bombay, India) were added to
promastigotes or purified amastigotes and incubated for
6 h at 22 ꢁC. Normal human PBMC were incubated with
Na251CrO4 for 2 h at 37 ꢁC with gentle shaking at 15 min
interval. Radiolabelled promastigotes, amastigotes, or
normal human PBMC were washed (4·) and resuspended
in Medium-199 supplemented with 8% FBS at a concen-
tration of 1.0 · 106 mlꢀ1. Various concentrations (10, 5,
2.5 mg mlꢀ1) each of compounds (1–5) or sodium anti-
mony gluconate were added to radiolabelled promastig-
otes or amastigotes (0.5 · 106) in a total volume of
1.0 ml in 5.0 ml tubes, and to radiolabelled normal
human PBMC (0.2 · 105) in a total volume of 0.2 ml in
microtitre plates. After 18 h of incubation at 37 ꢁC,
promastigotes or amastigotes were centrifuged, 0.5 ml ali-
quots of cell-free supernatants were collected and
counted in a gamma counter. For normal human PBMC,
0.1 ml of supernantants were collected from each well
without disturbing the cell pellet. The percentage 51Cr
release was calculated. The spontaneous release was that
obtained from promastigotes, amastigotes, or normal
human PBMC incubated with the medium alone, the

190
D. Mandal et al. / Phytochemistry 67 (2006) 183–190
Yoshikawa, M., Murakami, T., Matsuda, H., Yamahara, J., Murukami,
N., Kitagawa, I., 1996. Bioactive saponins and glycosides III. Horse
Chestnut. (1): The structures, inhibitory effects on ethanol absorption,
and hypoglycemic activity of Escins Ia, Ib, IIa, IIb and IIIa from the
seeds of Aesculus hippocastanum L. Chemical and Pharmaceutical
Bulletin 44, 1454–1464.