Halimodendrin I, a new acylated triterpene glycoside from Halimodendron halodendron (Fabaceae)

Article abstract of DOI:10.1016/j.phytol.2011.06.004

Halimodendrin I, a new acylated triterpene glycoside (1), was isolated and chemically characterized as 3β-O-palmitoyl-28-[3′-palmitoyl-β-d- glucopyranosyl]-olean-12-en-28-oic acid from the aerial part of Halimodendron halodendron (Fabaceae) by IR, 1D and 2D NMR, HR-ESI-MS and LR-ESI-MS experiments. In addition, seven known compounds were isolated and identified as: palmitic acid, glycerol-2-linoleneate, glycerol-1,3-dilinoleneate, ferulic acid, 3-O-methylquercetin, β-sitosterol, and β-sitosterol-3-O-β- d-glucopyranoside. Nine fatty acids were identified and quantified in the saponifiable matter of the hexane extract. These fatty acids are: myristic, n-pentadecanoic, palmitoleic, palmitic, linoleic, oleic, stearic, arachidic, and behenic acids. The volatile oil was isolated by hydrodistillation (0.013%, w/w) with unpleasant smell. Twenty-seven components were identified in the oil by GC/MS.

Full text of DOI:10.1016/j.phytol.2011.06.004

Phytochemistry Letters 4 (2011) 323–327
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Halimodendrin I, a new acylated triterpene glycoside from Halimodendron
halodendron (Fabaceae)
Zhanar A. Kozhamkulova a, Mohamed M. Radwan b,c, Galiya E. Zhusupova a,
Zharilkasin A. Abilov a, Samir A. Ross c,d,
*
a Al-farabi Kazakh National University, School of Chemistry, Department of Organic and Natural Products Chemistry, Kazakhstan
b Department of Pharmacognosy, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
c National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
d Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
Halimodendrin I, a new acylated triterpene glycoside (1), was isolated and chemically characterized as
3b b-D-glucopyranosyl]-olean-12-en-28-oic acid from the aerial part of
-O-palmitoyl-28-[30-palmitoyl-
Received 15 March 2011
Received in revised form 3 June 2011
Accepted 17 June 2011
Available online 1 July 2011
Halimodendron halodendron (Fabaceae) by IR, 1D and 2D NMR, HR-ESI-MS and LR-ESI-MS experiments. In
addition, seven known compounds were isolated and identified as: palmitic acid, glycerol-2-linoleneate,
glycerol-1,3-dilinoleneate, ferulic acid, 3-O-methylquercetin,
b-sitosterol, and b-sitosterol-3-O-b-D-
glucopyranoside. Nine fatty acids were identified and quantified in the saponifiable matter of the hexane
extract. These fatty acids are: myristic, n-pentadecanoic, palmitoleic, palmitic, linoleic, oleic, stearic,
arachidic, and behenic acids. The volatile oil was isolated by hydrodistillation (0.013%, w/w) with
unpleasant smell. Twenty-seven components were identified in the oil by GC/MS.
Keywords:
Halimodendron halodendron
Fabaceae
Halimodendrin I
Acylated triterpene glycoside
Flavonoid
Published by Elsevier Ireland Ltd on behalf of Phytochemical Society of Europe.
Volatile oil
Fatty acids
1. Introduction
The genus Halimodendron (Fabaceae) is a monotypic genus of
legume containing the single species Halimodendron halodendron,
which is known by several common names (Caragana argentea,
Halimodendron argenteum, and Robinia halodendron), including
common salt tree and Russian salt tree (Pavlov, HB., 1961). It is
native to Russia and southern Asia, but it can be found on other
continents where it is an introduced species, and one that is often a
noxious weed (Pavlov, 1961). Only one manuscript (Wang et al.,
2010) reported the separation of phenolic compounds from H.
halodendron, this encouraged us to carry out phytochemical
studies on the aerial parts of this plant. Herein, we report the
isolation and identification of one new acylated triterpene
glycoside (1) and seven known compounds from the above plant.
Also the chemical composition of the volatile oil and fatty acids
content were studied (Table 2).
2. Results and discussions
* Corresponding author at: National Center for Natural Products Research, School
of Pharmacy, The University of Mississippi, University, MS 38677, USA.
Tel.: +1 662 915 1031; fax: +1 662 915 7989.
Compound 1, named Halimodendrin I was obtained as a
white amorphous powder. The molecular formula was determined
as C68H118O10 by HR-ESI-MS, showing an [M + NH4]+ peak at
E-mail address: sross@olemiss.edu (S.A. Ross).
1874-3900/$ – see front matter. Published by Elsevier Ireland Ltd on behalf of Phytochemical Society of Europe.
doi:10.1016/j.phytol.2011.06.004

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Z.A. Kozhamkulova et al. / Phytochemistry Letters 4 (2011) 323–327
Table 1
1H and 13C NMR data for Halimodendrin I (1), in CDCl3.
a
Position
1
dC
dH
Key HMBC H ! C
Position
25
dC
dH
Key HMBC H ! C
38.3
1.04 m
1.60 m
1.85 m
2.00 m
4.48
15.6
0.92 s
2
3
25.4
80.8
26
27
16.9
25.8
0.74 s
1.12 s
C-100, C-23
t (8.0)
–
C-24, C-4, C-2
4
37.9
55.5
18.2
32.3
39.6
47.7
37.1
23.6
28
29
30
10
176.4
17.1
23.8
94.1
76.4
79.1
69.1
71.8
5
0.88
0.74 s
C-19, C-20
C-19, C-20
C-28
6
1.24
0.92
7
1.43
5.56 d (8)
3.51 m
8
–
20
9
1.44 m
–
30
4.92 t (8)
3.70 t (9.2)
3.62 (8.4)
C-l0, C-20, C-40, C-50
10
11
40
2.03
50
1.86
12
122.9
5.29 bs
60
62.1
3.87 (12, 2.4)
3.77 (12, 2.4)
13
14
15
143.4
41.4
27.8
–
100
1000
200
174.0
176.1
35.1
–
2.84 m
2.43
2.70 m
2.30
2.60
–
–
2.28 t (7.4)
C-l00, C-200, C-300
C-l00, C-200, C-300
16
23.2
2000
34.5
2.40 t (7.2)
17
18
19
20
21
47.1
41.4
45.9
30.8
34.5
300
3000
400-1300
4000-13000
1400, 14000
25.4
25.1
29.4
30.86
32.1
1.60 bs
1.60 bs
1.24 m
1.24 m
1.24 m
2.88 m
1.24 m
–
2.41 t
(7.2)
22
23
24
32.9
16.7
28.3
1500, 15000
1600, 16000
22.9
14.3
1.24 m
0.84 s
0.84 s
0.87 t (6.4)
Note:
d values are in ppm; coupling constants values (J) in Hz) are in parentheses.
a
Multiplicity is not clear for some proton signals due to ovelapping.
m/z = 1112.8977 (calcd. for C68H122O10N 1112.91056). The IR
spectrum of 1 indicated the presence of hydroxy (3438 cmꢀ1) and
ion at m/z 403 in the LRMS corresponding to hexose plus palmitoyl
moieties with loss of a methyl group. The above structure
elucidation of 1 was further supported by 1H–1H COSY and HMBC
data (Table 1). From these results, the structure of 1was established
as
12-en-28-oic acid.
The known compounds obtained in this study, palmityl alcohol
(Zongren and Huimin, 2009), glycerol-2-linoleneate (Milan et al.,
1987), glycerol-1,3-dilinoleneate (Milan et al., 1987), ferulic acid
ester (1724 cmꢀ1) groups. The seven methyl resonated at
dH 0.74 (s,
6H), 0.84 (s, 6H), 0.92 (s, 6H), and 1.12 (s, 3H), in the 1H NMR
spectrum(Table1)alongwithcharacteristic36carbonresonancesin
the 13C NMR indicating that 1 is an oleanene triterpene monoglyco-
side derivative (Kuiate et al., 2009). The 13C NMR of the aglycone
3b b-D-glucopyranosyl]-olean-
-O-palmitoyl-28-[30-palmitoyl-
showed two olefenic carbons at
C-12 and C-13 respectively (Kuiateet al., 2009; Machidaet al., 1997).
The presence of the anomeric proton at H 5.56 (d, J = 8.0 Hz) in the
1H NMR spectrum correlated to the anomeric carbon (
C 94.1) in the
HMQC spectrum confirmed the occurance of one hexose moiety and
the large coupling constant (J = 8.0 Hz) showed the -configuration
dC 122.9 and 143.3 characteristic for
d
(Kelly et al., 1976), 3-O-methylquercetin (Campos et al., 2002),
b-
d
sitosterol (Zhang et al., 2005), and -sitosterol-3-O- -glucopyr-
b
b-D
anoside (Flamini et al., 2001), were identified by comparison of
their physical and spectroscopic data with literature reports.
The unpleasant smell of H. halodendron and the absence of
any reports on its volatile oil contents encouraged us to study
these contents. The oil was obtained by hydrodistillation
(0.013%, w/w) and the GC/MS analysis of the oil resulted in
the identification of 27 components (Table 2). The components
of the oil were identified by comparing their retention time and
mass fragmentation patterns with those of the available
references and/or with published data (Adams, 1989) as well
as through GC/MS library search. Five aldehydes were identified
in the oil (nonanal, 2-nonenal, decanal, dodecanal and tetra-
decanal). They constitute 16.85% of the oil. Their presence in a
high percentage in the oil could explain why the oil has an
unpleasant smell. Five alcohols were identified in the oil (1-
octen-3-ol, octanol, p-menth-1-en-8-ol, E-2-tetradecen-1-ol,
and 3,7,11,15-tetramethyl-2-hexadecen-1-ol) and together con-
stitute 9.23% of the oil. The major constituents in the oil are:
octacosane (15.12%), linalyl anthranilate (9.01%), heneicosane
b
of the sugar (Avilov et al., 2003; Gao et al., 2008; Liu et al., 2009;
Machida et al., 1997; Velozo et al., 1999). This sugar was attached to
C-28 based on the HMBC correlation between the anomeric proton
at
NMR spectroscopic data (Table 1) also displayed characteristic
signalsat H 0.87(t, 6H)and H 1.24(m, nCH2), C 14.3, and C 22.9;dC
176.4 and C 174.0 for two acyl moieties. The hydrolysis of 1 with 5%
dH 5.56 and the ester carbonyl at d C
C 176.1 (Table 1). The 1H and 13
d
d
d
d
d
KOH/MeOH yielded oleanolic acid, palmitic acid and glucose
(oleanolic acid and glucose were identified by TLC and palmitic
acid was identified by GC after derivatization). The down field
chemical shift of H-3 (dH 4.48) and C-3 (dC 80.8) (Machida et al.,
1997; Van et al., 2009) indicated the placement of one palmitoyl
moiety at C-3 which was confirmed by the HMBC correlation H-3
(4.48) and the ester carbonyl at dC 174.0 (C-100). The sugar was
located at C-28 based on its 13C NMR chemical shift (dC 94.1)
(Machida et al., 1997; Park et al., 2005) and the HMBC correlation of
H-10 (
H-30
dH 5.56) with C-28 (dC 176.4). The HMBC correlation between
(dH 4.92) and the third ester carbonyl carbon (dC 176.1)
(10.39%), a-farensene (3.37%), and hexahydrofarnesyl acetone
(3.11%). This is the first report on the composition of the volatile
oil of H. halodendron.
indicating the location of the other palmitoyl moiety at C-30 of the
glucose(Table1)thatwasconfirmedbythepresenceofthefragment

Z.A. Kozhamkulova et al. / Phytochemistry Letters 4 (2011) 323–327
325
Table 2
Chemical composition of the volatile oil of H. halodendron.
Peak#
Retention time (min)
% of each peak
Molecular formula
Identification
1
2
6.708
6.914
7.077
7.68
2.85
C8H16
C8H16
C9H14
C8H16
O
O
O
O
1-Octen-3-ol
1.12
3-Heptanone, 5-methyl
Furan, 2-pentyl
Octanol
2.72
4
0.90
5
8.54
0.9
C10H14
m-Cymene
6
10.8
2.29
C8H16
Cyclopropane, pentyl
Linalyl anthranilate
Nonanal
7
12.41
12.7
9.01
C17H23NO2
8
4.899
2.102
1.789
2.201
4.842
2.184
1.786
3.873
1.586
1.588
3.374
1.292
3.774
3.107
2.396
10.393
0.543
0.651
15.123
0.380
C9H18
C9H16
O
O
9
15.94
18.05
18.85
24.44
28.05
29.85
32.13
34.45
36.21
41.45
44.68
50.57
57.38
70.63
77.00
77.79
77.91
78.96
79.36
2-Nonenal
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
C10H18
O
O
p-menth-1-en-8-ol
Decanal
C10H20
C13H22
C13H16
O
2,6,10,10-Tetramethyl-1-oxa-spiro [4.5]dec-6-ene
Naphthalene, 1,2-dihydro-1,1,6-trimethyl
C13H18
C12H24
C13H22
C13H20
C15H24
C14H28
O
O
O
O
Beta-D amascenone
Dodecanal
Cis-Geranylacetone
b-ionone
a-farnesene
O
E-2-Tetradecen-1-ol
C14H28
C18H36
O
O
Tetradecanal
Hexahydrofarnesyl acetone
3,7,11,15-Tetramethyl-2-hexadecen-1-ol
Heneicosane
C20H40
C21H44
C17H36
C21H44
C28H58
C17H36
O
Tetradecane, 2,6,10-trimethyl
Heptadecane, 2,6,10,15-tetramethyl
Octacosane
Tetradecane, 2,6,10-trimethyl
The chemical composition of the fatty acids in the aerial part of
the plant was determined by GC coupled with FID and MS
detectors. The identification of nine fatty acids was based on
comparison with authentic samples (retention time and mass
spectral fragmentation) as well as by using the computer library.
The percentages of fatty acids were determined following
normalization of all peaks in the flame ionization detector
chromatogram. The identified unsaturated fatty acids constitute
46.7% of the total fatty acids while the identified saturated fatty
acids represent 42.2% of the total fatty acids. Palmitic, oleic, linoleic
and stearic acids are the major fatty acids components (Table 3).
with a 3 mm inverse probe using residual solvent signal as
internal standard. The mass spectrometric analysis was performed
on an Agilent Series 1100 SL mass spectrometer equipped with
an ESI source. GC–MS analysis was carried out on a HP 6890
series GC, equipped with a split/splitless capillary injector, a HP
6890 Series injector autosampler, and
(30 m ꢁ 0.25 mm ꢁ 0.25 m, Agilent).The GC was interfaced to a
HP 5973 quadrupole mass selective detector through a transfer line
set at 240 8C. The injector temperature was 250 8C, and 1
a DB-5 ms column
m
mL
injections were performed in the split (1:10) mode. Column flow
was set at a constant pressure of 5.66 psi, giving an initial flow of
0.7 mL/min, using helium as carrier gas. The oven temperature was
raised from 70 8C to 200 8C at rate 2 8C/min. Then continue at 200 8C
for 15 min. The total run was 80 min. The filament was operated at
70 eV, with an emission current of 35 A. The multiplier voltage was
automatically set to 2247 V. The ion source and quadrupole
temperatures were 230 and 150 8C, respectively. The acquisition
range was m/z 30–800 at 1.95 scans per second, starting 3.5 min
after injection. Column chromatography was performed on silica gel
3. Experimental
3.1. General
Melting points were determined on a Thomas-Hoover capillary
melting point apparatus. Optical rotations were recorded on a
Rudolph Research Analytical Autopol IV automatic polarimeter
model 589-546. High-resolution mass spectra were measured using
a Bruker BioApex. IR spectra were recorded on a Bruker Tensor 27
instrument. 1H and 13C NMR spectra wererecordedon VarianAS 400
or Bruker DRx-400 NMR spectrometers in CDCl3 for compound 1,
˚
(EM Science, 60 A, 230–400 mesh ASTM, Bakerbond Polar Plus C18
bonded phase) and Sepahex LH-20 (25–100 lm, lipophilic, Sigma–
Aldrich). TLC was carried out on aluminum-backed plates pre-
˚
coated with silica gel F254 (20 ꢁ 20 cm, 200 lm, 60 A, Merck) and on
glass-backed plates pre-coated with silica gel F254 (10 ꢁ 20 cm,
glycerol-2-linoleneate, glycerol-1,3-dilinoleneate,
the fatty acids, and CD3OD for ferulic acid, C5D5N for
O- -glucopyranoside and DMSO-d6 for 3-O-methylquercetin,
b-sitosterol and
˚
200 lm, 60 A, Analtech). Visualizationwasaccomplished byspraying
b-sitosterol-3-
with p-anisaldehyde [0.5 mL in glacial acetic acid (50 mL) and
b-D
Table 3
Identified Fatty acids of H. halodendron aerial parts.
Peak #
Rt in minutes
% area peak
% in plant material (g%, w/w)
Identification
1
2
3
4
5
6
7
8
9
16.94
18.09
18.98
19.25
21.00
21.07
21.27
23.13
24.85
3.5
0.053
0.016
0.040
0.371
0.323
0.371
0.154
0.016
0.022
Myristic acid (14:0)
n-Pentadecanoic acid (15:0)
Palmitoleic acid (16:1)
Palmitic acid (16:0)
Linoleic acid (18:2)
Oleic acid (18:1)
1.1
2.9
23.0
20.7
23.1
9.9
Stearic acid (18:0)
1.1
Arachidic acid (20:0)
Behenic acid (22:0)
1.6

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Z.A. Kozhamkulova et al. / Phytochemistry Letters 4 (2011) 323–327
sulfuric acid (97%, 1 mL)] spray reagent followed by heating.
Preparative TLC was performed on Analtech Uniplates, having a
3.4. Preparation and analysis of the volatile oil
silica gel GF preparative layer with UV 254, 20 ꢁ 20 cm, 1500
m
m
The air-dried plant material (400 g) was subjected to steam
distillation (5 h) to yield 0.05 g of light-yellow oil with unpleasant
smell (0.013%, w/w yield). Reference Standards: Different mono-
terpenes, sesquiterpenes, and alkanes were obtained from Aldrich
Chemical Co. Inc. (Milwaukee, WI), Fluka Chemical Corp. (New
York, NY), Sigma Chemical Co. (St. Louis, MO), and Varian
Associates (Houston, TX). These reference standards include:
thick. High-performance flash chromatography (HPFC) was per-
formed using a Horizon Biotage system (Biotage, Inc., Charlottes-
villle, VA) with normal phase silica gel cartridges (KP-SIL, 10 g, 40–
˚
63 lm, 60 A, Biotage).
3.2. Plant material
tricyclene,
nene, fenchone,
trans-carveol,
-cedrene, (ꢀ)-isoledene, (ꢀ)-isolongifolene, (ꢀ)-
isolongifolol, -humulene, cuparene, myristyl alcohol, citronellyl
acetate, neryl acetate, geranyl acetate, camphene, alloaromaden-
drene, n-eicosane, n-heneicosane, and hydrocarbon references
(C9–C21). Solutions were prepared in methanol at concentrations
of 1 mg/mL. Volatile oil solution: For GCMS analysis the volatile oil
was dissolved in MeOH at a concentration of 10 mg/mL (Table 2).
a
-pinene, sabinene,
a-phellandrene, 1-decene, limo-
The aerial parts including flowers of the plant H. halodendron
(Pall) Vos. were collected on May 29th, 2010 from Almaty region in
mountain plain Zailiiskii Alatau. The plant was identified by Dr.
Gemedzhieva, professor of botany at Institute of plant biology, Al-
Farabi Kazakhstan National University, Almaty. A voucher speci-
men (HH-25) was kept at the Institute of Plant Biology, Almaty,
Khazakhstan.
a
-terpineol, terpinolene, cis-verbenol, cis-and
a
a
3.3. Extraction and isolation
3.5. Fatty acids of Halimodendron halodendron
Air-dried plant material (700 g) was extracted by maceration
with MeOH (3 L ꢁ 3) for 24 h at room temperature. The combined
MeOH extracts were concentrated under vacuo to afford 90 g
residue which was subjected to fractionation by vacuum liquid
chromatography (VLC) over a silica gel column (1.5 kg), eluting
with mixtures of hexanes–EtOAc (100:0–0:100), followed by
EtOAc–MeOH (100:0–0:100) and finally with H2O–MeOH (25:75).
Eleven fractions (A–K) were collected (2 L each). Fraction B: (1.9 g,
eluted with 20% EtOAc in hexane) was chromatographed over a
silica gel columm (60 g, 2 cm i.d ꢁ 65 cm) eluted with mixtures of
hexanes–EtOAc in a manner of increasing polarities. Forty-seven
subfractions were collected (50 mL each). Subfractions 19–20
(741 mg, eluted with 2% EtOAc in hexane) was purified on solid
phase extraction (SPE) amino column (20 g) using eluents: CHCl3,
1% isopropanol in CHCl3, 2% isopropanol in CHCl3, and finally 2%
Acetic acid in CHCl3 to afforded palmitic acid (691 mg). Subfrac-
tions 24–28 (201 mg, eluted with 5% EtOAc in hexane) gave
glycerol -1,3-dilinoleneate (201 mg). Fractions C–E were combined
(3.6 g) and were chromatographed over a silica gel columm (80 g,
2 cm i.d ꢁ 65 cm) eluted with mixtures of hexanes–EtOAc in a
manner of increasing polarities. Sixty-nine subfractions (50 mL
each) were collected. Subfractions 10–12 (720.2 mg, eluted with
10% EtOAc in hexane) was purified on SPE amino column (20 g)
using 1% isopropanol in CHCl3; 2% isopropanol in CHCl3, 2% acetic
Extraction: 0.5 gram of the aerial part of the plant was extracted
with hexane (2 mL ꢁ 3) and the hexane extracts were combined
and evaporated in vacuo to afford 8.0 mg residue.
Saponification of the lipoidal matter in the hexane extract: The
hexane extract (8.0 mg) was dissolved in hexane (0.5 mL) and
alcoholic KOH solution (2 mL) was added. The mixture was
refluxed for 1 h, then cooled and diluted with distilled H2O (4 mL)
and extracted with ether (4 mL ꢁ 2) to remove the unsaponifiable
matter. The ether layer was washed with water (1 mL ꢁ 5) and the
aqueous washes were added to the aqueous layer. The combined
aqueous phase was rendered acidic with 10% H2SO4 and extracted
with ether (8 mL ꢁ 2). The ether layer was dried over anhydrous
Na2SO4 and evaporated to dryness (fatty acids fraction).
Esterification of fatty acids: The fatty acid fraction was dissolved
in 0.5 mL dry ether (in
a GC vial) and freshly prepared
diazomethane solution was added (6 drops) twice (until efferves-
cence ceased). The ethereal reaction mixture was evaporated in the
GC vial and the residue was dissolved in 0.8 mL dry ether and the
vial was capped.
Reference standards: Reference standards of methyl esters of
fatty acids were obtained from Polyscience Corporation (Nile, IL,
USA) and Sigma Chemical Company (St. Louis, MO, USA). These
standards include: methyl hexanoate (caproate), methyl octanoate
(caprylate), methyl decanoate (caprate), methyl dodecanoate
(laurate), methyl tetradecanoate (myristate), methyl palmitoleate,
methyl hexadecanoate (palmitate), methyl oleate, methyl linole-
ate, methyl linolenate, methyl stearate, methyl arachidate, methyl
docasonate (behenate), and methyl (erucate). Standard solutions
were prepared in hexane at concentration 1 mg/mL.
acid in ether and finally MeOH to gave Ferulic acid (9.1 mg),
b-
sitosterol (69 mg) and glycerol-2-linoleneate (39.5 mg). Subfrac-
tions 40–41 (206.8 mg, eluted with 20% EtOAc in hexane), purified
on SPE amino column (20 g) using CHCl3 in isopropanol mixtures in
a manner of increasing polarities affording 38.2 mg of compound 1.
Fractions F (2.2 g, eluted with 50% EtOAc in hexanes) was
chromatographed over a silica gel columm (60 g) using mixtures of
hexanes–EtOAc in a manner of increasing polarities. Twenty one
subfractions (150 mL each) were collected. Subfraction 21
(191.7 mg, eluted with 40% EtOAc in hexanes) was purified on
SPE silica column (20 g) using with 3% MeOH in CHCl3, 5% MeOH in
CHCl3 and finally 6% MeOH in CHCl3 to afford 3-O-methyl-
quercetin (45 mg).
Fraction I (8.7 g, eluted with EtOAc) was mixed with 12 g
celite and 15 mL MeOH and dried. The dried slurry was applied
onto the top of VLC columm packed with silica gel (200 g). The
columm was eluted with CHCl3–MeOH (100:0–0:100). Ten
subfractions (1 L each) were collected. Subfraction 7–8 (3.6 g,
eluted with 15–20% MeOH in CHCl3) was purified on silica
column (170 g column) using CHCl3/MeOH mixtures in a manner
GC/MS analysis: The mixture was analysed by GCMS using the
same conditions used for the analysis of volatile oil. Results are
summarized in Table 3.
3.6. Alkaline hydrolysis of compound 1
5 mg of compound 1 in 5% dry KOH–MeOH (2 mL) was heated
under reflux for 4 h. The reaction mixture was neutralized with 2 N
HCl and extracted with CHCl3. The CHCl3 layer was subjected to TLC
and GC/MS analysis. The TLC to identify oleanolic acid [TLC; Rf 0.35
hexane/acetone (3:1)] and GC/MS after methylation with diazo-
methane to give palmitoyl methyl ester (Rt 19.25 min). The sugar
wasidentifiedas
D-glucosebycomparisonwithauthenticsamples of
D
-glucose and -galcatose through silica gel TLC of the aqueous layer
D
of increasing polarities to yield
anoside (297.8 mg).
b
-sitosterol-3-O-
b
-
D
-glucopyr-
using solvent system EtOAc/MeOH/HOAc/H2O (11:2:2:2) followed
by spraying with anisaldehyde/H2SO4 and heating.

Z.A. Kozhamkulova et al. / Phytochemistry Letters 4 (2011) 323–327
327
O-palmitoyl-28-[30-palmitoyl-
b-D-glucopyranosyl]-olean-12-en-
Gao, Z., Ali, Z., Khan, I.A., 2008. Glycerogalactolipids from the fruit of Lycium
barbarum. Phytochemistry 69, 2856–2861.
Kelly, C.J., Harruff, R.C., Carmack, M., 1976. Polyphenolic acids of Lithospermum
ruderale. II. Carbon-13 nuclear magnetic resonance of lithospermic and ros-
marinic acids. J. Org. Chem. 41, 4445–4449.
Kuiate, T.T., Joseph, J., Tanoli, S.A., Mitaine-Offer, A.C., Ngadjui, B.T., Ali, M.S., Luu, B.,
Lacaille-Dubois, M.A., 2009. Antimicrobial pentacyclic triterpenoids from Ter-
minalia superba. Planta Med. 75, 522–527.
28-oic acid (1): White amorphous powder; ½a D25
þ 37 (c 0.4, CHCl3);
ꢂ
IR (NaCl): 3438, 2940, 2836, 1724, 1615 cmꢀ1; 1H and 13C NMR, see
Table 1; HR-EI-MS: m/z = 1112.8977 [M + NH4]+ (calcd. for
C
68H122O10 N: 1112.91056).
Velozo, L.S.M., da Silva, B.P., Bernardo, R.R., Parente, J.P., 1999. Doratin 7-O-b-
Acknowledgments
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Liu, Y.B., Su, E.N., Li, J.B., Zhang, J.L., Yu, S.S., Qu, J., Liu, J., Li, Y., 2009. Steroidal
glycosides from Dregea sinensis var. corrugata screened by liquid chromatogra-
phy–electro-spray ionization tandem mass spectrometry. J. Nat. Prod. 72,
229–237.
The authors are thankful to Dr. Bharanti Avula for MS data. Z.A.K
is grateful to the Khazakhstan government for financial support.
Machida, K., Yamaguchi, T., Kamiya, Y., Kikuchi, M., 1997. Acetylated triterpeneoids
from Liguistrum alifolium. Phytochemistry 46, 977–979.
Appendix A. Supplementary data
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