Oleanane-type glycosides from Tremastelma palaestinum (L.) Janchen

Article abstract of DOI:10.1002/cbdv.201300121

Three new oleanane-type glycosides, 1-3, were isolated from the whole plant of Tremastelma palaestinum (L.) Janchen, along with eight known triterpene glycosides. The structures of the new compounds were established as 3-O-[β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3) -β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2) -α-l-arabinopyranosyl]hederagenin (1), 3-O-[β-d-glucopyranosyl- (1→3)-α-l-rhamnopyranosyl-(1→3)-β-d-glucopyranosyl- (1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl] hederagenin 28-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl ester (2), and 3-O-[α-l-rhamnopyranosyl-(1→3)-β-d- glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2) -α-l-arabinopyranosyl]oleanolic acid 28-O-β-d-glucopyranosyl- (1→6)-β-d-glucopyranosyl ester (3) by using 1D- and 2D-NMR techniques and mass spectrometry. This is the first report on the phytochemical investigation of a species belonging to Tremastelma genus. Copyright

Full text of DOI:10.1002/cbdv.201300121

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Oleanane-Type Glycosides from Tremastelma palaestinum (L.) Janchen
by Gçkhan S¸enela), Derya Gꢀlcemala), Milena Masullob), Sonia Piacente*b) and Tamer
Karayıldırım*a)
a) Chemistry Department, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey (phone:
þ902323882369; fax: þ902323888264; e-mail: tamer.karayildirim@ege.edu.tr
b
`
) Dipartimento di Farmacia, Universita degli Studi di Salerno, Via Giovanni Paolo II n.132, I-84084
Fisciano (phone: þ39089969763; fax: þ39089969602; e-mail: piacente@unisa.it)
Three new oleanane-type glycosides, 1–3, were isolated from the whole plant of Tremastelma
palaestinum (L.) Janchen, along with eight known triterpene glycosides. The structures of the new
compounds were established as 3-O-[b-d-glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!3)-b-d-
glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin (1), 3-O-[b-d-
glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-a-l-rhamnopyrano-
syl-(1!2)-a-l-arabinopyranosyl]hederagenin 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl es-
ter (2), and 3-O-[a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!
2)-a-l-arabinopyranosyl]oleanolic acid 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester (3)
by using 1D- and 2D-NMR techniques and mass spectrometry. This is the first report on the
phytochemical investigation of a species belonging to Tremastelma genus.
Introduction. – The genus Tremastelma (Dipsacaceae) is represented by one species
in Turkey [1]. T. palaestinum (Syn: Knautia palaestina L., Scabiosa brachiata Sm.,
Pterocephalus palaestinus Coult., Asterocephalus brachiatus Rchb., Callistemma
brachiatum Boiss., Callistemma palaestinum L.) is an annual plant which mainly grows
in West and Northwestern part of Anatolia, some of the Greek islands, Balkan
peninsula, and West of Syria [1]. From a phylogenetic point of view [2][3], Dip-
sacaceae are considered among the most advanced families within the Dicotyledons.
Most of the taxa are widespread over the Mediterranean region and the Middle East
[3][4]. Although there are no reports of the medicinal uses of genus Tremastelma, some
of the Dipsacaceae plants have been used for their antidermatosic, anti-eczematous,
antipyretic, antihypertensive, and pharyngeal antiseptic properties [5], as well as for
the treatment of breast cancer [6], diphtheria [7], hemorrhoids [8], cold, flu,
rheumatoid arthritis, and enteritis [9], and also as analgesic and anti-inflammatory
agent to treat spermatorrhea, pain, and fractures [10]. This is the first phytochemical
report on the genus Tremastelma.
Results and Discussion. – The MeOH extract of the air-dried and powdered whole
plant T. palaestinum was partitioned with hexane, CHCl3, and BuOH. The BuOH
extract was fractionated by vacuum liquid chromatography (VLC), followed by
column chromatography (CC) to yield compounds 1–3 (Fig.). The aglycones of the
isolated compounds were identified as oleanane-type triterpenes by 1H- and 13C-NMR
analysis (Table 1) [11][12].
ꢀ 2014 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Figure. Structures of compounds 1–3, isolated from Tremastelma palaestinum
The HR-MALDI-TOF mass spectrum of
1
(m/z 1243.6096 ([MþNa]þ ,
C59H96NaOþ26 ; calc. 1243.6088) indicated the molecular formula C59H96O26. The ESI-
MS showed a major ion peak at m/z 1243 ([MþNa]þ ). Significant fragment-ion peaks,
in MS/MS analysis, at m/z 1081 ([MþNaꢀ162]þ ), 935 ([MþNaꢀ162ꢀ146]þ ), 773
([MþNaꢀ162ꢀ146ꢀ162]þ ), 627 ([MþNaꢀ162ꢀ146ꢀ162ꢀ146]þ ), 477 ([Mþ
Naꢀ162ꢀ146ꢀ162ꢀ146ꢀ150]þ ), corresponding to the loss of one pentose unit and
four hexose units, were observed.
The 1H-NMR spectrum displayed signals for six tertiary Me groups at d 0.73, 0.87,
0.91, 0.97, 1.00, and 1.19, for an olefinic H-atom at d 5.25 (t, J ¼ 3.5), one oxygen-
bearing CH group at d 3.65 (dd, J ¼ 11.5, 4.2, HꢀC(3)), and one primary alcohol
function at d 3.36 and 3.61 (HꢀC(23)) (Table 1). These signals, along with the
resonances in the 13C-NMR spectrum for the Me groups at d 13.8, 18.0, 33.8, 24.0, 16.5,
and 26.5, and for the two olefinic C-atoms at d 122.6 and 145.9, suggested that
compound 1 possessed hederagenin as aglycone [13]. The downfield shift of C(3) (d
82.4) of the aglycone evidenced that compound 1 was a monodesmosidic glycoside. The
13C-NMR spectrum showed 59 C-signals, of which 30 were assigned to the aglycone
moiety and 29 to a sugar moiety containing of five sugar units. The 1H-NMR spectrum
displayed in the sugar region signals corresponding to five anomeric H-atoms at d 4.52
(d, J ¼ 3.7), 4.54 (d, J ¼ 7.5), 4.62 (d, J ¼ 7.5), 5.21 (d, J ¼ 1.2), and 5.22 (d, J ¼ 1.2),
which were unambiguously correlated by HSQC experiment to the corresponding C-
resonances at d 104.7, 105.2, 104.9, and 101.5 (2 C), respectively. The chemical shifts of
all the individual H-atoms of the five sugar units were ascertained from a combination
of 1D-TOCSY and DQF-COSY spectral analysis, and the 13C chemical shifts of their
respective attached C-atoms were assigned unambiguously on the basis of the HSQC
spectrum (Table 2). These data evidenced the presence of two b-glucopyranosyl units
(d 4.54 and 4.62), two a-rhamnopyranosyl units (d 5.21 and 5.22), and one a-
arabinopyranosyl unit (d 4.52). The b-configurations of the glucopyranosyl units were
deduced from the J values of the anomeric H-atom (J¼7.5 ) . The a-configuration of the

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rhamnopyranosyl units was established by the HꢀC(1)/C(1) J value of 169 Hz,
determined by the residual direct correlation observed in the HMBC spectrum, in
agreement with that reported for the a-anomer of rhamnopyranose [14]. For the
arabinopyranosyl unit, the coupling constant of the anomeric H-atom was reported not
to be diagnostic on its own, owing to the high conformational mobility of
1
arabinopyranosides (4C1 $ C4). Evidence supporting an a-l-arabinopyranoside con-
figuration in rapid conformational exchange was obtained from the ROESY experi-
ment which showed correlations between HꢀC(1) and HꢀC(2), and between HꢀC(1)
1
4
and HꢀC(3) as expected for C4 and C1, conformations, respectively. The ROESY
correlation between HꢀC(1) and HꢀC(3) would not be expected for both 1C4- and 4C1-
b-l-arabinopyranosides. A ROESY correlation was also observed between HꢀC(1)
and HꢀC(5) as expected for an a-l-arabinopyranoside in a 4C1 conformation [15].
An unambiguous determination of the sequence and linkage sites was achieved
from the HMBC spectrum, which showed key correlation peaks between HꢀC(1)(Ara)
(d 4.52) and C(3) (d 82.4), between HꢀC(1)(RhaI) (d 5.22) and C(2)(Ara) (d 76.6),
and between HꢀC(1)(GlcI) (d 4.54) and C(3)(RhaI) (d 82.6), between
HꢀC(1)(RhaII) (d 5.21) and C(3)(GlcI) (d 83.3), and between HꢀC(1)(GlcII) (d
4.62) and C(3)(RhaII) (d 82.8). The d-configuration of glucose and the l-
configurations of the arabinose and rhamnose units were established by hydrolysis of
1 with 1n HCl, trimethylsilylation, and determination of retention time on a chiral
column by GC [16]. On the basis of all these evidences, the structure of 1 was
established as 3-O-[b-d-glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!3)-b-d-glu-
copyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin.
The HR-MALDITOF mass spectrum of
2
(m/z 1567.7151 ([MþNa]þ ,
C71H116NaOþ36 ; calc. 1567.7144) indicated the molecular formula C71H116O36. The ESI-
MS showed a major ion peak at m/z 1567 ([MþNa]þ ), and significant fragment-ion
peaks, in MS/MS analysis, at m/z 1405 ([MþNaꢀ162]þ ), 1259 ([MþNaꢀ162ꢀ
146]þ ), 1097 ([MþNaꢀ162ꢀ146ꢀ162]þ ), 951 ([MþNaꢀ162ꢀ146ꢀ162ꢀ146]þ ),
801 ([MþNaꢀ162ꢀ146ꢀ162ꢀ146ꢀ150]þ ), 639 ([MþNaꢀ162ꢀ146ꢀ162ꢀ146ꢀ
150ꢀ162]þ ), 477 ([MþNaꢀ162ꢀ146ꢀ162ꢀ146ꢀ150ꢀ162ꢀ162]þ ), correspond-
ing to the loss of one pentose unit and six hexose units, were observed.
1
The H- and 13C-NMR chemical shifts of the aglycone moiety of 2 were almost
superimposable on those of 1 (Table 1), suggesting hederagenin as aglycone.
With respect to the sugar portion, the occurrence of two additional sugar units was
observed compared to 1. The 1H NMR spectrum displayed in the sugar region signals
corresponding to seven anomeric H-atoms at d 4.37 (d, J¼7.5), 4.52 (d, J¼3.7), 4.55 (d,
J¼7.5), 4.62 (d, J¼7.5), 5.21 (d, J¼1.2), 5.22 (d, J¼1.2), and 5.38 (d, J¼7.5) which
showed correlations in the HSQC spectrum with the anomeric C-atom signals at d
104.2, 104.7, 105.2, 105.1, 101.4 (2 C), and 95.3, respectively. A detailed analysis of
NMR data revealed the presence of four b-glucopyranosyl units (d 4.37, 4.55, 4.62, and
5.38), two a-rhamnopyranosyl units (d 5.21 and 5.22), and one a-arabinopyranosyl unit
(d 4.52). An unambiguous determination of the sequence and linkage sites was
obtained from the HMBC spectrum, which allowed us to identify at C(3) the same
sugar chain as in 1, and the occurrence of a further sugar chain at C(28), as indicated by
the upfield shift of C(28) at d 178.3 when compared to compound 1. Key HMBCs
between the resonance of HꢀC(1)(GlcIII) (d 5.38) and C(28) (d 178.3), and between

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the resonance of HꢀC(1)(GlcIV) (d 4.37) and C(6)(GlcIII) (d 69.5), confirmed this
hypothesis. On the basis of all these evidences, the structure of 2 was established as 3-O-
[b-d-glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranosyl-(1!
3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin 28-O-b-d-gluco-
pyranosyl-(1!6)-b-d-glucopyranosyl ester.
The HR-MALDI-TOF mass spectrum of
3
(m/z 1389.6677 ([MþNa]þ ,
C65H106NaOþ30 ; calc. 1389.6667) provided the molecular formula C65H106O30. The ESI-
MS exhibited the major ion peak at m/z 1389 ([MþNa]þ ), and significant fragment-
ion peaks, in MS/MS, analysis at m/z 1243 ([MþNaꢀ146]þ ), 1081 ([MþNaꢀ146ꢀ
162]þ ), 935 ([MþNaꢀ146ꢀ162ꢀ146]þ ), 785 ([MþNaꢀ146ꢀ162ꢀ146ꢀ150]þ ),
623 ([MþNaꢀ146ꢀ162ꢀ146ꢀ150ꢀ162]þ ), 461 ([MþNaꢀ146ꢀ162ꢀ146ꢀ150ꢀ
162ꢀ162]þ ), corresponding to the loss of one pentose unit and five hexose units were
observed.
The 1H-NMR spectrum of 3 displayed signals for seven tertiary Me groups at d 0.88,
0.94, 0.96, 0.97, 1.06, 1.18, and 0.82, and for a vinyl H-atom at d 5.31 (t, J¼3.5). These
signals, along with the resonances in the 13C-NMR spectrum for the Me groups at d 17.8,
17.3, 33.6, 24.0, 16.2, 28.2, and 33.6, and of the two olefinic C-atoms at d 123.3 and 144.4,
evidenced that compound 3 possessed an olean-12-ene skeleton as aglycone [17].
Detailed NMR studies allowed us to identify the aglycone as oleanolic acid [18]. The
downfield shifts of C(3) (d 90.5) and C(28) (d 178.3) of the aglycone indicated that
compound 3 was a bidesmosidic glycoside. The 13C-NMR spectrum showed 65 C-atom
signals of which 30 were assigned to the aglycone moiety (Table 1) and 35 to a sugar
portion. The 1H-NMR spectrum displayed in the sugar region signals corresponding to
six anomeric H-atoms at d 4.37 (d, J¼7.5), 4.49 (d, J¼3.7), 4.53 (d, J¼7.5), 5.16 (d, J¼
1.2), 5.20 (d, J¼1.2), and 5.38 (d, J¼7.5 ) .
Complete assignments of the resonances of each sugar unit were achieved by
extensive 1D- (1H, 13C, TOCSY) and 2D- (HSQC, HMBC) NMR analyses. These data
revealed the presence of three b-glucopyranosyl units (d 4.37, 4.53, and 5.38), two a-
rhamnopyranosyl units (d 5.16 and 5.20), and one a-arabinopyranosyl unit (d 4.49). A
detailed analysis of the HMBC spectrum indicated that compound 3 differed from 2,
only in the absence of the terminal glucose unit in the sugar chain linked at C(3). Thus,
compound 3 was identified as 3-O-[a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranosyl-
(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]oleanolic acid 28-O-b-d-
glucopyranosyl-(1!6)-b-d-glucopyranosyl ester.
Additionally, eight known oleanane-type glycosides, 3-O-a-l-arabinopyranosylhe-
deragenin 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester [19], 3-O-[b-d-
glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]oleanolic
acid 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester [20], 3-O-[a-l-rham-
nopyranosyl-(1!2)-a-l-arabinopyranosyl]oleanolic acid 28-O-b-d-glucopyranosyl-
(1!6)-b-d-glucopyranosyl ester [21], 3-O-[a-l-rhamnopyranosyl-(1!2)-a-l-arabi-
nopyranosyl]hederagenin 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester
[22], 3-O-[b-d-glucopyranosyl(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyra-
nosyl]hederagenin [23], 3-O-[a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]he-
deragenin [24], 3-O-[a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranosyl(1!3)-a-l-
rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin [25], and 3-O-[a-l-rham-
nopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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arabinopyranosyl]hederagenin 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl
ester [26], were isolated.
As mentioned above, compounds 1–3 have never been reported before. 3-O-[b-d-
Glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]oleanolic
acid 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester, 3-O-[a-l-rhamnopyr-
anosyl-(1!2)-a-l-arabinopyranosyl]oleanolic acid 28-O-b-d-glucopyranosyl-(1!6)-
b-d-glucopyranosyl ester, 3-O-[b-d-glucopyranosyl(1!3)-a-l-rhamnopyranosyl-(1!
2)-a-l-arabinopyranosyl]hederagenin, and 3-O-[a-l-rhamnopyranosyl-(1!3)-b-d-
glucopyranosyl(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederage-
nin are reported for the first time in a species belonging to the Dipsacaceae family. 3-O-
[a-l-Arabinopyranosyl]hederagenin 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyra-
nosyl ester and 3-O-[a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-a-l-
rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin 28-O-b-d-glucopyrano-
syl-(1!6)-b-d-glucopyranosyl ester have been found in Cephalaria gigantea and
Dipsacus asperoides [27–30], while 3-O-[a-l-rhamnopyranosyl-(1!2)-a-l-arabino-
pyranosyl]hederagenin 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester has
been previously isolated from the MeOH extract of Cephalaria pastricensis leaves [30]
and 3-O-[a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin from the
roots of Scabiosa soongorica [31]. The species cited above belong all to Dipsacaceae
family.
The authors are grateful to Ege University Research Foundation (2010 Fen044) for financial support.
Experimental Part
General. GC Analysis: ThermoFinnigan Trace GC apparatus; l-Chirasil-Val column (0.32 mmꢁ
25 m). Column chromatography (CC): silica gel 60 (0.063–0.200 mm; Merck) and LiChroprep RP-18
(25–40 mm, Merck). TLC: silica gel 60 F254 (Merck) and RP-18 F254s (Merck) plates. Optical rotations:
JASCO DIP 1000 polarimeter. IR Spectra: Bruker IFS-48 spectrometer; ˜n in cmꢀ1. NMR Spectra:
Bruker DRX-600 spectrometer (BrukerBioSpin, D-Rheinstetten) equipped with a Bruker 5-mm TCI
CryoProbeat 300 K; all 2D-NMR spectra were acquired in CD3OD (99.95%, SigmaAldrich), and
standard pulse sequences and phase cycling were used for DQF-COSY, HSQC, and HMBC spectra; the
NMR data were processed using UXNMR software. Exact masses were determined by a Voyager DE
mass spectrometer. Samples were analyzed by matrix-assisted laser desorption ionization time-of-flight
(MALDITOF) mass spectrometry. A mixture of analyte soln. and a-cyano-4-hydroxycinnamic acid
(Sigma) was applied to the metallic sample plate and dried. Mass calibration was performed with the ions
from ACTH (fragment 18–39) at 2465.1989 Da and angiotensin III at 931.5154 Da as internal standard.
ESI-MS: ThermoFinnigan LCQ Deca XP Max ion-trap mass spectrometer, equipped with Xcalibur
software.
Plant Material. Tremastelma palaestinum (L.) Janchen (whole plant) was collected from Bornova,
Izmir, Turkey, in June, 2011. Samples of plant material were identified by Assoc. Prof. Dr. S. G. S¸enol
(Ege University, Faculty of Science, Department of Biology). A voucher specimen (EGE 40858) was
deposited with the Ege University Botanical Garden & Herbarium Research and The Application
Center, Izmir, Turkey.
Extraction and Isolation. The air-dried and powdered plant material of T. palaestinum (whole plant;
400 g) was extracted with MeOH (3ꢁ3 l) at r.t. for 3 d. After filtration, the solvent was removed by
rotary evaporation to give a crude extract (40 g). The residue was dissolved in H2O (300 ml) and then
partitioned successively with hexane (3ꢁ250 ml), CHCl3 (3ꢁ250 ml), and BuOH saturated with H2O
(3ꢁ250 ml). The BuOH extract (5 g) was subjected to vacuum liquid chromatography (VLC) using

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reversed-phase (RP) material (Lichroprep RP-18, 25–40 mm, 150 g) employing H2O (1000 ml), H2O/
MeOH (8 :2, 1200 ml; 6 :4, 2400 ml; 4 :6, 3000 ml; 2 :8, 1600 ml) and MeOH (600 ml) to give ten main
fractions (A–J). Fr. D (500 mg) was subjected to CC SiO2 (60 g); CHCl3/MeOH/H2O 80 :20 :2, 1000 ml;
70 :30 :3, 500 ml; 61:32 :7, 750 ml) to yield 12 subfractions, and 3 (8 mg), 3-O-[a-l-arabinopyranosyl]-
hederagenin 28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester (6 mg), 3-O-[b-d-glucopyrano-
syl-(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]oleanolic acid 28-O-b-d-glucopyrano-
syl-(1!6)-b-d-glucopyranosyl ester (10 mg), 3-O-[b-d-glucopyranosyl(1!3)-a-l-rhamnopyranosyl-
(1!2)-a-l-arabinopyranosyl]hederagenin (10 mg) and 3-O-[a-l-rhamnopyranosyl-(1!3)-b-d-gluco-
pyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin 28-O-b-d-glucopyr-
anosyl-(1!6)-b-d-glucopyranosyl ester (10 mg). Subfr. 2 (40 mg) was submitted to CC (SiO2 (15 g);
CHCl3/MeOH/H2O 80 :20 :2, 500 ml; 70 :30 :3, 300 ml; 61:32 :7, 250 ml) to give 2 (7 mg). Subfr. 5
(35 mg) was chromatographed on RP material (Lichroprep RP-18, 25–40 mm, 10 g), employing MeOH/
H2O 6 :4 (500 ml) to yield arabinopyranosyl oleanolic acid 28-O-b-d-glucopyranosyl-(1!6)-b-d-
glucopyranosyl ester [20], 3-O-[a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]oleanolic acid 28-
O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester, (12 mg), and 3-O-[a-l-rhamnopyranosyl-(1!
2)-a-l-arabinopyranosyl]hederagenin-28-O-b-d-glucopyranosyl-(1!6)-b-d-glucopyranosyl ester
(9 mg). Subfr. 8 (80 mg) was subjected to CC (SiO2 (15 g); CHCl3/MeOH/H2O 80 :20 :2, 200 ml;
70 :30 :3, 200 ml; 61:32 :7, 250 ml) to yield 3-O-[a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]-
hederagenin (8 mg). Fr. E (850 mg) was subjected to CC (SiO2 (80 g); CHCl3/MeOH/H2O 80 :20 :2,
1000 ml; 70 :30 :3, 500 ml; 61:32 :7, 700 ml) to yield 9 subfractions. Subfr. 3 (80 mg) was submitted to CC
(SiO2 (15 g); CHCl3/MeOH/H2O 80 :20 :2, 300 ml; 70 :30 :3, 300 ml; 61:32 :7, 250 ml) to give 3-O-[a-l-
rhamnopyranosyl-(1!3)-b-d-glucopyranosyl(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-arabinopyrano-
syl]hederagenin (6 mg). Subfr. 4 (30 mg) was chromatographed on RP material (Lichroprep RP-18, 25–
40 mm; 10 g), employing MeOH/H2O (6 :4, 500 ml), to yield 1 (8 mg).
Acid Hydrolysis. A soln. (1.0 mg each) of compounds 1–3 in 1n HCl (0.25 ml) was stirred at 808 for
4 h. After cooling, the soln. was concentrated by blowing with N2. The residue was dissolved in 1-
(trimethylsilyl)-1H-imidazole and pyridine (0.1 ml), and the soln. was stirred at 608 for 5 min. After
drying the soln. with a stream of N2, the residue was partitioned between H2O and CH2Cl2 (1 ml; 1:1 (v/
v)). The CH2Cl2 layer was analyzed by GC using an l-Chirasil-Val column (0.32 mmꢁ25 m). Temps. of
the injector and detector were 2008 both. A temp. gradient system was used for the oven; the initial temp.
was maintained at 1008 for 1 min and then raised to 1808 at the rate of 58/min [16]. The peaks of d-glucose
(14.74 min), l-rhamnose (10.72 min), and l-arabinose (8.91 and 9.81 min) were detected in the
hydrolysate of 1.
Authentic samples treated in the same manner with 1-(trimethylsilyl)-1H-imidazole in pyridine were
detected at 14.71 (d-glucose), 10.70 (l-rhamnose), and 8.92 and 9.80 min (l-arabinose).
3-O-[b-d-Glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-a-l-
rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin (¼(3b)-3-{[b-d-Glucopyranosyl-(1!3)-
6-deoxy-a-l-mannopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-6-deoxy-a-l-mannopyranosyl-(1!2)-
a-l-arabinopyranosyl]oxy}-23-hydroxyolean-12-en-28-oic Acid; 1). Amorphous white solid. [a]D25
¼
þ19.8 (c¼0.1, MeOH). IR (KBr): 3428 (OH), 2934 (CH), 1680 (C¼O), 1658 (C¼C). 1H- and 13C-NMR
(CD3OD, 600 MHz): see Tables 1 and 2. ESI-MS: 1243 ([MþNa]þ ). ESI-MS/MS: 1081 ([MþNaꢀ
162]þ ), 935 ([MþNaꢀ162ꢀ146]þ ), 773 ([MþNaꢀ162ꢀ146ꢀ162]þ ), 627 ([MþNaꢀ162ꢀ146ꢀ
162ꢀ146]þ ), 477 ([MþNaꢀ162ꢀ146ꢀ162ꢀ146ꢀ150]þ ). HR-MALDITOF-MS: 1243.6096 ([Mþ
Na]þ , C59H96NaO2þ6 ; calc. 1243.6088).
3-O-[b-d-Glucopyranosyl-(1!3)-a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-a-l-
rhamnopyranosyl-(1!2)-a-l-arabinopyranosyl]hederagenin 28-O-b-d-Glucopyranosyl-(1!6)-b-d-
glucopyranosyl Ester (¼6-O-b-d-Glucopyranosyl-1-O-[(3b)-3-{[b-d-glucopyranosyl-(1!3)-6-deoxy-a-
l-mannopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-6-deoxy-a-l-mannopyranosyl-(1!2)-a-l-arabi-
nopyranosyl]oxy}-23-hydroxy-28-oxoolean-12-en-28-yl]-b-d-glucopyranose; 2). Amorphous white solid.
1
[a]2D5 ¼ þ22.5 (c¼0.1, MeOH). IR (KBr): 3442 (OH), 2930 (CH), 1670 (C¼O), 1655 (C¼C). H- and
13C-NMR (CD3OD, 600 MHz): see Tables 1 and 2. ESI-MS: 1567 ([MþNa]þ ). ESI-MS/MS: 1405 ([Mþ
Naꢀ162]þ ), 1259 ([MþNaꢀ162ꢀ146]þ ), 1097 ([MþNaꢀ162ꢀ146ꢀ162]þ ), 951 ([MþNaꢀ162ꢀ
146ꢀ162ꢀ146]þ ), 801 ([MþNaꢀ162ꢀ146ꢀ162ꢀ146ꢀ150]þ ), 639 ([MþNaꢀ162ꢀ146ꢀ162ꢀ

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
417
146ꢀ150ꢀ162]þ ), 477 ([MþNaꢀ162ꢀ146ꢀ162ꢀ146ꢀ150ꢀ162ꢀ162]þ ). HR-MALDITOF-MS:
1567.7151 ([MþNa]þ , C65H106NaO3þ0 ; calc. 1567.7144).
3-O-[a-l-Rhamnopyranosyl-(1!3)-b-d-glucopyranosyl(1!3)-a-l-rhamnopyranosyl-(1!2)-a-l-
arabinopyranosyl]oleanolic Acid 28-O-b-d-Glucopyranosyl-(1!6)-b-d-glucopyranosyl Ester (¼1-O-
[(3b)-3-{[6-Deoxy-a-l-mannopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-6-deoxy-a-l-mannopyra-
nosyl-(1!2)-a-l-arabinopyranosyl]oxy}-28-oxoolean-12-en-28-yl]-6-O-b-d-glucopyranosyl-b-d-gluco-
pyranose; 3). Amorphous white solid. [a]2D5 ¼ ꢀ10.5 (c¼0.1, MeOH). IR (KBr): 3450 (OH), 2938 (CH),
1
1680 (C¼O), 1660 (C¼C). H- and 13C-NMR (CD3OD, 600 MHz): see Tables 1 and 2. ESI-MS: 1389
([MþNa]þ ). ESI-MS/MS: 1243 ([MþNaꢀ146]þ ), 1081 ([MþNaꢀ146ꢀ162]þ ), 935 ([MþNaꢀ
146ꢀ162ꢀ146]þ ), 785 ([MþNaꢀ146ꢀ162ꢀ146ꢀ150]þ ), 623 ([MþNaꢀ146ꢀ162ꢀ146ꢀ150ꢀ
162]þ ), 461 ([MþNaꢀ146ꢀ162ꢀ146ꢀ150ꢀ162ꢀ162]þ ). HR-MALDITOF-MS: 1389.6677 ([Mþ
Na]þ , C71H116NaO3þ6 ; calc. 1389.6667).
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Received April 12, 2013