Spectral assignments and reference data
anomericprotons[δ 4.43(d, J = 8.0 Hz, H-1glc I), 4.49(d, J = 7.6 Hz,
H-1glc II), 5.02 (s, H-1rham), 5.27 (br s, H-1ara(f))]. The olefinic carbon
signals at δ 126.8 (C-12) and 143.5 (C-13) along with other carbon
signals in 13C-NMR led to the identification of aglycone as the
oleanolic acid derivative.[6] The characteristic signal of H-18 in 1H-
NMR appeared at δ 2.50 as doublet (J = 4.4 Hz) instead of double
doublet,showingthatoneoftheprotonsofC-19wasreplacedwith
hydroxyl group, and this was further confirmed by a deshielding
of C-19 at δ 83.3 in the 13C-NMR spectrum.[7] The chemical shifts
of the oxygenated methine carbons at δ 93.6 and 74.2 were
assigned to C-22 and C-21, respectively on the basis of HMQC
experiment and the literature data.[8,9] The coupling constants
of 9.9 Hz indicated that both protons at these positions were
trans to each other,[10,11] whereas the high frequency chemical
shift value at δ 92.0 was attributed to C-3 due to glycosidation
at this position.[12] The β orientation of the hydroxyl group at
C-3 was inferred from the chemical shift and coupling pattern
of H-3. Another methine at δ 68.4 was assigned to C-15 bearing
hydroxyl. The high-frequency shift of H3-27(δ 1.38) and those of
C-14 (δ 48.5) and C-8 (δ 42.6) as compared to reported chemical
shifts of oleanane[13] indicated the presence of a hydroxyl group at
C-15. The stereochemistry of C-15, C-19, and C-22 was established
with the help of Nuclear Overhauser Effect Spectroscopy (NOESY)
experiment. ThecrosspeaksobservedbetweenprotonsatH-18/H-
19, H-18/H-22 showed the α disposition of the hydroxyl group at
C-19 and C-22. The absence of H3-27 interaction with H-15 in
the NOESY spectrum revealed an α orientation of the hydroxyl
group at C-15. The stereochemistry at C-21 was established on the
basis of coupling constant (J = 9.9 Hz) between H-21 and H-22,
which indicated that both protons were trans to each other, and
confirmed that the hydroxyl group at C-21 was β oriented.
FAB-MS indicated the loss of a pentose (m/z 827 [M-H-C5H8O4]−
and di hexose (m/z 635 [M-H-C12H20O10]−). The 1H- and 13C-NMR
spectra of 2 (Table 1) indicated the presence of the following
functions: seven methyls [δ 0.84, 0.86, 0.97, 1.05, 1.07, 1.23, and
1.38 (each s, H-29, 24, 25, 30, 23, 26, and 27, respectively)], four
oxy-methine protons [δ 3.18 (d, J = 2.1 Hz, H-3), 3.60 (dd, J = 11.8,
5.8 Hz, H-21), 3.71 (m, H-15), 4.10 (m, H-2)], an olefinic proton
[δ 5.48 (br s, H-12)], three sugar moieties [4.48 (d, J = 7.8 Hz,
H-1glc I), 4.49(d, J = 7.4 Hz, H-1glc II), 5.27(s, H-1ara(f))]. The 13C-NMR
spectrum showed 30 signals for the aglycone moiety, which could
be correlated to the corresponding protons by HMQC experiment,
indicating that aglycone is an oleanolic acid derivative. The
chemical shifts at δ 3.18 (d, J = 2.1 Hz) and 4.10 (m) corresponded
to 91.8 (C-3) and 83.7 (C-2) in the 13C-NMR spectrum, ascribable
respectively to the 3α and 2α protons on carbons bearing a
hydroxyl function. While the signal at δ 3.60 (dd, J = 11.5, 5.8) with
a cross peak at δ 78.9 was assigned to C-21, the high-frequency
signal at δ 3.71 (m) corresponding to 68.4 was assigned to C-
15 due to hydroxyl substituents at these positions. Identification
of sugars was done by acid hydrolysis and their thiazolidine
derivatives were prepared in the same manner as in 1. The absence
of any 13C-NMR glycosidation shift for α-L-arabinofuranosyl and
β-D-glucopyraonsyl suggested that all three sugars were terminal
units. The position of each sugar unit was directly deduced from
HMQCandHMBCexperiments. Thecrosspeaksobservedbetween
δ 4.48 (H-1glc I) and 91.8 (C-3), and between δ 5.27 (H-1ara (f)) and
83.7 (C-2) indicated the attachment of β-D-glucopyranosyl and α-
L-arabinofuranosyl at C-3 and C-2, respectively. Similarly, the cross
peak between δ 4.49 (H-1glc II) and 78.9 (C-21) showed the linkage
site at C-21.[14] Consequently, the structure of 2 was proposed as
2β, 3β, 15α, 21β-tetrahydroxyolean-12-ene-28-oic acid 2-O-[α-L-
arabinofuranoside]-3, 21-bis-O-[β-D-glucopyranoside].
The configurations of the sugar units were assigned after
hydrolysis of 1 with 1 N HCl. The hydrolyzed sugars were
derivatized by preparing their thiazolidine derivatives, and the gas
chromatography (GC) retention time of each sugar was compared References
with those of authentic sugar samples prepared in the same
[1] Y. R. Chadha, TheWealthofIndia:ADictionaryofIndianRawMaterials
manner. The absence of any 13C-NMR glycosidation shift for α-
L-rhamnopyranosyl and α-L-arabinofuranosyl moieties suggested
that these sugars were terminal units. Glycosidation shifts were
observed for C-3glc I (δ 6.7) and C-3glc II (δ 85.2) (Table 1). Direct
evidence for the sugars sequence and their linkage site at C-22
was derived from the HMBC spectra where correlation peaks of
the signals at δ 4.49 (H-1glc II) with 93.6 (C-22), and δ 5.27 (H-
1ara (f)) with 85.2 (C-3glc II) were presented. Similarly, the sequence
of disaccharide moiety at C-3 was assigned by the cross peaks
between resonances at δ 4.43 (H-1glc I) with δ 92.0 (C-3) and δ 5.02
(H-1rha) to 86.7 (C-3glc I) in the HMBC experiment. On the basis of
the above evidence, compound 1 was unambiguously assigned
as 3β, 15α, 19α, 21β, 22α-pentahydroxyolean-12-ene-28-oic acid
3-O-{α-L-rhamnopyranosyl-(1 → 3)-β-D-glucopyranoside}-22-O-
{α-L-arabinofuranosyl-(1 → 3)-β-D-glucopyranoside}.
and Industrial Products, vol. X (Sp-Wp), Publication and Information
Directorate: New Delhi, 1977, p 26.
[2] V. U. Ahmad, S. Arshad, S. Bader, A. Ahmed, S. Iqbal, R. B. Tareen,
J. Asian Nat. Prod. Res. 2006, 8, 105.
[3] V. U. Ahmad, S. Arshad, S. Bader, A. Ahmed, S. Iqbal, A. Khan,
R. B. Tareen, Nat. Prod. Commun. 2007, 2, 889.
[4] Q. Ye, W. Zhao, Planta Med. 2002, 68, 723.
[5] C. Dubuisson, Y. Fukumoto, L. S. Hegedus, J. Am. Chem. Soc. 1995,
117, 3697.
[6] T. Miyase, K-I. Shiokawa, D. M. Zhang, A. Ueno, Phytochemistry
1996, 41, 1411.
[7] S. Miyase, K. Yoshikawa, S. Arihara, Chem. Pharm. Bull. 1992, 40,
2304.
[8] J. Kinjo, F. Kishida, K. Watanabe, F. Hashimoto, T. Nohara, Chem.
Pharm. Bull. 1994, 42, 1874.
[9] T. Araa, J. Kinjo, T. Nohara, R. Isobe, Chem. Pharm. Bull. 1995, 43,
1176.
[10] T. Taniyama, Y. Nagahama, M. Yoshikawa, I. Kitagam, Chem. Pharm.
Compound 2 was also isolated as a gummy material and
Bull. 1988, 36, 2829.
[11] B. C. Pal, T. Chaudhuri, K. Yoshikawa, S. Arihara, Phytochemistry
1994, 35, 1315.
[12] R. Cerri, R. Aquino, F. de Simone, C. Pizza, J. Nat. Prod. 1988, 51, 257.
[13] V. U. Ahmad, A. Basha, Spectroscopic Data of Saponins: The
Triterpenoid Glycosides, CRC Press: Boca Raton, 2000.
[14] J. Englert, B. Weniger, R. Lobstein, R. Anton, E. Krempp,
D. Guillaume, Y. Leroy, J. Nat. Prod. 1995, 58, 1265.
possessed a laevo optical rotation ([α]D
= −15◦, MeOH).
25
The IR spectrum of 2 showed absorption bands at 3431, 1735,
and 1032 cm−1, ascribable to hydroxyl, carbonyl, and ether
functionalities, respectively. The molecular formula, C47H76O20
of 2 was determined from the positive and negative ion FAB-MS
spectra at m/z 983 [M + Na]+ and 959 [M − H]−, respectively,
and by HRFAB-MS. The fragmentation pattern in the negative ion
c
Magn. Reson. Chem. 2008, 46, 986–989
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