New mono- and bisdesmosidic triterpene glycosides from Pittosporum angustifolium Lodd.

Article abstract of DOI:10.5560/ZNB.2014-4143

Fifteen new mono- and bisdesmosidic triterpene saponins, named pittangretosides J, K, M, Q-Z, A1, and B1, along with three known compounds were isolated from the leaves of Pittosporum angustifolium. By spectroscopic, mass spectrometric and chemical evidence, their structures were established as glycosides of A1- and R1-barrigenol, barringtogenol C and camelliagenin A backbones.

Full text of DOI:10.5560/ZNB.2014-4143

New Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum
angustifolium Lodd.
Christian Bäckera, Kristina Jenett-Siemsb, Karsten Siemsc, Martina Wurstera,
Anja Bodtked, Timo H. J. Niedermeyere, and Ulrike Lindequista
a
Department of Pharmaceutical Biology, Institute of Pharmacy, Ernst Moritz Arndt University
Greifswald, Friedrich-Ludwig-Jahn-Straße 17, 17489 Greifswald, Germany
Department of Pharmaceutical Biology, Institute of Pharmacy, Free University of Berlin,
b
Königin-Luise-Str. 2 + 4, 14195 Berlin, Germany
AnalytiCon Discovery GmbH, Hermannswerder Haus 17, 14473 Potsdam, Germany
Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Ernst Moritz
c
d
Arndt University Greifswald, Friedrich-Ludwig-Jahn-Straße 17, 17489 Greifswald, Germany
Interfaculty Institute of Microbiology and Infection Medicine, Eberhard Karls University
e
Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany
Reprint requests to Dipl.-Pharm. Christian Bäcker. Fax: +49(0)3834864885.
E-mail: cbaecker@uni-greifswald.de
Z. Naturforsch. 2014, 69b, 1026 – 1044 / DOI: 10.5560/ZNB.2014-4143
Received July 7, 2014
This work is respectfully dedicated to the loving memory of Adelheid Bombor, a unique character
and the most amiable and courageous person I have ever known (C. B.)
Fifteen new mono- and bisdesmosidic triterpene saponins, named pittangretosides J, K, M, Q–
Z, A1, and B1, along with three known compounds were isolated from the leaves of Pittosporum
angustifolium. By spectroscopic, mass spectrometric and chemical evidence, their structures were es-
tablished as glycosides of A1- and R1-barrigenol, barringtogenol C and camelliagenin A backbones.
Key words: Pittosporum angustifolium, Pittosporaceae, Triterpene Saponins, Pittangretosides
Introduction
pletely uninvestigated, triterpene glycosides could be
characterized as dominant secondary metabolites in
Pittosporum angustifolium Lodd. (Pittosporaceae) a few Pittosporum species [5 – 9], as well. Our ongo-
is a small tree, growing endemically in Australia’s in- ing phytochemical screening of the leaves of P. an-
land areas. The plant is colloquially known as “gumby gustifolium resulted in the identification of another
gumby”, and various medical preparations of the eighteen triterpene saponins. Fifteen of these are de-
leaves, seeds and fruits are used in the field of Aborig- scribed for the first time as natural products, and
inal ethnomedicine and further for the complementary their isolation and structural elucidation is herewith
treatment of malignant diseases [1, 2].
reported.
In recent phytochemical studies we have reported
the isolation and characterization of twelve new acy- Results and Discussion
lated triterpene saponins of the A1- and R1-barrigenol
type, named pittangretosides A–I and N–P, from the
The defatted crude 80% (v/v) ethanol extract of
leaves [2] and the seeds [3] of P. angustifolium. De- the leaves was purified and fractionated by chromato-
pending on the acylation pattern of those compounds, graphic procedures, using Sephadex LH20, silica gel
cytotoxic activity against different cancer cell lines and RP18 solid phase extraction. For the isolation of
has been observed [2, 3]. Additionally, five known compounds 1–18 (Figs. 1 and 2), obtained purified
polyphenolic constituents have been isolated from the subfractions were subjected to semipreparative HPLC.
leaves [4]. Although the polyphenolic phytochemistry New natural products 1–15 were named pittangreto-
of other Pittosporum species remains almost com- sides J, K, M, Q–Z, A1, and B1.
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1027
Fig. 2. Bisdesmosidic triterpene saponins isolated from the
leaves of Pittosporum angustifolium.
5.38 ppm in the 1H NMR spectrum (Table 1) and
further comparison of the NMR spectroscopic data
of 1 with those of known structures [2] confirmed
an A1-barrigenol (olean-12-ene-3β, 15α, 16α, 22α,
28-pentol) aglycone backbone. The 1H NMR spec-
trum showed four anomeric protons at δH = 4.49 (d,
J = 7.6), 4.87 (d, J = 7.0), 4.95 (d, J = 8.0), and
Fig. 1. Monodesmosidic triterpene saponins isolated from the
leaves of Pittosporum angustifolium.
According to their spectroscopic data, compounds 5.14 (br s) ppm, corresponding to δC = 105.7, 103.8,
16–18 were identified as the known R1-barrigenol 103.9, and 108.5 ppm in the HMQC spectrum (Ta-
glycosides 21β -angeloyloxy-22α -acetoxy-3β -[β - ble 2). As one of the anomeric carbon atoms (δC =
D -glucopyranosyl -(1→2)] -[α -L -arabinopyranosyl - 105.7) showed a cross peak with H-3 of the agly-
(1→3)] -[α -L -arabinofuranosyl -(1→4)] -β -D -gluc- cone part in the HMBC spectrum, and the chemi-
uronopyranosyloxyolean -12 -ene -15α,16α,28 -triol cal shift of C-3 at δC = 90.4 ppm was indicative of
(16) [5], 22α -(2-methylbutyroyloxy)-3β -[β -D-glu- a glycosylation, too, an attachment of the oligosac-
copyranosyl-(1→2)]-[α-L-arabinopyranosyl-(1→3)]- charide chain to C-3 of the aglycone could be as-
[α -L -arabinofuranosyl -(1→4)] -β -D -glucuronopyr- signed. By using extensive H-H COSY, HMBC, and
anosyloxyolean-12-ene-15α,16α,28-triol (17) [6], HMQC experiments, the glycoside moiety was deter-
and 21β -angeloyloxy-22α -angeloyloxy-3β -[β -D - mined as a 2,3,4-trisubstituted β-glucuronopyranosic
glucopyranosyl -(1→2)] -[α -L -arabinopyranosyl - acid, as recently identified for A1-barrigenol glyco-
(1→3)] -[α -L -arabinofuranosyl -(1→4)] -β -D -gluc- sides from P. angustifolium [2]. Unequivocal assign-
uronopyranosyloxyolean -12 -ene -15α,16α,28-triol ments were made by HMBC cross peaks between H-
(18) [5].
1 of a β-galactopyranose and δC = 80.8 ppm (GlcA-
Pittangretoside J (1) displayed in its high-resolution 2), H-1 of an α-arabinopyranose and δC = 79.9 ppm
ESI mass spectrum a quasimolecular ion [M–H]− at (GlcA-3), and H-1 of an α-arabinofuranose and δC =
m/z = 1091.5256 (neg. mode), predictive of a molec- 74.7 ppm (GlcA-4). Thiazolidine carboxylate analysis
ular formula of C52H84O24. Seven tertiary methyl by GC-MS revealed a D configuration for β-glucuronic
groups at δH = 0.92, 0.98, 0.99, 1.00, 1.04, 1.10, acid and β-galactose as well as an L configuration
and 1.39 ppm, an olefinic proton resonance at δH
=
for α-arabinose. In contrast to the A1-barrigenol gly-
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1
Table 1. 13C (125 MHz) and H (500 MHz) NMR spectroscopic data (ppm) of the aglycone moieties of compounds 1–3 in
CD3OD (J in Hz)a.
Position
1
2
3
13C
1H
13C
1H
13C
1H
1
2
3
4
5
6
7
8
39.7
26.0
90.4
39.5
55.5
18.3
35.8
41.4
47.9
36.9
23.7
124.7
143.8
47.9
67.3
72.5
44.3
42.0
46.8
31.6
45.1
73.0
27.8
15.3
16.1
16.8
19.4
67.5
32.8
24.5
1.64, 1.01
1.73, 1.94
3.20
39.9
n. d.
91.5
40.5
56.5
n. d.
36.9
42.4
48.3
38.0
n. d.
125.2
144.8
48.9
68.5
73.1
n. d.
43.3
47.5
32.3
45.4
73.9
28.2
16.8
16.1
17.7
20.7
68.7
33.5
24.6
0.98, 1.63
1.73, 1.95
3.17 dd (5.0;11.5)
39.5
23.5
91.2
39.4
55.6
18.6
36.2
41.2
47.2
36.8
23.5
126.1
143.0
47.5
67.6
73.0
48.7
40.4
46.6
35.5
76.5
83.9
27.4
15.8
15.1
16.8
19.7
60.8
28.8
18.2
1.01, 1.65
1.76, 1.65
3.23 dd (4.8;13.7)
–
–
–
0.79 d (11.7)
1.44, 1,58
1.70, 1.78
–
1.57
–
1.88, 1.96
5.38 brs
0.79 d (11.8)
1.39, 1.57
1.72, 1.77
0.80 d (12.0)
1.44, 1.55
1.75, n. d.
–
1.58
–
1.89, 1.94
5.46 br s
–
–
1.55
–
1.91
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.36 brs
–
–
–
–
–
3.66
3.65
3.94 d (4.4)
4.15 d (4.4)
3.93 d (4.5)
4.14 d (4.5)
–
2.16 dd (3.5;14.5)
1.01, 2.37 t (13.7)
–
1.44, 2.07 t (12.5)
4.05 dd (5.5;12.0)
1.10 s
–
2.15 dd (3.5;14.6)
1.00, 2.37 t (13.5)
–
1.43, 2.06 t (12.5)
4.05 dd (5.5;12.0)
1.08 s
2.56 brd (14.1)
2.46 t (13.4), 1.07
–
4.07
4.07
1.10 s
0.89 s
1.01 s
1.04 s
0.99 s
1.00 s
1.04 s
1.39 s
0.87 s
0.97 s
1.02 s
1.37 s
1.37 s
3.24 d (11.3), 3.65 d (11.3)
0.98 s
3.26 d (10.5), 3.55 d (10.5)
0.92 s
3.25 d (10.5), 3.53 d (10.5)
0.91 s
0.98 s
1.08 s
0.93 s
Acyl
–
–
–
–
Ac [Ara (p) II C-2]
1
2
–
–
–
–
–
–
–
–
169.6
20.8
–
2.15 s
–
–
–
–
Ac [Ara (p) II C-3]
1
2
–
–
–
–
–
–
–
–
170.2
20.6
–
1.98 s
–
–
–
–
Ac [Ara (p) II C-4]
1
2
–
–
–
–
–
–
–
–
170.4
20.7
–
2.11 s
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; Ara (p) II, second arabinopyranose at C-22; Ac, acetic acid.
cosides recently isolated from P. angustifolium [2], L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl-
no additional signals of acyl substituents were ob- (1→4)]-β -D -glucuronopyranosyloxyolean-12-ene-
served and H-15 (δH = 3.94), H-16 (δH = 4.15), H-22 15α,16α,22α,28-tetrol.
(δH = 4.05), and H-28 (δH = 3.26, 3.55 ppm) showed
The NMR spectroscopic data of pittangretoside K
normal shifts for unsubstituted hydroxymethine pro- (2) displayed a strong similarity to those of compound
tons and one primary alcoholic function, respectively. 1 (Tables 1 and 2). Since the ESI mass spectrum
Thus, the new natural product pittangretoside J (1) was revealed the same molecular formula of C52H84O24,
elucidated as 3β -[β -D-galactopyranosyl-(1→2)]-[α- which was deduced from a quasimolecular ion [M–
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
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Table 2. 13C (125 MHz) and 1H (500 MHz) NMR spectroscopic data (ppm) of the sugar moieties of compounds 1–3 in
CD3OD (J in Hz)a.
Position
1
2
3
13C
1H
13C
1H
13C
1H
C-3
GlcA
GlcA
GlcA
1
2
3
4
5
6
105.7
80.8
79.9
74.7
79.5
n. d.
4.49 d (7.6)
3.93
105.3
79.8
80.0
74.6
79.9
n. d.
4.45 d (7.6)
3.92
104.6
78.9
79.5
73.7
78.9
n. d.
4.57 d (6.5)
3.94
3.88
3.88
3.75
–
3.87
3.89
3.87
–
3.89
3.82
3.82
–
Gal
Glc
Glc
1
2
3
4
5
6
103.8
73.5
75.3
73.0
77.4
61.9
4.87 d (7.0)
3.58
3.49
3.65
3.48
3.67, 3.75
102.6
75.9
77.7
72.2
78.0
63.2
5.00 d (7 .0)
3.20
3.38
3.12 t (9.2)
3.30
101.9
74.9
77.2
71.4
77.8
63.4
5.03 d (7.8)
3.22
3.40 t (9.0)
3.12 t (9.6)
3.32
3.58, 3.82
3.67 dd (5.1;11.3), 3.87
Ara (p)
Ara (p)
Ara (p)
1
2
3
4
5
103.9
73.0
75.1
70.2
67.2
4.95 d (8.0)
3.65
3.52
3.75
3.57, 3.83
103.7
72.9
74.1
70.2
67.2
4.94 d (7.9)
3.58
3.51
3.75
3.55, 3.83
103.1
72.3
73.1
69.5
66.6
4.92 d (7.8)
3.60
3,52
3.79
3.52 dd (9.6;3.1), 3.84
Ara (f)
Ara (f)
Ara (f)
1
2
3
4
5
108.5
81.5
79.5
87.2
63.1
5.14 br s
3.95 br s
3.75
4.43 q (4.4)
3.66, 3.58
107.9
81.5
79.3
87.1
63.4
5.19 br s
3.96 br s
3.75
4.40 q (4.4)
3.66, 3.57
107.4
81.0
78.6
86.3
62.6
5.10 brs
3.97 brs
3.77
4.47 q (4.1)
3.71 dd (11.7;3.4); 3.57
Ara (p) II (C-22)
1
2
3
4
5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
102.5
70.5
71.3
68.6
64.3
4.73 d (7.7)
5.23 dd (8.4, 10.4)
5.15 dd (3.3, 10.0)
5.31 brs
4.05 m, 3.91
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; GlcA, glucuronopyranosic acid; Glc, glucopyranose; Gal, galactopyranose; Ara (p), arabinopyranose; Ara
(f), arabinofuranose; Ara (p) II, second arabinopyranose.
H]− at m/z = 1091.5263 (neg. mode), and as com- and GC-MS procedures, which gave signals of β-
pounds 2 and 1 were poorly separable by HPLC, their glucuronic acid, β-glucose and α-arabinose. The ab-
structural differences were assumed to be very small. solute configurations of sugars were determined as
Indeed, only the sugar resonances showed deviations, thiazolidine carboxylates, indicating the presence of
and no characteristic signals of a β-galactopyranose as β-D-glucuronic acid, β-D-glucose and α-L-arabinose.
found in compound 1 were observed. Instead, chem- The linkage of the oligosaccharide chain turned out
ical shifts for the proton and carbon resonances of to be the same as in 1, as HMBC cross peaks be-
a β-glucopyranose unit were assigned [C/H-1: δC = tween H-1 of the β-glucopyranose and δC = 79.8 ppm
102.6, δH = 5.00 (d, J = 7.0); C/H-2: δC = 75.9, (GlcA-2), H-1 of the α-arabinopyranose and δC
=
δH = 3.20; C/H-3: δC = 77.7, δH = 3.38; C/H-4: 80.0 ppm (GlcA-3), and H-1 of an α-arabinofuranose
δC = 72.2, δH = 3.12; C/H-5: δC = 78.0, δH = 3.30; and δC = 74.6 ppm (GlcA-4) were detected. The novel
C/H-6: δC = 63.2, δH = 3.58, 3.82 ppm]. This was compound pittangretoside K (2) was consequently
also supported by acid hydrolysis and subsequent TLC established as 3β -[β -D-glucopyranosyl-(1→2)]-[α-
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl- first bisdesmosidic triterpene saponin isolated from
(1→4)]-β -D -glucuronopyranosyloxyolean-12-ene- P. angustifolium, and its novel structure was thus
15α,16α,22α,28-tetrol.
elucidated as 3β -[β -D-glucopyranosyl-(1→2)]-[α-
The ESI mass spectrum of pittangretoside L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl-
M (3) showed quasimolecular ions [M–H]− at (1→4)] - β - D - glucuronopyranosyloxy - 22α - (2,3,4 -
m/z = 1365.5889 (neg. mode) and [M+2Na]2+ at triacetyloxy-α-L-arabinofuranosyloxy)-olean-12-ene-
m/z= 706.2785 (pos. mode) that were compatible 15α,16α,21β,28-tetrol.
with a molecular formula of C63H98O32. In the H
1
The ESI mass spectrum of pittangretoside Q (4)
NMR spectrum five resonances of anomeric protons displayed a quasimolecular ion [M–H]− at m/z =
were observed at δH = 4.57 (d, J = 6.5), 4.73 (d, 1157.5742 (neg. mode) predicting a molecular for-
J = 7.7), 4.92 (d, J = 7.8), 5.03 (d, J = 7.8), and 5.10 mula of C57H90O24, which meant one oxygen atom
(br s) ppm, and the corresponding carbon atom signals less than pittangretosides A and B which were recently
were assigned by HMBC correlations at δC = 104.6, isolated from the leaves of P. angustifolium [2]. Be-
102.5, 103.1, 101.9, and 107.4 ppm, respectively cause the NMR spectra of compound 4 were nearly
(Table 2). Furthermore, an olefinic proton resonance identical to those of pittangretoside B, again a strong
at δH = 5.46 ppm, seven signals of tertiary methyl structural similarity was proposed (Tables 3 and 4).
groups at δH = 0.89, 0.93, 0.98, 1.01, 1.04, 1.10, Characteristic signals of an angeloyl residue were ob-
and 1.37 ppm, six resonances for oxygenated carbons served at δH = 6.08 (q, J = 7.1) ppm for a methine
(Table 1) and comparison with literature data [3] proton resonance as well as a methyl doublet at δH =
identified compound 3 as a derivative of R1-barrigenol 1.98 ppm and another methyl singlet at δH = 1.91 ppm.
(olean-12-ene-3β, 15α, 16α, 21β, 22α, 28-hexol). The linking position at the aglycone moiety was deter-
Additionally, the NMR spectroscopic data included mined at C-22, because a long range correlation be-
characteristic signals of three acetyl residues. On tween H-22 (δH = 5.46 ppm) and the carbonyl car-
acid hydrolysis, 3 gave evidence for β-glucuronic bon atom of the angeloyl residue has been observed in
acid, β-glucose and α-arabinose. The corresponding the HMBC spectrum. The oligosaccharide part turned
thiazolidine carboxylates, analyzed by GC-MS, un- out to be same as in compound 1 and pittangreto-
derlined the absolute D configuration for β-glucuronic side B [2], consisting of a β-glucuronopyranosic acid,
acid and β-glucose, as well as the L configuration for a β-galactopyranose, an α-arabinopyranose, and an
α-arabinose. Interpretation of two-dimensional NMR α-arabinofuranose, which was also supported by hy-
spectra, including H-H COSY, HMBC, and HMQC, drolysis and subsequent TLC and GC-MS analysis.
led to the same oligosaccharide chain as well as the Again, the absolute configuration was determined as
same attachment at C-3 of the aglycone part as in D for β-glucuronic acid and β-galactose and L for
compound 2, because a long range correlation of one α-arabinose. In contrast to the similar compound pit-
of the anomeric carbon atoms (δC = 104.6 ppm) and tangretoside B [2], the proton and carbon resonances
H-3 (3.23 ppm) was clearly observed. The fifth sugar of compound 4 at C-15 were shifted upfield (δH
=
unit, identified as a second arabinopyranose (Ara 1.34, δC = 35.0 ppm), leading to the assumption, that
II), showed also a long range correlation between its this position was not oxidized as in pittangretoside
anomeric carbon atom (δC = 102.5 ppm) and the H-22 B. Thus, the aglycone part was identified as camel-
(δH = 4.07 ppm) of the aglycone which indicates an liagenin A (15-desoxy-A1-barrigenol), and the struc-
attachment at this position. The proton resonances of ture of pittangretoside Q was established as 22α-
H-2, H-3, and H-4 of that second arabinopyranose angeloyloxy-3β -[β -D-galactopyranosyl-(1→2)]-[α-
were suspiciously shifted downfield with δH = 5.23 L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl-
(dd, J = 8.4, 10.4), 5.15 (dd, J = 3.3, 10.0), and 5.31 (1→4)]-β -D -glucuronopyranosyloxyolean-12-ene-
(br s) ppm, respectively. Since HMBC correlations 16α,28-diol.
revealed cross peaks between H-2, H-3, and H-4
with a corresponding carbonyl carbon of one of the spectrum
Pittangretoside R (5) showed in its ESI mass
a
quasimolecular ion [M–H]− at
three acetyl groups, the second sugar attachment was m/z = 1239.5833 (neg. mode), corresponding to
unambiguously identified as a threefold acetylated a molecular formula of C57H92O29. From the 1H
arabinopyranose. So far, pittangretoside M (3) is the NMR spectrum five anomeric protons were assigned
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1031
1
Table 3. 13C (125 MHz) and H (500 MHz) NMR spectroscopic data (ppm) of the aglycone moieties of compounds 4–6 in
CD3OD (J in Hz)a.
Position
4
5
6
13C
1H
13C
1H
13C
1H
1
2
3
4
5
6
7
8
39.9
25.9
91.2
39.7
56.8
18.9
33.8
41.8
47.5
36.9
23.9
124.4
143.9
42.7
35.0
70.1
46.0
41.2
47.8
31.9
41.6
73.1
28.1
16.8
15.8
17.3
27.1
63.9
32.6
24.8
0.99, 1.64
1.73, 1.94
3.17 dd (4.1;10.1)
40.1
23.8
91.8
40.3
56.4
19.2
37.2
42.1
48.1
36.2
24.5
126.1
144.0
48.4
68.4
72.8
49.4
41.9
47.8
36.2
77.6
87.4
28.2
16.7
16.1
17.5
20.7
64.8
30.1
19.0
0.99, 1.64
1.72, 1.92
3.19
39.1
23.9
90.6
39.4
55.6
19.0
35.6
41.3
47.5
36.8
24.5
126.0
142.4
47.8
67.8
73.5
47.6
40.4
46.4
36.1
78.8
72.9
27.6
15.9
15.4
16.9
19.8
62.7
28.5
19.1
1.00, 1.64
1.70, 1.94
3.20 dd (3.4;12.4)
–
–
–
0.80 d (12.0)
1.41, 1.59
0.79 d (12.0)
1.36, 1.56
1.71, 1.76
0.81 d (11.8)
1.42, 1.60
1.72, 1.77
n. d.
–
1.57
–
1.95
5.37 br s
–
–
1.34
–
1.59
–
1.92
–
1.59
–
1.93
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.41 br s
5.50 br s
–
–
–
–
3.66
3.89
–
3.79 d
4.10 d (4.0)
4.13 br s
–
2.53 brd (15.2)
1.07, 2.47 t (12.5)
–
1.57, 2.28 t (11.6)
5.46 dd (5.2;12.0)
1.10 s
–
2.36 brd (15.0)
1.04 dd (3.0; 11.9), 2.45 t (13.1)
2.66
1.22 d (10.2), 2.66
–
5.97 d (10.7)
5.64 d (10.7)
1.11 s
–
4.03 d (9.5)
3.88 d (9.5)
1.07 s
0.87 s
0.99 s
1.02 s
1.36 s
2.31, 3.51
0.96 s
0.91 s
0.90 s
0.99 s
0.97 s
1.51 s
0.89 s
1.00 s
1.03 s
1.44 s
3.07 d (10.9), 3.29
0.93 s
3.06 d (10.7), 3.32
0.89 s
1.07 s
1.12 s
Acyl
Ang (C-21)
Ang (C-21)
1
2
3
4
5
169.1
129.8
137.3
15.4
–
–
–
–
–
–
–
–
–
–
–
–
168.4
128.3
138.2
15.2
–
–
6.08 q (7.1)
1.98
6.08 q (7.5)
1.94
20.9
1.91
19.6
1.86
Ang (C-22)
1
2
3
4
5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
168.4
128.3
138.2
15.2
–
–
6.08 q (7.5)
1.94
19.6
1.86
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; Ang, angelic acid.
at δH = 4.33 (d, J = 7.7), 4.51 (d, J = 7.6), 4.91 carboxylates of a hydrolyzed sugar portion showed
(d, J = 7.5), 5.01 (d, J = 7.7), and 5.13 (br s) ppm peaks for β-D-glucuronic acid, β-D-glucose and
(Table 4). A detailed look at H-H COSY, HMBC, and α-L-arabinose. The aglycone backbone was also
HMQC spectra revealed the same bisdesmosidic sugar established as R1-Barrigenol (Table 3). However,
linkage as in compound 3. Again, a tetrasaccharide compared to compound 3, the proton resonances of
chain was attached at C-3, and a second arabinopy- Ara II at H-2 (δH = 3.61 ppm), H-3 (δH = 3.51 ppm),
ranose (Ara II) was linked to C-22. Thiazolidine and H-4 (δH = 3.81 ppm) showed a normal shift for
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1032
C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
Table 4. 13C (125 MHz) and 1H (500 MHz) NMR spectroscopic data (ppm) of the sugar moieties of compounds 4–6 in
CD3OD (J in Hz)a.
Position
4
5
6
13C
1H
13C
1H
13C
1H
C-3
GlcA
GlcA
GlcA
1
2
3
4
5
6
105.6
80.0
79.0
75.0
78.2
n. d.
4.48 d (7.7)
3.94
105.3
79.3
80.1
74.8
77.5
n. d.
4.51 d (7.6)
3.92
104.7
79.3
79.9
73.7
79.0
n. d.
4.54 d (7.0)
3.91
3.90
3.91
3.78
–
3.89
3.90
3.78
–
3.89
3.82
3.77
–
Gal
Glc
Gal
1
2
3
4
5
6
103.3
72.5
76.4
73.0
76.3
62.0
4.88 d (6.5)
3.54
3.49
3.66
3.50
3.66, 3.82
102.6
75.9
77.8
72.3
78.2
63.2
5.01 d (7.7)
3.20 dd (7.9; 9.0)
3.38 t (9.0)
3.11 t (9.0)
3.30 m
102.3
72.2
76.0
72.5
76.1
61.6
4.88 d (6.8)
3.54
3.49
3.67
3.50
3.67, 3.82
3.83, 3.65
Ara (p)
Ara (p)
Ara (p)
1
2
3
4
5
103.6
72.9
73.0
69.9
67.4
4.94 d (7.5)
3.61
3.50
3.75
3.60, 3,84
103.9
72.8
72.8
70.4
67.4
4.91 d (7.5)
3.58
3.51
3.76
3.56, 3.83
102.9
72.3
72.6
69.7
66.8
4.94 d (7.6)
3.59
3.53
3.77
3.56, 3.84
Ara (f)
Ara (f)
Ara (f)
1
2
3
4
5
107.6
81.7
79.5
87.1
62.6
5.18 brs
3.98 brs
3.76
4.44 q (4.5)
3.68, 3.57
107.9
81.6
79.3
86.8
63.1
5.13 brs
3.96 brs
3.76
4.44 q (4.4)
3.67, 3.57
106.6
80.4
79.0
86.5
63.0
5.14 brs
3.97 brs
3.75
4.45 q (4.3)
3.66, 3.55
Ara (p) II (C-22)
1
2
3
4
5
–
–
–
–
–
–
–
–
–
–
106.3
72.8
74.4
69.8
67.7
4.33 d (7.7)
3.61
3.51
3.81
3.65, 3.92
–
–
–
–
–
–
–
–
–
–
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; GlcA, glucuronopyranosic acid; Glc, glucopyranose; Gal, galactopyranose; Ara (p), arabinopyranose; Ara
(f), arabinofuranose; Ara (p) II, second arabinopyranose.
unesterified hydroxyl groups. Furthermore, no signals 1271.6080 (neg. mode), leading to a molecular for-
of acetyl or other acyl groups were observed. This was mula of C62H96O27, which has already been assigned
also underlined by the lack of ATR-IR signals at 1737 for the known compound 18 [5, 10]. Since the sepa-
and 1228 cm−1 that were assigned for the carbonyl ration of both compounds, 6 and 18, by HPLC was
part of the threefold acetylated Ara II in compound a challenge, and because of a common ESI-MS frag-
3. The new structure of pittangretoside R was thus ment ion pattern and almost identical chromatographic
determined as 3β-[β-D-glucopyranosyl-(1→2)]-[α-L- behavior, it was presumed that compound 6 could be
arabinopyranosyl - (1→3)] - [α - L - arabinofuranosyl - an isomer of 18, in which the glucose of the tetrasac-
(1→4)] - β - D - glucuronopyranosyloxy - 22α - L - charide chain was again replaced by galactose. This
arabinofuranosyloxyolean-12-ene-15α,16α,21β,28- was confirmed by 1H NMR spectra, showing signals of
tetrol.
anomeric protons at δH = 4.54 (d, J = 7.0, GlcA), 4.94
The ESI mass spectrum of pittangretoside S (6) re- [d, J = 7.6, Ara (p)], 5.14 [br s, Ara (f)] and finally 4.88
vealed a quasimolecular ion peak [M–H]− at m/z = (d, J = 6.8) ppm for the galactose moiety (Table 4),
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1033
and, moreover, by TLC and GC-MS analysis. The ab- charide chain of compound 3 was again replaced
solute configuration was determined to be D for β- by a galactose moiety in compound 8. The second
glucuronic acid and β-galactose and L for α-arabinose. arabinopyranose (Ara II) at C-22 was also threefold
As the remaining part of the chemical structure of com- acetylated as found in 3, which was supported by
pound 6 was identical to that of glycoside 18 (Ta- NMR data and strong signals in the ATR-IR spec-
ble 3), the new natural product pittangretoside S was trum at 1736 and 1223 cm−1. On acid hydrolysis,
consequently elucidated as 21β -angeloyloxy-22α - followed by TLC and GC-MS procedures, 8 gave β-
angeloyloxy-3β -[β -D-galactopyranosyl-(1→2)]-[α- D-glucuronic acid, β-D-galactose and α-L-arabinose.
L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl- Thus, the new compound pittangretoside U was
(1→4)]-β -D -glucuronopyranosyloxyolean-12-ene- established as 3β-[β-D-galactopyranosyl-(1→2)]-[α-
15α,16α,28-triol.
L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl-
Pittangretoside T (7) had a molecular formula (1→4)] - β - D - glucuronopyranosyloxy - 22α - (2,3,4 -
of C62H96O26, deduced from a quasimolecular ion triacetyloxy-α-L-arabinofuranosyloxy)-olean-12-ene-
[M–H]− at m/z = 1255.6174 (neg. mode) in its ESI 15α,16α,21β,28-tetrol.
mass spectrum, just one oxygen atom less than the
Pittangretoside V (9) showed in its ESI mass
compounds 6 and 18. With respect to the NMR spectrum a quasimolecular ion peak [M–H]− at
spectroscopic data, compound 7 showed a strong m/z = 1281.5726 (neg. mode), which gave a molecu-
structural similarity to glycoside 6, and resonances lar formula of C59H94O30. The NMR data were nearly
for two angeloyl residues as well as for the same identical to those of compound 5 (Tables 5 and 6),
oligosaccharide chain as in 6, consisting of a β- except for three additional signals at δC = 171.6,
glucuronopyranosic acid, a β-galactopyranose, an 20.1 and δH = 2.14 (s) ppm corresponding to an
α-arabinopyranose, and an α-arabinofuranose, have acetyl group. Again, a backbone of R1-barrigenol
been assigned (Tables 5 and 6). GC-MS data corrob- was deduced from extensive H-H COSY, HMBC, and
orate an absolute configuration of β-D-glucuronic HMQC experiments, possessing the same branched
acid, β-D-galactose and α-L-arabinose. A detailed tetrasaccharide attached to C-3 and a second arabino-
look at the NMR data revealed again – as already furanose (Ara II) at C-22, including identical absolute
found for compound 4 – a lack of oxidation at configurations, as already described for compounds 3,
C-15 (δH = 1.39, 1.70, δC = 33.7 ppm) as the and 5. Nevertheless, H-3 of Ara II appeared downfield
only difference between both compounds, 6 and 7. shifted at δH = 4.77 (dd, J = 3.1, 10.0) ppm, and as
So, pittangretoside T is the first saponin isolated this proton displayed a long range correlation with
from P. angustifolium possessing a barringtogenol the carbonyl carbon atom of the acetyl residue in the
C
(15-desoxy-R1-barrigenol) aglycone. Its new HMBC spectrum, the acylation position was unequivo-
structure was elucidated as 21β -angeloyloxy-22α- cally determined to be at C-3 of Ara II. Consequently,
angeloyloxy-3β -[β -D-galactopyranosyl-(1→2)]-[α- the novel natural product pittangretoside V was
L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl- elucidated as 3β-[β-D-glucopyranosyl-(1→2)]-[α-L-
(1→4)]-β -D -glucuronopyranosyloxyolean-12-ene- arabinopyranosyl - (1→3)] - [α - L - arabinofuranosyl -
16α,28-diol.
(1→4)] - β - D - glucuronopyranosyloxy - 22α - (3 -
acetyloxy - α - L - arabinofuranosyloxy) - olean - 12 -
The ESI mass spectrum of pittangretoside U (8)
displayed a quasimolecular ion [M–H]− at m/z = ene-15α,16α,21β,28-tetrol.
1365.5968 (neg. mode) which substantiated a molec-
Pittangretoside W (10) had the same molecular for-
ular formula of C63H97O32, already found for the mula as compound 9, C59H94O30, substantiated by its
bisdesmosidic compound 3. The 1H NMR spectrum of ESI mass spectrum and a quasimolecular ion [M–H]−
8 also showed five anomeric protons at δH = 4.58 (d, at m/z = 1281.5757 (neg. mode). Again, nearly all
J = 6.8), 4.72 (d, J = 7.8), 4.89 (d, J = 6.8), 4.94 (d, resonances of recorded NMR spectra were identical
J = 7.5), and 5.11 (br s) ppm, which were assigned to those of compound 9 (Tables 7 and 8), which was
to their anomeric carbon atoms at δC = 104.7, 102.3, isolated from the same purified fraction. This time,
102.8, 104.0, and 107.4 ppm in the HMQC spectrum no differences between the sugar compositions were
(Table 6). Further analysis of two-dimensional NMR observed, since 10 displayed also anomeric protons
spectra revealed that the glucose of the tetrasac- at δH = 4.41 (d, J = 7.7), 4.53 (d, J = 6.5), 4.93 (d,
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1034
C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1
Table 5. 13C (125 MHz) and H (500 MHz) NMR spectroscopic data (ppm) of the aglycone moieties of compounds 7–9 in
CD3OD (J in Hz)a.
Position
7
8
9
13C
1H
13C
1H
13C
1H
1
2
3
4
5
6
7
8
38.9
23.9
90.9
39.6
55.9
18.8
32.9
39.9
47.9
36.3
23.8
124.3
142.0
41.6
33.7
68.7
47.2
40.4
46.9
36.0
78.7
73.2
27.4
16.0
15.6
16.3
26.5
63.6
28.5
19.2
0.99, 1.66
1.74, 1.94
3.19 brd (11.4)
39.0
25.7
90.7
39.3
55.7
19.2
35.8
40.8
47.2
36.5
23.9
126.3
143.0
47.6
67.7
73.0
48.9
40.4
46.5
35.3
76.6
83.8
27.3
16.3
15.9
16.9
19.7
60.8
28.7
18.0
0.98, 1.66
1.73, 1.88
3.20 dd (4.0;12.0)
38.5
24.9
91.1
39.1
55.7
19.8
35.9
41.4
47.4
36.8
23.9
126.0
143.0
47.4
67.5
72.5
n. d.
41.2
46.7
35.3
77.0
86.9
27.3
16.4
15.8
17.0
19.7
63.8
29.4
18.0
0.99, 1.65
1.74, 1.90
3.19
–
–
–
0.80 d (11.4)
1.46, 1.60
1.71, n. d.
0.79 d (11.5)
1.43, 1.53
0.80 d (11.9)
1.45, 1.55
1.75
–
1.59
–
1.92
5.44 br s
–
–
3.66
1.76
–
1.60
–
1.94
5.43 br s
–
–
3.81
–
1.56
–
1.94
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.41 brs
–
–
1.39, 1.70
4.02 brs
3.65
–
4.10 d (4.0)
–
2.39 brd (13.8)
1.07, 2.47 t (13.0)
–
–
2.65 brd (15.6)
1.22 dd (3.0;11.6), 2.66 t (14.2)
2.56 br d (14.4)
1.05, 2.46 t (13.1)
–
4.04 d (10.0)
4.07 d (10.0)
1.10 s
–
6.01 d (10.1)
5.59 d (10.1)
1.10 s
4.06 d (9.6)
3.93 d (9.6)
1.10 s
0.89 s
1.00 s
1.03 s
1.38 s
3.33, 3.54
0.99 s
0.93 s
0.89 s
1.00 s
0.96 s
1.51 s
0.89 s
1.00 s
1.03 s
1.36 s
2.97 d (10.9), 3.29 d (10.9)
0.89 s
3.25 d (10.8), 3.66
0.98 s
1.12 s
0.93 s
Acyl
Ang (C-21)
Ac [Ara (p) II C-2]
Ac [Ara (p) II C-3]
1
2
168.7
128.5
–
–
170.3
20.0
–
171.6
20.1
–
2.15 s
2.14 s
3
138.2
6.08 q (7.5)
Ac [Ara (p) II C-3]
–
–
4 (1)
5 (2)
15.0
19.5
1.94
1.85
170.6
19.6
–
–
–
–
–
1.98 s
Ang (C-22)
Ac [Ara (p) II C-4]
1
2
3
4
5
168.7
128.5
138.2
15.0
–
–
171.0
19.5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2.11 s
6.10 q (7.5)
1.94
–
–
–
19.5
1.85
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; Ara (p) II, second arabinopyranose at C-22; Ang, angelic acid, Ac, acetic acid.
J = 7.5), 5.04 (d, J = 7.7), and 5.15 (br s) ppm, and has been localized at C-22 by HMBC correlations,
GC-MS analysis of the corresponding thialzolidine showed a clearly downfield shifted resonance of
carboxylates confirmed a D configuration for β- H-4 at δH = 5.07 ppm (br s). Further analysis of the
glucuronic acid, and β-glucose, and an L configuration HMBC spectrum of compound 10 revealed a cross
for α-arabinose. Instead, the second arabinofuranose peak between this proton and the carbonyl carbon
(Ara II), whose attachment position to the aglycone atom of an acetyl group. Thus, pittangretosides V and
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1035
Table 6. 13C (125 MHz) and 1H (500 MHz) NMR spectroscopic data (ppm) of the sugar moieties of compounds 7–9 in
CD3OD (J in Hz)a.
Position
7
8
9
13C
1H
13C
1H
13C
1H
C-3
GlcA
GlcA
GlcA
1
2
3
4
5
6
104.3
79.6
79.8
72.7
79.0
n.d.
4.53 d (7.0)
3.91
104.7
79.4
79.6
72.8
78.6
n.d.
4.58 d (6.8)
3.92
105.1
80.3
78.4
74.1
78.7
n.d.
4.53 d (6.5)
3.92
3.88
3.78
3.78
–
3.90
3.79
3.80
–
3.91
3.92
3.73
–
Gal
Gal
Glc
1
2
3
4
5
6
102.6
72.0
75.8
72.2
75.8
61.3
4.89 d (6.9)
3.55
3.49
3.65
3.53
3.67, 3.77
102.8
72.0
75.5
72.2
75.6
62.1
4.89 d (6.8)
3.56
3.48
3.65
3.52
3.67, 3.81
101.7
75.2
76.8
71.6
77.3
61.9
5.04 d (7.7)
3.22 t (8.4)
3.39 t (9.0)
3.12 t (9.0)
3.30
3.59, 3.85
Ara (p)
Ara (p)
Ara (p)
1
2
3
4
5
102.9
72.7
72.3
69.0
66.5
4.94 d (7.7)
3.62
3.52
3.78
3.57, 3.85
103.0
72.5
72.2
69.4
66.3
4.94 d (7.5)
3.61
3.53
3.78
3.57, 3.85
102.9
72.5
72.1
69.6
66.8
4.93 d (7.5)
3.57
3.52
3.77
3.57, 3.85
Ara (f)
Ara (f)
Ara (f)
1
2
3
4
5
106.9
80.6
79.0
86.6
63.1
5.16 brs
3.96 brs
3.77
4.44 q (4.5)
3.66, 3.58
107.4
80.6
78.7
86.6
62.0
5.11 brs
3.97 brs
3.74
4.47 q (3.6)
3.68, 3.58
107.3
81.0
78.1
86.6
62.4
5.15 brs
3.97 brs
3.77
4.47
3.67 dd (12.1; 4.5), 3.57
Ara (p) II (C-22)
Ara (p) II (C-22)
1
2
3
4
5
–
–
–
–
–
–
–
–
–
–
102.3
70.4
70.9
68.5
64.2
4.72 d (7.8)
5.24 dd (7.8;10.2)
5.14 dd (3.3;10.2)
5.31 brs
105.1
70.3
76.4
66.9
66.6
4.45 d (7.7)
3.81
4.77 dd (3.1;10.0)
4.02 brs
3.89, 4.06
3.49, 3.93
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; GlcA, glucuronopyranosic acid; Glc, glucopyranose; Gal, galactopyranose; Ara (p), arabinopyranose; Ara
(f), arabinofuranose; Ara (p) II, second arabinopyranose.
W differ only in the acetylation position at Ara II, and and 8) confirmed the same bisdesmosidic structure
the new chemical structure of pittangretoside W was as found in compound 8, with galactose as part
determined as 3β-[β-D-glucopyranosyl-(1→2)]-[α-L- of the tetrasaccharide chain at C-3 and a second
arabinopyranosyl - (1→3)] - [α - L - arabinofuranosyl - arabinopyranose (Ara II) linked to C-22. The absolute
(1→4)] - β - D - glucuronopyranosyloxy - 22α - (4 - configuration of the sugars by their corresponding
acetyloxy - α - L - arabinofuranosyloxy) - olean - 12 - thiazolidine carboxylates revealed D for β-glucuronic
ene-15α,16α,21β,28-tetrol.
acid and β-galactose, and further L for α-arabinose.
From the ESI mass spectrum of pittangretoside Since the ATR-IR spectra of compound 11 showed
X (11) and a detected quasimolecular ion peak [M– strong signals at 1737 and 1229 cm−1, and the proton
H]− at m/z = 1349.5986, a molecular formula of resonances of H-2, H-3, and H-4 of Ara II appeared
C63H98O31 was predicted. In contrast to compounds downfield shifted at δH = 5.14 (dd, J = 7.7, 10.0),
3 and 8, this meant the loss of an oxygen atom. 5.11 (dd, J = 3.0, 10.0), and 5.28, (br s) ppm, re-
A more detailed look at the NMR data (Tables 7 spectively, each of those three positions were again
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1036
C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
Table 7. 13C (125 MHz) and 1H (500 MHz) NMR spectroscopic data (ppm) of the aglycone moieties of compounds 10–12 in
CD3OD (J in Hz)a.
Position
10
11
12
13C
1H
13C
1H
13C
1H
1
2
3
4
5
6
7
8
39.2
23.7
91.0
39.3
55.6
18.4
35.8
41.1
47.3
36.4
23.5
125.7
143.4
47.2
67.1
72.3
47.8
41.3
47.6
35.3
77.0
86.7
27.4
16.5
15.6
17.2
19.5
63.8
28.7
17.9
0.99, 1.61
1.75, 1.91
3.21
–
0.81 d (12.0)
1.43, 1.56
39.8
24.5
91.2
39.4
55.8
19.2
36.0
41.1
47.1
36.6
23.5
125.5
143.7
47.6
67.6
73.3
n.d.
41.1
46.8
31.4
43.3
78.9
27.6
16.0
15.9
16.6
20.0
61.5
32.4
24.0
0.97, 1.65
1.72, 1.94
3.18
–
0.79 d (11.9)
1.42, 1.55
1.74
–
1.56
–
1.93
5.43 brs
39.1
25.6
91.5
39.5
55.4
n.d.
35.4
41.2
47.4
36.8
24.0
125.0
143.7
47.7
67.5
73.2
n.d.
40.6
46.2
31.4
43.6
78.9
27.4
15.8
16.1
16.6
19.4
61.5
32.6
24.2
0.99, 1.62
1.74, 1.93
3.20 dd (4.5;11.0)
–
0.80 d (12.2)
1.43, 1.55
1.74, 1.77
1.76
–
1.56
–
1.95
–
1.59
–
1.92
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.43 br s
–
5.44 brs
–
–
3.84
3.66
–
–
–
–
3.81 d (4.4)
4.13 d (4.4)
–
3.68 d (4.5)
3.74 d (4.5)
–
2.38 brd (13.8)
1.07, 2.46 t (13.8)
–
2.52 brd (14.2)
1.00, 2.36 t (13.0)
–
1.70, 2.16 t (13.5)
4.25 dd (6.5;13.0)
1.10 s
2.53 brd (15.0)
1.02, 2.36 t (13.3)
–
1.71, 2.15 t (12.8)
4.25 dd (6.4;12.8)
1.09 s
4.06 d (9.7)
3.90
1.10 s
0.89 s
1.00 s
1.05 s
1.39 s
3.33, 3.52
0.98 s
0.93 s
0.88 s
1.00 s
1.04 s
1.37 s
0.89 s
1.01 s
1.05 s
1.37 s
3.20 d (11.2), 3.30
0.90 s
3.20 d (12.0), 3.30
0.91 s
0.97 s
0.98 s
Acyl
Ac [Ara (p) II C-4]
Ac [Ara (p) II C-2]
Ac [Ara (p) II C-2]
1
2
171.3
19.8
–
170.7
19.5
–
170.8
19.8
–
2.13 s
2.15 s
2.15 s
Ac [Ara (p) II C-3]
Ac [Ara (p) II C-3]
1
2
–
–
–
–
170.9
20.1
–
170.6
20.3
–
1.99 s
1.99 s
Ac [Ara (p) II C-4]
Ac [Ara (p) II C-4]
1
2
–
–
–
–
170.9
19.5
–
170.6
19.8
–
2.10 s
2.10 s
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; Ara (p) II, second arabinopyranose at C-22; Ac, acetic acid.
acylated with acetic acid. Cross peaks between these determined as A1-barrigenol, and the structure was
protons and the corresponding carbonyl carbon atoms elucidated as 3β -[β -D-galactopyranosyl-(1→2)]-[α-
have been detected in the HMBC spectrum. The L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl-
distinguishing feature to compound 8 (R1-barrigenol) (1→4)] - β - D - glucuronopyranosyloxy - 22α - (2,3,4 -
was found to be at C-21 of compound 11, show- triacetyloxy-α-L-arabinofuranosyloxy)-olean-12-ene-
ing shifts for a methylene group (δH = 1.70, 2.16, 15α,16α,28-triol.
δC = 43.3 ppm) instead of a hydroxymethin group.
Compared to compound 11, pittangretoside Y (12)
Thus, the aglycon part of pittangretoside X (11) was possesses the same molecular formula of C63H98O31,
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1037
1
Table 8. 13C (125 MHz) and H (500 MHz) NMR spectroscopic data (ppm) of the sugar moieties of compounds 10–12 in
CD3OD (J in Hz)a.
Position
C-3
10
11
12
13C
1H
13C
1H
13C
1H
GlcA
GlcA
GlcA
1
2
3
4
5
6
104.8
79.2
79.3
74.5
78.6
n.d.
4.53 d (6.5)
3.93
104.8
79.8
79.3
73.0
78.3
n.d.
4.48 d (7.7)
3.95
104.9
81.0
79.2
74.9
78.8
n.d.
4.53 d (7.0)
3.97
3.90
3.93
3.73
–
3.89
3.79
3.79
–
3.92
3.90
3.77
–
Glc
Gal
Glc
1
2
3
4
5
6
101.6
75.6
77.0
71.6
77.3
62.2
5.04 d (7.7)
3.23 t (8.5)
3.39 t (9.0)
3.12 t (9.3)
3.31
102.6
72.5
75.8
72.4
75.6
61.8
4.89 d (7.0)
3.54
3.48
3.66
3.52
3.67, 3.84
101.6
74.8
76.9
71.5
77.2
61.9
5.04 d (7.5)
3.21
3.38 t (9.0)
3.13 t (9.5)
3.30
3.58, 3.85
3.58, 3.84
Ara (p)
Ara (p)
Ara (p)
1
2
3
4
5
103.2
72.0
72.0
69.2
66.5
4.93 d (7.5)
3.60
3.533
3.77
3.55, 3.89
103.0
72.9
72.4
69.5
66.7
4.95 d (7.7)
3.64
3.52
3.78
3.54, 3.83
103.0
72.2
72.9
69.2
66.4
4.94 d (7.5)
3.61
3.52 dd (3.0;9.5)
3.78
3.56, 3.84
Ara (f)
Ara (f)
Ara (f)
1
2
3
4
5
107.2
81.2
78.5
86.8
62.3
5.15 brs
3.98 brs
3.79
4.46 q (4.0)
3.66, 3.58
107.2
90.7
78.5
86.7
62.2
5.17 brs
3.98 brs
3.77
4.46 q (4.1)
3.66, 3.60
107.0
81.4
79.2
87.0
62.5
5.16 brs
3.96 brs
3.75
4.46 q (4.0)
3.68, 3.57
Ara (p) II (C-22)
Ara (p) II (C-22)
Ara (p) II (C-22)
1
2
3
4
5
104.9
72.4
72.1
71.7
64.8
4.41 d (7.7)
3.65
3.72
5.07 brs
102.8
70.3
71.5
68.6
64.0
4.70 d (7.7)
5.14 dd (7.7;10.0)
5.11 dd (3.0;10.0)
5.28 brs
102.9
70.5
71.4
68.6
64.1
4.70 d (7.5)
5.15 dd (7.5;10.0)
5.11 dd (3.5;10.0)
5.29 brs
3.73, 3.99 brd (12.0)
3.80, 3.99
3.80, 3.99 brd (14.6)
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; GlcA, glucuronopyranosic acid; Glc, glucopyranose; Gal, galactopyranose; Ara (p), arabinopyranose; Ara
(f), arabinofuranose; Ara (p) II, second arabinopyranose.
as deduced from a quasimolecular ion [M–H]− at (1→4)] - β - D - glucuronopyranosyloxy - 22α - (2,3,4 -
m/z = 1349.5988. The ATR-IR, NMR (Tables 7 triacetyloxy-α-L-arabinofuranosyloxy)-olean-12-ene-
and 8) and HRMS data of 12 were again nearly 15α,16α,28-triol.
identical to those of 11. More detailed studies of
For pittangretoside Z (13), a molecular formula
the sugar components (NMR, hydrolysis, absolute of C57H90O26 was deduced from a quasimolecular
configuration) verified that galactose in compound 11 ion peak [M–H]− at m/z = 1189.5634 from its ESI
1
was replaced once more by glucose in compound 12. mass spectrum. The H NMR spectrum revealed four
So far, compounds 1 and 2, 3 and 8, 6 and 18, and anomeric proton resonances at δH = 4.51 (d, J = 6.5),
11 and 12 are pairs of isomers, differing only in the 4.93 (d, J = 7.5), 5.04 (d, J = 7.7), and 5.17 (br
presence of glucose or galactose in the tetrasaccharide s) ppm (Table 10), that turned out to belong to a β-
chain. Consequently, pittangretoside Y (12) was finally glucuronopyranosic acid, a β-glucopyranose, an α-
established as 3β -[β -D-glucopyranosyl-(1→2)]-[α- arabinopyranose, and an α-arabinofuranose unit of
L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl- the common tetrasaccharide chain as in compound 2.
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1038
C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
Table 9. 13C (125 MHz) and 1H (500 MHz) NMR spectroscopic data (ppm) of the aglycone moieties of compounds 13–15 in
CD3OD (J in Hz)a.
Position
13
14
15
13C
1H
13C
1H
13C
1H
1
2
3
4
5
6
7
8
38.7
24.8
91.0
39.5
55.6
n.d.
1.00, 1.65
1.73, 1.92
3.21
–
0.81 d (12.0)
1.44, 1.56
1.77
–
1.57
–
1.92
5.46 brs
39.3
24.1
90.3
39.6
56.3
18.9
32.8
40.3
46.9
36.3
23.9
124.0
141.9
41.6
33.7
68.3
46.4
40.5
0.97, 1.62
1.71, 1.97
3.19
–
0.81 d (11.7)
1.46, 1.60
n.d.
–
1.59
–
1.93
5.39 brs
38.4
n.d.
0.99, 1.65
1.75, 1.88
3.20 dd (4.5, 14.0)
91.1
39.7
55.8
19.2
32.9
40.3
47.0
36.5
23.7
123.8
142.0
41.2
33.7
68.4
47.4
40.4
–
0.81 d (11.9)
1.46, 1.58
35.6
41.1
47.1
36.6
23.7
125.8
143.0
47.3
67.3
72.2
48.2
41.5
47.1
35.5
81.2
72.1
27.5
16.1
15.8
17.1
19.5
64.6
28.9
19.2
1.75
–
1.58
–
1.94
5.40 brs
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
–
–
3.84
3.98
–
–
–
1.37, 1.74
4.03 brs
–
–
1.38, 1.74
4.04 brs
–
–
2.47 brd (14.2)
1.17 dd (4.0;13.3), 2.59 t 13.3)
2.60 brd (13.8)
2.59 brd (13.5)
46.9 1.20 dd (3.4;13.0), 2.69 t (13.8) 47.0 1.20 dd (3.7;12.7), 2.69 t (13.2)
–
5.61 d (10.2)
4.03 d (10.2)
1.10 s
0.89 s
1.01 s
1.04 s
1.41 s
3.28, 3.41
0.87 s
1.04 s
35.3
78.8
74.3
27.6
15.8
15.2
15.8
26.7
63.9
28.3
19.5
–
5.90 d (10.0)
5.50 d (10.0)
1.10 s
36.0
78.9
74.2
27.4
16.1
15.4
15.8
26.8
63.8
28.6
19.2
–
5.90 d (10.3)
5.52 d (10.3)
1.11 s
0.89 s
0.99 s
0.95 s
1.50 s
0.89 s
1.00 s
0.96 s
1.50 s
3.01 d (11.0), 3.26 d (11.0)
0.88 s
3.01 d (11.3), 3.27 d (11.3)
0.89 s
1.09 s
1.10 s
Acyl
Ang (C-21)
Ang (C-21)
Ang (C-21)
1
2
3
4
5
169.2
128.9
137.2
15.1
–
–
168.3
128.4
138.1
15.1
–
–
168.2
128.5
138.0
15.2
–
–
6.11 q (7.1)
2.00
6.13 q (7.2)
1.97
6.13 q (7.0)
1.97
20.1
1.95
19.7
1.87
19.9
1.87
Ac (C-22)
Ac (C-22)
1
2
–
–
–
–
172.1
20.0
–
172.3
20.0
1.97 s
1.97 s
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; Ang, angelic acid; Ac, acetic acid.
Again, GC-MS techniques confirmed the absolute con- tion with H-21 of the R1-barrigenol aglycone moiety
figuration of the corresponding thiazolidine carboxy- at δH = 5.61 (d, J = 10.2) ppm, whereby the attach-
lates of hydrolyzed sugars as D for β-glucuronic acid ment position of that residue was unambiguously as-
and β-glucose, and L for α-arabinose. A characteristic signed. Comparing compound 13 with the known gly-
proton resonance at 6.11 ppm (q, J = 7.1) and two ad- coside 16, the latter one possesses an additional acy-
ditional methyl signals at δH = 1.95 and 2.00 ppm indi- lation with acetic acid at C-22. Finally, the new struc-
cated an angeloyl moiety (Table 9). Its carbonyl carbon ture of pittangretoside Z (13) was established as 21β-
atom at δC = 169.2 ppm showed a long range correla- angeloyloxy-3β -[β -D -glucopyranosyl-(1→2)]-[α -
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1039
Table 10. 13C (125 MHz) and 1H (500 MHz) NMR spectroscopic data (ppm) of the sugar moieties of compounds 13–15 in
CD3OD (J in Hz)a.
Position
13
14
15
13C
1H
13C
1H
13C
1H
C-3
GlcA
GlcA
GlcA
1
2
3
4
5
6
104.8
79.0
79.6
75.8
78.5
n.d.
4.51 d (6.5)
3.93
104.8
79.6
78.6
72.7
79.0
n.d.
4.49 d (6.8)
3.90
104.8
81.0
79.1
73.6
78.2
n.d.
4.52 d (6.5)
3.95
3.90
3.91
3.77
–
3.88
3.79
3.77
–
3.92
3.95
3.71
–
Glc
Gal
Glc
1
2
3
4
5
6
101.8
75.0
77.5
72.2
77.5
61.9
5.04 d (7.7)
3.22 t (8.5)
3.38 t (9.0)
3.13 t (9.0)
3.30
102.9
72.0
75.9
72.4
75.7
61.7
4.90 d (7.0)
3.55
3.48
3.64
3.54
3.67, 3.75
101.8
75.5
76.8
71.3
77.8
62.4
5.04 d (7.8)
3.21 t (8.2)
3.39 t (9.0)
3.13 t (9.5)
3.29
3.59, 3.85
3.67, 3.84 dd (5.3; 12.3)
Ara (p)
Ara (p)
Ara (p)
1
2
3
4
5
103.0
72.1
73.5
69.5
66.3
4.93 d (7.5)
3.52
3.50 dd (3.3;9.7)
3.76
103.3
72.5
72.5
69.2
67.0
4.96 d (7.5)
3.63
3.52 dd (3.0;9.6)
3.78
103.3
72.2
72.5
69.2
66.6
4.93 d (7.4)
3.63
3.51 dd (3.0;9.5)
3.77
3.59, 3.76
3.61, 3.85
3.54, 3.84
Ara (f)
Ara (f)
Ara (f)
1
2
3
4
5
n.d.
80.7
78.8
86.3
62.4
5.17 brs
3.97 brs
3.78
4.46 q (4.3)
3.67, 3.58
107.1
80.8
78.8
86.9
63.1
5.14 brs
3.98 brs
3.77
4.44 q (4.8)
3.67, 3.58
107.0
80.8
78.8
86.0
63.0
5.14 brs
3.98 brs
3.78
4.46 q (4.0)
3.66, 3.58
a
Assignments were made by 1H-1H COSY, HMBC, and HMQC experiments; overlapped 1H resonances are reported without designated
multiplicity; n. d., not determined; GlcA, glucuronopyranosic acid; Glc, glucopyranose; Gal, galactopyranose; Ara (p), arabinopyranose; Ara
(f), arabinofuranose.
L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl- δH = 1.37 and 1.74 ppm for C-15 were observed
(1→4)]-β -D -glucuronopyranosyloxyolean-12-ene- (Table 9), indicating no oxidation at this position
15α,16α,22α,28-tetrol.
and, consequently, a barringtogenol C aglycon
In the ESI mass spectrum of pittangretoside A1 (14), moiety as already assigned for pittangretoside T
a [M–H]− quasimolecular ion at m/z = 1215.5881 (7). Thus, the novel natural product pittangretoside
substantiated a molecular formula of C59H92O26. A1 (14) was elucidated as 21β -angeloyloxy-22α-
Again, carefully evaluated one- and two-dimensional acetyloxy-3β -[β - D -galactopyranosyl-(1→2)]-[α -
NMR spectra revealed a strong structural similarity to L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl-
the known compound 16. However, the glycosidic part (1→4)]-β -D -glucuronopyranosyloxyolean-12-ene-
showed some differences, since distinctive resonances 16α,28-diol.
for a galactose moiety were observed [δH = 4.90,
For pittangretoside B1 (15), a molecular formula
d (7.0) ppm, H-1; 3.55 ppm, H-2; 3.48 ppm, H-3; identical to that of compound 14, C59H92O26, was
3.63 ppm, H-4, and 3.54 ppm, H-5] (Table 10), in- generated by a quasimolecular ion peak [M–H]− at
stead of a glucose unit as described for compound m/z = 1215.5864 in its ESI mass spectrum. Since the
16. This was also supported by TLC and GC-MS chromatographic interactions during the HPLC iso-
procedures, identifying β-D-galactose together with lation process were again very similar to those for
β-D-glucuronic acid, and α-L-arabinose. As an- compound 14, pittangretoside B1 (15) was proposed
other different point, resonances at δC = 33.7, and to be the glucose isomer of 14. Indeed, on acid hy-
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
drolysis and subsequent GC-MS and TLC examina- (1/2, 8/3, 6/18, 11/12, 14/15), the structures of an-
tions, 15 gave β-D-glucose, β-D-glucuronic acid, and other three pairs of such isomers from P. angustifolium
α-L-arabinose. Except for the NMR data of those have been previously published by us [2]. Furthermore,
exchanged sugars, the remaining data were basically since esterified R1- and A1-barrigenol structures have
identical (Tables 9 and 10). Resonances for both, an been characterized as aglycone skeletons of triterpene
acetyl and an angeloyl residue were observed as well saponins in a number of Pittosporum species, such as
as their linkage at C-22 and C-21, respectively, as P. tobira [5], P. undulatum [6], P. senacia [7], P. verti-
assigned by HMBC correlations. C-15 appeared also cillatum [8], and P. viridiflorum [9], their occurrence
at δC = 33.7, and δH = 1.38 and 1.74 ppm as ob- could be regarded as a chemotaxonomic marker. As
served for compound 14, proving that the aglycone a concluding remark, it is mentioned that the present
structure was barringtogenol C, as well. Pittangreto- work together with recent results [2, 3] witnesses the
side B1 was thus established as 21β -angeloyloxy- isolation and characterization of 28 triterpene saponins
22α-acetyloxy-3β -[β -D-glucopyranosyl-(1→2)]-[α- from the leaves and another seven from the seeds of
L-arabinopyranosyl-(1→3)]-[α -L-arabinofuranosyl- P. angustifolium, collectively representing 31 differ-
(1→4)]-β -D -glucuronopyranosyloxyolean-12-ene- ent compounds of the class of triterpene glycosides,
16α,28-diol.
of which 27 were described for the first time as natural
In summary, a total of eighteen triterpene saponins, products.
including fifteen new natural products, were isolated
from the leaves of P. angustifolium. With respect to Experimental Section
their structural features, among the elucidated eleven
monodesmosidic compounds 1, 2, 4, 6, 7, and 13–
General
NMR spectra were recorded in CD3OD on a Bruker DRX
18 as well as the bisdesmosidic ones 3, 5, 8, 9, 10,
11, and 12, aglycone moieties of A1-barrigenol (1, 2,
11, 12, 17), R1-barrigenol (3, 5, 6, 8–10, 13, 16, 18),
camelliagenin A (4) and barringtogenol C (7, 14, 15)
were assigned. To the best of our knowledge, the lat-
ter two backbones have not been described before as
constituents of original triterpene saponins from a Pit-
tosporum sp., but have been found in the hydrolyzate
of fractions from P. phillyraeoides [10] and P. undu-
latum [11]. So far, only two bisdesmosidic triterpene
saponins were isolated from the Pittosporum genus
(P. senacia [7]), consisting of oleanolic acid and R1-
barrigenol aglycones, of which the latter one was later
isolated from P. verticillatum [8] as well, and pos-
sesses also a second arabinopyranose linked to C-22,
as it is present in all described bisdesmosidic struc-
tures of this study. While the second sugar residue in
common bisdesmosidic structures is attached mostly
at C-28 of the aglycone backbone, the present attach-
ment at C-22 seems to be rarer and was confirmed only
for a few saponins isolated from Glycine max, Sophora
flavescens and Eryngium yuccifolium [12 – 14]. Inter-
estingly, a galactopyranose moiety, as it was found in
the compounds 1, 4, 6–8, 11, and 14, has not been de-
scribed as a part of the common branched tetrasac-
charide linked to C-3 in published work on phyto-
chemistry of investigated Pittosporum species. Be-
500 (Billerica, MA, USA) instrument. All semipreparative
HPLC procedures were carried out on a Shimadzu system
(Kyoto, Japan) with a two-channel UV detector by using one
of the following columns: RP18 phase with polar endcapping
250 × 10 mm, 4 µm (column I), RP18 phase 250 × 4.6 mm,
5 µm (column II) and 250×10 mm, 5 µm (column III) (Phe-
nomenex, Torrance, USA). For GC-MS analysis an Agilent
device (gas chromatograph, G1530N; mass selective detec-
tor, MSD G2588A; Santa Clara, CA, USA) together with
a DB-5MS column (30 m×0.25 mm×0.25µm; J & W Sci-
entific; Folsom, CA, USA) was used under conditions pre-
viously described [2]. Detected compounds were assigned
by mass spectral data compared with NIST database 2.0 d
(National Institute of Standards and Technology, Gaithers-
burg, MD, USA) and data generated by comparison of re-
tention times of the TIC (total ion chromatograms) of au-
thentic samples of D-glucose (Sigma-Aldrich, St. Louis, MO,
USA), D-galactose (Sigma-Aldrich), L-arabinose (Fluka, St.
Louis, MO, USA), and D-glucuronic acid (Sigma-Aldrich).
All LC-MS measurements were performed on a LC-MS-IT-
TOF device (Shimadzu) with a Chromolith SpeedRod RP18
column (50 mm × 4.6 mm, Merck; Darmstadt, Germany)
or a Kinetex C18 column (2.6 µm, 100 mm × 3 mm, Phe-
nomenex) and electrospray ionization (ESI). ATR-IR spec-
tra were recorded on a Thermo Scientific Nicolet IR 200
FT-IR spectrometer (Waltham, MA, USA), and optical ro-
tation values were determined on a Perkin Elmer 241 po-
larimeter (Waltham, MA, USA). Pre-coated silica gel 60
sides five galactose/glucose isomers reported herewith plates (Merck) were applied for thin layer chromatography
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1041
analysis, using a mixture of EtOAc-iso-PrOH-HOAc-H2O and 15 were collected unseparated), fraction A3_100% (com-
(4 : 2 : 2 : 1) and a detection reagent [0.25 g Thymol (Sigma- pounds 4, 7, 16, compounds 6 and 18 as well as 14 and 15
Aldrich), 2.5 mL H2SO4, 47.5 mL EtOH] to visualize sug- were collected unseparated and compound 17 was obtained
ars. Sprayed plates were heated at 135 ◦C for 5 min. Solid- only unpure); method 2 (isocratic 39.5% B, column III) used
phase extractions (SPE) were carried out with a vacuum man- for further separation of compounds 14 (tR = 29.80 min) and
ifold and RP18-cartridges (Strata C18E, 200 g/120 mL, Phe- 15 (tR = 31.42 min); method 3 (isocratic 50% B, column III)
nomenex).
used for further separation of compounds 6 (tR = 13.47 min)
and 18 (tR = 14.54 min); compound 17 (tR = 17.76 min) was
purified by method 4 (isocratic 44% B, column II); method
5 (0:30, 10:34, 11:36, 22:36, column I) used for fractions
A2_40% and A3_40% (compound 3, tR = 13.79 min; com-
pound 11, tR = 25.51 min; compound 12, tR = 26.27 min),
method 6 (isocratic 28.2% B, column I) used for frac-
Plant material
Leaves of P. angustifolium were collected in June 2008
on the grounds of Central Queensland GG foundation (K.
A. Amato and the Trustee for Milner Krasser Family Trust)
near Mount Morgan, Rockhampton, Queensland, Australia,
and were a gift of Dr. Kornelia Krasser and Mr. Klaus von
Gliszczynski, Australia. The plant material was authenti-
cated by Dr. Peter König, Curator of the Botanical Garden of
Greifswald and a voucher specimen (no. 20110013PA) was
deposited at the Institute of Pharmacy, Department of Phar-
maceutical Biology, Ernst Moritz Arndt University, Greifs-
wald, Germany.
tion A4_40% (compounds 8, tR = 27.09 min and 3, tR
=
28.84 min); method 7 (0:27, 25:27, 26:80, 27:27, 32:27, col-
umn III) used for 90 mg of fraction A4_60% to isolate com-
pound 13 (tR = 23.29 min); method 8 (0:22.5, 33:22.5, 37:24,
39:80, 40:22.5, 43:22.5, column I) used for fraction A5_30%
(compound 5, tR = 14.07 min; compound 9, tR = 29.13 min;
compound 10, tR = 34.05 min); method 9 (isocratic 27.5%
B, column I) used for fraction A5_100% (compound 1, tR
=
Extraction and isolation
21.13 min; compound 2, tR = 22.27 min). Total amounts: 1
(2.4 mg), 2 (1.5 mg), 3 (44.1 mg), 4 (3.0 mg), 5 (4.9 mg), 6
(4.1 mg), 7 (4.9 mg), 8 (7.3 mg), 9 (2.3 mg), 10 (2.8 mg), 11
(1.2 mg), 12 (1.7 mg), 13 (2.1 mg), 14 (1.6 mg), 15 (1.9 mg),
16 (10.2 mg), 17 (3.2 mg), 18 (45.2 mg.)
Pulverized, dried leaves (8.4 g) were defatted with
CH2Cl2 via Soxhlet for 24 h and then extracted three times
with 80% (v/v) EtOH under reflux. 2.8 g of the crude
extract were subjected to an open column chromatogra-
phy using Sephadex LH-20 (Sigma-Aldrich), eluting with
MeOH. A saponin-enriched fraction of 1.9 g was then ap-
plied for a subsequent fractionation on silica gel (60–40 µm,
Merck) using a stepwise gradient of CH2Cl2-MeOH-H2O
mixtures as recently reported [2] to yield saponin frac-
tions A1 (93 mg), A2 (168 mg), A3 (107 mg), A4 (405 mg),
A5 (183 mg). Afterwards, solid-phase extractions were per-
formed, using H2O as washing solvent for all fractions and
the following MeOH concentrations for precleaning and/or
fractionation: A1 (30%, 100%), A2 and A3 (40%, 100%),
A4 (40%, 60%), A5 (30%, 100%). Corresponding sub-
fractions were obtained (the MeOH concentration used for
elution is represented by additional subscript indices) as
A1_100% (27 mg), A2_40%(26 mg), A2_100% (63 mg), A3_40%
(24 mg), A3_100% (69 mg), A4_40% (139 mg), A4_60%
(231 mg), A5_30% (64 mg), and A5_100% (78 mg). HPLC
conditions used for isolation: solvent A (H2O), solvent
B (acetonitrile), each with 0.05% HCOOH, gradient elu-
tion or isocratic; flow rate: analytical column (column II)
1 mL min−1, semipreparative columns (columns I and III)
4 mL min−1; detection 206 nm; methods (expressed as time
[min]:concentration solvent B [%]: method 1 (0:41, 11:45,
21:46, 28:50, 29:41, 32:41, column I) used for fraction
A1_100% (compound 18, tR = 21.73 min), fraction A2_100%
(compound 4, tR = 16.56 min; compound 7, tR = 29.38 min;
Compound 1 (pittangretoside J)
Colorless amorphous powder. C52H84O24; [α]2D0 = −54.5
(c = 0.09, MeOH). – ATR-IR: νmax = 3351, 2945, 1598,
1465, 1390, 1256, 1150, 1072, 1050, 1005 cm−1. – 1H
and 13C NMR: see Tables 1 and 2. – HRMS ((+)-ESI-
IT-TOF): m/z(%) = 455.3584 (91.4) [(M+H)-GlcA-Gal-
2Ara-2H2O]+, 437.3428 (38.1) [(M+H)-GlcA-Gal-2Ara-
3H2O]+. – HRMS ((–)-ESI-IT-TOF): m/z(%) = 1091.5256
(100) (calcd. for C52H83O24, 1091.5280, monoisotopic
mass, [M–H]−).
Compound 2 (pittangretoside K)
Colorless amorphous powder. C52H84O24; [α]2D0 = −38.2
(c = 0.09, MeOH). – ATR-IR: νmax = 3367, 2953, 1605,
1458, 1412, 1359, 1136, 1070, 1039, 1007 cm−1. – 1H
and 13C NMR: see Tables 1 and 2. – HRMS ((+)-ESI-IT-
TOF): m/z(%) = 455.3584 (78.3) [(M+H)-GlcA-Glc-2Ara-
2H2O]+, 437.3426 (100) [(M+H)-GlcA-Glc-2Ara-3H2O]+.
– HRMS ((–)-ESI-IT-TOF): m/z(%) = 1091.5263 (100)
(calcd. for C52H83O24, 1091.5280, monoisotopic mass, [M–
H]−).
Compound 3 (pittangretoside M)
Colorless amorphous powder. C63H98O32; [α]2D0 = −24.0
compound 16, tR = 9.56 min; compound 18; compounds 14 (c = 0.41, MeOH). – ATR-IR: νmax = 3431, 2934, 1737,
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1370, 1228, 1073, 1051, 1006 cm−1. – 1H and 13C 1602, 1387, 1239, 1158, 1072, 1042 cm−1. – 1H and 13C
NMR: see Tables 1 and 2. – HRMS ((+)-ESI-IT-TOF): NMR: see Tables 5 and 6. – HRMS ((+)-ESI-IT-TOF):
m/z(%) = 706.2785 (24.9) [M+2Na]2+. – HRMS ((–)- m/z(%) = 537.3941 (4.3) [(M+H)-GlcA-Gal-2Ara-Ang-
ESI-IT-TOF): m/z(%) = 1365.5889 (100) (calcd. for 2H2O]+, 419.3352 (2.0) [(M+H)-GlcA-Gal-2Ara-2Ang-
C63H97O32, 1365.5968, monoisotopic mass, [M–H]−).
4H2O]+. – HRMS ((–)-ESI-IT-TOF): m/z(%) = 1255.6174
(100) (calcd. for C62H95O26, 1255.6176, monoisotopic
mass, [M–H]−).
Compound 4 (pittangretoside Q)
Colorless amorphous powder. C57H90O24; [α]2D0 = −8.2
(c = 0.16, MeOH). – ATR-IR: νmax = 3368, 2920, 1688,
Compound 8 (pittangretoside U)
1
1601, 1426, 1387, 1239, 1147, 1072, 1042 cm−1. – H and
Colorless amorphous powder. C63H98O32; [α]2D0 = −31.1
(c = 0.22, MeOH). – ATR-IR: νmax = 3402, 2953, 1736,
1619, 1371, 1223, 1049, 1006 cm−1. – 1H and 13C
NMR: see Tables 5 and 6. – HRMS ((+)-ESI-IT-TOF):
m/z(%) = 706.2858 (18.6) [M+2Na]2+. – HRMS ((–)-ESI-
13C NMR: see Tables 3 and 4. – HRMS ((+)-ESI-IT-TOF):
m/z(%) = 439.3547 (94.1) [(M+H)-GlcA-Gal-2Ara-Ang-
2H2O]+, 421.3446 (100) [(M+H)-GlcA-Gal-2Ara-Ang-
3H2O]+. – HRMS ((–)-ESI-IT-TOF): m/z(%) = 1157.5742
(100) (calcd. for C57H89O24, 1157.5749, monoisotopic
mass, [M–H]−).
IT-TOF): m/z(%) = 1365.5895 (2.4) (calcd. for C63H97O32
,
1365.5968, monoisotopic mass, [M–H]−).
Compound 5 (pittangretoside R)
Compound 9 (pittangretoside V)
Colorless amorphous powder. C57H92O29; [α]2D0 = −12.8
(c = 0.24, MeOH). – ATR-IR: νmax = 3372, 2931, 1608,
1414, 1373, 1256, 1072, 1043, 1006 cm−1. – 1H and 13C
NMR: see Tables 3 and 4. – HRMS ((+)-ESI-IT-TOF):
m/z(%) = 661.3956 (16.5) [(M+Na)-GlcA-Glc-Ara(p)-
Ara(f)]+, 643.2797 (68.1) [(M+Na)-GlcA-Glc-Ara(p)-
Ara(f)-H2O]+, 471.3484 (85.0) [(M+Na)-GlcA-Glc-
Ara(p)-Ara(f)-Ara(p)II-2H2O]+, 453.3400 (100) [(M+Na)-
Colorless amorphous powder. C59H94O30; [α]2D0 = −11.4
(c = 0.17, MeOH). – ATR-IR: νmax = 3372, 2923, 1719,
1591, 1377, 1235, 1074, 1041, 1007 cm−1. – 1H and 13C
NMR: see Tables 5 and 6. – HRMS ((+)-ESI-IT-TOF):
m/z(%) = 703.4145 (23.9) [(M+Na)-GlcA-Glc-Ara(p)-
Ara(f)]+, 664.2858 (100) [M+2Na]2+, 471.3440 (20.0)
[(M+H)-GlcA-Glc-Ara(p)-Ara(f)-Ara(p)II-2H2O]+,
453.3312 (23.8) [(M+H)-GlcA-Glc-Ara(p)-Ara(f)-Ara(p)II-
3H2O]+, 435.3312 (23.9) [(M+H)-GlcA-Glc-Ara(p)-
Ara(f)-Ara(p)II-4H2O]+, 417.3183 (2.5) [(M+H)-GlcA-
Glc-Ara(p)-Ara(f)-Ara(p)II-5H2O]+. – HRMS ((–)-ESI-IT-
GlcA-Glc-Ara(p)-Ara(f)-Ara(p)II-3H2O]+,
(87.9) [(M+Na)-GlcA-Glc-Ara(p)-Ara(f)-Ara(p)II-
4H2O]+, 417.3197 (48.3) [(M+Na)-GlcA-Glc-Ara(p)-
Ara(f)-Ara(p)II-5H2O]+.
HRMS ((–)-ESI-IT-TOF):
435.3293
–
TOF): m/z(%) = 1281.5726 (35.6) (calcd. for C59H93O30
,
m/z(%) = 1239.5833 (13.9) (calcd. for C57H91O29
,
1281.5757, monoisotopic mass, [M–H]−).
1239.5804, monoisotopic mass, [M–H]−).
Compound 10 (pittangretoside W)
Compound 6 (pittangretoside S)
Colorless amorphous powder. C59H94O30; [α]2D0 = −8.7
(c = 0.21, MeOH). – ATR-IR: νmax = 3355, 2925, 1730,
1600, 1374, 1250, 1073, 1024, 1001 cm−1. – 1H and 13C
NMR: see Tables 7 and 8. – HRMS ((+)-ESI-IT-TOF):
m/z(%) = 703.4235 (11,7) [(M+Na)-GlcA-Glc-Ara(p)-
Ara(f)]+, 664.2943 (100) [M+2Na]2+, 471.3315 (4.9)
[(M+H)-GlcA-Glc-Ara(p)-Ara(f)-Ara(p)II-2H2O]+,
Colorless amorphous powder. C62H96O27; [α]2D0 = −17.5
(c = 0.33, MeOH). – ATR-IR: νmax = 3364, 2923, 1693,
1591, 1354, 1240, 1155, 1071, 1041, 997 cm−1. – 1H and
13C NMR: see Tables 3 and 4. – HRMS ((+)-ESI-IT-
TOF): m/z(%) = 571.3992 (33.6) [(M+H)-GlcA-Gal-2Ara-
Ang-H2O]+, 553.3849 (100) [(M+H)-GlcA-Gal-2Ara-Ang-
2H2O]+, 535.3722 (39.4) [(M+H)-GlcA-Gal-2Ara-Ang-
3H2O]+, 453.3331 (17.7) [(M+H)-GlcA-Gal-2Ara-2Ang-
3H2O]+, 435.3284 (26.5) [(M+H)-GlcA-Gal-2Ara-2Ang-
4H2O]+. – HRMS ((–)-ESI-IT-TOF): m/z(%) = 1271.6080
(100) (calcd. for C62H95O27, 1271.6066, monoisotopic
mass, [M–H]−).
453.3407 (40.8) [(M+H)-GlcA-Glc-Ara(p)-Ara(f)-Ara(p)II-
3H2O]+, 435.3409 (27.5) [(M+H)-GlcA-Glc-Ara(p)-
Ara(f)-Ara(p)II-4H2O]+.
–
HRMS ((–)-ESI-IT-TOF):
m/z(%) = 1281.5808 (20.0) (calcd. for C59H93O30
,
1281.5757, monoisotopic mass, [M–H]−).
Compound 11 (pittangretoside X)
Compound 7 (pittangretoside T)
Colorless amorphous powder. C62H96O26; [α]2D0 = −19.6
Colorless amorphous powder. C63H98O31; [α]2D0 = −6.5
(c = 0.25, MeOH). – ATR-IR: νmax = 3409, 2922, 1701, (c = 0.09, MeOH). – ATR-IR: νmax = 3390, 2940, 1737,
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C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
1043
1602, 1371, 1229, 1066, 1047, 1008 cm−1. – 1H and 13C (100) (calcd. for C57H89O26, 1189.5648, monoisotopic
NMR: see Tables 7 and 8. – HRMS ((+)-ESI-IT-TOF): mass, [M–H]−).
m/z(%) = 771.4248 (16.6) [(M+Na)-GlcA-Gal-Ara(p)-
Compound 14 (pittangretoside A1)
Ara(f)]+, 731.4386 (6.8) [(M+H)-GlcA-Gal-Ara(p)-
Ara(f)-H2O]+, 713.4176 (27.1) [(M+H)-GlcA-Gal-Ara(p)-
Colorless amorphous powder. C59H92O26; [α]2D0 = −12.0
Ara(f)-2H2O]+, 698.2894 (100) [M+2Na]2+, 455.3557
(c = 0.12, MeOH). – ATR-IR: νmax = 3371, 2929, 1712,
(14.5) [(M+H)-GlcA-Gal-Ara(p)-Ara(f)-Ara(p)II-2H2O]+,
437.3466 (30.2) [(M+H)-GlcA-Gal-Ara(p)-Ara(f)-Ara(p)II-
3H2O]+, 419.3355 (16.6) [(M+H)-GlcA-Gal-Ara(p)-
1602, 1372, 1256, 1155, 1072, 1041 cm−1. – 1H and
13C NMR: see Tables 9 and 10. – HRMS ((–)-ESI-IT-
TOF): m/z(%) = 1215.5881 (24.4) (calcd. for C59H91O26
,
Ara(f)-Ara(p)II-4H2O]+.
–
HRMS ((–)-ESI-IT-TOF):
1215.5804, monoisotopic mass, [M–H]−).
m/z(%) = 1349.5986 (100) (calcd. for C63H97O31
,
1349.6019, monoisotopic mass, [M–H]−).
Compound 15 (pittangretoside B1)
Colorless amorphous powder. C59H92O26; [α]2D0 = −14.8
(c = 0.14, MeOH). – ATR-IR: νmax = 3414, 2945, 1715,
Compound 12 (pittangretoside Y)
Colorless amorphous powder. C63H98O31; [α]2D0 = −9.0 1604, 1369, 1253, 1159, 1074, 1022, cm−1. – 1H and
(c = 0.13, MeOH). – ATR-IR: νmax = 3423, 2925, 1740, 13C NMR: see Tables 9 and 10. – HRMS ((–)-ESI-IT-
1609, 1369, 1228, 1050, 1001 cm−1. – 1H and 13C TOF): m/z(%) = 1215.5864 (15.4) (calcd. for C59H91O26
NMR: see Tables 7 and 8. – HRMS ((+)-ESI-IT-TOF): 1215.5804, monoisotopic mass, [M–H]−).
m/z(%) = 771.4241 (18.9), [(M+Na)-GlcA-Glc-Ara(p)-
,
Acidic hydrolysis
Ara(f)]+, 731.4386 (8.2) [(M+H)-GlcA-Glc-Ara(p)-Ara(f)-
H2O]+, 713.4177 (31.4), [(M+H)-GlcA-Glc-Ara(p)-
0.4 – 1.0 mg of each compound were treated as recently
described [3] and applied for TLC and GC-MS procedures.
Bisdesmosidic structures, acylated at the second arabinopy-
ranose linked to C-22 of the aglycon moieties (compounds 3,
8, 9–12), were pretreated with 1 mL of 1 M NaOH (Merck)
at 80 ◦C for 4 h, then neutralized with 1 M HCl (VWR Inter-
national, Darmstadt, Germany). The mixture was purified by
a solid-phase extraction, using conditioned and equilibrated
SPE cartridges (C18E, 500 mg, Phenomenex) by eluting with
H2O. A washing step, using MeOH, was performed for the
recovery of deacetylated compounds. The further procedure
corresponds to that of the non-acylated compounds. The ab-
solute configuration of sugars was confirmed by their thia-
zolidine carboxylates [15], comparing obtained GC-MS data
with those of authentic samples of L-Ara (tR = 36.538 min),
D-Glc (tR = 37.647 min), D-Gal (tR = 40.383 min) and D-
GlcA (tR = 41.109 min).
Ara(f)-2H2O]+,698.2890 (100) [M+2Na]2+
, 455.3557
(17.1) [(M+H)-GlcA-Glc-Ara(p)-Ara(f)-Ara(p)II-2H2O]+,
437.3466 (35.1) [(M+H)-GlcA-Glc-Ara(p)-Ara(f)-Ara(p)II-
3H2O]+, 419.3354 (19.3) [(M+H)-GlcA-Glc-Ara(p)-
Ara(f)-Ara(p)II-4H2O]+.
–
HRMS ((–)-ESI-IT-TOF):
m/z(%) = 1349.5988 (100) (calcd. for C63H97O31
,
1349.6019, monoisotopic mass, [M–H]−).
Compound 13 (pittangretoside Z)
Colorless amorphous powder. C57H90O26; [α]2D0 = −6.0
(c = 0.17, MeOH).
– ATR-IR: νmax = 3346, 2908,
1603, 1387, 1243, 1158, 1075, 1041, 1005 cm−1. – 1H
and 13C NMR: see Tables 9 and 10. – HRMS ((+)-
ESI-IT-TOF): m/z(%) = 647.4031 (13.8) [(M+H)-Glc-
2Ara-Ang-2H2O]+, 629.3905 (2.1) [(M+H)-Glc-2Ara-Ang-
3H2O]+, 471.3552 (74.3) [(M+H)-GlcA-Glc-2Ara-Ang-
2H2O]+, 453.3457 (100) [(M+H)-GlcA-Glc-2Ara-Ang-
3H2O]+, 435.3351 (86.0) [(M+H)-GlcA-Glc-2Ara-Ang-
4H2O]+, 417.3246 (59.3) [(M+H)-GlcA-Glc-2Ara-Ang-
Acknowledgement
We wish to sincerely thank Dr. Rudolf Kunze, Berlin, Ger-
5H2O]+, 399.3108 (4.9) [(M+H)-GlcA-Glc-2Ara-Ang- many, Dr. Cornelia Krasser and Mr. Klaus von Gliszczynski,
6H2O]+. – HRMS ((–)-ESI-IT-TOF): m/z(%) = 1189.5634 Australia, for the provision of the plant material.
[1] L. W. Cayzer, M. D. Crisp, I. R. H. Telford, Aust. Syst.
Bot. 2000, 13, 845 – 902.
[2] C. Bäcker, K. Jenett-Siems, K. Siems, M. Wurster,
A. Bodtke, C. Chamseddin, M. Crüsemann, U. Lindeq-
uist, Planta Med. 2013, 79, 1461 – 1469.
[3] C. Bäcker, K. Jenett-Siems, K. Siems, M. Wurster,
A. Bodtke, U. Lindequist, Z. Naturforsch. 2014, 69c,
1 – 8.
[4] C. Bäcker, K. Jenett-Siems, A. Bodtke, U. Lindequist,
Biochem. Syst. Ecol. 2014, 55, 101 – 103.
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1044
C. Bäcker et al. · Mono- and Bisdesmosidic Triterpene Glycosides from Pittosporum angustifolium Lodd.
[5] I. D’Acquarica, M. C. Di Giovanni, F. Gasparrini, [10] S. G. Errington, P. R. Jefferies, Phytochemistry 1988,
D. Misiti, C. D’Arrigo, N. Fagnano, D. Guarnieri, 27, 543 – 545.
G. Iacono, G. Bifulco, R. Riccio, Tetrahedron 2002, [11] R. Higuchi, T. Komori, T. Kawasaki, E. V. Lassak, Phy-
58, 10127 – 10136. tochemistry 1983, 22, 1235 – 1237.
[6] R. Higuchi, T. Fujioka, M. Iwamoto, T. Komori, [12] I. Kitagawa, M. Saito, T. Taniyama, M. Yoshikawa,
T. Kawasaki, E. V. Lassak, Phytochemistry 1983, 22,
2565 – 2569.
[7] J. Linnek, A. C. Mitaine-Offer, T. Paululat, M. A. La-
caille-Dubois, Magn. Reson. Chem. 2012, 50, 798 –
802.
[8] M. J. Manase, A.-C. Mitaine-Offer, T. Miyamoto, C.
Tanaka, S. Delemasure, P. Dutartre, M.-A. Lacaille-Du-
bois, Fitoterapia 2013, 91, 231 – 235.
[9] Y. Seo, J. M. Berger, J. Hoch, K. M. Neddermann,
I. Bursuker, S. W. Mamber, D. G. I. Kingston, J. Nat.
Prod. 2002, 65, 65 – 68.
Chem. Pharm. Bull. 1985, 33, 598 – 608.
[13] M. Yoshikawa, H. K. Wang, H. Kayakiri, T. Taniyama,
I. Kitagawa, Chem. Pharm. Bull. 1985, 33, 4267 –
4274.
[14] Z. Zhang, S. Y. Li, S. Ownby, P. Wang, W. Yuan, W. L.
Zhang, R. S. Beasley, Phytochemistry 2008, 69, 2070 –
2080.
[15] S. Hara, H. Okabe, K. Mihashi, Chem. Pharm. Bull.
1987, 35, 501 – 506.
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