A furostanol saponin and phytoecdysteroid from roots of Helleborus orientalis

Article abstract of DOI:10.1007/s10600-007-0039-8

A furostanol saponin mixture and a known phytoecdysteroid were isolated from the roots of Helleborus orientalis Lam. Their structures were established as 26-[(β-D-glucopyranosyl)oxy]-22α-hidroxyfurosta-5,25(27)-dien- 1β,3β,11α-triol (1a), 26-[(β-D-glucopyranosyl)oxy]- 22α-methoxyfurosta-5,25(27)-dien-1β,3β,11α-triol (1b), and 20-hydroxy-β-ecdyson-3-O-β-D-glycoside (2). Acid hydrolysis of 1a,b gave (1β,3β,11α,22α)-22,26-dimethoxyfurosta-5,25(27)-dien- 1,3,11-triol (aglycone 1) and of 2 gave 20-hydroxy-β-ecdyson (aglycone 2). Their structures were elucidated by spectral analysis. Springer Science+Business Media, Inc. 2007.

Full text of DOI:10.1007/s10600-007-0039-8

Chemistry of Natural Compounds, Vol. 43, No. 1, 2007
A FUROSTANOL SAPONIN AND PHYTOECDYSTEROID
FROM ROOTS OF Helleborus orientalis
Sebahat Akin1* and Huseyin Anil2
UDC 547.926
A furostanol saponin mixture and a known phytoecdysteroid were isolated from the roots of Helleborus
orientalis Lam. Their structures were established as 26-[(β-D-glucopyranosyl)oxy]-22α-hidroxyfurosta-
5,25(27)-dien-1β,3β,11α-triol (1a), 26-[(β-D-glucopyranosyl)oxy]-22α-methoxyfurosta-5,25(27)-dien-
1β,3β,11α-triol (1b), and 20-hydroxy-β-ecdyson-3-O-β-D-glycoside (2). Acid hydrolysis of 1a,b gave
(1β,3β,11α,22α)-22,26-dimethoxyfurosta-5,25(27)-dien-1,3,11-triol(aglycone1)andof2gave 20-hydroxy-β-
ecdyson (aglycone 2). Their structures were elucidated by spectral analysis.
Key words: Helleborus orientalis, furostanol saponin, phytoecdysteroid.
Helleborus extracts exhibit high biological activity with antialgic, myorelaxant, antirheumatic, and blood vessel
regulating actions [1]. Previously isolated classes of constituents, furostanol saponin [2–5], phytoecdysteroids [6–9], and
bufadienolides [10], were characterized from Helleborus species.
This study describes the isolation and the exact structure determination of two glycosides, a furostanol saponin 1a,b
and a phytoecdysteroid 2, from Helleborus orientalis Lam roots.
Compounds 1a,b was obtained as a mixture of the C-22 hydroxyl and C-22 methoxyl forms. It gave two spots at TLC
due to the existence of 22-hydroxy and 22-methoxy derivatives of the glycoside. Acid hydrolysis of the mixture gave aglycone
1. The 1HMRspectral data ofaglycone1 contained signals of olefinic protons at 5.62, 4.78, and 4.83 ppm, twomethoxyl protons
at 3.21 and 3.63 ppm, and methyl groups at 0.96 (21-CH3), 0.79 (18-CH3), and 1.15 ppm (19-CH3). Furthermore, the presence
of a semiketal carbon signal at 109.4 ppm suggested the furostanol nature of the aglycone 1. The EI-MS spectrum showed an
ion peak at m/z: 491 [M+H]+ for C29O6H46. The above 1H NMR spectral data and a comparison of the 13C NMR signals (Table
1) and of – 85.92 of aglycone 1 with those described in the literature [1, 2] showed the structure of the aglycone1 to be
(1β,3β,11α,22α)-22,26-dimethoxyfurosta-5,25(27)-dien-1,3,11-triol.
The furostanol glycosidic nature of compounds 1a,b was evidenced by the furostan steroidal structure (916 and
833 cm–1) in the IR spectra. The 1H NMR spectral data of the mixture of compounds 1a,b contained a signal for one anomeric
proton at 4.24 ppm (d, J = 7.3 Hz, H-1′). The 13C NMR spectral data (Table 1) contained an anomeric carbon signal at
104.0 ppm. Furthermore, the presence of a semiketal carbon signal at 110.4 and 110.7 ppm suggested the furostanol nature of
compounds 1a,b. The peak intensities of these semiketal carbon signals were 0.775 and 0.742, respectively. The ratio of these
components (1a and 1b) in the mixture of was 1:1. The negative FAB-MS showed an ion peak at m/z: 571.4 [M-OCH3-2H2O]–,
suggesting the molecular formula as C34O11H54. The structure of 1a was established as 26-[(β-D-glucopyranosyl)oxy]-22α-
hydroxyfurosta-5,25(27)-dien-1β,3β,11α-triol and 1b as 26-[(β-D-glucopyranosyl)oxy]-22α-methoxyfurosta-5,25(27)-dien-
1β,3β,11α-triol. In this paper, the NMRdata of 1a and 1b are reported for the first time. Compounds 1a,b are presumed to exist
as C-22 hydroxyl and C-22 methoxyl derivatives. It is thought that furostanol saponin exists in the form of just 1a in the roots
of Helleborus orientalis Lam and that compound 1b may be artifacts of the extraction and isolation procedure. Such
interconversion between hydroxyl and methoxyl groups at C-22 furostanol glycosides has alreadybeen reported in the literature
[1, 4].
1)Balikesir University, FacultyofArtsandSciences, Department ofChemistry, Balikesir, Turkey, fax0(266)2493360,
e-mail: sakin@balikesir.edu.tr; 2) Ege University, Faculty of Sciences, Department of Chemistry, Izmir, Turkey. Published in
Khimiya Prirodnykh Soedinenii, No. 1, pp. 75-77, January-February, 2007. Original article submitted December 1, 2005.
0009-3130/07/4301-0090 ©2007 Springer Science+Business Media, Inc.
90

TABLE 1. Chemical Shifts of 13C Atom of Glycosides and of Aglycone (δ, ppm)
1a
1b
1
1a
1b
1
Aglycone
C atom
Aglycone
C atom
1
2
78.6
35.5
75.5
40.9
147.2
126.2
33.3
31.8
50.4
47.8
75.9
32.7
40.7
58.1
33.2
81.4
63.6
78.6
35.5
75.5
40.9
147.2
126.2
33.3
31.8
50.4
47.8
75.9
32.7
40.7
58.1
33.2
81.4
63.6
75.1
35.1
66.2
40.6
143.4
125.9
32.7
31.0
52.1
42.5
67.7
32.0
40.2
57.0
35.0
81.1
62.0
18
19
17.2
16.3
42.1
13.9
110.4
28.4
37.9
140.5
66.1
112.1
-
17.2
16.3
42.1
13.9
110.7
28.4
37.9
140.5
66.1
112.1
50.5
104.0
71.7
75.0
68.5
72.1
62.8
17.0
14.5
3
20
41.6
4
21
13.3
5
22
109.4
28.5
6
23
7
24
39.7
8
25
138.5
63.0
9
26
10
11
12
13
14
15
16
17
27
113.0
-OCH3
Glc-1′
2′
49.4 and 51.5
104.0
71.7
75.0
68.0
72.1
62.8
3′
4′
5′
6′
OH
6'
5'
O
HO
OH
HO
O
3'
1'
21
18
OH
OH
21
26
20
17
R
25
27
25
23
18
OH
HO
HO
OH
19
1
23
13
17
27
11
9
6'
HO
O
19
13
5'
O
15
HO
9
1
10
1'
OH
15
10
7
3'
3
O
5
OH
7
OH
5
3
HO
O
1a, b
1b:
2
1a:
R = OH;
R = OCH3
The 1H NMR spectral data of compound 2 contained signals for one anomeric proton at 4.72 ppm (d, J = 6.7 Hz, H′).
The 13C NMR spectral data contained one anomeric carbon signal at 105.5 ppm. The negative FAB-MS showed an ion peak
at m/z: 623.3[M-H O-H]–. 1H NMR spectral data and comparison of the 13C NMR signals of 2 with those described in the
2
literature [6, 7] showed the structure of 2 to be 20-hydroxy-β ecdyson-3-O-β-D glycoside.
EXPERIMENTAL
The plant material from Helleborus orientalis Lam was collected in Bursa Uludag. A plant specimen was deposited
under registration number BULU 26118 in the Herbarium of Uludag University (Bursa, Turkey).
Dried and powdered roots of the plant (1000 g) were extracted with 80% MeOH (4×5000 mL) at room temperature
for about 15 days. The mixture was filtered. The combined extracts were evaporated to dryness under vacuum at 40°C. The
residue (25 g) was extracted with hexane and partitioned between n-BuOH and H O phases. The n-butanol portion (16 g) was
2
column chromatographed (Silica gel 60, 0.063–0.200 mm) with CHCl3–MeOH (10:1) and three different CHCl3–MeOH–H2O
solventsystems [(65:20:10); (65:25:10); (65:30:10)]. The fractionswereseparatedusingpreparativeTLC[CHCl3–MeOH-H2O,
(65:32:10)]. Preparative TLC was achieved using 20×20 cm glass plates coated with 0.5 mm layers ofsilicagel (Merck Kieselgel
60 PF254+366). Glycosides 1a,b (450 mg) and glycoside 2 (180 mg) were isolated from the root samples.
Glycosides 1a,b (90 mg) and glycoside 2 (60 mg) were dissolved in 10 mL MeOH and 10 mL 2M HCI added to this.
After refluxing the mixture for 4 hours, H2O was added and the aglycones were extracted with CHCl3 and purified on a silica
91

gel column using CHCl3–MeOH (9:1). After acid hydrolysis ofglycosides 1a,b and glycoside 2, aglycone 1 and aglycone 2 were
obtained. After that, sugar analysis was performed using TLC by comparison with authentic samples.
Glycosides 1a,b (120 mg) were acetylated with pyridine–Ac2O and purified by preparative TLC using CHCl3–MeOH
(97:3).
IR spectra were recorded on a Bruker IFS-48 FTIRspectrometer (KBr disks). The 1H NMR and 13C NMR spectra were
recorded on a Bruker 200 MHz spectrometer with TMS as an internal standard. Optical rotations were measured on a
Schmidt+Haensch Polartonic E polarimeter. EI-MS, CI-MS spectra were recorded on a Kratos MS-80, and negative ion FAB-
MS spectra were recorded on a Kratos Concept-II H MS instrument using Cs ion (6KV) in glycerin.
20
Compounds (1a,b) (amorphous ): [α]D –67.41° (c 0.6, MeOH); IR (νmaxKBr, cm–1): 1076 and 3382 (OH), 1558
(=CH-),1652(=CH2), 916,833 (furostanol nature). 1H NMR (δ, pyridine-d5, J/Hz): 0.94 (s, 18-CH3), 1.26 (d, J = 6.7, 21-CH3),
1.42 (s, 19-CH3), 3.24 (22-OCH3), 4.42 (q, H-16) 4.24 (d, J = 7.3, anomeric proton), 5.61 (d, J = 4.9, H-6). The 13C NMR
spectral data of acetylation product of 1a,b: δ = 169.40–176.0 (7×acetyl C=O) and 13.9–21.2 (7×acetyl CH3). FAB-MS (m/z):
571.4 [M-OCH3-2H2O]–.
20
Aglycone 1 (amorphous): [α]D –85.92° (c 0.34, CHCl3); ([α]D18 –86.9°) [lit. 2]. 1H NMR (δ, CDCl3, J/Hz): 0.79
(s, 18-CH3), 0.96 (d, J = 6, 21-CH3), 1.15 (s, 19-CH3), 3.21 (s, 22-OCH3), 3.63 (s, 26-OCH3), 4.39 (q, H-16), 4.78 (d, J = 6.6,
H-27a), 4.83 (d, H-27b), 5.62 (d, J = 5.2, H-6). EI-MS (m/z): 491[M+H]+, 477, 459, 426, 409, 391, 345, 211,84.
Compound 2. IR (νmaxKBr, cm–1): 1087 and 3404 (OH), 1770 (C=O), 1577(C=C). 1H NMR (δ, pyridine-d5, J/Hz):
0.86 (s, 18-CH3), 1.10 (s, 19-CH3), 1.25 (s, 26-CH3), 1.27 (s, 27-CH3), 1.43 (s, 21-CH3), 4.72 (d, J = 6.7, anomeric proton),
5.63 (d, J = 5.1, H-7). 13C NMR (δ, pyridine-d5): 140.4 (=C, C-8), 120.5 (=CH-, C-7), 167.0 (C=O, C-6), 105.5 (anomeric
carbon), 84.0–62.7 (3C-O-, 7CH-O-), 62.2 (1CH -O-, C-6′), 58.0–32.0 (2 quaternary C-, 3 methine C, 8 methylene C), 29.9,
2
16.8, 16.3, 15.0, 13.8 (5-methyl groups). EIS-MS (m/z): 647.3 [M+Na-H2O]+, 503.2 [20-hydroxyecdysone+Na]+; FAB-MS
(m/z): 623.3[M-H2O-H]–.
Aglycone 2. C27O7H44. 13C NMR (δ, CDCl3): 138.6 (=C , C-8 ), 125.8 (=CH-, C-7), 168.0 (C=O, C-6), 84–66.5
(3C-O-, 3CH-O-), 61.4–26.9 (2 quaternary C-, 3 methine C, 8 methylene C), 24.7, 16.7 14.7, 14.4, 13.3 (5 methyl groups).
CI-MS (m/z): 444 [M-2H2O]+, 426,391 (100), 331, 279, 58.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
A. Braca, J. M. Prieto, N. D. Tommasi, F. Tome′, and I. Morelli, Phytochemistry, 65, 2921 (2004).
H. F. G. Linde, D. Isaac, H. H. A. Linde, and D. Zivanov, Helv. Chim. Acta, 54 (6), 1703 (1971).
S. M. Vladimirov, D. S. Zivanovstakic, and B. J. Ribar, Phytochemistry, 30, 1724 (1991).
R. Tschesche, B. T. Tjoa, G. Wulff, and R. V. Noronha, Tetrahedron Let., 49, 5141 (1968).
W. Wisner and H. Kating, Planta Medica, 26, 228 (1974).
R. Hardman and T. V. Benjamin, Planta Medica, 39, 148 (1980).
B. Kismer and M. Wichtl, Arch. Pharm., 320, 541 (1987).
S. Liedtke, E. Lorch, P. Goedings, and M. Wichtl, Pharmazie, 52, 964 (1997).
D. Louden, A. Handley, S. Taylor, E. Lenz, S. Miller, I. D. Wilson, A. Sage, and R. Lafon, J. Chromatogr., A 910,
237 (2001).
10.
L. Krenn and B. Kopp, Phytochemistry, 1, 1 (1998).
92