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DOI: 10.1055/s-2002-35654
© Georg Thieme Verlag Stuttgart · New York
Cycloartane and Oleanane Saponins from Egyptian Astragalus spp. as Modulators of Lymphocyte Proliferation
Dr. Luisella Verotta
Dipartimento di Chimica Organica e Industriale
Università degli Studi di Milano
via Venezian 21, 20133 Milano
Italy
Fax: +39 02 5031 4106.
Email: luisella.verotta@unimi.it
Publication History
Received: March 18, 2002
Accepted: May 25, 2002
Publication Date:
26 November 2002 (online)
Abstract
From the roots of Astragalus kahiricus DC., three known saponins, namely, astraversianin VI, astraversianin X, astragaloside VIII, and a new saponin were isolated and identified by spectral data. The structure of the latter was elucidated by spectral means and assigned as cycloastragenol 3-O-[β-D-(2′,3′-diacetyl, 4′-trans-2-butenoyl)-xylopyranosyl], 6-O-β-D-xylopyranoside (kahiricoside I). From the aerial parts of A. hamosus L., the known compounds azukisaponin V and peregrinoside I were isolated. As judged by in vitro tests, the saponins isolated from Astragalus spp. endemic to Egypt were not cytotoxic against a variety of human cancer cells. However, dose-related modulation of lymphocyte proliferation was observed, and structure-activity relationships are described.
Key words
Astragalus spp. - Leguminosae - cycloartane-type saponins - oleanane-type saponins - lymphocytes proliferation modulators
Introduction
Several Astragalus species are used as medicinal plants, especially A. membranaceous (Radix Astragali), which is listed in the Chinese Pharmacopoeia, as are A. complanatus and A. mongholicus. Recent pharmacological studies have shown that Radix Astragali possesses an anti-influenza action and may prevent respiratory infections and both effects are believed to be related to the enhancement of immunological functions. The compounds responsible for the immunomodulating properties of Astragalus preparations are polysaccharides and saponins [1].
In the course of our structural studies on metabolites from Astragalus species endemic to Egypt, a number of cycloartane-type triterpene saponins have been isolated [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. They are mainly derivatives of cycloastragenol [20(R),24(S)-epoxy-9β,19cyclolanostan-3β,6α,16β,25-tetrol] [2], [3], [11], but examples of 20(S),24(R)-epoxy-9β,19cyclolanostan-3β,6α,16β,25-tetrol (cyclogalegenin, A. sieberi) [4], 9β,19-cyclolanostan-3β,6α,16β,24(R),25 pentol (cycloasgenin, A. alexandrinus) [5], [24,25,26,27-tetranor]-16β,23(R)-epoxy-9β,19-cyclolanostan-3β,6α,23α triol (A. tomentosus, A. spinosus) [6], [7], 9β,19-cyclolanost-24-ene-3β,6α,16β-triol (A. trigonus) [7], [8], 9β,19-cyclolanost-24-oxo-25-ene-3β,6α,16β-triol (A. trigonus) [10] oligosaccharides have been isolated as well. In addition, oleanane-type triterpene saponins have been isolated. They are 3β,22β,24-trihydroxy-olean-12-ene (soyasapogenol B) oligosaccharides, namely, azukisaponin V from A. trigonus [10] and A. tribuloides [12], and azukisaponin II from A. tribuloides [12].
We currently report investigations conducted with two additional species, A. kahiricus DC. and A. hamosus L. and the potential of the isolates to stimulate lymphocyte proliferation.
#Materials and Methods
#Plant material
A. kahiricus DC. and A. hamosus L. were collected from El-Arish, Sinai, Egypt, in March 1998. Voucher specimens have been deposited at the herbarium of the Department of Botany, Faculty of Science, University of Alexandria, Egypt .
#Isolation procedures and physical properties
The freshly sliced roots of A. kahiricus DC. (3.75 kg ) were extracted by maceration in 90 % EtOH, the ethanolic extract was concentrated (200 mL), added slowly to 300 mL of distilled water and partitioned successively into petrol, CH2Cl2, EtOAc, and n-BuOH. The CH2Cl2 extract was concentrated, the residue (10 g) was chromatographed on silica gel (400 g), eluting with CH2Cl2-MeOH mixtures (49 : 1, 1.2 L; 48 : 2, 0.5 L; 47 : 3, 1 L; 23 : 2, 1.2 L; 9 : 1, 1.2 L). Fractions 18 - 27 (1.62 g; eluent 47 : 3) yielded kahiricoside I (26) (80 mg), and fractions 28 - 39 (1.8 g; eluent 23 : 2) yielded compound 7 (240 mg). The EtOAc extract was concentrated, the residue (2 g) was chromatographed on silica gel (100 g) eluted with CH2Cl2-MeOH mixtures (19 : 1, 0.8 L; 37 : 3, 0.3 L; 9 : 1, 2 L). Fractions 9 - 11 (700 mg; eluent 37 : 3) yielded compound 6 (350 mg). The concentrated n-BuOH extract (8 g) was also chromatographed on silica gel (350 g), eluting with EtOAc-MeOH mixtures (17 : 3, 6.5 L; 11 : 1, 1 L; 4 : 1, 2.5 L) to yield astragaloside VIII (500 mg , 17.5 % MeOH ).
Kahiricoside I (26): m. p. 190 - 192 °C; [α]D 20: + 28.3° (c 0.9, MeOH); FAB+ MS: m/z = 929 [M + Na]+; 1H-NMR (1H: 500 MHz; 13C: 125 MHz, C5D5N) : see Tables [1] and [2].
Astraversianin VI (7): FAB+ MS: m/z = 819 [M + Na]+; FAB- MS: m/z = 795 [M - H]-; m. p. 245 - 248 °C; [α]D 20: + 30.4° (c , MeOH); 1H- and 13C-NMR (1H: 200 MHz; 13C: 50.2 MHz, C5D5N) data as reported previously [13].
Astraversianin X (6): FAB+ MS: m/z = 777 [M + Na]+; FAB- MS: m/z = 753 [M - H]-; m. p. 245 °C; [α]D 20: + 25.2° (c 0.15, MeOH); 1H- and 13C-NMR (1H: 200 MHz; 13C: 50.2 MHz, C5D5N) data as reported previously [13].
Astragaloside VIII: m. p. 260 °C. 30 mg were disolved in MeOH, added to sulphonic acid resin, and treated with CH2N2. The crude residue was purified on silica gel eluted with 10 % MeOH in CHCl3 (20 mL). 12 mg of astragaloside VIII methyl ester were recovered. [α]D 20: -11.9° (c 1, MeOH); lit [14] [α]D 20: -14° (c 0.71, MeOH); 1H- and 13C-NMR (1H: 200 MHz; 13C: 50 MHz, C5D5N) data as reported previously [14].
The air-dried defatted powdered fruiting aerial parts of A. hamosus L. (1.7 kg ) were extracted and fractionated as with A. kahiricus DC. The CH2Cl2 extract was concentrated and the residue (13 g) was chromatographed on silica gel (450 g) eluting with CH2Cl2-CH3OH mixtures (24 : 1, 3 L; 19 : 1, 2 L). Fractions 21 - 27 (315 mg; eluent 19 : 1) were combined and subjected to further purification by preparative TLC on SiO2 (EtOAc-CH3COOH-H2O, 5 : 1 : 1). A band revealed under UV light by quenching at 366 nm afforded peregrinoside I (2) (17 mg; Rf 0.4) . The concentrated EtOAc extract (3 g) was chromatographed on silica gel (100 g), eluting with EtOAc-MeOH mixtures (39 : 1, 2 L; 19 : 1, 2 L; 37 : 3, 1.4 L; 9 : 1, 0.5 L). Fractions 37 - 40 were combined (750 mg; eluent 37 : 3) and subjected to preparative TLC (EtOAc-CH3COOH-H2O, 5 : 1:1) to give azukisaponin V (25) (13 mg, Rf 0.22).
Peregrinoside I (3): m. p., [α]D 20 , 1H- and 13C-NMR (1H 600 MHz; 13C 150 MHz) were identical with data in [11].
Azukisaponin V (25): (Rf 0.22, EtOAc-CH3COOH-H2O, 5 : 1:1), (Rf 0.35, CH3Cl3-MeOH-H2O, 5 : 5 : 3), m. p. 244 - 246 °C. Methyl ester m. p. 273 °C; [α]D 20: + 13.1 (c 0.7, MeOH) were identical with data in [10].
Compound 20 was isolated from the saponins of A. trigonus [10] and identified as a mixture of diastereoisomers by spectral means (unpublished data) [15]. Cycloastragenol (1) was obtained by enzymatic hydrolysis from astragaloside IV isolated from A. spinosus [7]. The other saponins have been described in the corresponding papers [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12].
δH | δC | |
1ax | 1.504 | 31.8 (t) |
1eq | 1.258 | |
2eq | 2.174 | 29.7 (t) |
2ax | 1.90 | |
3ax | 3.34 (dd, 4.6, 11.7) | 89.3 (d) |
4 | 42.3 (s) | |
5ax | 1.825 (d, ) | 51.7 (d) |
6ax | 3.76 m | 78.2 (d) |
7eq | 2.114 | 34.0 (t) |
7ax | 1.906 | |
8ax | 2.01 | 44.8 (d) |
9 | 21.3 (s) | |
10 | 28.6 (s) | |
11ax | 1.72 | 26.4 (t) |
11eq | 1.36 | |
12eq | 1.65 | 33.5 (t) |
12ax | 1.556 | |
13 | 45.3 (s) | |
14 | 46.3 (s) | |
15α | 2.294 | 46.1 (t) |
15β | 1.821 | |
16ax | 5.04 m | 73.2 (d) |
17ax | 2.55 (d, 7.8) | 58.3 (d) |
18 | 1.392 s | 20.8 (q) |
19B | 0.56( d, ) | 27.6 (t) |
19A | 0.18 (d, ) | |
20 | 87.4 (s) | |
21 | 1.312 (s) | 28.7(q) |
22α | 3.097 | 35.1 (t) |
22β | 1.673 | |
23α | 2.27 | 26.6 (t) |
23β | 2.02 | |
24α | 3.89 (dd, 5.4, 8.9) | 81.8 (d) |
25 | 71.2 (s) | |
26 | 1.580 (s) | 28.3 (q) |
27 | 1.312 (s) | 27.2 (q) |
28 | 1.666 (s) | 28.1 (q) |
29 | 1.20(s) | 16.6 (q) |
30 | 1.06 (s) | 19.9 (q) |
16OH | ||
a J values are in parentheses. |
δC | δH | Mult. | J (Hz) | significant BH-C carbon connections | ||
3-O-Xyl | 1′ | 103.6 | 4.87 | d | 7.6 | C-3 |
2′ | 72.4 | 5.45 | dd | 7.4, 9.3 | COCH3 | |
3′ | 72.8 | 5.75 | t | 9.3 | COCH3 | |
4′ | 69.9 | 5.35 | ddd | 5.4, 9.8, 9.0 | COCH = CHCH3 | |
5′ | 62.7 | 4.37A | dd | 5.5, 11.6 | ||
3.68B | m | |||||
2′COCH3 | 20.7 | 2.01 | s | |||
2′COCH3 | 169.9 | |||||
3′COCH3 | 20.7 | 2.03 | s | |||
3′COCH3 | 170.5 | |||||
4′COCH = CHCH3 | 18.0 | 1.64 | dd | 6.9, 1.6 | ||
4′COCH = CHCH3 | 146.9 | 7.02 | dq | 15.5, 6.9 | ||
4′COCH = CHCH3 | 122.2 | 5.85 | dd | 15.5, 1.7 | ||
4′COCH = CHCH3 | 165.5 | |||||
6-O-Xyl | 1¿ | 105.7 | 4.84 | d | 7.3 | C-6 |
2¿ | 75.5 | 4.00 | t | 7.8 | ||
3¿ | 78.7 | 4.14 | t | 8.4 | ||
4¿ | 71.5 | 4.17 | m | |||
5¿ | 67.1 | 4.30A | dd | 4.7, 11.1 | ||
3.68B | m |
Biological Evaluation
Cytotoxicity with cultured mammalian cells: Isolates were evaluated for cytotoxic potential as described previously [16]. Experiments were performed with the following panel of cell lines: BC1 (human breast cancer), Lu1 (human lung cancer), Col2 (human colon cancer), LNCaP (human prostate cancer), and KB cells. In brief, cultured cells were treated with various concentrations of test compounds for a period of 3 days at 37 °C in a 5 % CO2 incubator. The cells were then fixed with trichloroacetic acid, washed, stained with sulforhodamine B, and washed once again. Finally, residue dye was solubilized and used to quantify cell survival. In all cases, no significant activity was observed (ED50 values >20 μg/mL), although positive controls such as taxol and vinblastine mediated intense cytotoxicity.
Immunomodulatory effects: Female CD-1 mice were received from Charles River Breeding Laboratories at 8-weeks-of-age. Animals were fed Prolab RMH 4020 Rat/Mouse Chow (blox) and maintained on a 12 h light/dark cycle. For the isolation of target cells, mice were anesthetized with ether and spleens were removed by surgical dissection. The tip of the spleen was cut off and the remaining portion was placed on a stainless steel filter. The spleen was pressed against the filter using a pestle-like device. The filter was rinsed with Hank"s solution to collect splenocytes in a Petri dish. The cells were then resuspended with 5 mL of complete RPMI-1640 medium, and the cell concentration was adjusted to 5 × 106/mL. The potential of test samples to stimulate cell proliferation was determined as described previously [17]. Briefly, test samples (initially dissolved in DMSO) were added to 96-well plates (in triplicate) to yield final test concentrations of 0 (control), 0.01, 0.1, 1, 10, or 100 μg/mL. This was followed by the addition of 0.1 mL of the cell suspension, and 0.1 mL of medium or medium supplemented with Con A (final concentration, 8 μg/mL). Plates were then incubated at 37 °C with 5 % CO2 in a water-jacketed incubator (100 % humidity) for 70 h. [3 H]Thymidine (1 μCi, 65 Ci/mmol) was then added, and the mixtures were incubated for an additional 18 h. Following this incubation, cells were harvested onto a glass fiber mat, washed for the removal of unincorporated thymidine, and counted by liquid scintillation spectroscopy. Incorporation of [3H]thymidine was calculated as [dpm (test sample) - dpm (control)]/dpm (control). In this assay system, Con A serves both as a co-stimulator and a positive control. In each case, relative to control incubations, statistical analyses were performed using Student’s t-test. Significant differences were indicated by p < 0.05.
#Results and Discussion
From the fresh roots of A. kahiricus and the dried aerial parts of A. hamosus, two known soyasapogenol B derivatives, astragaloside VII and azukisaponin V (25), and three known cycloastragenol oligosaccharides, peregrinoside I (3), astraversianin X (6), and astraversianin VI (7), were isolated, and their structures elucidated by spectral means and comparison with known compounds. The structure of the new saponin 26, isolated from A. kahiricus, was elucidated by standard spectral means (1D- and 2D-NMR experiments at 500 MHz [10] and FAB MS). A molecular formula of C48H74O16 was deduced from the FAB spectrum (m/z 929 [M + Na]+). The GHSQC, GHMQC, COSY and TOCSY experiments allowed identification of the cycloastragenol skeleton [12], and the presence of two sugar units. They were identified as two β-D-xylopyranose units, respectively linked to aglycone positions 3 and 6; the two acetyl and the 2-E-butenoyl groups were located on the same 3-O-linked sugar moiety, as deduced by the proton-carbon long-range correlations (see Table [2]). Compound 26 was assigned the structure of cycloastragenol 3-O-[β-D-(2′,3′-diacetyl-4′-trans-2-butenoyl)-xylopyranosyl] 6-O-β-D-xylopyranoside (kahiricoside I).
All the available saponins isolated in our previous studies [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12] and those here described (compounds 1 - 25) were evaluated for cytotoxic potential with a panel of human cancer cell lines, and immunomodulatory potential using mouse splenocytes. Compound 26 was not tested because of its close relationship with 9 and the rare occurrence in Astragalus species with respect to all the other saponins (1 - 25).[*] [] [*] [*]
When evaluated with cultured BC1 (human breast cancer), Lu1 (human lung cancer), Col2 (human colon cancer), LNCaP (human prostate cancer), and KB cells, no significant growth inhibitory activity was observed with these test compounds (ED50 values >20 μg/mL).
Next, based on analogous studies that have been reported with related compounds [18], the potential of the isolated compounds to stimulate the proliferation of mouse splenocytes was examined in the presence and absence of Con A (Table [3]). These products allow comparison with the same cycloartane-type aglycones, by taking into consideration branched or closed side chains, and, in the case of the latter, the stereochemistries at C20 and C24 (cycloastragenol or cyclogalegenin).
The triterpenoid cycloastragenol (1) [20(R),24(S)-epoxy-9β,19-cyclolanostan-3β,6α,16β,25-tetrol] stimulates the proliferation of mouse splenocytes only in absence of Con A, as well as its 6-O-xyloside (trigonoside I, 4). Cycloastragenol 6-O-glucoside (5), on the contrary, shows only a weak increase in splenocyte proliferation at 0.1 μg/mL, higher at 1 μg/mL, in the absence of Con A. In the presence of Con A, the effect is much more evident. Cycloastragenol 3-O-glycosides (2 and 3) maintain activity at 0.1 μg/ml, both in presence or absence of Con A. These data lead to the conclusion that 3-O- and 6-O-cycloastragenol derivatives are more active than the aglycone itself, and that the 6-O-glucoside is more active than the 6-O-xyloside.
Variations in activity can be noted between the 3-O-glycosides of cycloastragenol 6-O-glucoside (B series) and the series of the 3-O-glycosides of cycloastragenol 6-O-xyloside (A series). Within the A series, the activity decreases by conversion of the 3-O-xyloside (6) and its 2′-OAc (7), to its 2′-O-arabinoside (8), its 2′-O-arabinoside, 3′-OAc (9), or its 2′-O-rhamnoside (10), in the absence of Con A. In the presence of Con A, activity is maintained, and the effect is opposite, e. g., 2′-O-substituted 3-O-xylosides are more active than the parent 3-O-xyloside. A comparison within the B series shows that the activity decreases by conversion from the 3-O-xyloside of cycloastragenol 6-O-glucoside (11) to its 2′-OAc (12) and 2′,3′di-OAc (13) in the absence of Con A, but activity is completely lost in the presence of Con A.
A change in the stereochemistry of the tetrahydrofuran ring [20(S),24(R)-epoxy-9β,19-cyclolanostan-3β,6α,16β,25-tetrol = cyclogalegenin] (14) maintains the activity if compared to cycloastragenol (1), in absence of Con A, and activity is increased in the presence of Con A. Its 3-O-glucoside (15) lacks activity in the absence of Con A, while the di-glucoside (16) has a slightly higher activity; both these compounds stimulate the proliferation of mouse splenocytes in presence of ConA.
Cycloasgenin [9β,19-cyclolanostan-3β,6α,16β,24(R),25 pentol] derivatives (17 - 21), stimulate the proliferation of mouse splenocytes both in the presence or absence of Con A, except for the 6-oxo derivative (17) and (21). A 6 α-hydroxy configuration seems to be preferred for the maintainance of the activity.
Tetranorcycloartane (22, 23) and oleanane type saponins (24, 25) show significant proliferative responses only in the presence of Con A.
Recently, employing THP-1 cells transiently transfected with a luciferase construct in which expression was dependent on nuclear factor kappa B activation, a series of cycloartane-type triterpene glycosides obtained from Astragalus species were tested and found to be inactive [19]. Although directed toward the establishment of an immunostimulatory response, it would be fortuitous if data obtained with this highly specific and well-defined test system correlated with the results reported herein. First, there are fundamental differences in terms of cell types, treatment times, anticipated dose-response relationships and endpoints. Moreover, when monitoring cellular proliferation in the presence and absence of a known activator, such as LPS or Con A, a variety of mechanisms may yield a positive or negative response, and it is common to observe stimulation followed by inhibition as a function of test compound concentration.
From our data, although not exhaustive, some conclusions about structure-activity relationships for the modulation of lymphocyte proliferation of the most recurrent saponins in Astragalus sp. can be drawn:
1. Cycloastragenol 3-O-glycosides (2, 3, 7 - 10) preferentially carrying a substituent in position 2 of the 3-O-linked sugar are the most active compounds in the presence of a growth stimulator.
2. Taking into account the same substituent at C3, cycloastragenol 6-O-xylosides generally maintain activity both in the presence or absence of Con A; cycloastragenol 6-O-glucosides lack activity in the presence of Con A (compare 6 to 11 and 7 to 12).
Our data indicate that the saponins characterizing Astragalus species are indeed stimulators of an immune response in the concentration range 0.01 - 10 μM and support previous findings on related structures [18]. Since the reported cycloastragenol saponins represent chemical structures peculiar to the genus Astragalus [1], it is reasonable to postulate the species used in the pharmacopeas of Eastern countries for the treatment of conditions such as viral infection or cancer may function by modulation of an immune response, since specific cytotoxic and antiviral properties are weak [1]. It would be of value to further test these agents for potential to modulate immunological factors with additional in vivo and in vitro systems.




μg/ml | No Con A | Con A (8μg/ml) | No Con A | Con A (8μg/ml | ||||||||
x ± S.D. | ± % | P | x ± S.D. | ± % | P | x ± S.D. | ± % | P | x ± S.D. | ± % | P | |
Cycloastragenol (1) | Peregrinoside II (2) | |||||||||||
0 | 618 ± 76 | 47443 ± 3473 | 398 ± 25 | 58887 ± 5805 | ||||||||
0.01 | 1411 ± 33 | 128 | 0.000 | 31751 ± 3649 | -33 | 0.001 | 449 ± 69 | 13 | 0.082 | 102819 ± 4914 | 75 | 0.000 |
0.1 | 1615 ± 136 | 161 | 0.000 | 38439 ± 4994 | -19 | 0.032 | 479 ± 45 | 20 | 0.013 | 109867 ± 14038 | 87 | 0.000 |
1 | 1560 ± 35 | 152 | 0.000 | 36000 ± 4957 | -25 | 0.003 | 300 ± 33 | -25 | 0.009 | 95553 ± 5672 | 45 | 0.000 |
10 | 1875 ± 105 | 203 | 0.000 | 67478 ± 7525 | 42 | 0.000 | 199 ± 56 | -50 | 0.000 | 71095 ± 7223 | 21 | 0.057 |
100 | 422 ± 76 | -32 | 0.050 | 368 ± 80 | -99 | 0.000 | 108 ± 11 | -73 | 0.000 | 2223 ± 484 | -96 | 0.000 |
Peregrinoside I (3) | Trigonoside I (4) | |||||||||||
0 | 115 ± 38 | 44123 ± 4765 | 730 ± 85 | 48428 ± 4496 | ||||||||
0.01 | 246 ± 26 | +114 | 0.002 | 105495 ± 6818 | 139 | 0.000 | 2301 ± 905 | +215 | 0.006 | 55790 ± 2880 | +15 | 0.411 |
0.1 | 422 ± 99 | +267 | 0.000 | 117576 ± 1090 | 166 | 0.000 | 1726 ± 161 | +136 | 0.010 | 43495 ± 7110 | -10 | 0.217 |
1 | 231 ± 69 | +101 | 0.004 | 122046 ± 1214 | 177 | 0.000 | 1727 ± 122 | +137 | 0.007 | 41872 ± 1257 | -14 | 0.114 |
10 | 103 ± 23 | -10 | 0.738 | 82476 ± 1109 | 87 | 0.000 | 1176 ± 43 | +61 | 0.171 | 36648 ± 3003 | -24 | 0.011 |
100 | 148 ± 11 | +29 | 0.334 | 102 ± 27 | -100 | 0.000 | 147 ± 5 | -80 | 0.081 | 211 ± 94 | -99 | 0.000 |
Cycloastragenol 6-O-Glucoside (5) | Astraversianin X (6) | |||||||||||
0 | 414 ± 45 | 25134 ± 1694 | 730 ± 85 | 48428 ± 4496 | ||||||||
0.01 | 505 ± 61 | +24 | 0.322 | 54288 ± 5575 | +116 | 0.000 | 1199 ± 175 | +64 | 0.000 | 49754 ± 3996 | +0.6 | 0.993 |
0.1 | 677 ± 62 | +64 | 0.013 | 94244 ± 12014 | +275 | 0.000 | 1486 ± 329 | +104 | 0.000 | 40328 ± 3098 | -17 | 0.021 |
1 | 1254 ± 345 | +202 | 0.000 | 62078 ± 7540 | +147 | 0.000 | 1010 ± 105 | +38 | 0.013 | 39570 ± 6505 | -18 | 0.140 |
10 | 692 ± 61 | +67 | 0.01 | 56087 ± 1703 | +123 | 0.000 | 806 ± 44 | +10 | 0.448 | 28127 ± 5061 | -42 | 0.000 |
100 | 201 ± 51 | -34 | 0.043 | 1181 ± 42 | -95 | 0.000 | 196 ± 42 | -73 | 0.000 | 372 ± 98 | -98 | 0.000 |
Astraversianin VI (7) | Astraversianin X (8) | |||||||||||
0 | 1149 ± 116 | 53741± 11284 | 395 ± 25 | 56395 ± 4484 | ||||||||
0.01 | 1617 ± 166 | +40 | 0.003 | 120976 ± 11112 | +125 | 0.000 | 383 ± 63 | -3 | 0.719 | 111044 ± 12793 | +97 | 0.000 |
0.1 | 2353 ± 167 | +105 | 0.000 | 124037 ± 12459 | +131 | 0.000 | 520 ± 16 | +32 | 0.004 | 90855 ± 3374 | +61 | 0.000 |
1 | 1724 ± 67 | +50 | 0.012 | 105926 ± 13141 | +97 | 0.000 | 337 ± 53 | -15 | 0.124 | 87922 ± 5214 | +60 | 0.000 |
10 | 1658 ± 313 | +44 | 0.023 | 107606 ± 10272 | +100 | 0.000 | 367 ± 66 | -7 | 0.432 | 68309 ± 787 | +21 | 0.099 |
100 | 136 ± 27 | -88 | 0.000 | 722 ± 71 | -99 | 0.000 | 141 ± 31 | -64 | 0.000 | 336 ± 76 | -99 | 0.000 |
Trigonoside III (9) | Astraversianin XV (10) | |||||||||||
0 | 395 ± 25 | 57996 ± 3423 | 718 ± 24 | 59074 ± 1430 | ||||||||
0.01 | 541 ± 74 | +37 | 0.003 | 109371 ± 12573 | +89 | 0.000 | 768 ± 178 | +6 | 0.705 | 103344 ± 6307 | 75 | 0.000 |
0.1 | 422 ± 17 | +7 | 0.526 | 116696 ± 5194 | +101 | 0.000 | 561 ± 120 | -32 | 0.232 | 106874 ± 12737 | 81 | 0.000 |
1 | 315 ± 61 | -21 | 0.052 | 101216 ± 14318 | +81 | 0.000 | 602 ± 60 | -16 | 0.369 | 117939 ± 4820 | 100 | 0.000 |
10 | 180 ± 61 | -54 | 0.000 | 65033 ± 5991 | +12 | 0.415 | 238 ± 23 | -67 | 0.002 | 79369 ± 7197 | 35 | 0.003 |
100 | 57 ± 13 | -86 | 0.000 | 149 ± 21 | -99 | 0.000 | 627 ± 224 | -13 | 0.476 | 649 ± 98 | -99 | 0.000 |
Astraversianin XIV (11) | Astragaloside II (12) | |||||||||||
0 | 730 ± 85 | 48428 ± 4496 | 618 ± 76 | 47443 ± 3473 | ||||||||
0.01 | 1113 ± 72 | +52 | 0.002 | 51360 ± 1483 | +6 | 0.438 | 725 ± 60 | +17 | 0.021 | 42360 ± 4301 | -11 | 0.246 |
0.1 | 1799 ± 176 | +146 | 0.000 | 34366 ± 2831 | -29 | 0.001 | 1124 ± 176 | +82 | 0.000 | 46800 ± 4680 | -1 | 0.975 |
1 | 1699 ± 206 | +133 | 0.000 | 43431 ± 7703 | -10 | 0.124 | 848 ± 59 | +37 | 0.009 | 43300 ± 4484 | -9 | 0.337 |
10 | 745 ± 16 | +2 | 0.091 | 36765 ± 1948 | -24 | 0.003 | 856 ± 81 | +39 | 0.008 | 40056 ± 8273 | -16 | 0.127 |
100 | 357 ± 102 | -51 | 0.003 | 2741 ± 10 | -94 | 0.000 | 337 ± 60 | -45 | 0.001 | 33717 ± 5902 | -29 | 0.070 |
Astragaloside I (13) | Cyclogalegenin (14) | |||||||||||
0 | 618 ± 76 | 47443 ± 3473 | 390 ± 65 | 23662 ± 1835 | ||||||||
0.01 | 923 ± 173 | +49 | 0.012 | 46120 ± 5945 | -3 | 0.680 | 550 ± 131 | +41 | 0.012 | 46949 ± 4911 | +100 | 0.054 |
0.1 | 907 ± 253 | +47 | 0.154 | 40764 ± 6886 | -14 | 0.074 | 877 ± 68 | +125 | 0.000 | 94863 ± 5729 | +312 | 0.000 |
1 | 1126 ± 172 | +82 | 0.012 | 39205 ± 2224 | -17 | 0.033 | 550 ± 76 | +51 | 0.42 | 60525 ± 4696 | +156 | 0.001 |
10 | 1018 ± 243 | +65 | 0.039 | 37350 ± 4376 | -21 | 0.015 | 415 ± 68 | +6 | 0.783 | 20089 ± 3994 | -15 | 0.470 |
100 | 174 ± 32 | -72 | 0.018 | 4081 ± 194 | -92 | 0.000 | 244 ± 29 | -37 | 0.041 | 12981 ± 3823 | -45 | 0.060 |
Sieberoside I (15) | Sieberoside II (16) | |||||||||||
0 | 3037 ± 784 | 51728 ± 6352 | 369 ± 42 | 44711 ± 4676 | ||||||||
0.01 | 2738 ± 425 | -10 | 0.000 | 80220 ± 6202 | +55 | 0.003 | 954 ± 243 | +158 | 0.000 | 88686 ± 4822 | +98 | 0.000 |
0.1 | 1447 ± 201 | -51 | 0.000 | 80050 ± 19500 | +56 | 0.003 | 1278 ± 111 | +246 | 0.000 | 90096 ± 5628 | +101 | 0.000 |
1 | 1104 ± 11 | -64 | 0.000 | 91234 ± 12022 | +76 | 0.000 | 591 ± 87 | +60 | 0.067 | 109866 ± 11147 | +145 | 0.000 |
10 | 534 ± 113 | -82 | 0.000 | 70680 ± 11928 | +37 | 0.030 | 513 ± 30 | +39 | 0.205 | 53136 ± 5807 | +19 | 0.275 |
100 | 168 ± 60 | -94 | 0.000 | 0 ± 0 | 0 | 573 ± 240 | +55 | 0.092 | 0 ± 0 | 0 | 0.000 | |
6-Oxo-cycloastragenol (17) | Compound 18 | |||||||||||
0 | 618 ± 76 | 47443 ± 3473 | 312 ± 68 | 52687 ± 6271 | ||||||||
0.01 | 711 ± 103 | +15 | 0.5 | 37605 ± 3649 | -4 | 0.21 | 664 ± 44 | +113 | 0.035 | 102134 ± 4024 | +94 | 0.000 |
0.1 | 1149 ± 163 | +86 | 0.001 | 43811 ± 4473 | -8 | 0.335 | 1044 ± 300 | +235 | 0.000 | 108781 ± 6690 | +106 | 0.633 |
1 | 893 ± 47 | +44 | 0.050 | 43551 ± 3119 | -8 | 0.112 | 1580 ± 601 | +406 | 0.000 | 113620 ± 21758 | +114 | 0.002 |
10 | 1688 ± 312 | +173 | 0.000 | 66222 ± 7268 | 40 | 0.000 | 572 ± 72 | +83 | 0.102 | 69817 ± 2972 | +33 | 0.010 |
100 | 297 ± 16 | -52 | 0.015 | 3306 ± 724 | -93 | 0.000 | 220 ± 16 | -29 | 0.551 | 5804 ± 1618 | -89 | 0.000 |
Compound 19 | Compound 20 | |||||||||||
0 | 392 ± 96 | 54191 - 6838 | 3326 ± 163 | 61644 ± 9466 | ||||||||
0.01 | 625 ± 14 | +59 | 0.000 | 79217 - 7324 | +46 | 0.000 | 2222 ± 539 | -33 | 0.000 | 145664 ± 5766 | +136 | 0.000 |
0.1 | 445 ± 25 | +14 | 0.114 | 104663 - 8674 | +93 | 0.000 | 3993 ± 323 | +20 | 0.008 | 136842 ± 11102 | +122 | 0.000 |
1 | 370 ± 74 | +6 | 0.847 | 72310 - 6002 | +87 | 0.005 | 838 ± 195 | -75 | 0.000 | 116250 ± 13747 | +80 | 0.000 |
10 | 222 ± 20 | +43 | 0.000 | 52851 - 7664 | -3 | 0.804 | 298 ± 132 | -91 | 0.000 | 107498 ± 8889 | +74 | 0.000 |
100 | 36 ± 15 | +91 | 0.000 | 0 - 0 | 0 | 0.000 | 184 ± 124 | -94 | 0.000 | 1305 ± 66 | -99 | 0.000 |
Compound 21 | Compound 22 | |||||||||||
0 | 730 ± 85 | 48428 - 4496 | 398 ± 25 | 56158 ± 4399 | ||||||||
0.01 | 1062 ± 88 | +45 | 0.019 | 31744 - 1677 | -34 | 0.006 | 403 ± 65 | +1 | 0.710 | 96526 ± 3826 | +72 | 0.000 |
0.1 | 1495 ± 249 | +105 | 0.000 | 46923 - 12085 | -3 | 0.726 | 310 ± 30 | -22 | 0.012 | 91241 ± 8904 | +62 | 0.000 |
1 | 1218 ± 82 | +67 | 0.002 | 35784 - 2774 | -26 | 0.024 | 330 ± 53 | -17 | 0.046 | 85633 ± 5606 | +52 | 0.000 |
10 | 796 ± 37 | +9 | 0.605 | 40978 - 7975 | -15 | 0.158 | 196 ± 22 | -51 | 0.000 | 32965 ± 4999 | +41 | 0.000 |
100 | 679 ± 62 | -7 | 0.681 | 5502 - 750 | -89 | 0.000 | 46 ± 12 | -88 | 0.000 | 566 ± 59 | -99 | 0.000 |
Compound 23 | Azukisaponin II (24) | |||||||||||
0 | 398 ± 25 | 61490 - 6111 | 507 ± 76 | 58202 ± 7390 | ||||||||
0.01 | 413 ± 26 | +4 | 0.192 | 104684 - 10107 | +70 | 0.000 | 666 ± 158 | +31 | 0.039 | 83038 ± 6654 | +43 | 0.000 |
0.1 | 488 ± 65 | +22 | 0.002 | 96317 - 6374 | +57 | 0.000 | 498 ± 45 | -2 | 0.984 | 84960 ± 7203 | +46 | 0.000 |
1 | 233 ± 38 | -41 | 0.000 | 69119 - 7216 | +12 | 0.158 | 458 ± 2 | -10 | 0.481 | 98661 ± 8886 | +70 | 0.000 |
10 | 165 ± 12 | -59 | 0.000 | 20171 - 1376 | -67 | 0.000 | 318 ± 99 | -37 | 0.017 | 60443 ± 2703 | +4 | 0.670 |
100 | 39 ± 11 | -90 | 0.000 | 896 - 71 | -99 | 0.000 | 192 ± 30 | -62 | 0.001 | 1091 ± 0 | -98 | 0.000 |
Azukisaponin V (25) | ||||||||||||
0 | 392 ± 96 | 58189 ± 2958 | ||||||||||
0.01 | 572 ± 24 | +46 | 0.000 | 74087 ± 5067 | +27 | 0.001 | ||||||
0.1 | 404 ± 47 | +3 | 0.633 | 97137 ± 8168 | +67 | 0.000 | ||||||
1 | 530 ± 21 | +35 | 0.002 | 83045 ± 8092 | +43 | 0.000 | ||||||
10 | 281 ± 60 | -28 | 0.010 | 37912 ± 7264 | -35 | 0.000 | ||||||
100 | 74 ± 21 | -81 | 0.000 | 0 ± 0 | 0 | 0.000 |
Acknowledgements
Work supported by MURST and CNR of Italy.
#References
- 1 Verotta L, El-Sebakhy N A. Cycloartane and Oleanane Saponins from Astragalus sp. In: Studies in Natural Products Chemistry, Bioactive Natural Products (Part F). Ed. Atta-ur-Rahman, Elsevier Science Publishers- Amsterdam,. 2001; 25 179-234
- 2 Abdallah R M, Ghazy N M, El-Sebakhy N A, Pirillo A, Verotta L. Astragalosides from Egyptian Astragalus spinosus Vahl. Pharmazie. 1993; 48 452-4
- 3 Gariboldi P, Pelizzoni F, Tatò M, Verotta L, El-Sebakhy N A, Asaad A M, Abdallah R M, Toaima S M. Cycloartane triterpene glycosides from Astragalus trigonus . Phytochemistry. 1995; 40 1755-60
- 4 Verotta L, Tatò M, El-Sebakhy N A, Toaima S M. Cycloartane triterpene glycosides from Astragalus sieberi . Phytochemistry. 1998; 48 1403-9
- 5 Orsini F, Verotta L, Barboni L, El-Sebakhy N A, Asaad A M, Abdallah R M, Toaima SM Soad M. Cycloartane triterpene glycosides from Astragalus alexandrinus . Phytochemistry. 1994; 35 745-9
- 6 El-Sebakhy N A, Harraz F M, Abdallah R M, Asaad A M, Orsini F, Pelizzoni F, Sello G, Verotta L. Cycloartane triterpene glycosides from Egyptian Astragalus species. Phytochemistry. 1990; 29 3271-4
- 7 Abdallah R M, Ghazy N M, Assad A M, El-Sebakhy N A, Pirillo A, Verotta L. Constituents of the Egyptian Astragalus tomentosus . Pharmazie. 1994; 49 377-8
- 8 Verotta L, Orsini F, Tatò M, El-Sebakhy N A, Toaima SM Soad M. A cycloartane triterpene 3β,16β diglucoside from Astragalus trigonus and its non natural 6-hydroxy epimer. Phytochemistry. 1998; 49 845-52
- 9 El-Sebakhy N A, Waterman P G. 6-Oxocycloartan-3β,16β-diglucoside: a new cycloartane diglucoside from Astragalus trigonus . Planta Med. 1985; 4 350-2
- 10 Pelizzoni F, Verotta L, Nicastro G, Tatò M, El-Sebakhy N A, Asaad A M, Abdallah R M, Toaima SM Soad M. A minor cycloartan-3β,16β-diglucoside from Astragalus trigonus . Gazz Chim Ital. 1996; 126 657-61
- 11 Verotta L, Guerrini M, El-Sebakhy N A, Asaad A M, Toaima S M, Abou-Sheer M E, Luo Y ing-De, Pezzuto J M. Cycloartane saponins from Astragalus peregrinus as modulators of lymphocyte proliferation. Fitoterapia. 2001; 72 894-905
- 12 El-Sebakhy N A, Asaad A M, Abdallah R M, Toaima S M, Soad M, Verotta L, Orsini F. Constituents of Egyptian Astragalus tribuloides Del. Natural Product Sciences. 2000; 6 1-5
- 13 Gan L i-Xian, Han X iao-Bing, Chen Y u-Qun. The chemical investigation of Astragalus sieverianus Pall. Part 3. The structures of thirteen astrasieversianins from Astragalus sieversianus . Phytochemistry. 1986; 25 2389-93
- 14 Konoshima T, Kozuka M, Haruna M, Ito K, Kimura T, Tokuda H. Studies on the constituents of leguminous plants. XII. The structures of new triterpenoid saponins from Wistaria brachybotrys Sieb. et Zucc. Chem Pharm Bull. 1989; 37 2731-5
- 15 All the NMR spectra recorded at 500 MHz are available on request.
- 16 Likhitwitayawuid K, Angerhofer C K, Cordell G A, Pezzuto J M, Ruangrungsi N. Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from Stephania erecta . J Nat Prod. 1993; 56 30-8
- 17 Mar W, Tan G R, Cordell G A, Pezzuto J M, Jurcic K, Redl K, Steinke B, Wagner H. Biological activity of novel macrocyclic alkaloids (budmunchiamines) from Albizia amara detected on the basis of interaction with DNA. J Nat Prod. 1991; 54 1531-42
- 18 Calis I, Yuruker A, Tasdemir D, Wright A D, Sticher O, Luo Y D, Pezzuto J M. Cycloartane triterpene glycosides from the roots of Astragalus melanophrurius . Planta Med. 1997; 63 183-6
- 19 Bedir E, Pugh N, Calis I, Pasco D S, Khan I A. Immunostimulatory effects of cycloartane-type triterpene glycosides from Astragalus species . Biol Pharm Bull. 2000; 23 834-7
Dr. Luisella Verotta
Dipartimento di Chimica Organica e Industriale
Università degli Studi di Milano
via Venezian 21, 20133 Milano
Italy
Fax: +39 02 5031 4106.
Email: luisella.verotta@unimi.it
References
- 1 Verotta L, El-Sebakhy N A. Cycloartane and Oleanane Saponins from Astragalus sp. In: Studies in Natural Products Chemistry, Bioactive Natural Products (Part F). Ed. Atta-ur-Rahman, Elsevier Science Publishers- Amsterdam,. 2001; 25 179-234
- 2 Abdallah R M, Ghazy N M, El-Sebakhy N A, Pirillo A, Verotta L. Astragalosides from Egyptian Astragalus spinosus Vahl. Pharmazie. 1993; 48 452-4
- 3 Gariboldi P, Pelizzoni F, Tatò M, Verotta L, El-Sebakhy N A, Asaad A M, Abdallah R M, Toaima S M. Cycloartane triterpene glycosides from Astragalus trigonus . Phytochemistry. 1995; 40 1755-60
- 4 Verotta L, Tatò M, El-Sebakhy N A, Toaima S M. Cycloartane triterpene glycosides from Astragalus sieberi . Phytochemistry. 1998; 48 1403-9
- 5 Orsini F, Verotta L, Barboni L, El-Sebakhy N A, Asaad A M, Abdallah R M, Toaima SM Soad M. Cycloartane triterpene glycosides from Astragalus alexandrinus . Phytochemistry. 1994; 35 745-9
- 6 El-Sebakhy N A, Harraz F M, Abdallah R M, Asaad A M, Orsini F, Pelizzoni F, Sello G, Verotta L. Cycloartane triterpene glycosides from Egyptian Astragalus species. Phytochemistry. 1990; 29 3271-4
- 7 Abdallah R M, Ghazy N M, Assad A M, El-Sebakhy N A, Pirillo A, Verotta L. Constituents of the Egyptian Astragalus tomentosus . Pharmazie. 1994; 49 377-8
- 8 Verotta L, Orsini F, Tatò M, El-Sebakhy N A, Toaima SM Soad M. A cycloartane triterpene 3β,16β diglucoside from Astragalus trigonus and its non natural 6-hydroxy epimer. Phytochemistry. 1998; 49 845-52
- 9 El-Sebakhy N A, Waterman P G. 6-Oxocycloartan-3β,16β-diglucoside: a new cycloartane diglucoside from Astragalus trigonus . Planta Med. 1985; 4 350-2
- 10 Pelizzoni F, Verotta L, Nicastro G, Tatò M, El-Sebakhy N A, Asaad A M, Abdallah R M, Toaima SM Soad M. A minor cycloartan-3β,16β-diglucoside from Astragalus trigonus . Gazz Chim Ital. 1996; 126 657-61
- 11 Verotta L, Guerrini M, El-Sebakhy N A, Asaad A M, Toaima S M, Abou-Sheer M E, Luo Y ing-De, Pezzuto J M. Cycloartane saponins from Astragalus peregrinus as modulators of lymphocyte proliferation. Fitoterapia. 2001; 72 894-905
- 12 El-Sebakhy N A, Asaad A M, Abdallah R M, Toaima S M, Soad M, Verotta L, Orsini F. Constituents of Egyptian Astragalus tribuloides Del. Natural Product Sciences. 2000; 6 1-5
- 13 Gan L i-Xian, Han X iao-Bing, Chen Y u-Qun. The chemical investigation of Astragalus sieverianus Pall. Part 3. The structures of thirteen astrasieversianins from Astragalus sieversianus . Phytochemistry. 1986; 25 2389-93
- 14 Konoshima T, Kozuka M, Haruna M, Ito K, Kimura T, Tokuda H. Studies on the constituents of leguminous plants. XII. The structures of new triterpenoid saponins from Wistaria brachybotrys Sieb. et Zucc. Chem Pharm Bull. 1989; 37 2731-5
- 15 All the NMR spectra recorded at 500 MHz are available on request.
- 16 Likhitwitayawuid K, Angerhofer C K, Cordell G A, Pezzuto J M, Ruangrungsi N. Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from Stephania erecta . J Nat Prod. 1993; 56 30-8
- 17 Mar W, Tan G R, Cordell G A, Pezzuto J M, Jurcic K, Redl K, Steinke B, Wagner H. Biological activity of novel macrocyclic alkaloids (budmunchiamines) from Albizia amara detected on the basis of interaction with DNA. J Nat Prod. 1991; 54 1531-42
- 18 Calis I, Yuruker A, Tasdemir D, Wright A D, Sticher O, Luo Y D, Pezzuto J M. Cycloartane triterpene glycosides from the roots of Astragalus melanophrurius . Planta Med. 1997; 63 183-6
- 19 Bedir E, Pugh N, Calis I, Pasco D S, Khan I A. Immunostimulatory effects of cycloartane-type triterpene glycosides from Astragalus species . Biol Pharm Bull. 2000; 23 834-7
Dr. Luisella Verotta
Dipartimento di Chimica Organica e Industriale
Università degli Studi di Milano
via Venezian 21, 20133 Milano
Italy
Fax: +39 02 5031 4106.
Email: luisella.verotta@unimi.it



