Planta Med 2002; 68(11): 986-994
DOI: 10.1055/s-2002-35654
Original Paper
Pharmacology
© Georg Thieme Verlag Stuttgart · New York

Cycloartane and Oleanane Saponins from Egyptian Astragalus spp. as Modulators of Lymphocyte Proliferation

Luisella Verotta1 , Marco Guerrini2 , Nadia A. El-Sebakhy3 , Aya M. Assad3 , Soad M. Toaima3 , Mohamed M. Radwan3 , Ying-De Luo4 , John M. Pezzuto4
  • 1Dipartimento di Chimica Organica e Industriale, Milano, Italy
  • 2Istituto di Chimica e Biochimica ”G. Ronzoni”, Milano, Italy
  • 3Department of Pharmacognosy, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
  • 4Department of Medicinal Chemistry and Pharmacognosy, and Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA
Further Information

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)

Table of Contents #

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.

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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.

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Materials and Methods

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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 .

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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].

Table 1 1H- and 13C-NMR data of compound 26 [500 MHz, pyridine-d 5, δ in ppm from internal TMS]a
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.
Table 2 1H- and 13C-NMR assignments [in ppm, J (Hz), significant BH-C connections as determined from a GHMQC experiment] of the sugar moieties in compound 26
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
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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.

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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.

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Table 3 Effect of samples on lymphocyte transformation
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

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