Further Saponins and Flavonoids from Astragalus verrucosus Moris

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1 Pharmaceutical Biology 2003, Vol. 41, No. 8, pp Further Saponins and Flavonoids from Astragalus verrucosus Moris Luisa Pistelli, Isa Giachi, Elena Lepori and Alessandra Bertoli Dipartimento di Chimica Bioorganica e Biofarmacia, Università degli Studi di Pisa, via Bonanno 33, Pisa, Italy Abstract From the aerial parts of Astragalus verrucosus Moris, a novel cycloartane-type triterpene glycoside, named astraverrucin VII, was isolated along with cycloaraloside D (peregrinoside II) and cycloaraloside C (astrailienin A). The flavonoid composition was investigated for the first time, and fifteen known flavonoids were isolated and identified. All structural elucidation were performed by spectral means. The chemiotaxonomic importance of these findings is discussed. Keywords: Astragalus verrucosus Moris, Leguminosae, aerial parts, cycloartane-type triterpene, astraverrucin VII, flavonoids, spectral data. Introduction Astragalus L. is the largest genus in the Leguminosae family and one of the largest genera in the vascular plants, comprising about 2500 species of herbs or shrubs, mostly perennial, widely distributed throughout the temperate region of the world. Astragalus species are divided in two main groups: the medicinal plants and the poisonous species. The biologically active constituents of Astragalus spp. consist of polysaccharides, saponins and phenolics, while the toxic compounds includes imidazoline alkaloids, nitro-toxins and selenium derivatives. Astragali Radix, the dried roots of various Astragalus spp., is one of the most famous oriental crude drugs used in traditional medicine (it is officially listed in the Chinese Pharmacopoeia) as an antiperspirant, a diuretic or a tonic, but also for its hepatoprotective, antioxidative, immunostimulant and antiviral properties (Tang & Eisenbrand, 1992). Astragalus verrucosus Moris (Leguminosae) is an endemic perennial herb in Sardinia (Italy) (Tutin et al., 1972; Pignatti, 1982). Previously our phytochemical investigations afforded to six saponins, named astraverrucins I-VI from the aerial parts of this species (Pistelli et al., 1997; 1998). This plant is not used in folk medicine in the region where it grows, but the more polar extracts exhibited promising antifungal activity against the selected fungi and only astraverrucin II showed a significant inhibitory effect among tested pure compounds (Pistelli et al., 2002a) We have further examined the aerial parts of the titled plant and the present work describes the isolation and structure elucidation of a novel cycloartane triterpene glycoside, named astraverrucin VII (1) along with two known saponins, cycloaraloside D (also called peregrinoside II) (2) and cycloaraloside C (named also astrailienin A) (3). Fifteen flavonoids, sorted into two flavons, four flavonol glycosides, eight isoflavones and the pterocarpan maakiain, were also isolated and identified on the basis of their spectral data and in comparison with the literature values. Materials and methods Optical rotation were measured at 20 using Perkin-Elmer 141 polarimeter with a 1 dm cell; IR spectra as nujol mulls were registered with Mattson 3000 FTIR spectrometer (Unicam); 1 H and 13 C NMR spectra were recorded on a Bruker AC-300 spectrometer. FAB-MS was recorded on a VG ZAB instrument (Xe atoms of energy of 2.6 KV) in a dithiodiethanol matrix; EI-MS were determined by direct inlet (20 ev) on an HP 5988 A. TLC was carried out on silica Accepted: March 3, 2004 Address correspondence to: Luisa Pistelli, Dipartimento di Chimica Bioorganica e Biofarmacia, via Bonanno 33, Pisa, Italy. luipi@farm.unipi.it DOI: / Taylor & Francis Ltd.

2 A. verrucosus Moris saponins and flavonids 569 gel (Merck). Sephadex LH-20 (Pharmacia) and Si gel 60 ( and mesh, Merck) were used for CC. SPE were performed with SepPak Waters RP-18 column ( mm). HPLC apparatus consisted of Waters W600E pumps (supported by Millennium 32 Software) and Waters UV W486 detector equipped with a reverse phase column (Supelcosil SPLC-18; mm id). Plant material Astragalus verrucosus Moris is a perennial herb, located in a restricted area of the Sardinia Island, in Italy (Tutin et al., 1972). The aerial plant material, without flowers, was harvested at Is Pisittus (Sardinia Island) in June 1996 and authenticated by Prof A. Manunta of the Istituto di Botanica ed Orto Botanico, Università di Urbino, Italy, where a voucher specimen was deposited in the Herbarium (Register No: URB-96/357). Extraction and isolation of saponins Air-dried powdered aerial parts of Astragalus verrucosus Moris (1100 g) were defatted with n-hexane and extracted in a Soxhlet apparatus with CHCl 3 and Me in turn, to give the respective extracts (H: 7.9 g; C: 29.4 g; M: g). The M residue was suspended in H 2 O and shaken with EtOAc followed by n-bu to give, on evaporation of the solvents, the corresponding residues (E: 60.6 g; B: g; W: 120.9). All the saponins have been isolated from E and B extracts g of the E residue was chromatographed on a Sephadex LH-20 column (Me as eluent) to give six crude fractions EA-EF. Part of the fr. EB (1.4 g), after silica gel CC (CHCl 3 - Me 8.5:1.5Æ6 :4) furnished 23 subfrs. EB 1 -EB 23. Astraverrucin VII, the new saponin 1 (31 mg), was purified from subfrs. EB 11, while saponin 2 (31.3 mg) was obatined from subfr. EB 21 after silica gel CC (CHCl 3 -Me 8 :2). The column chromatografy of B extract (only 15.3 g) on Sephadex LH-20 (Me-H 2 O 8 :2 as eluent) afforded to nine main fractions (frs. B 1 -B 9 ). The fr. B 2 (9.6 g), rich in saponins, was further subjected to CC on silica gel (CHCl 3 - Me-H 2 O 7:3:0.3Æ6 :4:1), to obtain 24 subfractions: B 2(I) -B 2(XXIV). Saponins 2 (8 mg) and 3 (130 mg) were obtained as pure compounds from frs. B 2(XIII), and B 2(XVII) respectively. Astraverrucin VII (1). [a] D 20 =+5.95 (pyridine, c 0.47); TLC (CHCl 3 -Me-H 2 O 6:2:0.2) R f 0.58; FAB-MS (positive ion mode) m/z: 717 [M + Na] +, 695 [M + H] +, 472 [Mglc-OAc] +, 143; EIMS m/z (rel. int.): 454 (1.18), 187 (1.80), 159 (2.19), 143 (100), 125 (9.56), 109 (9.09), 85 (14.28), 59 (24.44), 43 (29.33); 1 H NMR (Py-d 5 ): d 6.59 (1H, br s, rha- 1), 4.92 (1H, d, J = 7.8 Hz, glc-1), 3.86 (1H, dd, J = 8.8 and 5.4 Hz, H-24), 2.50 (1H, d, J = 7.8Hz, H-17), 2.02 (3H, s, COMe), 1.98 (3H, s, H 3-28), 1.55 (3H, s, H 3-26), 1.39 (3H, s, H 3-18),1.29 (6H, s, H 3-21 and H 3-27), 1.27 (3H, s, H 3-29), 0.98 (3H, s, H 3-30), 0.24 (1H, d, J = 3.9Hz, H-19b), 0.22 (1H, d, J = 3.9 Hz, H-19a); 13 C NMR (Py-d 5 ): d (MeCO), (glc-1), 89.2 (C-3), 87.2 (C-20), 81.6 (C-24), 78.4 (glc-3), 75.6 (glc-2), 74.4 (glc-5), 73.4 (C-16), 71.6 (glc-4), 71.2 (C-25), 67.9 (C-6), 64.4 (glc-6), 58.3 (C-17), 54.0 (C-5), 47.0 (C-8), 46.5 (C-15), 46.0 (C-14), 45.0 (C-13), 42.5 (C-4), 38.6 (C-7), 34.8 (C-22), 33.3 (C-12), 32.4 (C-1), 30.6 (C-19), 30.2 (C-2), 29.5 (C-10), 28.8 (C-28), 28.5 (C- 27), 28.1 (C-26), 27.0 (C-21), 26.3 (C-23), 26.1 (C-11), 21.5 (C-18), 21.4 (MeCO), 20.9 (C-9), 20.1 (C-30), 16.5 (C-29). Cycloaraloside D (peregrinoside II) (2). TLC (CHCl 3 - Me-H 2 O 8:2:0.2) R f 0.14; 1 H NMR (Py-d 5 ): d ppm 6.59 (1H, br s, rha-1), 5.00 (1H, d, J = 7.3 Hz, glc-1), 3.87 (1H, m, H-24), 3.08 (2H, m, H-22), 2.52 (1H, d, J = 7.8 Hz, H- 17), 2.52 (3H, s, H-28), 1.72 (3H, d, J = 6.3 Hz, rha-6), 1.55 (3H, s, H-26), 1.50 (3H, s, H-18), 1.37 (3H, s, H-29), 1.27 (3H, s, H-21), 1.28 (3H, s, H-27), 0.99 (3H, s, H-30), 0.52 (3H, s, H-19b), 0.16 (3H, s, H-19a); 13 C NMR (Py-d 5 ): d ppm (glc-1), (rha-1), 88.6 (C-3), 87.2 (C-20), 81.6 (C-24), 79.9 (glc-2), 78.1 (glc-5), 78.0 (glc-3), 74.1 (rha-4), 73.4 (C-16), 72.5 (rha-2 and rha-3), 72.1 (glc-4), 71.2 (C- 25), 69.6 (rha-5), 62.8 (glc-6); 67.7 (C-6), 58.3 (C-17), 54.1 (C-5), 46.7 (C-8), 46.5 (C-15), 46.1 (C-14), 44.9 (C-13), 42.5 (C-4), 38.4 (C-7), 34.8 (C-12), 33.3 (C-22), 32.5 (C-1), 30.3 (C-2 and C-19), 29.3 (C-10), 28.6 (C-28), 28.5 (C-27), 28.1 (C-26), 27.1 (C-21), 26.4 (C-11), 26.2 (C-23), 21.3 (C-9), 20.7 (C-18), 20.1 (C-30), 18.7 (rha-6), 16.7 (C-29). Cycloaraloside C (astrailienin A) (3). TLC (CHCl 3 /Me/H 2 O 6:2:0.2), R f = 0.274; 1 H NMR (Py-d 5 ): 5.35 (1H, d, J = 2.0 Hz, api-1), 4.26 (1H, d, J = 7.8 Hz, glc- 1) 3.86 (1H, m, H-24), 2.52 (1H, d, J = 7.3Hz, H-17), 1.98, 1.56, 1.44, 1.40, 1.28, 1.28, 0.99 (3H each, s, 7 Me) and 0.55 (3H, s, H-19b), 0.22 (3H, s, H-19a); 13 C NMR (Py-d 5 ) d ppm: (api-1), (glc-1), 88.8 (C-3), 87.1 (C-20), 81.5 (C-24), 80.5 (api-3), 79.3 (glc-2), 78.6 (api-2), 78.2 (glc-5), 77.8 (glc-3), 75.5 (api-4), 73.3 (C-16), 71.8 (glc-4), 71.1 (C-25), 67.8 (C-6), 66.0 (api-5), 62.7 (glc-6), 58.2 (C- 17), 53.8 (C-5), 46.8 (C-8), 46.5 (C-15), 45.9 (C-14), 44.8 (C-13), 42.5 (C-4), 38.5 (C-7), 34.7 (C-22), 33.2 (C-12), 32.4 (C-1), 30.3 (C-19), 30.1 (C-2), 29.3 (C-10), 28.6 (C-28), 28.4 (C-27), 28.1 (C-26), 26.9 (C-21), 26.3 (C-23), 26.1 (C-11), 21.4 (C-9), 20.7 (C-30), 20.0 (C-18), 16.5 (C-29). Isolation of flavonoids All the flavonoids have been isolated from C, E and B extracts. Part of the C residue (18.9 g) was chromatographed on Sephadex LH-20 (Me-CHCl 3 7 :3 as eluent) to obtain six fractions (frs. C A -C F ). Nine subfractions were obtained (C E1 -C E9 ) from fr. C E (200 mg) after silical gel CC (CHCl 3 - EtOAc 8 :2). Subfrs. C E3 and C E6 contained pure 18 (11.3 mg) and 15 (13.3 mg), respectively; subfrs. C E8 and C E4, further purified by preparative TLC (CHCl 3 -EtOAc-H 2 O 8:2:0.1 and CHCl 3 -Me 8 : 2) gave compounds 4 (2.1 mg) and 17 (5.6 mg). Fr. C D (400 mg) was fractioned by silica gel column using CHCl 3 -EtOAc as mobile phase to obtain sixteen frs a- r. Fr. q contained pure 10 (8.9 mg); from fr. n pure compound

3 570 L. Pistelli et al. 13 (10.3 mg) was purified after prep.tlc over silica gel (toluene-etoac-hco 5 :4:1), while crystallization of fr. h afforded 14 (11.5 mg). Fr. ED (736 mg), obtained from E extract after Sephadex LH-20 gel filtration (Me as eluent), was chromatographed on silica gel column (CHCl 3 -Me 9.5 :0.5Æ7 :3). Twenty subfractions were obtained (ED 1 -ED 20 ) and after crystallization compounds 12 (8.7 mg), 11 (12.1 mg), and 6 (10 mg) were purified from subfrs. ED 10, ED 15 and ED 20, respectively. Fr. EF (904 mg) furnished pure 5 (100 mg) after crystallization. The mother liquid, subjected first to silica gel CC (CHCl 3 -Me 8 :2 followed by Me) then purified by SPE (eluted with Me-H 2 O 8 :2) followed by preptlc (CHCl 3 -EtOAc 7 : 3), gave 15 (7.2 mg) and 4 (8 mg). From the B extract, the fr. B 7 (110 mg) was chromatographed on silica gel column (CHCl 3 -Me-H 2 O 6 :2:0.2), followed by HPLC separation using Me-H 2 O 4:6 as eluent (flow rate 1.2 ml/min) to afford 8 (8.5 mg) and 6 (5.7 mg). Rutin 7 (7.7 mg) was also obtained from the same fraction, after preptlc (CHCl 3 -Me-H 2 O 6:2:0.2). Fr. B 9 (80 mg) was subjected to column chromatography on silica gel (CHCl 3 - Me-H 2 O 6 :2:0.2) and afforded thirteen subfractions (subfrs. B 9I -B 9XIII ). Subfrs. B 9VII and B 9XIII were further subjected to preptlc (EtOAc :HCO :H 2 O 10:2.2:1) to obtain 16 (3.7 mg) and 9 (9.0 mg), respectively. O O CH 2 OR O OR R R 1 H Ac 2 rhamnose H 3 apiose H Results and discussion Extracts of the aerial parts of Astragalus verrucosus Moris were reinvestigated to isolate and identify other compounds not yet evidenced in our previous works (Pistelli et al., 1997; 1998). Three cycloartane-type saponins were isolated and purified by a combination of chromatographic methods from the EtOAc and n-bu: Astraverrucin VII (1), Cycloaraloside D (2), and Cycloaraloside C (3). The new compound (1) showed a molecular ion at m/z 695 [M + 1] + on FAB-MS, which is compatible with the molecular formula C 38 H 62 O 11. Taking into account the results of our comprenhensive 1 H and 13 C-NMR studies (Pistelli et al., 1997; 1998) and previous knowledge derived from metabolites isolated from the genus Astragalus, the main features of cycloartane-type triterpene were evident: characteristics signals due to cyclopropanemethylene protons as an AX system (d 0.23 and 0.51, J AX = 3.9 Hz, H 2-19), seven tertiary methyl groups in a high field (d 0.98, 1.22, 1.27, 1.29, 1.39, 1.55, 1.97) and H-17 at d 2.50 (J = 7.8 Hz). In the mass spectrum of compound (1) a maximum peak at m/z 143 was observed indicating the presence of a tetrahydrofuran side chain. This fact, in addition to the presence of a triplet signal at d 3.85 (J = 8.8 Hz) belonging to H-24 in the 1 H NMR spectrum, permitted the conclusion that 1 had a cycloastragenol as aglycone. The chemical shifts of the C-20 and C-24 carbon atoms of the 20,24-epoxy-25-hydroxy function were superimposable to those of the corresponding atoms of cycloastragenol, showing that the stereochemistry of these Figure 1. Structures of saponins 1 3 from A. verrucosus Moris. asymmetric carbon atoms was also identical (Hirotani et al., 1994). Additionally the resonance of only one anomeric proton was clearly observed in the low field region in the 1 H NMR spectrum at d 4.92 (d, J = 7.8 Hz), indicative of the presence of one b-linked sugar unit. This was supported by the 13 C- NMR spectrum, which showed one anomeric carbon signal at 106.9ppm. Thus 1 was considered to be a cycloastragenol glucoside in which the sugar residue was linked to C-3 (d 89.2) of the aglycone by means of the diagnostic glycosidation shift of this carbon atom. The 1 H and 13 C-NMR spectra also contained a 3H singlet at d 2.05 and signals at 21.4 and ppm; consequently 1 had one acetyl group. The position of the acetyl residue was found by a comparative study of the 13 C-NMR spectra of glucoside (1) and astraverrucin I (Pistelli et al., 1997), where the signals of C-5 and C-6 carbon atoms of the b-d-glucose residue appeared at 74.4 and 64.4ppm, while in the spectrum of 1 the same signals appeared at 74.4 and 64.4ppm. These changes in the chemical shifts showed that the acetyl group had substituted the group at C-6 of the glucose. The 13 C NMR spectrum of 1 exhibited 38 signals: thirty signals were accounted for the aglycone moiety, the remaining signals were in accord with the presence of one hexose and an acetyl unit. Based on a DEPT experiments, 1 H- 1 H and 1 H- 13 C COSY spectra and by comparison with 13 C NMR data of the related astraverrucin II-III (Pistelli et al., 1997) all signals could be assigned. Thus

4 A. verrucosus Moris saponins and flavonids 571 astraverrucin VII (1) resulted as the 6 -O-acetyl isomer of astraverrucin I. Cycloaraloside D (2) (also named peregrinoside II) and Cycloaraloside C (named also astrailienin A) (3) were also isolated for the first time from this source, although they were previously isolated from other Astragalus spp.: A. amarus (Isaev, 1991) and A. peregrinus (Verotta et al., 2001; 2002) for (2), and A. amarus (Isaev & Abubakirov, 1990) and A. iliensis (Yu-Cun et al., 1990) for (3). These saponins showed the typical spectra of cycloastragenol diglycoside (two anomeric protons at d 5.0 and 6.59 for (2) and 4,26 and 5.35 for (3) in the 1 H spectrum, also supported by the presence of two anomeric carbons in the 13 C NMR spectra). Since only C-3 of both aglycones showed a glycosylation shifts, these glycosides resulted to have a monodesmosidic structure, and differ for the 3-O-linked saccharidic moiety. Both disaccharide portions of the two saponins contained a glucose moiety, with rhamnose as second sugar in (2) [confirmed by the further methyl signal as a doublet at d 1.72 (3H, J = 6.3 Hz) in the 1 H NMR spectrum] and apiose in (3) [on the basis of the coupling constant of anomeric proton d 5.35 (J = 2.0 Hz) and by the presence of five signals in the 13 C NMR spectrum, superimposable to those reported in the literature] (Isaev & Abubakirov, 1990; Yu-Qun et al., 1990). Rhamnose and apiose had to be attached to hydroxyl group in C-2 of glucose both in Cycloaraloside D (2) and in Cycloaraloside C (3), respectively, as showed by the glycosilation shifts of C-2 and by the shifts of C -1 and C -3 (see data reported in Materials an Methods). All the data obtained for compounds (2) and (3) were unambiguously assigned on the basis of a combination of 1D and 2D NMR techniques and confirmed by comparison with the corresponding literature data (Isaev, 1991; Verotta et al., 2001; Isaev & Abubakirov, 1990; Yu-Qun et al., 1990). The analysis of flavonoidic content yielded fifteeen known compounds. They were identified as two flavones: apigenin (4) (Agrawal & Markham, 1989) and apigenin 7-O-b-D-(6- O-p-coumaroyl)glucoside (5) (Itokawa et al., 1981; Nauwar et al., 1989); four flavonol derivatives as kaempferol 3-Orutinoside or nicotiflorin (6) (Bader et al., 1993), quercetin 3-O-rutinoside or rutin (7) (Markham & Ternai, 1976), kaempferol 3-O-robinobioside (8), and quercetin 3-O-robinobioside (9) (Brasseur & Angeuot, 1986), respectively, and eight isoflavones: daidzein (10), daidzin (11) (Agrawal & Markham, 1989), ononin (12) (Cui et al., 1992), calycosin (13) (Harborne & Baxter, 1999), psudobaptigenin (14) (Markham & Mabry, 1968), genistein (15) (Agrawal & Markham, 1989), genistin (16) (Hasegawa, 1957) and pratensein (17) (Agrawal & Markham, 1989). Maackiain (18), the only pterocarpan derivative isolated herein, was previously isolated in A. cicer and A. trojanus. (Martin et al., 1994; Bedir et al., 1999). The structures of all these constituents were confirmed by comparison with authentic samples or published data. All isolated flavonoids are uncommon in Astragalus genus: nicotiflorin, kaempferol 3-O-robinobioside and quercetin 3-O-robinobioside were identified here for the first time, while this is the first report of apigenin 7-O-b-D-(6-Op-coumaroyl)glucoside presence in the Leguminosae family. But isoflavones constituted the prominent fraction of A. verrucosus flavonoids, and among these daidzin, genistein, genistin and pratensein represent previously unreported natural products in the Astragalus genus, while daidzein, calycosin, ononin and psudobaptigenin were identified only in a few Astagalus spp. (A. sinicus, A. membranaceus, A. complanatus, and A. cicer) (Pistelli, 2002b). Acknowledgements We acknowledge Prof Antonio Manunta (Istituto di Botanica ed Orto Botanico, Università di Urbino, Italy) for collection and identification of the plant material. 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