Phytochemistry 71 (2010) 956–963
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Triterpene glycosides from Astragalus icmadophilus _ Ibrahim Horo a, Erdal Bedir b,*, Angela Perrone c, Fevzi Özgökçe d, Sonia Piacente c, Özgen Alankusß-Çalısßkan a,* _ Ege University, Faculty of Science, Department of Chemistry, Bornova, 35100 Izmir, Turkey _ Ege University, Faculty of Engineering, Department of Bioengineering, Bornova, 35100 Izmir, Turkey c Salerno University, Department of Pharmaceutical Sciences, 84084 Fisciano (Salerno), Italy d Yüzüncü Yıl University, Faculty of Science and Letters, Department of Biology, 65180 Van, Turkey a
b
a r t i c l e
i n f o
Article history: Received 21 January 2010 Received in revised form 10 February 2010 Available online 16 March 2010 Keywords: Astragalus icmadophilus Saponin 20,24-Epoxycycloartane glycosides 20,25-Epoxycycloartane glycosides
a b s t r a c t Six cycloartane-type triterpene glycosides were isolated from Astragalus icmadophilus along with two known cycloartane-type glycosides, five known oleanane-type triterpene glycosides and one known flavonol glycoside. The structures of the six compounds were established as 3-O-[a-L-arabinopyranosyl(1 ? 2)-O-3-acetoxy-a-L-arabinopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,24(S),25-pentahydroxycycloartane, 3-O-[a-L-rhamnopyranosyl-(1 ? 2)-O-a-L-arabinopyranosyl-(1 ? 2)-O-b-D-xylopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,24(S),25-pentahydroxy cycloartane, 3-O-[a-L-arabinopyranosyl(1 ? 2)-O-3,4-diacetoxy-a-L-arabinopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,24(S),25-pentahydroxycycloartane, 3-O-[a-L-arabinopyranosyl-(1 ? 2)-O-3-acetoxy-a-L-arabinopyranosyl]-6-O-b-Dglucopyranosyl-3b,6a,16b,25-tetrahydroxy-20(R),24(S)-epoxycycloartane, 3-O-[a-L-arabinopyranosyl(1 ? 2)-O-b-D-xylopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,24a-tetrahydroxy-20(R),25-epoxycycloartane, 3-O-[a-L-rhamnopyranosyl-(1 ? 2)-O-a-L-arabinopyranosyl-(1 ? 2)-O-b-D-xylopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,24a-tetrahydroxy-20(R),25-epoxycycloartane by the extensive use of 1D- and 2D-NMR experiments along with ESIMS and HRMS analysis. The first four compounds are cyclocanthogenin and cycloastragenol glycosides, whereas the last two are based on cyclocephalogenin as aglycone, more unusual in the plant kingdom, so far reported only from Astragalus spp. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Astragalus L., one of the largest genera of flowering plants with about 2000 different species belonging to the family Leguminosae, is represented by 380 species in the flora of Turkey (Davis, 1970). The roots of Astragalus membranaceus are used in traditional Chinese medicine as an antiperspirant, diuretic and tonic drug. It has also been used in the treatment of diabetes mellitus, nephiritis, leukemia and uterine cancer (Tang and Eisenbrand, 1992). In the district of Anatolia, located in South Eastern Turkey, an aqueous extract of the roots of Astragalus species is traditionally used against leukemia and for its wound healing properties. Astragalus species are known to be rich in two major classes of biologically active compounds, polysaccharides and saponins. (Ríos and Waterman, 1997; Bedir et al., 2000; Li, 2000). Astragalus saponins have shown interesting pharmacological properties, including immunostimulating (Yesßilada et al., 2005; Çalısß et al., 1997; Bedir et al.,
* Corresponding authors. Tel./fax: +90 232 388 4955 (E. Bedir), tel./fax: +90 232 388 8264 (O.A.-Çalısßkan). E-mail addresses:
[email protected] (E. Bedir), ozgen.alankus.caliska@ege. edu.tr (Ö. Alankusß-Çalısßkan). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.02.014
2000), anti-protozoal (Özipek et al., 2005), antiviral (Gariboldi et al., 1995), cytotoxic (Radwan et al., 2004), and cardiotonic activities (Khushbaktova et al., 1994). Moreover, Astragaloside IV, a widely encountered 20,24-epoxy cycloartane glycoside found in Astragalus species, has been proven to be a neuroprotective agent and proposed as a potential agent in the treatment of Parkinson’s disease (Luo et al., 2004; Chan et al., 2009). Several cycloartane- and oleanane-type triterpene glycosides were isolated from Turkish Astragalus species (Bedir et al., 1998a,b, 1999a,b, 2000; Çalısß and Sticher, 1996; Çalısß et al., 1997, 1999; Özipek et al., 2005). As a part of our ongoing research of new bioactive compounds from Turkish Astragalus species, we carried out a study on Astragalus icmadophilus Hand.-Mazz. (Leguminosae). This paper reports the isolation of six new cycloartane-type triterpene glycosides (1–6) from the methanol extract of the whole plant of A. icmadophilus along with two known cycloartane-type glycosides (7, 8), five known oleanane-type triterpene glycosides (9–13) and one known flavonol glycoside (14). Their structures were elucidated by extensive spectroscopic methods including 1D- (1H, 13C and TOCSY) and 2D-NMR (DQF-COSY, HSQC, HMBC, and ROESY) experiments as well as ESIMS and HRMS analysis.
_ Horo et al. / Phytochemistry 71 (2010) 956–963 I.
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J = 4.2 Hz), six tertiary methyl groups at d 1.26 (3H, s), 1.20 (3H, s), 1.18 (6H, s), 1.03 (3H, s) and 1.01 (3H, s), a secondary methyl group at d 0.97 (d, J = 6.5 Hz), and four methine proton signals at d 4.47 (ddd, J = 8.0, 8.0, 5.2 Hz), 3.57 (ddd, J = 9.5, 9.5, 4.5 Hz), 3.41 (dd, J = 10.5, 2.4 Hz) and 3.22 (dd, J = 11.3, 4.0 Hz), which were indicative of secondary alcoholic functions (Table 1). The NMR data of the aglycone moiety of 1 were in good agreement with those reported for cyclocanthogenin (Bedir et al., 1998a) with glycosidation shifts for C-3 (d 90.0) and C-6 (d 79.8) (Table 1). In addition a singlet signal at d 2.13 (3H, s) ascribable to the further methyl group of an acetoxy group, along with signals for three anomeric protons at d 4.73 (d, J = 4.3 Hz), 4.42 (d, J = 6.3 Hz) and 4.36 (d, J = 7.5 Hz) were also present. The chemical shifts of all the individual protons of the three sugar units were ascertained from a combination of 1D-TOCSY and DQF-COSY spectral analysis, and the 13C
2. Results and discussion The HRMALDITOF mass spectrum of 1 (m/z 983.5198 [M+Na]+, calcd for C48H80O19Na, 983.5192) supported a molecular formula of C48H80O19. The ESIMS mass spectrum showed the major ion peak at m/z 983.5 which was assigned to [M+Na]+. The MS/MS of this ion showed a peak at m/z 803.5 [M+Na180]+, corresponding to the loss of an hexose unit. In the MS3 spectrum peaks at m/z 743.4 [M+Na18060]+, corresponding to the loss of an acetate molecule, and 671.4 [M+Na180132]+, due to the loss of a pentose unit, were observed. A detailed comparison of the aglycone moiety NMR data (1H, 13C, HSQC, HMBC, COSY) of compounds 1–3 showed that the aglycone moiety was identical in the three compounds (Fig. 1). In particular, the 1H NMR spectrum of 1 showed signals due to a cyclopropane methylene at d 0.60 and 0.27 (each 1H, d,
OH
OH
OH
O
OH
OH
RO RO
O
OH
O OH
O
HO HO
β-D-glc
O
HO HO
OH
OH
β-D-glc
1
Compound
OH
2 α-L-araI
AcO OH α-L-araII O HO OH
O
HO HO
O
5
Compound
OAc α-L-araI O
β-D-xyl
O
R
3
β-D-xyl
O
OH α-L-araII O HO OH
O
HO
O
OH α-L-ara O HO OH
O
R
β-D-xyl
O
HO HO
AcO
OH α-L-ara
6
O
OH α-L-ara
O
HO
O
O
HO HO
HO
OH
OH
O OH
OH
O α-L-rha
α-L-rha
HO
O
HO HO
α-L-araI O
O AcO
O
OH O
OH O
O
HO HO
OH
HO OH
β-D-glc
α-L-araII
4 Fig. 1. Structures of compounds 1–6.
OH
O
_ Horo et al. / Phytochemistry 71 (2010) 956–963 I.
958
Table 1 13 C and 1H NMR data (J in Hz) of the aglycon moieties of compounds 1, 4 and 5 (600 MHz, d ppm, in CD3OD). 1a
a b
5b
4
dC
dH
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
32.6 30.0 90.0 43.2 53.1 79.8 34.5 46.3 21.9 29.3 26.6 33.6 46.7 47.7 47.8
16 17 18 19
73.1 57.8 17.9 28.7
20 21 22 23 24 25 26 27 28 29 30
29.4 18.1 33.8 28.1 78.3 74.0 25.2 25.2 28.1 16.0 19.8
1.57, 1.97, 3.22, – 1.64, 3.57, 1.92, 1.90, – – 1.90, 1.66, – – 2.13, 1.41, 4.47, 1.73, 1.18, 0.60, 0.27, 1.93, 0.97, 1.82, 1.65, 3.41, – 1.18, 1.20, 1.26, 1.03, 1.01,
1.30, m 1.72, m dd (11.3, 4.0) d (9.5) ddd (9.5, 9.5, 4.5) 1.63, m dd (11.9, 4.2)
1.38, m (2H) m
dd (12.7, 8.0) dd (12.7, 5.2) ddd (8.0, 8.0, 5.2) dd (9.9, 8.0) s d (4.2) d (4.2) m d (6.5) 1.24, m 1.44, m dd (2.4, 10.5) s s s s s
dC
dH
dC
dH
33.3 30.4 89.3 42.8 54.7 69.3 38.7 48.3 21.4 30.4 26.9 34.0 45.9 46.8 46.2
1.61, 1.24, m 2.10, 1.70, m 3.27, dd (11.3, 4.0) – 1.40, d (9.5) 3.49, ddd (9.5, 9.0, 4.5) 1.51, 1.41, m 1.84, dd (11.9, 4.2) – – 2.06, 1.24, m 1.71, (2H) m – – 1.98, dd (12.7, 8.0) 1.45, dd (12.7, 5.2) 4.69, ddd (8.0, 8.0, 5.2) 2.39, d (8.0) 1.31, s 0.59, d (4.2) 0.41, d (4.2) – 1.26, s 2.58,1.68, m 2.18, 2.02, m 3.86, dd (8.2, 6.0) – 1.26, s 1.41, s 1.33, s 1.07, s 1.03, s
32.9 30.2 89.5 43.2 53.4 80.4 35.3 46.8 22.0 30.4 27.1 34.7 46.1 47.0 47.7
1.54, 1.93, 3.22, – 1.61, 3.56, 1.91, 1.87, – – 1.95, 1.86, – – 2.06, 1.47, 4.65, 2.01, 1.46, 0.61, 0.32, – 1.53, 2.63, 2.22, 3.51, – 1.22, 1.32, 1.32, 1.04, 0.99,
74.5 58.6 22.0 31.9 88.3 27.8 35.4 26.2 82.9 79.8 23.1 25.3 28.6 16.9 20.1
75.2 61.2 20.3 29.9 78.7 28.3 26.9 23.7 69.7 76.9 28.1 28.1 28.1 16.1 20.0
1.26, m 1.69, m dd (11.3, 4.0) d (9.5) ddd (9.5, 9.5, 4.5) 1.53, m dd (11.9, 4.2)
1.31, m 1.71, m
dd (12.7, 8.0) dd (12.7, 5.2) ddd (8.0, 8.0, 5.2) dd (9.9, 8.0) s d (4.2) d (4.2) s 1.20, m 1.71, m br s s s s s s
The chemical shift values of the aglycon moieties of 2 and 3 were superimposable with those reported for 1. The chemical shift values of the aglycon moiety of 6 were superimposable with those reported for 5.
chemical shifts of their relative attached carbons were assigned unambiguously from the HSQC spectrum (Table 2). These data showed the presence of one b-glucopyranosyl unit (d 4.36) and two a-arabinopyranosyl units (d 4.73 and 4.42). Glycosidation shift was observed for C-2araI (d 76.2). The downfield shifts observed for H-2araI (d 3.76) and H-3araI (d 4.98) and the upfield shift observed for C-2araI (d 76.2) in comparison with those reported for a 2substituted arabinose unit suggested the presence of the acetoxy group at C-3araI. This evidence was confirmed by the HMBC correlations between the proton signal at d 4.98 (H-3araI) and the carbon resonance at d 172.2 (COCH3). An unambiguous determination of the sequence and linkage sites was obtained from the HMBC spectrum, which showed key correlation peaks between the proton signal at d 4.73 (H-1araI) and the carbon resonance at d 90.0 (C-3), d 4.42 (H-1araII) and d 76.2 (C-2araI), and the proton signal at d 4.36 (H-1glc) and the carbon resonance at d 79.8 (C-6). The D-configuration of glucose unit and the L-configuration of arabinose units were established after hydrolysis of 1 with 1 N HCl, trimethylsilation and determination of retention time on a chiral column by GC. On the basis of all these evidence, the structure of the new compound 1 was established as 3-O-[a-L-arabinopyranosyl-(1 ? 2)O-3-acetoxy-a-L-arabinopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a, 16b,24(S),25-pentahydroxycycloartane. The molecular formula of compound 2 was established as C52H88O22 by HRMALDITOFMS analysis (m/z 1087.5669 [M+Na]+, calcd for C52H88O22Na, 1087.5665). The 1H NMR spectrum of 2 showed signals for four anomeric protons at d 5.06 (d, J = 1.2 Hz), 4.99 (d, J = 4.3 Hz), 4.50 (d, J = 7.5 Hz) and 4.36 (d, J = 7.5 Hz), which were identified by the combination of HSQC, HMBC and COSY data as a-rhamnopyranose (d 5.06), one a-arabinopyranose (d 4.99), one b-xylopyranose (d 4.50) and one b-glucopyranose (d 4.36).
The sugar sequence and the linkage sites were determined by the HMBC correlations between the proton signal at d 5.06 (H-1rha) and the carbon resonance at d 76.0 (C-2ara), d 4.99 (H-1ara) and d 80.2 (C-2xyl), d 4.50 (H-1xyl) and d 90.0 (C-3) and the proton signal at d 4.36 (H-1glc) and the carbon resonance at d 79.8 (C-6). The Dconfiguration of glucose and xylose units and the L-configuration of arabinose and rhamnose units were established after hydrolysis of 2 followed by GC analysis on a chiral column. Thus, the new compound 2 was identified as 3-O-[a-L-rhamnopyranosyl(1 ? 2)-O-a-L-arabinopyranosyl-(1 ? 2)-O-b-D-xylopyranosyl]-6O-b-D-glucopyranosyl-3b,6a,16b,24(S),25-pentahydroxycycloartane. The HRMALDITOF mass spectrum of 3 (m/z 1025.5294 [M+Na]+, calcd for C50H82O20Na, 1025.5297) supported a molecular formula of C50H82O20. The ESIMS mass spectrum showed the major ion peak at m/z 1025.6 which was assigned to [M+Na]+. In the MS/MS spectrum a peak at m/z 845.5 [M+Na180]+, corresponding to the loss of an hexose unit, was observed. The MS3 spectrum displayed a peak at m/z 785.5 [M+Na18060]+, due to the loss of an acetate molecule. Finally, in the MS4 spectrum peaks at m/z 725.5 [M+Na1806060]+, indicating the loss of a second acetate molecule, at m/z 653.4 [M+Na18060132]+, due to the loss of a pentose unit and at m/z 593.4 [M+Na18060192]+, corresponding to the loss of an acetylated pentose unit, were observed. The 1H NMR spectrum of 3 displayed, in addition to signals of the aglycone moiety, signals ascribable to two methyl groups of acetoxy group at d 2.11 (3H, s) and 2.07 (3H, s) and signals for three anomeric protons at d 4.78 (d, J = 4.3 Hz), 4.42 (d, J = 6.3 Hz) and 4.35 (d, J = 7.5 Hz). On the basis of HSQC, HMBC, DQF-COSY and 1D-TOCSY correlations two a-arabinopyranose units (d 4.78 and 4.42) and one b-glucopyranose (d 4.35) were identified (Fig. 2). Key correlation peaks in the HMBC spectrum were observed between the
Table 2 13 C and 1H NMR data (J in Hz) of the sugar portions of compounds 1–3 and 5 (600 MHz, d ppm, in CD3OD). 1a dC
5
63.5
3-COCH3 3-COCH3 4-COCH3 4-COCH3
172.2 20.9
1 2 3 4 5
a-L-Ara (at C-2xyl) 105.0 72.0 73.9 69.4 66.9
dH
dC
4.73, 3.76, 4.98, 3.66,
b-D-Xyl (at C-3) 106.2 80.2 77.2 70.9
d (4.3) dd (6.3, 4.3) t (6.3) m
4.09, dd (11.9, 4.2) 3.44, dd (11.9, 5.8) – 2.13, s
4.42, 3.57, 3.56, 3.83, 3.92, 3.55,
d (6.3) dd (8.5, 6.3) dd (8.5, 3.0) m dd (12.7, 3.0) dd (12.7, 2.3)
66.0
a-L-Ara (at C-2xyl) 101.9 76.0 72.2 67.7 63.1
3 dH
dC
4.50, 3.42, 3.54, 3.54,
3,4-diAc-a-L-AraI (at C-3) 104.0 76.0 71.5 69.8
d (7.5) dd (9.2, 7.5) t (9.2) m
3.89, dd (11.7, 5.2) 3.23, t (11.7)
4.99, 3.91, 3.83, 3.84, 3.99, 3.48,
d (4.3) dd (8.5, 4.3) dd (8.5, 3.0) m dd (11.9, 6.3) dd (11.9, 2.4)
5.06, 3.90, 3.72, 3.44, 3.90, 1.30,
d (1.2) dd (1.2, 3.2) dd (3.2, 9.3) t (9.3) m d (6.5)
60.5 171.6 21.0 172.0 20.7 a-L-AraII (at C-2ara) 104.1 72.0 74.3 69.3 66.5
5 dH 4.78, d (4.3) 3.81, dd (6.3, 4.3) 5.13, t (6.3) 4.82, ddd (6.3, 5.8, 4.2) 4.16, dd (11.9, 4.2) 3.53, dd (11.9, 5.8) – 2.11, s – 2.07, s
dC
dH
b-D-Xyl (at C-3) 106.0 83.2 76.7 70.9
4.51, 3.46, 3.55, 3.55,
65.9
d (7.5) dd (9.2, 7.5) t (9.2) m
3.89, dd (11.7, 5.2) 3.23, t (11.7)
a-L-Ara (at C-2xyl) 4.42, 3.56, 3.56, 3.83, 3.90, 3.54,
d (6.3) dd (8.5, 6.3) dd (8.5, 3.0) m dd (12.7, 3.0) dd (12.7, 2.3)
106.0 73.4 73.9 69.4 67.0
4.50, 3.67, 3.60, 3.83, 3.92, 3.54,
d (6.3) dd (8.5, 6.3) dd (8.5, 3.0) m dd (12.7, 3.0) dd (12.7, 2.3)
a-L-Rha (at C-2ara) 1 2 3 4 5 6
a b
1 2 3 4 5
b-D-Glc (at C-6) 105.0 75.4 78.3 71.6 77.5
6
62.8
4.36, d (7.5) 3.22, dd (7.5, 9.0) 3.36, t (9.0) 3.32, t (9.0) 3.28, ddd (3.5, 4.5, 9.0) 3.88, dd (3.5, 12.0) 3.70, dd (4.5, 12.0)
102.1 72.0 71.9 73.8 70.1 17.6 b-D-Glc (at C-6) 104.8 75.6 78.3 71.6 77.5 62.9
4.36, d (7.5) 3.22, dd (7.5, 9.0) 3.38, t (9.0) 3.32, t (9.0) 3.28, ddd (3.5, 4.5, 9.0) 3.88, dd (3.5, 12.0) 3.71, dd (4.5, 12.0)
b-D-Glc (at C-6) 104.5 75.1 78.0 71.3 77.4 63.0
4.35, d (7.5) 3.21, dd (7.5, 9.0) 3.36, t (9.0) 3.31, t (9.0) 3.27, ddd (3.5, 4.5, 9.0) 3.88, dd (3.5, 12.0) 3.69, dd (4.5, 12.0)
b-D-Glc (at C-6) 104.9 75.6 78.5 71.7 77.6 63.0
_ Horo et al. / Phytochemistry 71 (2010) 956–963 I.
1 2 3 4
3-Ac-b-D-AraI (at C-3) 104.4 76.2 74.1 68.1
2b
4.37, d (7.5) 3.21, dd (7.5, 9.0) 3.36, t (9.0) 3.31, t (9.0) 3.28, ddd (3.5, 4.5, 9.0) 3.87, dd (3.5, 12.0) 3.70, dd (4.5, 12.0)
The chemical shift values of the sugar portion of 4 were superimposable with those reported for 1. The chemical shift values of the sugar portion of 6 were superimposable with those reported for 2.
959
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960
proton signal at d 4.78 (H-1araI) and the carbon resonance at d 90.2 (C-3), d 4.42 (H-1araII) and d 76.0 (C-2ara) and between the proton signal at d 4.35 (H-1glc) and the carbon resonance at d 79.8 (C-6). As suggested by the downfield shifts of H-3araI (d 5.13) and H-4araI (d 4.82) and by their HMBC correlations with the carbon resonances at d 171.6 (COCH3) and 172.0 (COCH3), respectively, it was evident that the two acetoxy groups were located at C-3 and C-4 of the inner arabinopyranose unit. Therefore, the structure of 3 was established as 3-O-[a-L-arabinopyranosyl-(1 ? 2)-O-3,4diacetoxy-a-L-arabinopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a, 16b,24(S),25-pentahydroxycycloartane. Compound 4 showed in the positive ESIMS a major ion peak at m/z 981.8 [M+Na]+ and a significant fragment in MS/MS analysis at m/z 801.5 [M+Na180]+, ascribable to the loss of an hexose unit. The MS3 fragmentation showed peaks at m/z 741.5 [M+Na18060]+ due to the loss of an acetate molecule and m/z 669.4 [M+Na180132]+, corresponding to the loss of one pentose unit. Its molecular formula was established unequivocally as C48H78O19 by HRMALDITOF mass spectrum (m/z 981.5041 [M+Na]+, calcd for C48H78O19Na, 981.5035). The 1H NMR spectrum of 4 showed signals due to a cyclopropane methylene at d 0.59 and 0.41 (each 1H, d, J = 4.2 Hz), seven tertiary methyl groups at d 1.41 (3H, s), 1.33 (3H, s), 1.31 (3H, s), 1.26 (6H, s), 1.07 (3H, s) and 1.03 (3H, s) and four methine proton signals at d 4.69 (ddd, J = 8.0, 8.0, 5.2 Hz), 3.86 (dd, J = 8.2, 6.0 Hz), 3.49 (ddd, J = 9.5, 9.0, 4.5 Hz) and 3.27 (dd, J = 11.3, 4.0 Hz) which were indicative of secondary alcoholic functions (Table 1). The NMR data of aglycone of compound 4 were superimposable with those reported for cycloastragenol (Kitagawa et al., 1983a). Additionally, in the 1H spectrum signal ascribable to a methyl of an acetoxy function at d 2.13 (3H, s) and signals for three anomeric protons at d 4.73 (d, J = 4.3 Hz), 4.42 (d, J = 6.3 Hz) and 4.35 (d, J = 7.5 Hz) were observed. The comparison of NMR data of the sugar region of compound 4 with those of compound 1 allowed us to determine that the two sugar chain were identical. On the basis of these evidence, the structure of compound 4 was established as 3-O-[a-L-arabinopyranosyl(1 ? 2)-O-3-acetoxy-a-L-arabinopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,25-tetrahydroxy-20(R),24(S)-epoxycycloartane.
The molecular formulas of compounds 5 and 6 were established as C46H76O18 and C52H86O22 by HRMALDITOFMS analysis (m/z 939.4932 [M+Na]+, calcd for C46H76O18Na, 939.4929, and m/z 1085.5511 [M+Na]+, calcd for C52H86O22Na, 1085.5508), respectively. It was apparent from their 1H and 13C NMR data that 5 and 6 possessed the same aglycone moiety. In particular, the 1H NMR spectrum of 5 showed signals due to a cyclopropane methylene at d 0.61 and 0.32 (each 1H, d, J = 4.2 Hz), six tertiary methyl groups at d 1.53 (3H, s), 1.46 (3H, s), 1.32 (6H, s), 1.22 (3H, s) and 0.99 (3H, s) and four methine proton signals at d 4.65 (ddd, J = 8.0, 8.0, 5.2 Hz), 3.56 (ddd, J = 9.5, 9.5, 4.5 Hz), 3.51 (br s) and 3.22 (dd, J = 11.3, 4.0 Hz). The NMR data of compound 5 were in agreement with those reported for 3b,6a,16b,24a-tetrahydroxy-20(R),25-epoxycycloartane named cyclocephalogenin, previously reported from Astragalus spp. (Bedir et al., 1998b; Çalısß et al., 2001; Alaniya et al., 2008). For the sugar region in the 1H NMR spectrum, three anomeric protons at d 4.51 (d, J = 7.5 Hz), 4.50 (d, J = 6.3 Hz) and 4.37 (d, J = 7.5 Hz) were observed. Complete assignments of the 1H and 13C NMR signals of the sugar portion were accomplished by 1D-TOCSY, HSQC, HMBC and DQF-COSY experiments which led to the identification of one b-xylopyranosyl unit (d 4.51), one b-glucopyranosyl unit (d 4.37), and one a-arabinopyranosyl unit (d 4.50). The determination of the sequence and linkage sites was obtained from the HMBC correlations which showed key correlation peaks between the proton signals at d 4.51 (H-1xyl) and the carbon resonance at d 89.5 (C-3), d 4.50 (H-1ara) and d 83.2 (C-2xyl) and the proton signal at d 4.37 (H-1glc) and the carbon resonance at d 80.4 (C-6). Thus, the compound 5 was identified as 3-O-[a-L-arabinopyranosyl(1 ? 2)-O-b-D-xylopyranosyl]-6-O-b-D-glucopyranosyl3b,6a,16b,24a-tetrahydroxy-20(R),25-epoxycycloartane. The comparison of the sugar region NMR data of compound 6 with those of compound 2 showed that the sugar chains of two compounds were superimposable; therefore, the structure of compound 6 was established as 3-O-[a-L-rhamnopyranosyl-(1 ? 2)-O-a-Larabinopyranosyl-(1 ? 2)-O-b-D-xylopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,24a-tetrahydroxy-20(R),25-epoxycycloartane.
(O) 22
21 18
11
12
(O)
1
10
2
α-L-araI O
3 (O)
15
8
5
7
4
30
6
(O) HO
28 O HO
HO
α-L-araII O
O
HO OH
(O)
29 OH
HO
β-D-glu
26 25
(O)
16
14
9 O
23
17
13
19
20
24
OH
Fig. 2. Partial structures deduced from 2D-NMR (COSY and HSQC) and key HMBC of 3.
(O)
27
_ Horo et al. / Phytochemistry 71 (2010) 956–963 I.
Additionally, two known cycloartane-type glycosides, oleifolioside B (7) (Özipek et al., 2005) and astragaloside I (8) (Kitagawa et al., 1983a), five known oleanene-type triterpene glycosides, azukisaponin V (9) (Kitagawa et al., 1983b), azukisaponin V methyl ester (10) (Mohamed et al., 1995), astragaloside VIII (11) (Kitagawa et al., 1983c), astragaloside VIII methyl ester (12) (Cui et al., 1992), 22-O-[b-D-glucopyranosyl-(1 ? 2)-O-a-L-arabinopyranosyl]-3b,22b,24-trihydroxy-olean-12-ene (13) (Yoshikawa et al., 1985) and the flavonol glycoside narcissin (14) (Senatore et al., 2000) were isolated. Compounds 1–4 are based on cyclocanthogenin and cycloastragenol, two of the most common aglycones in Astragalus genus, whereas compounds 5 and 6 are cyclocephalogenin glycosides, which are uncommon in the plant kingdom and have so far been reported only in Astragalus spp. (Sukhina et al., 2007; Bedir et al., 1998b; Agzamova and Isaev, 1999; Semmar et al., 2001). 3. Experimental 3.1. General Optical rotations were measured on a JASCO DIP 1000 polarimeter. IR measurements were obtained on a Bruker IFS-48 spectrometer. NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker BioSpinGmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbeat 300 K. All 2DNMR spectra were acquired in CD3OD (99.95%, SigmaAldrich) and standard pulse sequences and phase cycling were used for DQFCOSY, HSQC, and HMBC spectra. The NMR data were processed using UXNMR software. Exact masses were measured by a Voyager DE mass spectrometer. Samples were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry. A mixture of analyte solution and a-cyano-4-hydroxycinnamic acid (Sigma) was applied to the metallic sample plate and dried. Mass calibration was performed with the ions from ACTH (fragment 18–39) at 2465.1989 Da and angiotensin III at 931.5154 Da as internal standard. ESIMS analyses were performed using a ThermoFinnigan LCQ Deca XP Max iontrap mass spectrometer equipped with Xcalibur software. GC analysis was performed on a Termo Finnigan Trace GC apparatus using a l-Chirasil-Val column (0.32 mm 25 m). 3.2. Plant material A. icmadophilus Hand.-Mazz. was collected from Gürpınar Village, Eskinus (Nebirnav) hill, from altitude of 2963 m, Van, East Anatolia, Turkey in 09.07.2008. Samples of plant material were identified by F. Özgökçe (Department of Biology, Faculty of Science and Art, Yüzüncü Yıl University, Van, Turkey). Voucher specimen has been deposited in the Herbarium of Yüzüncü Yıl University, Van, Turkey (VANF 14727). 3.3. Extraction and isolation Air-dried and powdered plant material of A. icmadophilus (whole plant; 1200 g) was extracted with MeOH (3 3 l) at room temperature for 72 h. After filtration, the solvent was removed by rotary evaporation to give a crude extract (112 g). An aliquot of the crude extract (60 g) was dissolved in water (600 ml) and successively partitioned with n-hexane (250 ml 3), Et2O (250 ml 3), CH2Cl2 (250 ml 3), and n-BuOH (250 ml 3). After evaporation of the organic solvents, 1.57 g, 1.59 g, 370 mg and 28 g of extracts were obtained respectively. The BuOH fraction (16 g) rich in saponins was subjected to VLC using reverse-phase material (Lichroprep. RP-18, 25–40 lm, 300 g) eluting with water (2200 ml), H2O–MeOH (8:2,
961
2000 ml; 6:4, 3600 ml; 5:5, 200 ml; 4:6, 4500 ml; 3:7, 2400 ml; and 2:8, 1200 ml) to give 19 main fractions (Fr1–Fr19). An aliquot of Fr14 (1.1 g) was further subjected to High Performance Flash Chromatography (HPFC, Biotage, Inc., A Dyax Corp. Company; BiotageSI 40 M column), using CHCl3:MeOH mixtures (85:15, 80:20, 70:30, 60:40, 50:50) to yield compound 1 (34 mg) and compound 7 (84 mg). Fr12 (830 mg) was subjected to High Performance Flash Chromatography (HPFC, Biotage, Inc., A Dyax Corp. Company; BiotageSI 40 M column), using CHCl3:MeOH mixtures (85:15, 80:20, 70:30, 60:40, 50:50) to yield 280 fractions (Fr12–1/Fr12–280). Fr12–230 and Fr12–260 were combined (300 mg) and applied to open column chromatography using silica gel as a stationary phase (Merck. 7734, 55 g) under isocratic solvent elution using CHCl3:MeOH:H2O (80:20:2). While subfractions 200–230 gave compound 5 (37 mg), subfractions 515–535 and 730–750 afforded compound 6 (36 mg) and compound 2 (57 mg), respectively. Fr15 (2.30 g) was precipitated using MeOH and the precipitate (315 mg) was subjected to open column chromatography using silica gel as a stationary phase (Merck. 7734, 65 g) under isocratic solvent elution using CHCl3:MeOH:H2O (70:30:3). Fr15– 39–42 gave compounds 10 and 12 as a mixture (5 mg), whereas Fr15–134–156 provided compounds 9 and 11 as a mixture (114 mg). Fr17 (500 mg) was further separated over a normal phase silica gel (Merck. 7734, 55 g) under gradient solvent elution using CHCl3:MeOH:H2O (90:10:0.5 ? 80:20:2). While the subfractions 196–203 afforded compound 8 (3 mg), subfractions 254–264, 403–419, 461–495 gave compounds 13 (3 mg), 3 (36 mg) and 4 (53 mg), respectively. The main fractions 7 and 8 were combined (600 mg) and fractionated over an open column chromatography using polyamide as stationary phase. Elution was performed by MeOH to give 40 fractions. Subfraction 24–27 (22 mg) was subjected to Si gel (20 g) column chromatography by using CHCl3:MeOH:H2O (80:20:2) to yield compound 14 (14 mg). 3.4. 3-O-[a-L-arabinopyranosyl-(1 ? 2)-O-3-acetoxy-a-Larabinopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,24(S),25pentahydroxycycloartane (1) Amorphous white solid; C48H80O19; ½a25 D +28.4° (c 0.1 MeOH); IR m cm1: 3477 (>OH), 3041 (cyclopropane ring), 2940 (>CH), 1739 (C@O), 1264 and 1059 (C–O–C); for the 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data see Tables 1 and 2; ESI-MS m/z 983.5 [M+Na]+; MS/MS m/z 803.5 [M+Na180]+, MS3 m/z 743.4 [M+Na18060]+, 671.4 [M+Na180132]+, MS4 m/z 611.3 [M+Na18013260]+, 479.2 [M+Na180132132]+; HRMALDITOFMS [M+Na]+ m/z 983.5198 (calcd for C48H80O19Na, 983.5192). KBr max
3.5. 3-O-[a-L-rhamnopyranosyl-(1 ? 2)-O-a-L-arabinopyranosyl(1 ? 2)-O-b-D-xylopyranosyl]-6-O-b-D-glucopyranosyl3b,6a,16b,24(S),25-pentahydroxycycloartane (2) Amorphous white solid; C52H88O22; ½a25 D +37.2° (c 0.1 MeOH); IR m cm1: 3484 (>OH), 3037 (cyclopropane ring), 2949 (>CH), 1260 and 1064 (C–O–C); for the 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data see Tables 1 and 2; ESI-MS m/z 1087.6 [M+Na]+, MS/MS m/z 761.5 [M+Na-146]+, MS3 m/z 629.4 [M+Na-146–132]+; HRMALDITOFMS [M+Na]+ m/z 1087.5669 (calcd for C52H88O22Na, 1087.5665). KBr max
3.6. 3-O-[a-L-arabinopyranosyl-(1 ? 2)-O-3,4-diacetoxy-a-Larabinopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,24(S),25pentahydroxycycloartane (3)
m
Amorphous white solid; C50H82O20; ½a25 D +31.8° (c 0.1 MeOH); IR cm1: 3471 (>OH), 3032 (cyclopropane ring), 2931 (>CH),
KBr max
962
_ Horo et al. / Phytochemistry 71 (2010) 956–963 I.
1741 (C@O), 1258 and 1050 (C–O–C); for the 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data see Tables 1 and 2; ESI-MS m/ z 1025.6 [M+Na]+, MS/MS m/z 845.5 [M+Na180]+, MS3 m/z 785.5 [M+Na18060]+, MS4 m/z 725.5 [M+Na1806060]+, 653.4 [M+Na18060132]+, 593.4 [M+Na18060192]+; HRMALDITOFMS [M+Na]+ m/z 1025.5294 (calcd for C50H82O20Na, 1025.5297). 3.7. 3-O-[a-L-arabinopyranosyl-(1 ? 2)-O-3-acetoxy-a-Larabinopyranosyl]-6-O-b-D-glucopyranosyl-3b,6a,16b,25tetrahydroxy-20(R),24(S)-epoxycycloartane (4) Amorphous white solid; C48H78O19; ½a25 D +22.1° (c 0.1 MeOH); IR KBr mmax cm1: 3488 (>OH), 3035 (cyclopropane ring), 2927 (>CH), 1732 (C@O), 1275 and 1030 (C–O–C); for the 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data see Tables 1 and 2; ESI-MS m/z 981.8 [M+Na]+, MS/MS m/z 801.5 [M+Na-180]+, MS3 m/z 741.5 [M+Na-180–60]+, 669.4 [M+Na-180–132]+, MS4 m/ z 609.4 [M+Na-180–132-60]+; HRMALDITOFMS [M+Na]+ m/z 981.5041 (calcd for C48H78O19Na, 981.5035). 3.8. 3-O-[a-L-arabinopyranosyl-(1 ? 2)-O-b-D-xylopyranosyl]-6-Ob-D-glucopyranosyl-3b,6a,16b,24a-tetrahydroxy-20(R),25epoxycycloartane (5) Amorphous white solid; C46H76O18; ½a25 D +8.5° (c 0.1 MeOH); IR m cm1: 3481 (>OH), 3045 (cyclopropane ring), 2934 (>CH), 1280–1040 (C-O-C); for the 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data see Tables 1 and 2; ESI-MS m/z 939.5 [M+Na]+, MS/MS m/z 759.5 [M+Na180]+, MS3 m/z 627.4 [M+Na180132]+; HRMALDITOFMS [M+Na]+ m/z 939.4932 (calcd for C46H76O18Na, 939.4929). KBr max
3.9. 3-O-[a-L-rhamnopyranosyl-(1 ? 2)-O-a-L-arabinopyranosyl(1 ? 2)-O-b-D-xylopyranosyl]-6-O-b-D-glucopyranosyl3b,6a,16b,24a-tetrahydroxy-20(R),25-epoxycycloartane (6) Amorphous white solid; C52H86O22; ½a25 D +11.7° (c 0.1 MeOH); IR 1 mKBr : 3474 (>OH), 3049 (cyclopropane ring), 2937 (>CH), max cm
1266–1052 (C–O–C); for the 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data see Tables 1 and 2; ESI-MS m/z 1085.6 [M+Na]+, MS/MS m/z 905.6 [M+Na180]+, MS3 m/z 759.5 [M+Na180146]+, MS4 m/z 627.4 [M+Na180146132]+; HRMALDITOFMS [M+Na]+ m/z 1085.5511 (calcd for C52H86O22Na, 1085.5508). 3.10. Acid hydrolysis A solution (1 mg each) of compounds 1 and 2 in 1 N HCl (0.5 ml) was stirred at 80 °C for 4 h. After cooling, the solution was concentrated by evaporation under a stream of N2. The residue was dissolved in 1-(trimethylsilyl)-imidazole and pyridine (0.1 ml), and the solution was stirred at 60 °C for 5 min. After drying the solution with a stream of N2, the residue was partitioned between H2O and CH2Cl2 (1 ml, 1:1 v/v). The CH2Cl2 layer was analyzed by GC using an L-Chirasil-Val column (0.32 mm 25 m). Temperatures of the injector and detector were 200 °C for both. A temperature gradient system was used for the oven, starting at 100 °C for 1 min and increasing up to 180 °C at a rate of 5 °C/min. The peaks of the hydrolysate of 1 were detected at 14.70 min (D-glucose) and 8.93 and 9.79 (L-arabinose). The peaks of L-arabinose (8.91 and 9.80 min), 9.68 and 10.71 (L-rhamnose), D-xylose (10.96 and 12.02 min) and D-glucose (14.72 min) were detected in the hydrolysate of 2. Retention times for authentic samples after being treated in the same manner with 1-(trimethylsilyl)-imidazole in pyridine were detected at 14.71 min
(D-glucose), 10.98 and 12.00 min (D-xylose), 9.67 and 10.70 (Lrhamnose) and 8.92 and 9.80 (L-arabinose). Acknowledgements This project was supported by TUBITAK (109T425) and also Ege University Research Foundation (2009 Fen 090). References Agzamova, M.A., Isaev, M.I., 1999. Triterpene glycosides of Astragalus and their genins LIX. Structure of cyclocanthoside F. Chem. Nat. Compd. 35, 314–319. Alaniya, M.D., Kavtaradze, N.Sh., Faure, R., Debrauwer, L., 2008. Cycloascauloside B from Astragalus caucasicus. Chem. Nat. Compd. 44, 324–326. _ Aquino, R., Piacente, S., Pizza, C., 1998a. Cycloartane triterpene Bedir, E., Çalısß, I., glycosides from the roots of Astragalus brachypterus and Astragalus microcephalus. J. Nat. Prod. 61, 1469–1472. _ Zerbe, O., Sticher, O., 1998b. Cyclocephaloside I: a novel Bedir, E., Çalısß, I., cycloartane-type glycoside from Astragalus microcephalus. J. Nat. Prod. 61, 503–505. _ Aquino, R., Piacente, S., Pizza, C., 1999a. 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