Cycloartane glycosides from Astragalus stereocalyx Bornm.

Phytochemistry 73 (2012) 119–126

Contents lists available at SciVerse ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Cycloartane glycosides from Astragalus stereocalyx Bornm. _ Funda Nuray Yalçın a,⇑, Sonia Piacente b,⇑, Angela Perrone b, Anna Capasso b, Hayri Duman c, Ihsan Çalısß d a

Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Ankara, Turkey Dipartimento di Scienze Farmaceutiche e Biomediche, Università degli Studi di Salerno, Via Ponte Don Melillo, I-84084 Fisciano, Italy c Gazi University, Faculty of Arts and Sciences, Department of Biology, Ankara, Turkey d Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Nicosia, Cyprus b

a r t i c l e

i n f o

Article history: Received 10 May 2011 Accepted 27 September 2011 Available online 25 October 2011 Keywords: Astragalus stereocalyx Cycloartane saponins Cycloasgenin C

a b s t r a c t Six cycloartane-type triterpene glycosides were isolated from Astragalus stereocalyx along with six known cycloartane-type glycosides. Their structures were established by the extensive use of 1D and 2D-NMR experiments along with ESIMS and HRMS analysis. Three compounds are based on an aglycon characterized by the occurrence of an unusual hydroxyl group at position 20, whereas three other compounds are based on cycloasgenin C as aglycon, so far reported from Astragalus spp. All the compounds were tested for their cytotoxic activity against a number of cancer cell lines. One compound exhibited activity versus human cervical cancer (Hela) with an IC50 value = 10 lM. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The genus Astragalus L., which includes 10 subgenera and approximately 150 sections, is the largest genus of Leguminosae family, containing about 2500 species and subspecies according to some sources (Maassoumi, 1998) and 3000 according to some other sources (Heywood, 1978). The members of this genus are represented in the flora of Turkey by 58 sections and about 452 taxa, mostly growing in steppes and high mountains (Chamberlain and Matthews, 1970; Davis et al., 1988; Aytaç, 2000; Ekici and Ekim, 2004). Astragalus species growing wild in Turkey, are economically important for the production of gum tragacanth, a very well-known foodstuff and pharmaceutical emulsifier (Çalısß and Sticher, 1996). In Turkish folk medicine, the aqueous extracts of some Astragalus species are used to treat leukemia as well as for wound healing (Çalısß et al., 1997; Bedir et al., 2000a). Polysaccharides and saponins are the major classes of chemical compounds isolated from Astragalus species. Cycloartanes are produced only by photosynthesizing organisms, and one of the initial representatives of this class of compounds, cycloartenol, serves as key link in the biosynthesis of different phytosterols. The plants of Astragalus genus proved to be the richest source of this class of ⇑ Corresponding authors. Tel.: +90 312 3051089; fax: +90 312 3114777 (F.N. Yalçın), tel.: +39 089 969763; fax: +39 089 969602 (S. Piacente). E-mail addresses: [email protected] (F.N. Yalçın), [email protected] (S. Piacente). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.09.011

compounds. Cycloartanes from Astragalus genus are found to possess cardiotonic, hypocholesteremic, anti-depressive and antiblastic actions as well as immunomodulatory activity (Çalısß et al., 1997; Bedir et al., 2000a; Mamedova and Isaev, 2004). This promising spectrum of pharmacological effects led us to further search for structurally interesting cycloartane glycosides from the genus Astragalus. Under this perspective, we studied the roots of Astragalus stereocalyx belonging to the section stereocalyx, endemic in Turkey, morphologically unique because of woody and inflated calyx shapes. This paper reports the isolation of six cycloartane-type triterpene glycosides (1–6) from the methanol extract of A. stereocalyx along with six known cycloartane-type glycosides. Compounds 1–3 show a aglycon characterized by an unusual free hydroxyl group at position 20. The structures were elucidated by extensive spectroscopic methods including 1D (1H, 13C and TOCSY) and 2DNMR (DQF-COSY, HSQC, HMBC and ROESY) experiments as well as ESIMS and HRMS analysis. Additionally, all the compounds were screened for their antiproliferative activities versus a number of cancer cell lines.

2. Results and discussion The roots of A. stereocalyx were extracted with MeOH. The methanolic extract was subjected to vacuum column chromatography and purified by different chromatographic steps to yield six compounds 1–6.

120

F.N. Yalçın et al. / Phytochemistry 73 (2012) 119–126

R1

OR4 OR3

OH

R2O OH

1

R1 = OH

R2 =

O

R1 = OH

HO β-D-glc

α-L-ara

R4 = H

OH

β-D-xyl

O

HO R2 = HO OH

R3 = R4 = H

O O

α-L-ara

OH

OH

R1 = OH

O

R3 = HO

OH

HO

3

OH

O

HO

2

β-D-xyl

O

HO HO OH

O

HO

R2 = OH HO

β-D-glc

R3 = R4 = H

O

O HO

OH

α-L-ara

β-D-glc

OH

4

R1 = R3 = H

R2 =

R1 = R4 = H

R2 =

β-D-glc

β-D-glc

OH

O

R3 = HO

HO

O O

β-D-glc

α-L-ara OH O HO

α-L-rha

6

R1 = R3 =R4 = H

R2 =

O

HO

The HRMALDITOF mass spectrum of 1 (m/z 957.5038 [M+Na]+, calcd. for C46H78O19Na, 957.5035) supported a molecular formula of C46H78O19. The ESIMS mass spectrum showed the major ion peak at m/z 957 which was assigned to [M+Na]+. The MS/MS of this ion showed a peak at m/z 777 [M+Na-180]+, corresponding to the loss of a hexose unit. In the MS3 spectrum peaks at m/z 645

HO

OH

OH

O

HO HO OH HO

HO

α-L-ara

OH

OH

5

O

R4 = HO

O O

HO

OH

O

HO HO OH

OH

OH

β-D-glc O O O

HO

OH

α-L-ara

OH

[M+Na-180–132]+, corresponding to the loss of a pentose unit, and 495 [M+Na-180–132-150]+, due to the loss of a further pentose unit, were observed. A detailed comparison of the aglycon moiety NMR data (1H, 13C, HSQC, HMBC, COSY) of compounds 1–3 showed that the aglycon moiety was identical in all three compounds. In particular, the 1H

121

F.N. Yalçın et al. / Phytochemistry 73 (2012) 119–126 Table 1 13 C NMR data of the aglycon moieties of compounds 1–6 (150 MHz, CD3OD). Position

1

2a

4

5

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

32.7 30.4 89.3 42.9 54.3 69.4 38.5 48.4 21.6 30.5 27.2 33.5 47.3 47.7 48.5 86.3 57.9 20.2 31.8 78.6 28.2 39.9 26.3 80.2 74.0 25.1 25.2 28.3 16.0 20.2

33.2 30.5 89.4 43.0 54.4 69.4 38.7 48.2 21.7 30.4 27.1 33.8 47.3 47.7 49.0 74.9 56.8 20.2 31.6 78.5 27.9 39.9 26.3 80.4 74.0 25.1 25.5 28.3 15.7 20.1

33.1 30.4 89.5 43.3 54.5 69.3 38.5 48.4 22.2 30.5 26.7 33.7 46.9 47.8 48.4 72.9 57.8 19.0 31.3 32.4 18.2 34.2 30.1 91.8 75.0 23.7 26.0 28.4 16.2 20.3

32.8 30.3 89.5 43.2 54.0 69.8 38.1 48.5 21.9 30.4 26.9 33.2 46.4 47.5 48.4 83.5 57.7 19.3 31.1 31.9 17.4 34.5 30.3 80.5 73.5 24.2 25.3 28.1 15.7 19.9

33.2 30.4 89.6 42.8 54.6 69.5 38.7 48.5 22.2 30.5 26.8 33.5 46.6 47.5 48.5 72.9 57.7 19.0 31.5 32.0 18.2 34.9 29.5 80.4 73.8 24.6 25.5 28.5 16.3 20.3

a The chemical shift values of the aglycon moiety of 3 were superimposable with those reported for 2.

NMR spectrum of 1 showed signals due to a cyclopropane methylene at d 0.56 and 0.40 (each 1H, d, J = 4.2 Hz), seven tertiary methyl groups at d 1.47 (3H, s), 1.39 (3H, s), 1.32 (3H, s), 1.19 (6H, s), 1.04 (3H, s) and 0.97 (3H, s), and four methine proton signals at d 4.57 (ddd, J = 7.5, 7.5, 5.2 Hz), 3.47 (ddd, J = 9.5, 9.5, 4.5 Hz), 3.29 (dd, J = 10.5, 1.6 Hz) and 3.22 (dd, J = 11.3, 4.0 Hz), which were indicative of secondary alcoholic functions. The NMR data (Tables 1 and 2) of the aglycon moiety of 1 differed from those reported for cycloasgenin C (Kucherbaev et al., 2002) by the occurrence of the hydroxyl function at position 20. The location of the hydroxyl function at C-20 has been deduced from the HMBC correlations between the proton signals at d 1.39 (Me-21), d 2.13 (H-17), d 1.88 and 1.76 (H2-22), d 1.78 and 1.41 (H2-23) and the carbon signal at d 78.6 (C-20). The MS data in combination with NMR data allowed us to rule out that the aglycon was a 20,24-epoxycycloartane and thus it was identified as a cycloartane with the acyclic side chain and two hydroxyl groups at C-20 and C-24. Glycosidation shifts were observed for C-3 (d 89.3) and C-16 (d 86.3) (Table 1). The 13C NMR chemical shift for C-24 can be regarded as a characteristic parameter in the determination of the configurations of C-24. In the case of the 24R configuration, the chemical shift for C-24 gives resonance at 79.9–80.6 ppm (Isaev et al., 1983a,b, 1982; Agzamova and Isaev, 1998), while for the 24S configuration the chemical shift for C-24 gives resonance at 77.0–78.2 ppm (Isaev et al., 1992; Bedir et al., 2000b; Fadeev et al., 1988). The 13C NMR data for C-24 of compound 1 are comparable to those reported for analogous compounds having a 24R configuration. The relative configurations of the oxygenated atoms were determined by the magnitude of the vicinal proton-proton coupling constants to be b-OH for C-3 (d 3.22, dd, J = 11.3, 4.0 Hz. Hax-3), a-OH for C-6 (d 3.47, ddd, J = 9.5, 9.5, 4.5 Hz, Hax-6) and b-OH for C-16 (d 4.57, ddd, J = 7.5, 7.5, 5.2 Hz, Hax-16). Additionally, the relative

Table 2 1 H NMR data (J in Hz) of the aglycon moieties of compounds 1–6 (600 MHz, CD3OD). Position

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1.55, 1.94, 3.22, – 1.38, 3.47, 1.50, 1.88, – – 2.02, 1.92, – – 2.14, 1.83, 4.57, 2.13, 1.47, 0.56, 0.40, – 1.39, 1.88, 1.78, 3.29, 1.19, 1.19, 1.32, 1.04, 0.97,

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a

2a 1.24, m 1.69, m dd (11.3, 4.0) d (9.5) ddd (9.5, 9.5, 4.5) 1.36, m dd (11.9, 4.2)

1.25, m 1.81, m

dd (12.4, 7.5) dd (12.4, 5.2) ddd (7.5, 7.5, 5.2) d (7.5) s d (4.2) d (4.2) s 1.76, m 1.41, m dd (10.5, 1.6) s s s s s

1.56, 1.95, 3.23, – 1.38, 3.48, 1.48, 1.87, – – 2.01, 1.92, – – 2.05, 1.57, 4.73, 2.00, 1.48, 0.58, 0.40, – 1.43, 1.88, 1.76, 3.28, 1.19, 1.20, 1.33, 1.05, 0.97,

4 1.25, m 1.71, m dd (11.3, 4.0) d (9.5) ddd (9.5, 9.5, 4.5) 1.39, m dd (11.9, 4.2)

1.26, m 1.78, m

dd (12.4, 7.5) dd (12.4, 5.2) ddd (7.5, 7.5, 5.2) d (7.5) s d (4.2) d (4.2) s 1.74, m 1.46, m dd (10.5, 1.6) s s s s s

1.58, 2.08, 3.29, – 1.39, 3.48, 1.49, 1.82, – – 2.01, 1.70, – – 2.05, 1.41, 4.52, 1.71, 1.18, 0.55, 0.40, 1.79, 0.97, 2.13, 1.77, 3.39, 1.22, 1.20, 1.34, 1.05, 0.99,

5 1.25, m 1.71, m dd (11.3, 4.0) d (9.5) ddd (9.5, 9.5, 4.5) 1.38, m dd (11.9, 4.2)

1.24, m 1.67, m

dd (12.7, dd (12.7, ddd (8.5, dd (10.3, s d (4.2) d (4.2) m d (6.5) 1.08, m 1.27, m dd (10.5,

6.8) 5.2) 6.8, 5.2) 8.5)

1.6)

s s s s s

The chemical shift values of the aglycon moiety of 3 were superimposable with those reported for 2.

1.58, 2.07, 3.28, – 1.38, 3.46, 1.50, 1.81, – – 2.00, 1.70, – – 2.12, 1.72, 4.24, 1.84, 1.19, 0.54, 0.39, 1.95, 0.96, 1.94, 1.93, 3.19, 1.17, 1.19, 1.34, 1.05, 0.98,

6 1.23, m 1.71, m dd (11.3, 4.0) d (9.5) ddd (9.5, 9.5, 4.5) 1.36, m dd (11.9, 4.2)

1.23, m 1.67, m

dd (12.7, dd (12.7, ddd (8.5, dd (10.3, s d (4.2) d (4.2) m d (6.5) 1.03, m 1.20, m dd (10.5, s s s s s

6.8) 5.2) 6.8, 5.2) 8.5)

1.6)

1.58, 2.09, 3.28, – 1.40, 3.48, 1.50, 1.82, – – 2.02, 1.71, – – 2.05, 1.43, 4.46, 1.73, 1.19, 0.55, 0.40, 1.85, 0.97, 1.93, 1.84, 3.26, 1.17, 1.20, 1.34, 1.06, 0.99,

1.28, m 1.71, m dd (11.3, 4.0) d (9.5) ddd (9.5, 9.5, 4.5) 1.38, m dd (11.9, 4.2)

1.24, m 1.67, m

dd (12.7, dd (12.7, ddd (8.5, dd (10.3, s d (4.2) d (4.2) m d (6.5) 1.04, m 1.22, m dd (10.5, s s s s s

6.8) 5.2) 6.8, 5.2) 8.5)

1.6)

122

Table 3 13 C and 1H NMR data (J in Hz) of the sugar portions of compounds 1–6 (600 MHz, CD3OD). Position

1a dC

dH

b-D-Xyl (at C-3) 105.7 83.2 76.8 70.5 65.8 3.22 t (11.7)

dC 4.48 d (7.5) 3.46 dd (9.2, 7.5) 3.55 t (9.2) 3.53 m 3.88 dd (11.7, 5.2)

6

a-L-Ara

a

b-D-Glc (at C-3) 105.0 83.5 77.8 71.0 77.1 62.4

1 2 3 4 5

105.8 73.6 73.8 69.6 66.9

1 2 3 4 5 6

105.7 75.0 78.3 71.1 77.8 62.5

5 dH

dC 4.47 3.45 3.57 3.36 3.27

d (7.5) dd (9.0, 7.5) t (9.0) t (9.0) ddd (9.0, 4.5, 2.5)

3.87 dd (11.0, 2.5) 3.69 dd (11.0, 4.5)

a-L-Ara 4.50 d (6.8) 3.68 dd (8.5, 6.8) 3.59 dd (8.5, 3.0) 3.82 m 3.91 dd (11.9, 2.0) 3.55 dd (11.9, 3.0) b-D-Glc (at C-16) 4.38 3.19 3.38 3.32 3.30 3.89 3.71 dd (11.0, 4.5)

d (7.5) dd (9.0, 7.5) t (9.0) t (9.0) ddd (9.0, 4.5, 2.5) dd (11.0, 2.5)

106.2 73.4 73.7 69.3 67.1

105.9 75.7 77.8 71.0 78.0 62.3

6 dH

b-D-Glc (at C-3) 104.9 83.4 77.9 70.9 77.6 62.4

dC 4.48 3.44 3.57 3.37 3.26

d (7.5) dd (9.0, 7.5) t (9.0) t (9.0) ddd (9.0, 4.5, 2.5)

3.85 dd (11.0, 2.5) 3.69 dd (11.0, 4.5)

a-L-Ara 4.53 d (6.8) 3.69 dd (8.5, 6.8) 3.59 dd (8.5, 3.0) 3.82 m 3.92 dd (11.9, 2.0) 3.55 dd (11.9, 3.0) b-D-Glc (at C-24) 4.54 3.26 3.37 3.35 3.30 3.90 3.74 dd (11.0, 4.5)

d (7.5) dd (9.0, 7.5) t (9.0) t (9.0) ddd (9.0, 4.5, 2.5) dd (11.0, 2.5)

106.2 73.7 74.1 69.6 66.8

106.0 74.9 78.5 71.0 77.6 62.0

dH

b-D-Glc (at C-3) 104.9 83.5 76.7 78.7 73.5 62.4

4.49 3.50 3.69 3.61 3.34

d (7.5) dd (9.0, 7.5) t (9.0) t (9.0) ddd (9.0, 4.5, 2.5)

3.86 dd (11.0, 2.5) 3.70 dd (11.0, 4.5)

a-L-Ara 4.53 d (6.8) 3.70 dd (8.5, 6.8) 3.59 dd (8.5, 3.0) 3.82 m 3.91 dd (11.9, 2.0) 3.54 dd (11.9, 3.0) b-D-Glc (at C-16) 4.30 3.17 3.33 3.33 3.26 3.87 3.70 dd (11.0, 4.5)

The chemical shift values of the sugar portion at C-3 of compounds 2 and 3 were superimposable with those reported for 1 and 4, respectively.

106.3 73.4 74.2 69.5 67.0

4.55 d (6.8) 3.69 dd (8.5, 6.8) 3.60 dd (8.5, 3.0) 3.83 m 3.92 dd (11.9, 2.0) 3.55 dd (11.9, 3.0)

a-L-Rha d (7.5) dd (9.0, 7.5) t (9.0) t (9.0) ddd (9.0, 4.5, 2.5) dd (11.0, 2.5)

102.3 72.1 72.0 73.5 70.3 17.7

4.90, 3.86, 3.66, 3.43, 4.00, 1.29,

d (1.2) dd (1.2, 3.2) dd (3.2, 9.3) t (9.3) m d (6.5)

F.N. Yalçın et al. / Phytochemistry 73 (2012) 119–126

1 2 3 4 5

4a

F.N. Yalçın et al. / Phytochemistry 73 (2012) 119–126

configuration of C-20 was derived by the ROESY spectrum which showed key correlation peaks between Me-30a (d 0.97) and H16a (d 4.57) and H-17a (d 2.13) signals, between H-16a (d 4.57) and Me-21a (d 1.39) and H-17a (d 2.13) signals and between H17a (d 2.13) and Me-21a (d 1.39) signals. For the sugar region of compound 1 three anomeric protons at d 4.50 (d, J = 6.8 Hz), 4.48(d, J = 7.5 Hz) and 4.38 (d, J = 7.5 Hz) were observed. The chemical shifts of all the individual protons of the four sugar units were ascertained from a combination of 1D-TOCSY and DQF-COSY spectral analysis, and the 13C chemical shifts of their relative attached carbons were assigned unambiguously from the HSQC spectrum (Table 3). These data showed the presence of one a-arabinopyranosyl unit (d 4.50), one b-xylopyranosyl unit (d 4.48) and one b-glucopyranosyl units (d 4.38). Glycosidation shift was observed for C-2xyl (d 83.2). 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 4.48 (H-1xyl) and the carbon resonance at d 89.3 (C-3), d 4.50 (H-1ara) and d 83.2 (C-2xyl) and the proton signal at d 4.38 (H1glc) and the carbon resonance at d 86.3 (C-16). The D configuration of xylose and glucose units and the L configuration of arabinose unit were established after hydrolysis of 1 with 1 N HCl, trimethylsilation and determination of the retention times by GC (De Marino et al., 2003). On the basis of all these evidence, the structure of the compound 1 was established as 3-O-[a-L-arabinopyranosyl-(1 ? 2)-bD-xylopyranosyl]-16-O-b-D-glucopyranosyl-3b,6a,16b,20(S),24(R), 25-hexahydroxycycloartane. The molecular formula of compound 2 was established as C40H68O14 by HRMALDITOFMS analysis (m/z 795.4509 [M+Na]+, calcd. for C40H68O14Na, 795.4507). The 1H NMR spectrum for the sugar region of compound 2 showed signals for two anomeric protons at d 4.50 (d, J = 6.8 Hz) and 4.49 (d, J = 7.5 Hz). The NMR data of 2 in comparison with those of compound 1 showed that the two compounds differed only by the absence of the glucopyranosyl unit at C-16. Thus, the compound 2 was identified as 3-O-[a-l-arabinopyranosyl-(1 ? 2)-b-D-xylopyranosyl]-3b,6a,16b,20(S),24(R),25hexahydroxycycloartane. The HRMALDITOF mass spectrum of 3 (m/z 825.4615 [M+Na]+, calcd. for C41H70O15Na, 825.4612) supported a molecular formula of C41H70O15. The 1H NMR spectrum of 3 displayed, in addition to signals of the aglycon moiety, signals for two anomeric protons at d 4.52 (d, J = 6.8 Hz) and 4.47 (d, J = 7.5 Hz). On the basis of HSQC, HMBC, DQF-COSY and 1D-TOCSY correlations one a-arabinopyranosyl unit (d 4.52) and one b-glucopyranosyl unit (d 4.47) were identified. Glycosidation shifts were observed for C-3 (d 89.5) and C-2glc (d 83.6). Key correlation peaks in the HMBC spectrum were observed between the proton signal at d 4.47 (H-1glc) and the carbon resonance at d 89.5 (C-3) and between the proton signal at d 4.52 (H-1ara) and the carbon resonance at d 83.6 (C2glc). Therefore, the structure of 3 was established as 3-O-[a-L-arabinopyranosyl-(1 ? 2)-b-D-glucopyranosyl]-3b,6a,16b,20(S),24(R), 25-hexahydroxycycloartane. The molecular formula of 4 was established as C47H80O19 by HRMALDITOFMS analysis (m/z 971.5196 [M+Na]+, calcd. for C47H80O19Na, 971.5192). It was apparent from their 1H and 13C NMR data that 4–6 possessed the same aglycon moiety. In particular, the 1H NMR spectrum of 4 showed signals due to a cyclopropane methylene at d 0.55 and 0.40 (each 1H, d, J = 4.2 Hz), six tertiary methyl groups at d 1.34 (3H, s), 1.22 (3H, s), 1.20 (3H, s), 1.18 (3H, s), 1.05 (3H, s) and 0.99 (3H, s) and a secondary methyl group at d 0.97 (3H, d, J = 6.5 Hz), and four methine proton signals at d 4.52 (ddd, J = 8.5, 6.8, 5.2 Hz), 3.48 (ddd, J = 9.5, 9.5, 4.5 Hz), 3.39 (dd, J = 10.5, 1.6 Hz) and 3.29 (dd, J = 11.3, 4.0 Hz), which were indicative of secondary alcoholic functions. The NMR data (Tables 1 and 2) of the aglycon moiety of 4 were in good agreement with

123

those reported for cycloasgenin C (Kucherbaev et al., 2002). The H NMR spectrum of 4 displayed, in the sugar region, three anomeric proton signals at d 4.53 (d, J = 6.8 Hz), 4.54 (d, J = 7.5 Hz) and 4.47 (d, J = 7.5 Hz). The HSQC, HMBC, DQF-COSY and 1D-TOCSY data led to identify these sugar units as one a-L-arabinopyranosyl (d 4.53) and two b-glucopyranosyl (d 4.54 and 4.47) units. The comparison of the NMR data of compound 4 with those of compound 3 allowed us to determine the same sugar chain at C-3 and the occurrence in 4 of a b-glucopyranosyl unit at C-24. Thus, compound 4 was established as 3-O-[a-L-arabinopyranosyl(1 ? 2)-b-D-glucopyranosyl]-24-O-b-D-glucopyranosyl-3b,6a,16b, 24(R),25-pentahydroxycycloartane. The HRMALDITOF mass spectrum of 5 (m/z 971.5194 [M+Na]+, calcd. for C47H80O19Na 971.5192) supported a molecular formula of C47H80O19. It was evident by the comparison of the NMR data of compound 5 with those of compound 4 that the two compounds differed only by the glycosidation site of the second b-glucopyranosyl unit, linked at C-16 in 5 and at C-24 in 4, allowing us to identify compound 5 as 3-O-[a-L-arabinopyranosyl-(1 ? 2)-b-Dglucopyranosyl]-16-O-b- D -glucopyranosyl-3b,6 a,16b,24(R),25pentahydroxycycloartane. The molecular formula of 6 was established as C47H80O18 by HRMALDITOFMS analysis (m/z 955.5246 [M+Na]+, calcd. for C47H80O18Na, 955.5242). The ESIMS mass spectrum showed the major ion peak at m/z 955 which was assigned to [M+Na]+. In the MS/MS spectrum a peak at m/z 809 [M+Na-146]+, corresponding to the loss of a deoxy-hexose unit was observed. Moreover, the MS3 spectrum showed a peak at m/z 677 [M+Na-146–132]+, corresponding to the loss of a pentose unit. The 1H NMR spectrum of 6 displayed, in addition to signals of the aglycon moiety, signals for three anomeric protons at d 4.90 (d, J = 1.2 Hz), 4.55 (d, J = 6.8 Hz) and 4.49 (d, J = 7.5 Hz). Complete assignments of the 1H and 13C NMR signals of the sugar portion were accomplished by 1D-TOCSY, HSQC and DQF-COSY experiments which led to the identification of one rhamnopyranosyl unit (d 4.90), one a-arabinopyranosyl (d 4.55) and one b-glucopyranosyl unit (d 4.49). The a configuration of the rhamnopyranosyl unit was deduced from the H-1/C-1 J value = 169 Hz, measured from the residual direct correlation observed in the HMBC spectrum, in agreement with that reported for the a anomer of rhamnopyranose (Kasai et al., 1979). 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 4.49 (H-1glc) and the carbon resonance at d 89.6 (C-3), the proton signal at d 4.90 (H1rha) and the carbon resonance at d 78.7 (C-4glc) and between the proton signal at d 4.55 (H-1ara) and the carbon resonance at d 83.5 (C-2glc). Thus, compound 6 was elucidated as 3-O-{a-Lrhamnopyranosyl-(1 ? 4)-[a-L-arabinopyranosyl-(1 ? 2)]-b-D-glucopyranosyl}-3b,6a,16b,24(R),25-pentahydroxycycloartane. Compounds 4–6 are based on cycloasgenin C as aglycon, so far reported from Astragalus spp. (Isaev et al., 1982, 1983a,b; Isaev, 1995, 1996; Agzamova and Isaev, 1998; Kucherbaev et al., 2003, 2002), whereas compounds 1–3 show as aglycon a cycloasgenin C derivative, characterized by the occurrence of the hydroxyl group at position 20 which not is involved in the usual 20,24 epoxy function reported for cycloartane of Astragalus spp. Additionally, six known cycloartane-type glycosides, askendoside C (7) (Isaev et al., 1983a), askendoside F (8) (Isaev, 1995), askendoside G (9) (Isaev, 1996), 3-O-b-D-glucopyranosyl-16-O-b-Dglucopyranosyl-3b,6a,16b,24(R),25-pentahydroxycycloartane (10) (Verotta et al., 2002), elongatoside (11) (Çalısß et al., 2008) and trojanoside H (12) (Bedir et al., 1999) were isolated. Cycloartane glycosides have been reported for their cytotoxic activity on several cancer lines including solid tumor (HepG2), blood tumor (HL-60) and drug resistant tumor (R-HepG2) (Kikuchi et al., 2007). Moreover cancer chemopreventive effects of natural 1

124

F.N. Yalçın et al. / Phytochemistry 73 (2012) 119–126

and semisynthetic cycloartane-type and related triterpenoids have been reported (Tian et al., 2005). On the basis of the interesting activities reported for cycloartane glycosides, the antiproliferative activity of compounds 1–12 was tested in different cancer cell lines including Hela (human cervical cancer), HT-29 (human colon cancer), U937 (human leukemia) and H446 (human lung cancer). Compounds 1–12 were tested in a range of concentrations between 1 and 50 lM, but only few compounds showed a weak activity and only Hela cell line was responsive to the treatment of some analyzed compounds. In particular, compound 10 exhibited an IC50 value of 10 lM against Hela cells, while compounds 6, 7 and 9 exhibited IC50 values of 29.9, 31.5 and 24.4 lM, respectively. For all the other compounds higher IC50 values were observed. 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%, Sigma Aldrich) and standard pulse sequences and phase cycling were used for DQF-COSY, HSQC, HMBC and ROESY 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 (fragments 18–39) at 2465.1989 Da and a-cyano-4-hydroxycinnamic acid at 190.0504 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. stereocalyx Bornm. was collected from Tasßkent, Konya, Turkey (20.5.2009) and identified by Prof. Dr. Hayri Duman (Department of Biology, Faculty of Arts and Sciences, Gazi University, Ankara, Turkey). Voucher specimen has been deposited in the Herbarium of Gazi University, Ankara, Turkey (H Duman 10191). 3.3. Extraction and isolation Air-dried and powdered roots of A. stereocalyx (300 g) were extracted with MeOH (3  2.5 l) at 40 °C. After filtration, the solvent was removed by rotary evaporation yielding 50 g of extract. The MeOH extract (50 g) was subjected to vacuum liquid chromatography (VLC) using reversed-phase material (Lichroprep RP-18, 25–40 lm, 250 g) eluting with H2O/MeOH (100:0 to 0:100) to give thirteen main fractions (A–L). Fraction B (1.0 g) was submitted to MPLC on reversed phase silica gel (LiChrosorb C-18, 36  460 mm) using stepwise MeOH–H2O gradient (20–70% MeOH, 35 mL/fraction) to give 39 fractions. Fraction 10 (29 mg) was further purified on a silica gel column (7 g) eluting with CHCl3–MeOH–H2O (90:10:1, 85:15:1.5, 80:20:2 and 75:25:2.5, 250 mL each) to yield pure 1 (6 mg). Fraction 13 (97 mg) was applied to silica gel (13 g) column with the solvent system CHCl3– MeOH (95:5, 100 mL; 90:10, 100 mL) and CHCl3–MeOH–H2O

(90:10:1, 200 mL; 80:20:2, 200 mL; 75:25:2.5, 200 mL; 70:30:3, 200 mL) yielding 2 (20 mg). Fraction 9 (25.9 mg) consisted of crude compound 3 and was purified by silica gel (7 g) column chromatography eluted with CHCl3–MeOH–H2O (90:10:1, 85:15:1.5, 80:20:2 and 75:25:2.5, 250 mL each) to yield pure 3 (2.9 mg). Fraction F (3.3 g) was subfractionated by silica gel (300 g) column chromatography eluted with CHCl3–MeOH–H2O (85:15:1.5, 80:20:2, 75:25:2.5, 70:30:3, 60:40:4 1000 mL each) to give 15 subfractions (F1–15). Fraction F10 (800 mg) was applied to MPLC on reversed phase silica gel (LiChrosorb C-18, 36  460 mm) using stepwise MeOH–H2O gradient (20–80% MeOH, 35 mL/fraction) to give 20 fractions (F10A–U). Fraction F10H (120 mg) was purified by silica gel (10 g and 7 g) column chromatography systems with the solvent system, respectively CHCl3–MeOH–H2O (80:20:2, 700 mL) to yield 6 (39 mg) and CHCl3–MeOH–H2O (75:25:2.5, 600 mL) to yield 4 (19 mg) and 11 (7.9 mg). Fraction F10K (68 mg) was subjected to silica gel (7.5 g) column with the solvent CHCl3–MeOH–H2O (80:20:2, 800 mL) yielding 5 (15 mg). Fraction H (3 g) was subjected to vacuum liquid chromatography (VLC) using reversedphase material (Lichroprep RP-18, 25–40 lm, 100 g) eluting with H2O/MeOH (50:50 to 0:100) to give 10 fractions (H1–10). Fraction H3 (900 mg) was subjected to silica gel (120 g) column eluting with CHCl3–MeOH–H2O (90:10:1, 85:15:1.5, 80:20:2, 1000 mL each) to give 7 (21 mg), 8 (20 mg), 9 (8 mg) and 12 (57 mg). Fraction 11 (87 mg) was purified by silica gel (7 g) column chromatography eluted with CHCl3–MeOH–H2O (80:20:2, 300 mL) to give 10 (13 mg). 3.4. 3.4.3-O-[a-L-arabinopyranosyl-(1 ? 2)-b-D-xylopyranosyl]-16O–D-glucopyranosyl-3b,6a,16b,20(S),24(R),25hexahydroxycycloartane (1) 

Amorphous white solid; C46H78O19; ½a25 D þ 27:2 (c 0.1 MeOH); IR KBrmax cm1: 3480 (>OH), 3039 (cyclopropane ring), 2961 (>CH), 1250 and 1072 (C–O–C); for the 13C (CD3OD, 150 MHz) and 1H NMR (CD3OD, 600 MHz) data of the aglycon moiety see Tables 1 and 2, respectively; for the 13C NMR (CD3OD, 150 MHz) and 1 H (CD3OD, 600 MHz) data of the sugar portion see Table 3; ESIMS m/z 957 [M+Na]+; MS/MS m/z 777 [M+Na-180]+, MS3 m/z 645 [M+Na-180–132]+, 495 [M+Na-180–132-150]+; HRMALDITOFMS [M+Na]+ m/z 957.5038 (calcd. for C46H78O19Na, 957.5035). 3.5. 3-O-[a-L-arabinopyranosyl-(1 ? 2)-b-D-xylopyranosyl]3b,6a,16b,20(S),24(R),25-hexahydroxycycloartane (2) 

Amorphous white solid; C40H68O14; ½a25 D þ 25:2 (c 0.1 MeOH); IR mKBrmax cm1: 3475 (>OH), 3048 (cyclopropane ring), 2955 (>CH), 1260 and 1057 (C–O–C); for the 13C (CD3OD, 150 MHz) and 1H NMR (CD3OD, 600 MHz) data of the aglycon moiety see Tables 1 and 2, respectively; for the 13C NMR (CD3OD, 150 MHz) and 1 H (CD3OD, 600 MHz) data of the sugar portion see Table 3; ESIMS m/z 795 [M+Na]+; MS/MS m/z 663 [M+Na-132]+, 513 [M+Na-132– 150]+; HRMALDITOFMS [M+Na]+ m/z 795.4509 (calcd. for C40H68O14Na, 795.4507). 3.6. 3-O-[a-L-arabinopyranosyl-(1 ? 2)-b-D-glucopyranosyl]3b,6a,16b,20(S),24(R),25-hexahydroxycycloartane (3) 

Amorphous white solid; C41H70O15; ½a25 D þ 28:6 (c 0.1 MeOH); IR mKBrmax cm1: 3477 (>OH), 3036 (cyclopropane ring), 2948 (>CH), 1252 and 1067 (C–O–C); for the 13C (CD3OD, 150 MHz) and 1H NMR (CD3OD, 600 MHz) data of the aglycon moiety see Tables 1 and 2, respectively; for the 13C NMR (CD3OD, 150 MHz) and 1 H (CD3OD, 600 MHz) data of the sugar portion see Table 3; ESIMS m/z 825 [M+Na]+; MS/MS m/z 693 [M+Na-132]+, 513 [M+Na-132–

F.N. Yalçın et al. / Phytochemistry 73 (2012) 119–126

180]+; HRMALDITOFMS C41H70O15Na, 825.4612).

[M+Na]+

m/z

825.4615

(calcd.

for

3.7. 3-O-[a-L-arabinopyranosyl-(1 ? 2)-b-D-glucopyranosyl]-24-O-ba,16b,24(R),25-pentahydroxycycloartane (4)

D-glucopyranosyl-3b,6



Amorphous white solid; C47H80O19; ½a25 D þ 12:7 (c 0.1 MeOH); IR KBrmax cm1: 3482 (>OH), 3043 (cyclopropane ring), 2952 (>CH), 1263 and 1060 (C–O–C); for the 13C (CD3OD, 150 MHz) and 1H NMR (CD3OD, 600 MHz) data of the aglycon moiety see Tables 1 and 2, respectively; for the 13C NMR (CD3OD, 150 MHz) and 1 H (CD3OD, 600 MHz) data of the sugar portion see Table 3; ESIMS m/z 971 [M+Na]+; MS/MS m/z 839 [M+Na-132]+, 659 [M+Na-132– 180]+; HRMALDITOFMS [M+Na]+ m/z 971.5196 (calcd. for C47H80O19Na, 971.5192). 3.8. 3-O-[a-L-arabinopyranosyl-(1 ? 2)-b-D-glucopyranosyl]-16-O-ba,16b,24(R),25-pentahydroxycycloartane (5)

D-glucopyranosyl-3b,6

125

heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin at 37 °C in an atmosphere of 95% O2 and 5% CO2. The cells were used up to a maximum of 10 passages. Human cancer cells (3  103) were plated in 96-well culture plates in 90 lL of culture medium and incubated at 37 °C in humidified 5% CO2. The next day, 10 lL aliquots of serial dilutions of each tested compound (1–50 lM) were added to the cells and incubated for 48 h. Cell viability was assessed through the MTT assay. Briefly, 25 lL of MTT (5 mg/mL) were added and the cells were incubated for an additional 3 h. Thereafter, cells were lysed and the dark blue crystals solubilized with 100 lL of a solution containing 50% N,N-dimethylformamide, 20% SDS (sodium dodecyl sulfate) with an adjusted pH of 4.5. The optical density (OD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan MCC/340) equipped with a 620 nm filter. Cells viability in response to treatment was calculated as percentage of control cells treated with solvent DMSO at the final concentration 0.1%:% viable cells = (100  OD treated cells)/OD control cells.



Amorphous white solid; C47H80O19; ½a25 D þ 10:8 (c 0.1 MeOH); IR mKBrmax cm1: 3488 (>OH), 3045 (cyclopropane ring), 2940 (>CH), 1258 and 1064 (C–O–C); for the 13C (CD3OD, 150 MHz) and 1H NMR (CD3OD, 600 MHz) data of the aglycon moiety see Tables 1 and 2, respectively; for the 13C NMR (CD3OD, 150 MHz) and 1 H (CD3OD, 600 MHz) data of the sugar portion see Table 3; ESIMS m/z 971 [M+Na]+; MS/MS m/z 839 [M+Na-132]+, 791 [M+Na-180]+, MS3 m/z 659 [M+Na-132–180]+; HRMALDITOFMS [M+Na]+ m/z 971.5194 (calcd. for C47H80O19Na, 971.5192). 3.9. 3-O-{a-L-rhamnopyranosyl-(1 ? 4)-[a-L-arabinopyranosyl(1 ? 2)]-b-D-glucopyranosyl}-3b,6a,16b,24(R),25pentahydroxycycloartane (6) 

Amorphous white solid; C47H80O18; ½a25 D þ 14:5 (c 0.1 MeOH); IR mKBrmax cm1: 3470 (>OH), 3051 (cyclopropane ring), 2943 (>CH), 1268 and 1054 (C–O–C); for the 13C (CD3OD, 150 MHz) and 1H NMR (CD3OD, 600 MHz) data of the aglycon moiety see Tables 1 and 2, respectively; for the 13C NMR (CD3OD, 150 MHz) and 1 H (CD3OD, 600 MHz) data of the sugar portion see Table 3; ESIMS m/z 955 [M+Na]+; MS/MS m/z 809 [M+Na-146]+, MS3 m/z 677 [M+Na-146–132]+; HRMALDITOFMS [M+Na]+ m/z 955.5246 (calcd. for C47H80O18Na, 955.5242). 3.10. Acid hydrolysis The configuration of sugar units were established after hydrolysis of 1 and 6 with 1 N HCl, trimethylsilation and determination of the retention times by GC operating in the experimental conditions previously reported by De Marino et al. (2003). The peaks of the hydrolysate of 1 were detected at 8.93 and 9.81 min (L-arabinose), 10.97 and 12.01 (D-xylose) and 14.72 min (D-glucose). The peaks of L-arabinose (8.91 and 9.80 min), L-rhamnose (9.69 and 10.71 min) and D-glucose (14.70 min) were detected in the hydrolysate of 6. Retention times for authentic samples after being treated in the same manner with 1-(trimethylsilyl)-imidazole in pyridine were detected at 8.92 and 9.80 (L-arabinose), 9.67 and 10.70 (L-rhamnose), 10.98 and 12.00 min (D-xylose), and 14.71 min (D-glucose). 3.11. Antiproliferative activity Hela (human cervical cancer), HT-29 (human colon cancer), U937 (human leukemia) and H446 (human lung cancer) cells obtained from the European Collection of Cell Cultures were cultured in DMEM medium supplemented with 2 mM L-glutamine, 10%

Acknowledgement The authors are grateful to Elif Yılmaz for her help with the MPLC experiments.

References Agzamova, M.A., Isaev, M.I., 1998. Triterpene glycosides of Astragalus and their genins. Chem. Nat. Compd. 1998 (34), 155–159. Aytaç, Z., 2000. Astragalus L. In: Güner, A., Özhatay, N., Ekim, T., Basßer, K.H.C. (Eds.), Flora of Turkey and the East Aegean Islands, vol. 11. University Press, Edinburgh., pp. 79–88. _ Aquino, R., Piacente, S., Pizza, C., 1999. Trojanoside H: a Bedir, E., Çalısß, I., cycloartane-type glycoside from the aerial parts of Astragalus trojanus. Phytochemistry 51, 1017–1020. _ Pasco, D.S., Khan, I.A., 2000a. Immunostimulatory effects Bedir, E., Pugh, N., Çalısß, I., of cycloartane-type triterpene glycosides from Astragalus species. Biol. Pharm. Bull. 23, 834–837. _ Khan, I.A., 2000b. Macrophyllosaponin E: a novel compound from Bedir, E., Çalısß, I., the roots of Astragalus oleifolius. Chem. Pharm. Bull. 48, 1081–1083. _ Sticher, O., 1996. Triterpene saponins from plants of the flora of Turkey. In: Çalısß, I., Waller, C.R., Yamasaki, K. (Eds.), Saponins Used in Traditional Medicine Advances in Experimental Medicine and Biology, vol. 404. Plenum Press, New York., pp. 485–500. _ Yuruker, A., Tasßdemir, D., Wright, A.D., Sticher, O., Luo, Y.D., Pezzuto, J.M., Çalısß, I., 1997. Cycloartane triterpene glycosides from the roots of Astragalus melanophrurius. Planta Med. 63, 183–186. _ Barbic, M., Jurgenliemk, G., 2008. Bioactive cycloartane-type triterpene Çalısß, I., glycosides from Astragalus elongatus. Z. Naturforsch. C: J. Biosci. 63, 813–820. Chamberlain, D.F., Matthews, V.A., 1970. Astragalus L. In: Davis, P.H. (Ed.), Flora of Turkey and East Aegean Islands, vol. 3. University Press, Edinburgh., pp. 49–254. Davis, P.H., Mill, R.R., Tan, K., 1988. Flora of Turkey and the East Aegean Islands, vol. 10, University Press, Edinburgh. pp. 114–124. De Marino, S., Borbone, N., Iorizzi, M., Esposito, G., McClintock, J.B., Zollo, F., 2003. Bioactive asterosaponins from the starfish Luidia quinaria and Psilaster cassiope. Isolation and structure characterization by two-dimensional NMR spectroscopy. J. Nat. Prod. 66, 515–519. Ekici, M., Ekim, T., 2004. Revision of the section Holoeuce Bunge of the genus Astragalus L. (Leguminosae) in Turkey. Turk. J. Bot. 28, 307–347. Fadeev, Y.M., Isaev, M.I., Akimov, Y.A., Kintya, P.K., Gorovits, M.B., Abubakirov, N.K., 1988. Triterpene glycosides and their genins from Astragalus. XXV. Cyclocanthoside D from Astragalus tragacantha. Khim. Prir. Soedin. 1, 73–76. Heywood, V.H., 1978. Flowering Plants of the World. Oxford University Press, London. Isaev, M.I., 1995. Triterpene glycosides of Astragalus and their genins. LII. Askendoside F from Astragalus taschkendicus. Khim. Prir. Soedin. 6, 820–823. Isaev, M.I., 1996. Triterpene glycosides of Astragalus and their genins LIV. Askendoside G from Astragalus taschkendicus. Khim. Prir. Soedin. 5, 723–727. Isaev, M.I., Gorovits, M.B., Abdullaev, N.D., Abubakirov, N.K., 1982. Triterpenoid glycosides of Astragalus and their genins. VI. Cycloasgenin C from Astragalus taschkendicus. Khim. Prir. Soedin. 4, 458–464. Isaev, M.I., Gorovits, M.B., Gorovits, T.T., Abdullaev, N.D., Abubakirov, N.K., 1983a. Astragalus triterpenoid glycosides and their genins VIII. Askendoside C from Astragalus taschkendicus. Khim. Prir. Soedin. 2, 173–180. Isaev, M.I., Gorovits, M.B., Abdullaev, N.D., Abubakirov, N.K., 1983b. Triterpene glycosides of Astragalus and their genins. XIV. Askendoside A from Astragalus taschkendicus. Khim. Prir. Soedin. 5, 587–592.

126

F.N. Yalçın et al. / Phytochemistry 73 (2012) 119–126

Isaev, M.I., Imomnazarov, B.A., Fadeev, Y.M., Kintya, P.A., 1992. Triterpene glycosides of Astragalus and their genins. XLII. Cycloartanes of Astragalus tragacantha. Khim. Prir Soedin. 3 (4), 360–367. Kasai, E., Ohikara, M., Asakawa, J., Mizutani, K., Tanaka, O., 1979. 13 C NMR Study of a- and b-anomeric pairs of D-mannopyranosides and L-rhamnopyranosides. Tetrahedron 35, 1427–1432. Kikuchi, T., Akihisa, T., Tokuda, H., Ukiya, M., Watanabe, K., Nishino, H., 2007. Cancer chemopreventive effects of cycloartane-type and related triterpenoids in in vitro and in vivo models. J. Nat. Prod. 70, 918–922. Kucherbaev, K.Dzh., Uteniyazov, K.K., Kachala, V.V., Saatov, Z., Shashkov, A.S., 2002. Triterpene glycosides of plants of the Astragalus genus. IV. Structure of cyclounifolioside D from Astragalus unifoliolatus. Chem. Nat. Compd. 38, 574– 576. Kucherbaev, K.Dzh., Uteniyazov, K.K., Kachala, V.V., Saatov, Z., Shashkov, A.S., 2003. Triterpene glycosides from plants of the Astragalus genus. III. Structure of

cyclounifolioside C from Astragalus unifoliolatus. Chem. Nat. Compd. 38, 447– 449. Maassoumi, A.A., 1998. Astragalus in the Old World. Islamic Republic of Iran Ministry of Jahad-e-Sazandegi Research Inst. of Forests and Rangelands CheckList, Iran. Mamedova, R.P., Isaev, M.I., 2004. Triterpenoids from Astragalus plants. Chem. Nat. Compd. 40, 303–357. Tian, Z., Yang, M., Huang, F., Li, K., Si, J., Shi, L., Chen, S., Xiao, P., 2005. Cytotoxicity of three cycloartane triterpenoids from Cimicifuga dahurica. Cancer Lett. 226, 65– 75. Verotta, L., Guerrini, M., El-Sebakhy, N.A., Assad, A.M., Toaima, S.M., Radwan, M.M., Luo, Y.-D., Pezzuto, J.M., 2002. Cycloartane and oleanane saponins from Egyptian Astragalus spp. As modulators of lymphocyte proliferation. Planta Med. 68, 986–994.