Steroidal saponins from the aerial parts of Tribulus pentandrus Forssk

PHYTOCHEMISTRY Phytochemistry 65 (2004) 2935–2945 www.elsevier.com/locate/phytochem

Steroidal saponins from the aerial parts of Tribulus pentandrus Forssk Arafa I. Hamed a, Wieslaw Oleszek b, Anna Stochmal b, Cosimo Pizza

c,* ,

Sonia Piacente

c

a

Department of Botany, Faculty of Science, South Valley University, Aswan 81528, Egypt Department of Biochemistry, Institute of Plant Cultivation and Environmental Science, ul. Czartoryskich 8, 24-100 Pulawy, Poland c Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Salerno, Via Ponte Don Melillo, I-84084 Fisciano, Salerno, Italy

b

Received in revised form 5 July 2004 Available online 3 August 2004 Dedicated to Prof. Dr. Kurt Hostettmann on the occasion of his 60th birthday

Abstract Seven new steroidal glycosides named pentandrosides A(1)–G(7) were isolated from the EtOH extract of the aerial parts of Tribulus pentandrus. Pentandrosides A(1)–E(5) possess cholestane aglycones, pentandroside F(6) a furostan-type aglycone and pentandroside G(7) an unusual acyloxypregnane aglycone probably derived from the degradation of a furostan skeleton. Structure elucidation of 1–7 was accomplished through the extensive use of 1D- and 2D NMR experiments including 1H–1H (DQF-COSY, 1D-TOCSY) and 1H–13C (HSQC, HMBC) spectroscopy along with ESIMS and HRESIMS.
2004 Elsevier Ltd. All rights reserved. Keywords: Tribulus pentandrus; Zygophyllaceae; Cholestane glycosides; Furostan glycoside; Pregnane glycoside; Pentandrosides A–G

1. Introduction As a part of our ongoing investigations of wild medicinal plants growing in Egyptian Desert, we initiated a phytochemical study of Tribulus pentandrus (Zygophyllaceae). The genus Tribulus L. (Tribulaceae) consists of about 25 species which grow in the arid and semi-arid zones (El-Hadidi and Fayed, 1994). Among them is T. pentandrus Forssk. (syns = T. longipetalus), a green or grayish prostrate hairy herb, fruitless with broad wings forming a few spinescent teeth as broad as the wings, known with the Arabic name of ÔGoodoobÕ (Tackholm, 1974). Only some flavonoid glycosides have been reported from T. pentandrus (Saleh

*

Corresponding author. Tel.: +390-89-962813; fax: +390-89962828. E-mail address: [email protected] (C. Pizza). 0031-9422/$ - see front matter
2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2004.07.003

et al., 1982). We selected this plant on the basis of the great interest which another species of the same genus, T. terrestris, has received. T. terrestris L. is a famous pharmaceutical herb of world wild distribution. Earlier investigations performed on T. terrestris and T. cistoides resulted in the isolation of steroidal saponins (Tomova et al., 1974, 1977; Mahato et al., 1981; Achenbach et al., 1994, 1996; Wu et al., 1996; Wang et al., 1996, 1997; Xu et al., 1998; Cai et al., 1999, 2001; Bedir and Khan, 2000; Sun et al., 2002; Kostova et al., 2002), lignanamides (Li et al., 1998), alkaloids (Wo et al., 1999) and flavonoids (Bhutani et al., 1969; Saleh et al., 1982). Some sex function-enhancing preparations, namely Tribestan, Vitanone, and Tribusaponin based on saponins of the aerial parts of T. terrestris are commercially available (Tomova et al., 1981; Qian, 1990). More recently it has been reported that an extract of T. terrestris appeared to possess aphrodisiac properties probably due

2936

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945

to androgen increasing properties (Adaikan et al., 2000; Gauthaman et al., 2002).

Table 1 13 C NMR data of the sugar portions of compounds 1–2 and 4–7 in CD3ODa Position

2. Results and discussion An ethanolic extract of the aerial parts of T. pentandrus growing in Egypt was subjected to repeated chromatographic purifications, affording seven new glycosides which showed positive Liebermann–Burchard reactions and were suggested to be steroidal glycosides. Compound 1 had a molecular formula C38H62O13, as determined from 13C and 13C DEPT NMR, ESIMS in negative-ion mode, and HRESIMS in positive-ion mode. The HRESIMS spectrum of 1 showed the [M + H]+ ion at m/z 727.4293; the ESIMS spectrum of 1 exhibited the [M
H]
ion at m/z 725 and prominent fragments at m/z 563 [(M
H) – 162]
and 431 [(M
H) – (162 + 132)]
attributable to the sequential loss of a hexose and a pentose residue. The 13C- and DEPT 13C NMR spectra showed 38 carbon signals, of which 27 carbon signals were assigned to a cholestane aglycone and 11 carbon signals to a sugar portion. The 1H NMR spectrum (CD3OD) of 1 showed for the aglycone moiety two tertiary methyl proton signals at d 0.98 and 1.25 (each s), two methyl doublets at d 0.93 (J = 6.5) and 0.94 (J = 6.5) and 2 methine proton signals indicative of secondary alcoholic functions at d 4.23 (1H, ddd, J = 9.0, 7.5 and 4.5 Hz, H-16) and 3.77 (1H, br d, J = 8.5 Hz, H-22). Signals attributable to H2-26 resonated at d 3.39 (dd, J = 6.0 and 11.0) and d 3.47 (dd, J = 5.0 and 11.0). A further signal ascribable to an olefinic proton was evident at d 5.73 (br s). The 13C NMR spectrum of 1 also suggested for the aglycone moiety a cholestane skeleton with two secondary alcoholic functions (d 83.1 and 74.4), one primary alcoholic function (d 68.3) and an a,b-unsaturated carbonyl group (d 202.0, 175.1 and 124.1). On the basis of HSQC and HMBC data the aglycone of 1 was identified as (22S,25S)-16b,22,26-trihydroxycholest-4-en-3-one previously identified as the aglycone of glycosides of T. cistoides and characterized on the basis of NMR data and chemical conversions (Achenbach et al., 1996). The 13C NMR spectrum of 1 showed, in addition to the aglycone signals, 11 signals ascribable to a sugar portion made up of one bxylopyranosyl unit and one b-glucopyranosyl unit (Table 1); the anomeric protons of the two sugars resonated in the 1H NMR spectrum at d 4.22 (d, J = 7.5 Hz, H-1 of xyl) and 4.58 (d, J = 7.5 Hz, H-1 of glc), respectively (Table 2). 1D-TOCSY spectra showed the proton signals of each sugar unit which were correlated to the corresponding carbon signals by a HSQC experiment. The downfield shift exhibited by C-3 of the xylopyranosyl unit (d 88.3) allowed us to

1

2

4

5

6

7

Xyl 106.9 74.6 88.3 70.0 66.5

Xyl 106.9 74.0 89.5 69.7 66.0

GlcI 106.7 75.1 88.7 70.2 77.7 62.7

Gal 102.8 73.0 74.9 78.9 75.3 61.4

Gal 102.5 72.4 73.7 79.8 75.6 61.1

Gal 100.4 77.1 76.9 82.0 74.9 60.7

Glc 105.3 75.6 78.0 71.8 78.2 62.7

Glc 105.1 75.0 77.4 71.3 75.5 64.5 172.6 21.1

GlcII 105.6 75.5 78.0 71.5 78.2 62.7

Glc 106.0 75.3 78.0 71.7 77.9 62.7

GlcI 104.2 81.1 87.9 71.2 78.2 62.9

Rha 102.1 72.4 72.3 73.9 69.7 17.9

Xyl 107.2 75.1 77.9 71.8 66.5

GlcII 104.3 75.3 78.0 70.7 78.0 62.0

GlcI 105.5 81.8 88.4 70.4 78.0 63.2

1 2 3 4 5 6

GlcIII 104.3 75.2 78.1 71.5 78.1 62.3

XylI 105.4 75.9 78.2 70.9 67.1

1 2 3 4 5 6

GlcIV 104.7 75.3 77.9 71.4 77.9 62.6

XylII 104.9 75.4 78.4 71.0 67.2

1 2 3 4 5 6

1 2 3 4 5 6 COMe COMe

1 2 3 4 5 6

GlcII 104.6 75.1 77.4 71.7 77.9 62.8

1 2 3 4 5 6 a

Assignments were confirmed by HSQC and HMBC experiments.

deduce that this carbon was a glycosidation site. The HMBC experiment showed long range correlations between the proton signal at d 4.58 (H-1 of glc) and the carbon signal at d 88.3 (C-3 of xyl), and between the proton signal at d 4.22 (H-1 of xyl) and the carbon signal at d 83.1 (C-16). The configurations of the sugar units were assigned after hydrolysis of 1 with 1

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945

N HCl. The hydrolysate was trimethylsilated, and GC retention times of each sugar were compared with those of the authentic samples prepared in the same manner. In this way the sugar units of 1 were determined to be D -glucose and D -xylose in the ratio 1:1. Thus the structure of compound 1 was established as (22S,25S)-16b,22,26-trihydroxycholest-4-en-3-one 16-O-b-D -glucopyranosyl-(1!3)-b-D -xylopyranoside, named pentandroside A. Compound 2 had a molecular formula C40H64O14, as determined from 13C and 13C DEPT NMR, ESIMS in negative-ion mode, and HRESIMS in positive ion mode. Analysis of 1H and 13C NMR data (CD3OD) clearly suggested that compound 2 differed from 1 only for the occurrence of an acetylation at C-600 of the glucose unit as suggested by the downfield shifts of H2-6 (d 4.18 and 4.48 in 2 vs. d 3.66 and 3.91 in 1) (Table 2) and C-6 (d 64.5 in 2 vs. 62.7 in 1) of the glucose (Table 1). Compound 2 subjected to alkaline hydrolysis afforded 1. Thus the structure of 2 was established as (22S,25S)-16b,22,26-trihydroxycholest-4-en-3-one 16-O-[6-O-acetyl-b-D -glucopyranosyl)-(1!3)-b-D -xylopyranoside], named pentandroside B. Compound 3 had a molecular formula C38H62O14, as determined from 13C and 13C DEPT NMR, ESIMS in negative-ion mode, and HRESIMS in positive-ion mode. The HRESIMS spectrum of 3 exhibited the [M + H]+ ion at m/z 743.4292; The ESIMS spectrum of 3 showed the [M
H]
ion at m/z 741 and prominent fragments at m/z 579 [(M
H) – 162]
and 447 [(M
H) – (162 + 132)]
attributable to the sequential loss of a hexose and a pentose residue. Analysis of 1H and 13C NMR data (CD3OD) of 3 in comparison with those of 1 suggested that the two compounds differed only for the occurrence of a further hydroxyl group in 3 which was located at C-11 (d 68.9) on the basis of the carbon resonances of ring C. In particular the chemical shifts of C-9 and C-12 were assigned on the basis of the HMBC experiment in which correlations were observed from Me-19 (d 1.38) to C-9 (d 60.3), and from Me-18 (d 0.99) to C-12 (d 52.2). The downfield shifts exhibited by C-9 and C-12 in 3 if compared to the same carbons in 1 (C-9: d 55.1, C-12: d 40.7), suggested that the hydroxy group should be located at C-11. The configuration of the hydroxy group at C-11 was deduced as a on the basis of the coupling constants exhibited by the signal at d 4.01 (J = 4.5, 10.0, 10.0 Hz, H-11) which showed this proton to be axial. Thus the aglycone of 3 was (22S,25S)11a,16b,22,26-tetrahydroxycholest-4-en-3-one (3a) and 3 (22S,25S)-11a,16b,22,26-tetrahydroxycholest-4-en-3one 16-O-b-D -glucopyranosyl-(1!3)-b-D -xylopyranoside, named pentandroside C. Compound 4 (C39H64O13) showed in the HRESIMS the [M + H]+ ion peak at m/z 741.4473. It gave a quasi-molecular ion peak in its negative ion ESIMS

2937

at m/z 739 and prominent fragments at m/z 577 [(M
H) – 162]
and 415 [(M
H) – (162 + 162)]
attributable to the sequential loss of two hexose units. The 13C- and DEPT 13C NMR spectra showed 39 carbon signals, of which 27 were assigned to a cholestane aglycone and 12 to a sugar portion. The 1H NMR spectrum (CD3OD) of 4 showed for the aglycone moiety two methyl singlets at d 0.99 and 1.24, one methyl doublet at d 1.05 (J = 6.5 Hz), a further doublet for two methyl groups at d 0.93 (J = 6.5), the methine proton signal attributable to H-16 at d 4.15 (1H, ddd, J = 9.0, 7.5 and 4.5 Hz, H-16) and the signal ascribable to the olefinic proton at C-4 at d 5.73 (br s). A signal indicative of a further secondary alcoholic function at d 4.06 (1H, dd, 2.5 and 4.5 Hz) was also evident. The HMBC correlation between the methyl signal at d 0.99 (Me-18) and the carbon at d 73.5 (C-12) along with the resonances of ring C allowed to locate the secondary alcoholic function at C-12. The a-configuration of the hydroxy group at C-12 was deduced on the basis of the coupling constants exhibited by the signal at d 4.06 (J = 2.5 and 4.5 Hz, H-12) which showed this proton to be equatorial. On the basis of these data the aglycone of compound 4 was identified as the new (25S)-12a,16b-dihydroxycholest-4-en-3-one. The 13C NMR data clearly showed that the sugar chain was made up of two b-glucopyranosyl units. 1D-TOCSY experiments obtained by irradiating the anomeric signals at d 4.27 (H-1 of glcI) and d 4.58 (H-1 of glcII) showed the spin system of a b-glucopyranose in both cases. The downfield shift exhibited by C-3 of glcI (d 88.3) suggested that this carbon was a glycosidation site. Thus the two b-glucopyranosyl units were connected via a 1–3 glycosidic linkage (Tables 1, 2). Also in this case, the D configuration of the two glucose units was determined by acid hydrolysis of 4 followed by GC analysis. Thus 4 is (25S)-12a,16b-dihydroxycholest-4-en-3-one 16-Ob-D -glucopyranosyl-(1!3)-b-D -glucopyranoside, named pentandroside D. Being the aglycones of 3 and 4 never reported in literature, they were obtained by acid hydrolysis, characterized by NMR and designated respectively as pentandrogenin A (3a) and pentandrogenin B (4a). Compound 5 had a molecular formula C44H76O18, as determined from 13C and 13C DEPT NMR, ESIMS in negative-ion mode, and HRESIMS in positive-ion mode. The HRESIMS of 5 gave the quasi-molecular ion peak [M + H]+ at m/z 893.5142. The ESIMS spectrum showed the [M
H]
ion at m/z 891 and prominent fragments at m/z 729 [(M
H) – 162]
, 759 [(M
H) – 132]
, 597 [(M
H) – (162 + 132)]
and 435 [(M
H) – (162 + 162 + 132)]
attributable to the sequential loss of two hexoses and a pentose residue. The 13C- and DEPT 13C NMR spectra showed

2938

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945

Table 2 1 H NMR data of the sugar portions of compounds 1–2 and 4–7 in CD3ODa Position

1 2 3 4 5

dH (J in Hz) 1

2

4

5

6

7

Xyl 4.22 3.36 3.46 3.59 3.91 3.23

Xyl 4.18 3.35 3.40 3.56 3.91 3.22

GlcI 4.27 d (7.5) 3.39 dd (7.5, 9.0) 3.52 dd (9.0, 9.0) 3.43 dd (9.0, 9.0) 3.28 m

Gal 4.37 3.51 3.58 4.07 3.54

Gal 4.42 3.70 3.85 4.07 3.58

Gal 4.46 3.69 3.68 3.97 3.52

3.88 dd (2.5, 12.0) 3.71 dd (4.5, 12.0)

3.90 dd (2.0, 12.0) 3.70 dd (4.5, 12.0)

3.90 dd (2.0, 12.0) 3.70 dd (5.0, 12.0)

3.97 dd (2.0, 12.0) 3.63 dd (4.5, 12.0)

GlcII 4.58 d (7.5) 3.29 dd (7.5, 3.39 dd (9.0, 3.29 dd (9.0, 3.34 m 3.89 dd (2.5, 3.65 dd (4.5,

Glc 4.54 3.30 3.38 3.22 3.30 3.91 3.60

d (7.5) dd (7.5, dd (9.0, dd (9.0, m dd (2.5, dd (4.5,

GlcI 4.66 d (7.5) 3.74 dd (7.5, 3.81 dd (9.0, 3.77 dd (9.0, 3.77 m 3.96 dd (2.5, 3.63 dd (4.5,

Rha 5.21 d (1.5) 3.93 dd (1.5, 2.5) 3.67 dd (2.5, 8.9) 3.42 t (8.9) 4.22 m 1.21 d (6.5)

Xyl 4.16 3.16 3.33 3.48 3.86 3.18

d (7.5) dd (7.5, 9.0) dd (9.0, 9.0) m dd (4.5, 10.5) t (10.5)

d (7.5) dd (7.5, 9.0) dd (9.0, 9.0) m dd (4.5, 11.0) t (11.0)

d (7.5) dd (7.5, 9.0) dd (9.0, 9.0) m dd (4.5, 11.0) t (11.0)

6

Glc 4.58 3.30 3.39 3.29 3.34 3.91 3.66

1 2 3 4 5 6 COMe

1 2 3 4 5 6

d (7.5) dd (7.5, dd (9.0, dd (9.0, m dd (2.5, dd (4.5,

9.0) 9.0) 9.0) 12.0) 12.0)

Glc 4.50 3.30 3.41 3.30 3.58 4.48 4.18 2.07

d (7.5) dd (7.5, dd (9.0, dd (9.0, m dd (2.5, dd (4.5, s

9.0) 9.0) 9.0) 12.0) 12.0)

9.0) 9.0) 9.0) 12.0) 12.0)

d (7.8) dd (7.8, 8.5) dd (2.9, 8.5) dd (2.9, 1.2) m

9.0) 9.0) 9.0) 12.0) 12.0)

d (7.8) dd (7.8, 8.5) dd (2.9, 8.5) dd (2.9, 1.2) m

GlcII 4.94 d (7.5) 3.20 dd (7.5, 3.38 dd (9.0, 3.40 dd (9.0, 3.38 m 3.93 dd (2.5, 3.65 dd (4.5,

9.0) 9.0) 9.0) 12.0) 12.0)

9.0) 9.0) 9.0) 12.0) 12.0)

GlcIII 4.72 d (7.5) 3.31 dd (7.5, 9.0) 3.39 dd (9.0, 9.0) 3.30 dd (9.0, 9.0) 3.39 m

1 2 3 4 5 6

d (7.8) dd (7.8, 8.5) dd (2.9, 8.5) dd (2.9, 1.2) m

GlcI 4.49 d (7.5) 3.72 dd (7.5, 3.72 dd (9.0, 3.30 dd (9.0, 3.37 m 3.93 dd (2.5, 3.59 dd (4.5,

9.0) 9.0) 9.0) 12.0) 12.0)

XylI 4.85 d (7.5) 3.12 dd (7.5, 9.0) 3.31 dd (9.0, 9.0) 3.57 m 4.20 dd (4.5, 10.5) 3.17 t (10.5)

3.93 dd (2.5, 12.0) 3.67 dd (4.5, 12.0) GlcIV 4.27 d (7.5) 3.22 dd (7.5, 9.0) 3.30 dd (9.0, 9.0) 3.37 dd (9.0, 9.0) 3.29 m

1 2 3 4 5 6

XylII 4.63 d (7.5) 3.27 dd (7.5, 9.0) 3.32 dd (9.0, 9.0) 3.55 m 3.94 dd (4.5, 11.0) 3.30 t (11.0)

3.90 dd (2.5, 12.0) 3.70 dd (4.5, 12.0) GlcII 4.26 d (7.5) 3.21 dd (7.5, 3.38 dd (9.0, 3.31 dd (9.0, 3.30 m 3.63 dd (2.5, 3.97 dd (4.5,

1 2 3 4 5 6 a

Assignments were confirmed by DQF-COSY, 1D-TOCSY, HSQC and HMBC experiments.

9.0) 9.0) 9.0) 12.0) 12.0)

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945

44 carbon signals, of which 27 carbon signals were assigned to a cholestane aglycone and 17 carbon signals to a sugar portion. Analysis of 1H and 13C NMR data (CD3OD) of the aglycon portion of 5 in comparison with those of 1 suggested that the a,b-unsaturated carbonyl function of 1 gave place to a b-hydroxy group in 5. Thus the aglycone of 5 was identified as (22S,25S)-5a-cholestan-3b,16b,22,26-tetraol, named agavegenin, recently isolated for the first time by Agave americana (Jin et al., 2004). The C-3 and C-16 carbon signals exhibited both glycosidation shifts (d 79.3 and 83.1, respectively). The 13C NMR spectrum of 5 showed 17 signals ascribable to a b-D -xylopyranosyl unit, a b-D -glucopyranosyl unit and a b-D -galactopyranosyl unit (Table 1); the anomeric protons of the three sugars resonated in the 1H NMR spectrum at d 4.16 (d, J = 7.5 Hz, H-1 of xyl), 4.37 (d, J = 7.8 Hz, H-1 of gal) and 4.54 (d, J = 7.5 Hz, H-1 of glc) (Table 2). The downfield shift exhibited by C-4 of the galactopyranosyl unit (d 78.9) allowed to deduce that this carbon was a glycosidation site. On the basis of the HMBC experiment in which long range correlations were observed from the proton signal at d 4.54 (H-1 of glu) to the carbon signal at d 78.9 (C-4 of gal), the proton signal at d 4.37 (H-1 of gal) to the carbon signal at d 79.3 (C-3), and from the proton signal at d 4.16 (H-1 of xyl) to the carbon signal at d 83.1 (C-16), it was possible to deduce the occurrence of a b-xylopyranosyl unit at C16 and a disaccharide b-glucopyranosyl-(1!4)-b-galactopyranoside at C-3. The configurations of the sugar units were assigned after hydrolysis of 5 with 1 N HCl. The hydrolysate was trimethylsilated, and GC retention times of each sugar were compared with those of the authentic samples prepared in the same manner. In this way the sugar units of 5 were determined to be D -xylose, D -galactose and D -glucose. Thus the structure of compound 5 was established as (22S,25S)-16-O-b-D xylopyranosyl-5a-cholestan-3b,16b,22,26-tetraol 3-O-bD -glucopyranosyl-(1!4)-b-D -galactopyranoside, named pentandroside E. The HRESIMS of 6 (C57H96O30) gave the quasi-molecular ion peak [M + H]+ at m/z 1261.6123. The ESIMS showed the [M
H]
ion at m/z 1259 and prominent fragments at m/z 1097 [(M
H) – 162]
, 935 [(M
H) – (162 · 2)]
, 773 [(M
H) – (162 · 3)]
, 611 [(M
H) – (162 · 4)]
, 449 [(M
H) – (162 · 5)]
, attributable to the sequential loss of five hexose residues. The 13C- and DEPT 13C NMR spectra of the aglycone portion suggested the occurrence of a furostan skeleton with the usual hemiketalic function at C-22 (d 111.0) and two secondary alcoholic functions (d 84.6 and 70.3). The 1H NMR spectrum (CD3OD) of 6 showed for the aglycone moiety two methyl singlets at d 0.84 (Me-18) and 0.93 (Me-19), two methyl doublets at d 0.98 (J = 6.5, Me-27) and 1.03 (J = 6.5, Me-21) and two methine proton signals

2939

indicative of secondary alcoholic functions at d 3.89 (1H, m, H-2) and 3.52 (1H, m, H-3). Signals attributable to H2-26 resonated at d 3.42 (dd, J = 7.0 and 11.5) and d 3.77 (dd, J = 6.0 and 11.5). On the basis of HSQC and HMBC data the aglycone of 6 was identified as (25S)-5a-furostan-2a,3b,22a,26-tetraol. The a configuration of the hydroxy group at C-22 was deduced from the ROESY experiment, in which a diagnostic correlation was observed from the H-20 proton (d 2.21 m) with the H-23 protons (d 1.87 m, 1.67 m). The 25S configuration was deduced by comparison with 25S furostan glycosides isolated from T. terrestris (Cai et al., 2001; Bedir and Khan, 2000) and T. cistoides (Achenbach et al., 1994). The structure elucidation of the sugar portion was achieved by 1D- TOCSY, DQF-COSY, HSQC and HMBC experiments (Tables 1, 2). The isolated anomeric proton signals resonating at the uncrowded region of the spectrum (between d 4.27 and 4.94) were the starting point for the 1DTOCSY experiments. The 1D-TOCSY subspectra obtained by irradiating the anomeric proton signals at d 4.27, 4.66, 4.72 and 4.94 showed the spin system of b-D -glucose units while the 1D-TOCSY spectrum obtained by irradiating the signal at d 4.42 showed three signals (d 4.07, 3.85 and 3.70). Identification of each proton signal in 1D-TOCSY spectra was deduced by a DQF-COSY experiment which allowed the sequential assignments of all proton resonances within each sugar residue, starting from the well isolated anomeric proton signals (Table 2). The b-configurations of the five sugar units were shown by the large coupling constants of the anomeric proton signals (Agrawal, 1992). A HSQC experiment which correlated each hydrogen signal to the corresponding carbon signal allowed the assignment of all the carbon resonances and therefore the identification of the sugars as three terminal b-glucopyranosyl units (H-1: d 4.27, 4.72 and 4.94), a 2,3-disubstituted b-glucopyranose (H-1= d 4.66) and a b-galactopyranose glycosidated at C-4 (H-1: d 4.42). The sugar sequence was deduced from the HMBC spectrum, in which long-range correlations were observed from H-1 of gal (d 4.42) to C-3 of the aglycone (d 84.6), H-1 of glcI (d 4.66) to C-4 of gal (d 79.8), H-1 of glcII (d 4.94) to C-2 of glcI (d 81.1), H-1 of glcIII (d 4.72) to C-3 of glcI (d 87.9), and from H-1 of glcIV (d 4.27) to C-26 of the aglycone (d 76.1). Also in this case, the configuration of the sugar units was determined by acid hydrolysis of 6 followed by GC analysis. In this way the sugar units of 6 were determined to be D -galactose and D glucose in the ratio 1:4. On the basis of these data 6 was identified as (25S)-26-O-b-D -glucopyranosyl-5afurostan-2a, 3b,22a,26-tetraol 3-O-{b-D -glucopyranosyl-(1!2)-O-[b- D -glucopyranosyl-(1!3)]-O-b- D -glucopyranosyl-(1!4)- b- D -galactopyranoside}, named pentandroside F.

2940

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945

Compound 7 had a molecular formula C61H100O32, as determined from 13C and 13C DEPT NMR, ESIMS in negative-ion mode, and HRESIMS in positive-ion mode. The HRESIMS spectrum showed the [M + H]+ ion peak at m/z 1345.6312; The ESIMS showed the [M
H]
ion at m/z 1343 and prominent fragments at m/z 1181 [(M
H) – 162] attributable to the loss of a hexose residue, m/z 1067 [(M
H) – (162 + 114)]
attributable to the sequential loss of a side chain, m/z 935 [(M
H) – (162 + 132 + 114)]
, and 803 [(M
H) – (162 + 132 · 2 + 114)]
due to the cleavage of one and two pentose units respectively. Also evident were peaks at m/z 641, 479 and 333 arising from the sequential losses of two hexose and one deoxyhexose units from the ion at m/z 803. The 13Cand DEPT 13C NMR spectra showed 61 carbon signals, of which 21 carbon signals were assigned to a pregnane aglycone, 6 to an acyl moiety and 34 carbon signals to a sugar portion. The 1H NMR spectrum of 7 (CD3OD) showed for the aglycone moiety three tertiary methyl proton signals at d 0.86, 1.03 and 2.10 (each s), and two methine proton signals indicative of secondary alcoholic functions at d 3.70 (1H, br m, H-3) and 5.55 (1H, ddd, J = 9.0, 8.0 and 4.5 Hz, H-16). Also evident were the signals of a methyl doublet at d 0.96 (d, J = 6.5 Hz) and a primary alcoholic function at d 3.40 (dd, J = 6.0 and 11.5) and d 3.78 (dd, J = 5.0 and 11.5). The 13C NMR spectrum of 7 also suggested for the aglycon moiety a pregnane skeleton with two secondary alcoholic functions at C-3 (d 78.7) and C-16 (d 76.1). The downfield shift exhibited by H-16 proton signal (d 5.55) suggested that this proton should be involved in an ester linkage with an acyl group which resulted to be a (4S)-5-hydroxy-4methylpentanoyl moiety from NMR data (Zhu et al., 2001). In the case of acyloxypregnanes the resonances of the protons and carbons (C-3 0 , C-4 0 , C-5 0 , and C-6 0 ) around the C-4 0 centre in the 5-hydroxy-4methylpentanoyl moiety and in particular the 3JHH (5.0 and 6.0 Hz) between H-4 and H2-5 provided the evidence for the C-4 S configuration as described in previous reports (Dong et al., 2001). Thus the aglycone of 7 was identified as 3b-hydroxy-16b-[(4 0 S)-5 0 hydroxy-4 0 -methylpentanoyloxy]-5a-pregnan-20-one. The 13C NMR spectrum of 7 showed, in addition to the aglycone signals, 34 signals ascribable to a sugar portion made up of three hexose units, one 6-deoxyhexose and two pentose units (Table 1). In the 1H NMR spectra (Table 2) six anomeric proton signals resonated at d 5.21 (J = 1.2), 4.85 (J = 7.5), 4.63 (J = 7.5), 4.49 (J = 7.5), 4.46 (J = 7.8), and 4.26 (J = 7.5). A methyl doublet ascribable to Me-6 of the deoxyhexose sugar at d 1.21 (J = 6.5) was also evident. The structure elucidation of the sugar portion was achieved by 1D- TOCSY, DQF-COSY, HSQC and HMBC experiments as reported for 6 (Table 1).

The 1D-TOCSY subspectra obtained by irradiating the anomeric proton signals at d 4.85 and 4.63 allowed us to establish these protons as belonging to two b-D -xylose units while the subspectra obtained by irradiating the signals at d 4.49 and 4.26 showed the typical spin system of b-glucose units. In the case of the 6-deoxyhexose (H-1 = 5.21), an easier identification of an a-L -rhamnose unit was obtained by recording the 1D-TOCSY experiments also irradiating the methyl doublet at d 1.21. The 1D-TOCSY spectrum obtained by irradiating the signal at d 4.46 showed three signals (d 3.68, 3.69 and 3.97) corresponding to H-2, H-3 and H-4 of a b-galactopyranosyl unit. The HSQC experiment allowed the identification of the sugars as a terminal b-glucopyranose (H-1: d 4.26), a terminal a-rhamnopyranose unit (H-1: d 5.21) and b-xylopyranose units (H-1 = d 4.63; H1 = d 4.85), a 2,3-disubstituted b-glucopyranose (H-1: d 4.49) and a 2,4-disubstituted b-galactopyranose (H1: d 4.46). The sugar sequence was deduced from the HMBC spectrum, in which long-range correlations were observed from H-1 of gal (d 4.46) to C-3 of the aglycone (d 78.7), H-1 of rha (d 5.21) to C-2 of gal (d 77.1), H-1 of glcI (d 4.49) to C-4 of gal (d 82.0), H-1 of xylI (d 4.85) to C-2 of glcI (d 81.8), H-1 of xylII (d 4.63) to C-3 of glcI (d 88.4), and from H-1 of glcII (d 4.26). to C-5 0 of the aglycone (d 75.4). The sugar units determined by acid hydrolysis followed by GC analysis resulted to be D -galactose, D glucose, L -rhamnose and D -xylose in the ratio 1:2:1:2. On the basis of these data compound 7 was identified as 16b-[(4 0 S)-5 0 -(b-D -glucopyranosyloxy)-4 0 methylpentanoyloxy]-3b-hydroxy-5a-pregnan-20-one 3-O-{a- L -rhamnopyranosyl-(1!2)-O]-b- D -xylopyranosyl-(1!2)-O-[b-D -xylopyranosyl-(1!3)]-O-b-D -glucopyranosyl-(1!4)]-b-D -galactopyranoside}, named pentandroside G. Pentandroside A (1), the main constituent of the aerial parts of T. pentandrus, and pentandroside B (2) are closely related to steroidal glycosides previously isolated from T. cistoides (Achenbach et al., 1996); Compounds 1–2 present as aglycone (22S,25S)-16b,22,26-trihydroxy-cholest-4-en-3-one which occurred only in T. cistoides (Achenbach et al., 1996). Compounds 3–4 possess new aglycones while compound 7 is, to our knowledge, the first example of acyloxypregnane glycoside isolated from a Tribulus species. Acyloxypregnane glycosides along with furostan saponins have been previously reported in the genera Dioscorea (Kiyosawa et al., 1977) and Solanum (Zhu et al., 2001). Steroidal saponins are believed to be responsible for the sex function-enhancing properties of T. terrestris preparations (Tomova et al., 1981; Qian, 1990; Gauthaman et al., 2002). An investigation aimed to evaluate the aphrodisiac properties of the glycosidic fraction of the aerial parts of T. pentandrus is in progress.

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945 27

HO 21

25

18

22

11 19

H

1 10

H

17

OH

24

HO

OH

14

H

OH H

H

O

OR

O

H OH

O

HO

26

23

20

H

H

O

4

OH HO O

HO HO

R=

3

OH

OR

H

O

HO O O RO

2941

OH

H

HO O

HO HO

R=

4

OH

3a R =

OH

OH

O O

O O OH

OH

H

4a R =

HO HO

1 2

R=H R = Ac HO

OH

OH O HO HO

H

19

OH

O

H

OH

O

HO

O

H

O

O

OH

OH

HOHO

5

GlcIV

OH

O HO HO

O OH OH

21

OH HO HO

O

OH GlcIII

OH

HO

H

HO

OH

14

O

16

H

O O

HO

OH

4

H

H OH

HO HO

HO HO

O

GlcI

21

HO O HO HO

O O

O O

OH

OH

Xil II

H

O O

HO

O

4

1'

17

H

OH

5'

O 14

4'

2' 20

11 19

O

6'

OH

O 18

OH

GlcII O

6

GlcII

24

15

O O

17

19

O

25

26

22

12

O

HO O

20

18

GlcI

OH

27

23

3'

O

16

H

H

O HO HO Xil I

HO HO

OH

7

3. Experimental 3.1. General NMR spectra in CD3OD were obtained using a Bruker DRX-600 spectrometer, operating at 599.19 MHz for 1H and 150.86 MHz for 13C. 2D experiments:

1

H–1H DQF-COSY (double filtered direct chemical shift correlation spectroscopy), inverse detected 1 H–13C HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiple bond connectivity), were obtained using UXNMR software. Selective excitation spectra, 1D-TOCSY were acquired using waveform generator-based GAUSS shaped pulses,

2942

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945

mixing time ranging from 100 to 120 ms and a MLEV17 spin-lock field of 10 kHz preceded by a 2.5 ms trim pulse. ESIMS were performed on a Finnigan LC-Q Deca Ion Trap mass spectrometer scanned from 150 to 1200 Da. The mass spectral data were acquired and processed using Xcalibur software. Samples were dissolved in MeOH and infused in the ESI source by using a syringe pump at a flow rate of 3 ll/min. The capillary voltage was 5 V, the spray voltage 5 kV and the tube lens offset 50 V. The capillary temperature was 220 C. Exact masses were measured by a Q-Star Pulsar (Applied Biosystems, Foster City, CA) triplequadrupole orthogonal time-of-flight (TOF) instrument. Electrospray ionization was used in TOF mode at 10.000 resolving power. Samples were dissolved in TFA 0.1% acetonitrile/water (1:1), mixed with the internal calibrant, and introduced directly into the ion source by direct infusion. Optical rotations were measured on a Perkin–Elmer 192 polarimeter equipped with a sodium lamp (589 nm). GC analysis was performed on a Termo Finnigan Trace GC apparatus using a 1Chirasil-Val column (0.32 mm · 25 m). Silica gel (0.063–0.200 mm, E-Merck), was used for column chromatography, silica gel GF254 was used for TLC and C18 was used for ODS column. 3.2. Plant material The aerial parts of T. pentandrus were collected in December 2001 from Wadi Allaqi, south of Aswan, Egypt and identified according to Boulos (2000). The voucher specimens are deposited in Botany Department herbarium, Aswan Faculty of Science, Egypt. 3.3. Extraction and isolation of the compounds The aerial parts of T. pentandrus (200 g) were powdered and exhaustively extracted with 80% EtOH by maceration at room temperature. The crude extract was concentrated under reduced pressure to a syrupy consistence (15 g). The crude extract was dissolved with MeOH–H2O (2:1) and extracted by hexane to give 2.5 g. The mother liquor was loaded on a water preconditioned short C18 column (6x10 cm, 60 lm C18, Backer) and eluted with H2O (T-1), 40% MeOH (T-2) and 80% MeOH (T-3) respectively. T-3 fraction (saponins) was loaded on a C18 column (3 · 30 cm, 60 lm C18, Backer) with gradient elution with 60–65% MeOH to give two fractions (T-3A and T-3B). Fraction T-3A was subjected to a C18 (3 · 30 cm, LiChroprep RP-18, 25-40 lm, Merck) column chromatography by eluting with 20–25% CH3CN to give compounds 3 (10 mg ), 5 (15 mg), 6 (20 mg ) and 7 (10 mg). Fraction T-3B was loaded on a silica gel column (2 · 30 cm, LiChroprep Si 60, 25-40 lm, Merck) eluted with CHCl3:MeOH

(9:1) to give compounds 1 (600 mg), 2 (10 mg) and with CHCl3:MeOH (85:15) to give compound 4 (10 mg). 3.3.1. Pentandroside A (1) 25 Amorphous powder ½aD þ18 (MeOH; c 0.1); ESIMS in negative ion mode (m/z ): 725 [M
H]
, 563 [(M
H) – 162]
, 431 [(M
H) – (162 + 132)]
; HRESIMS: m/z [M + H]+ calcd. for C38H62O13 727.4269, found 727.4293; 1H NMR of the aglycone moiety (CD3OD): d 0.93 (3H, d, J = 6.5 Hz, Me-27), 0.94 (3H, d, J = 6.5 Hz, Me-21), 0.98 (3H, s, Me-18), 1.25 (3H, s, Me-19),3.39 (1H, dd, J = 6.0 and 11.0 Hz, H-26a),3.47 (1H, dd, J = 5.0 and 11.0 Hz, H-26b), 3.77 (1H, br d, J = 8.5 Hz, H-22), 4.23 (1H, ddd, J = 9.0, 7.5 and 4.5 Hz, H-16),5.73 (br s, H-4); 13C NMR of the aglycone portion (CD3OD): d 11.9 (C-21), 13.5 (C18), 17.3 (C-27), 17.7 (C-19), 21.9 (C-11), 31.0 (C-24), 33.2 (C-23), 33.3 (C-7), 33.9 (C-2), 34.7 (C-6), 36.6 (C1), 36.0 (C-20), 36.7 (C-8), 37.1 (C-15), 37.2 (C-25), 39.8 (C-10), 40.7 (C-12), 43.1 (C-13), 55.1 (C-9), 55.2 (C-14), 58.6 (C-17), 68.3 (C-26), 74.4 (C-22), 83.1 (C16), 124.1 (C-4), 175.1 (C-5), 202.0 (C-3); 1H and 13C NMR of the sugar portion: Tables 1, 2. 3.3.2. Pentandroside B (2) 25 Amorphous powder ½aD þ25 (MeOH; c 0.1); ESIMS in negative ion mode (m/z): 767 [M
H]
, 725 [(M
H) – 42]
, 563 [(M
H) – (42 + 162)], 431 [(M
H) – (42 + 162 + 132)]
; HRESIMS: m/z [M + H]+ calcd. for C40H65O14 769.4374, found 769.4398; 1H NMR and 13C NMR of the aglycone portion (CD3OD): superimposable on those reported for compound 1. 1H and 13C NMR of the sugar portion: Tables 1, 2. 3.3.3. Pentandroside C (3) 25 Amorphous powder ½aD þ27:3 (MeOH; c 0.1); ESIMS in negative ion mode (m/z): 741 [M
H]
, 579 [(M
H) – 162]
, 447 [(M
H) – (162 + 132)]
; HRESIMS: m/z [M + H]+ calcd. for C38H63O14 743.4218, found 743.4292; 1H NMR of the aglycone moiety (CD3OD): d 0.94 (3H, d, J = 6.5 Hz, Me-27), 0.96 (3H, d, J = 6.5 Hz, Me-21), 0.99 (3H, s, Me-18), 1.38 (3H, s, Me-19), 3.39 (1H, dd, J = 7.0 and 11.5 Hz, H-26a), 3.47 (1H, dd, J = 6.0 and 11.5 Hz, H-26b), 3.77 (1H, br d, J = 8.0 Hz, H-22), 4.01 (1H, ddd, J = 4.5, 10.0, 10.0 Hz, H-11), 4.22 (1H, ddd, J = 9.0, 7.0 and 4.5 Hz, H-16), 5.73 (br s, H-4); 13C NMR of the aglycone portion (CD3OD): d 12.1 (C-21), 14.0 (C-18), 16.9 (C-27), 18.3 (C-19), 31.2 (C-24), 32.7 (C-7), 33.2 (C-23), 34.5 (C-6), 34.6 (C-2), 35.9 (C-20), 37.0 (C-8), 37.0 (C-15), 37.2 (C25), 38.7 (C-1), 39.8 (C-10), 43.8 (C-13), 52.2 (C-12), 54.9 (C-14), 58.8 (C-17), 60.3 (C-9), 68.3 (C-26), 68.9 (C-11), 74.1 (C-22), 82.7 (C-16), 125.0 (C-4), 175.9 (C5), 202.2 (C-3); 1H and 13C NMR of the sugar portion: superimposable on those reported for compound 1.

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945

3.3.4. Pentandroside D (4) 25 Amorphous powder ½aD þ12 (MeOH; c 0.1); ESIMS in negative ion mode (m/z): 739 [M
H]
, 577 [(M
H) – 162], 415 [(M
H) – (162 + 162)]
; HRESIMS: m/z [M + H]+ calcd. for C39H65O13 741.4425, found 741.4473; 1H NMR of the aglycone moiety (CD3OD): d 0.93 (6H, d, J = 6.5 Hz, Me-26, Me-27), 0.99 (3H, s, Me-18), 1.05 (3H, d, J = 6.5 Hz, Me-21), 1.24 (3H, s, Me-19), 4.06 (1H, dd, J = 2.5 and 4.5 Hz, H-12), 4.15 (1H, ddd, J = 9.0, 7.0 and 4.5 Hz, H16),5.73 (br s, H-4); 13C NMR of the aglycone portion (CD3OD): d 14.5 (C-18), 17.0 (C-21), 17.5 (C-19), 23.0 (C-26, C-27), 26.5 (C-23), 29.1 (C-25), 29.5 (C-11), 30.7 (C-20), 31.0 (C-8), 33.3 (C-7), 34.5 (C-6), 34.6 (C2), 36.5 (C-1), 36.9 (C-15), 37.3 (C-22), 39.4 (C-10), 40.9 (C-24), 46.5 (C-14), 47.3 (C-13), 48.9 (C-9), 54.3 (C-17), 73.5 (C-12), 83.2 (C-16), 125.0 (C-4), 175.4 (C5), 202.2 (C-3); 1H and 13C NMR of the sugar portion: Tables 1, 2. 3.3.5. Pentandroside E (5) 25 Amorphous powder ½aD þ41 (MeOH; c 0.1); ESIMS in negative ion mode (m/z): 891 [M
H]
, 759 [(M
H) – 132]
, 729 [(M
H) – 162]
, 597 [(M
H) – (162 + 132)]
, 567 [(M
H) – 162 · 2]
, 435 [(M
H) – (162 + 132)]
; HRESIMS: m/z [M + H]+ calcd. for C44H77O18 893.5110, found 893.5142; 1H NMR of the aglycone moiety (CD3OD): d 0.87 (3H, Me-19), 0.94 (3H, d, J = 6.5 Hz, Me-21), ), 0.97 (3H, s, Me-18), 0.98 (3H, d, J = 6.5 Hz, Me-27), 3.71 (1H, m, H-3), 3.39 (1H, dd, J = 6.0 and 11.5 Hz, H-26a), 3.48 (1H, dd, J = 5.0 and 11.5 Hz, H-26b), 3.77 (1H, br d, J = 8.0 Hz, H-22), 4.22 (1H, ddd, J = 9.0, 7.0 and 4.5 Hz, H-16); 13C NMR of the aglycone portion (CD3OD): d 12.1 (C-21), 12.8 (C-19), 13.5 (C-18), 17.3 (C-27), 21.7 (C-11), 29.8 (C-6), 30.5 (C-2), 31.0 (C-24), 33.0 (C-23), 33.0 (C-7), 35.1 (C-4), 36.2 (C-8), 36.6 (C-20), 36.9 (C-10), 37.1 (C-15), 37.3 (C-25), 38.3 (C-1), 40.2 (C-12), 43.1 (C-13), 45.7 (C-5), 55.3 (C-14), 55.6 (C-9), 58.6 (C-17), 68.3 (C-26), 74.3 (C-22), 79.3 (C-3), 83.1 (C-16); 1H and 13C NMR of the sugar portion: Tables 1, 2. 3.3.6. Pentandroside F (6) 25 Amorphous powder ½aD
53:0 (MeOH; c 0.1); ESIMS in negative ion mode (m/z): the [M
H]
ion at m/z 1259 [M
H]
, 1097 [(M
H) – 162]
, 935 [(M
H) – (162 · 2)]
, 773 [(M
H) – (162 · 3)]
, 611 [(M
H) – (162 · 4)]
, 449 [(M
H) – (162 · 5)]
; HRESIMS: m/z [M + H]+ calcd. for C57H97O30 1261.6065, found 1261.6123; 1H NMR of the aglycone moiety (CD3OD): d 0.84 (3H, Me-18), 0.93 (3H, s, Me-19), 0.98 (3H, d, J = 6.5 Hz, Me-27), 1.03 (3H, d, J = 6.5 Hz, Me-21), 3.42 (1H, dd, J = 7.0 and 11.5 Hz, H-26a), 3.52 (1H, m, H-2), 3.77 (1H, dd, J = 6.0 and 11.5 Hz, H-26b), 3.89 (1H, m, H-2), 4.39

2943

(1H, ddd, J = 9.0, 7.0 and 4.5 Hz, H-16); 13C NMR of the aglycone portion (CD3OD): d 13.4 (C-19), 15.8 (C21), 16.7 (C-18), 17.1 (C-27), 22.1 (C-11), 28.9 (C-6, C24), 31.1 (C-23), 32.5 (C-15), 33.2 (C-7), 34.0 (C-4), 34.8 (C-25), 35.7 (C-8), 37.8 (C-10), 41.0 (C-12), 41.1 (C-20), 41.4 (C-13), 45.6 (C-1, C-5), 55.6 (C-9), 57.3 (C-14), 65.2 (C-17), 70.3 (C-2), 76.1 (C-26), 82.4 (C16), 84.6 (C-3), 111.0 (C-22); 1H and 13C NMR of the sugar portion: Tables 1, 2. 3.3.7. Pentandroside G (7) 25 Amorphous powder ½aD
13 (MeOH; c 0.1); ESIMS in negative ion mode (m/z): 1343 [M
H]
, 1181 [(M
H) – 162]
, 1067 [(M
H) – (162 + 114)]
, 935 [(M
H) – (162 + 132 + 114)]
, 803 [(M
H) – (162 + 132 · 2 + 114)]
, 641 [(M
H) – (162 · 2 + 132 · 2 + 114)]
, 479 [(M
H) – (162 · 3 + 132 · 2 + 114)]
, 333 [(M
H) – (162 · 3 + 132 · 2 + 146 + 114)]
; HRESIMS: m/z [M + H]+ calcd. for C61H101O32 1345.6276, found 1345.6312. 1H NMR of the aglycone moiety (CD3OD): d 0.86 (3H, Me-19), 0.96 (3H, d, J = 6.5 Hz, Me-6 0 ), 1.03 (3H, s, Me-18), 2.10 (3H, s, Me-21), 3.70 (1H, m, H-3), 3.40 (1H, dd, J = 6.0 and 11.5 Hz, H-26a), 3.78 (1H, dd, J = 5.0 and 11.5 Hz, H26b), 5.55 (1H, ddd, J = 9.0, 8.0 and 4.5 Hz, H-16); 13C NMR of the aglycone portion (CD3OD): d 12.8 (C-19), 14.3 (C-18), 16.9 (C-6 0 ), 21.7 (C-11), 29.6 (C-3 0 ), 29.7 (C-6), 30.5 (C-2), 30.8 (C-21), 33.0 (C-7), 33.1 (C-2 0 ), 34.1 (C-4 0 ), 35.1 (C-4), 35.8 (C-8), 36.1 (C-15), 36.9 (C10), 38.3 (C-1), 39.2 (C-12), 43.4 (C-13), 45.9 (C-5), 55.0 (C-14), 55.8 (C-9), 67.7 (C-17), 75.4 (C-5 0 ), 76.1 (C-16), 78.7 (C-3), 175.1 (C-1 0 ), 208.7 (C-20); 1H and 13C NMR of the sugar portion: Tables 1, 2. 3.4. Acid hydrolysis of compounds 3 and 4 Compounds 3 and 4 (4 mg) were refluxed with a mixture of 1 ml of 1 M HCl and 1 ml of dioxane at 80 for 4 h. The reaction mixture was neutralizated with 1 M NaOH and then evaporated to dryness. The dried reaction mixture was extracted with CHCl3. The CHCl3 extract was concentrated and chromatographed on silica gel with CHCl3–MeOH 9:1 to give the pure aglycone 3a from 3 and with CHCl3 to give the pure aglycone 4a from 4. 3.4.1. Pentandrogenin A (3a)  Amorphous powder ½a25 (CHCl3; c 0.1); D þ52:4 + HRESIMS: m/z [M + H] calcd. for C27H45O5 449.3267, found 449.3259; 1H NMR (CD3OD): d 0.92 (3H, d, J = 6.5 Hz, Me-27), 0.97 (3H, d, J = 6.5 Hz, Me-21), 0.99 (3H, s, Me-18), 1.39 (3H, s, Me-19), 3.36 (1H, dd, J = 7.0 and 11.5 Hz, H-26a), 3.44 (1H, dd, J = 6.0 and 11.5 Hz, H-26b), 3.90 (1H, br d, J = 8.0 Hz, H-22), 4.02 (1H, ddd, J = 4.5, 10.0, 10.0 Hz, H11), 4.10 (1H, ddd, J = 9.0, 7.0 and 4.5 Hz, H-16), 5.73

2944

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945

(br s, H-4); 13C NMR (CD3OD): d 13.1 (C-21), 14.3 (C18), 16.6 (C-27), 18.2 (C-19), 31.2 (C-24), 32.9 (C-7), 33.2 (C-23), 34.5 (C-6), 34.3 (C-2), 35.7 (C-20), 37.0 (C-25), 37.2 (C-8), 37.9 (C-15), 38.8 (C-1), 39.8 (C-10), 43.6 (C-13), 52.5 (C-12), 55.2 (C-14), 59.5 (C-17), 60.0 (C-9), 68.3 (C-26), 68.9 (C-11), 73.2 (C-16), 75.8 (C22), 125.0 (C-4), 175.9 (C-5), 202.2 (C-3). 3.4.2. Pentandrogenin B (4a) 25 Amorphous powder ½aD þ37:2 (CHCl3; c 0.1); HRESIMS: m/z [M + H]+ calcd. for C27H45O3 417.3369, found 417.3361; 1H NMR (CD3OD): d 0.92 (6H, d, J = 6.5 Hz, Me-26, Me-27), 0.99 (3H, s, Me18), 1.06 (3H, d, J = 6.5 Hz, Me-21), 1.25 (3H, s, Me19), 4.06 (1H, dd, J = 2.5 and 4.5 Hz, H-12), 4.01 (1H, ddd, J = 9.0, 7.0 and 4.5 Hz, H-16), 5.73 (br s, H-4); 13 C NMR of the aglycone portion (CD3OD): d 14.3 (C-18), 17.0 (C-21), 17.7 (C-19), 23.0 (C-26, C-27), 26.2 (C-23), 29.1 (C-25), 29.4 (C-11), 30.7 (C-20), 31.2 (C-8), 33.3 (C-7), 34.5 (C-6), 34.6 (C-2), 36.4 (C-1), 37.7 (C-15), 37.3 (C-22), 39.4 (C-10), 40.9 (C-24), 46.5 (C-14), 47.3 (C-13), 48.9 (C-9), 55.4 (C-17), 73.0 (C16), 73.5 (C-12), 125.0 (C-4), 175.4 (C-5), 202.2 (C-3). 3.5. Alkaline hydrolysis of 2 A solution of 2 (4 mg) in 3% KOH–MeOH (1 ml) was refluxed for 15 min. The solution was neutralized with 1 M HCl–MeOH, and evaporated to dryness in vacuo. The residue was dissolved in MeOH and was subjected to Sephadex LH-20 using MeOH as eluent to give 1. 3.6. GC analysis to determine absolute configuration of sugars A solution (1.5 mg each) of compounds 1 and 4–7 in 1 N HCl (0.5 ml) was stirred at 80 C for 4 h. After cooling, the solution was concentrated by blowing with N2. The residue was dissolved in 1-(trimethylsilyl)-imidazole and pyridine (0.2 ml), and the solution was stirred at 60 C for 5 min. After drying the solution with a stream of N2, the residue was separated by water 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. Peaks of the hydrolysate of 1 were detected at 10.96 and 12.00 min (D -xylose), and 14.72 min (D -glucose). The peak of the hydrolysate of 4 was detected at 14.74 min (D -glucose). Peaks of the hydrolysate of 5 were detected at 10.96 and 11.98 min (D -xylose), 13.98 and 14.95 (D -galactose), and 14.74 min (D -glucose). Peaks of the hydrolysate of 6 were detected at 10.95 and 11.98 min (D -xylose), 13.96 and 14.96 (D -galactose), and 14.73 min (D -glucose). Peaks

of the hydrolysate of 7 were detected at 9.66 and 10.70 (L -rhamnose), 10.97 and 11.98 min (D -xylose), 13.94 and 14.95 (D -galactose), and 14.73 min (D -glucose). Retention times for authentic samples after being treated simultaneously with 1-(trimethylsilyl)-imidazole in pyridine were detected at 9.68 and 10.72 (L -rhamnose), 10.98 and 12.00 min (D -xylose), 13.98 and 14.95 min (D -galactose), 14.74 min (D -glucose).

Acknowledgement The first author (AIH) acknowledges the fellowship from the Institute of Soil Science and Plant Cultivation, Pulawy, Poland.

References Achenbach, H., Hubner, H., Brand, W., Reiter, M., 1994. Cardioactive steroid saponins and other constituents from the aerial parts of Tribulus cistoides. Phytochemistry 35, 1527–1543. Achenbach, H., Hubner, H., Reiter, M., 1996. Cholestane and pregnane type glycosides from the roots of Tribulus cistoides. Phytochemistry 41, 907–919. Adaikan, P.G., Gauthaman, K., Prasad, R.N.V., 2000. Proerectile pharmacological effects of Tribulus terrestris extract on the rabbit corpus cavernosum. Annals of the Academic Medicine of Singapore 29, 22–26. Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 31, 3307–3330. Bedir, E., Khan, I.A., 2000. New steroidal glycosides from the fruit of Tribulus terrestris. Journal of Natural Products 63, 1699–1701. Bhutani, S.P., Chibber, S.S., Seshadri, T.R., 1969. Flavonoids of the fruits and leaves of Tribulus pentandrus: constitution of tribuloside. Phytochemistry 8, 299–303. Boulos, L., 2000. Flora of Egypt, (Geraniaceae-Boraginaceae), vol. 2. Al Hadara Publishing, Cairo, Egypt. Cai, L.F., Jing, F.Y., Zhang, J.G., Pei, F.K., Xu, Y.J., Liu, S.Y., Xu, D.M., 1999. Studies on the chemical components of Tribulus terrestris. Acta Pharmaceutica Sinica 34, 759–761. Cai, L.F., Wu, Y., Zhang, J.G., Pei, F.K., Xu, Y.J., Xie, S.G., Xu, D.M., 2001. Steroidal saponins from Tribulus terrestris. Planta Medica 67, 196–198. Dong, M., Feng, X., Wang, B., Wu, L., Ikejima, T., 2001. Two novel furostanol saponins from the rhizomes of Dioscorea penthaica Prain et Burkill and their cytotoxic activity. Tetrahedron 57, 501– 506. El-Hadidi, M.N., Fayed, A., 1994. Materials for excursion flora of Egypt. Tackholmia 15, 118. Gauthaman, K., Adaikan, P.G., Prasad, R.N.V., 2002. Aphrodisiac properties of Tribulus terrestris extract (protodioscin) in normal and castrated rats. Life Sciences 71, 1385–1396. Jin, J.M., Zhang, Y.J., Yang, C.R., 2004. Four new steroid constituents from the waste residue of fibre separation from Agave americana leaves. Chemical Pharmaceutical Bulletin 52, 654–658. Kiyosawa, S., Goto, K., Owashi, R., 1977. Glycosides of 20,22-secofurostane derivatives. Tetrahedron Letters 52, 4599–4602. Kostova, I., Dinchev, D., Rentsch, G.H., Dimitrov, V., Ivanova, A., 2002. Two new sulfated furostanol saponins from Tribulus terrestris. Zeitschrift fur Naturforschung C 57, 33–38. Li, J.X., Shi, Q., Xiong, Q.B., Prasain, J.K., Tezuka, Y., Hareyama, T., Wang, Z.T., Tanaka, K., Namba, T., Kadota, S., 1998.

A.I. Hamed et al. / Phytochemistry 65 (2004) 2935–2945 Tribulusamide A and B, new hepatoprotective lignanamides from the fruits of Tribulus terrestris: Indications of cytoprotective activity in murine hepatocyte culture. Planta Medica 64, 628–663. Mahato, S.B., Sahu, N.P., Ganguly, A.N., Miyahara, K., Kawasaki, T., 1981. Steroidal glycosides of Tribulus terrestris Linn. Journal of Chemical Society Perkins Transactions 1, 2405–2410. Qian, B.Y., 1990. Review on the research of Tribulus terrestris. Zhong Cheng Yao 12, 34. Saleh, N.A.M., Ahmed, A.A., Abdalla, M.F., 1982. Flavonoid glycosides of Tribulus pentandrus and Tribulus terrestris. Phytochemistry 21, 1995–2000. Sun, W., Gao, J., Tu, G., Guo, Z., Zhang, Y., 2002. A new steroidal saponin from Tribulus terrestris Linn. Natural Product Letters 16, 243–247. Tackholm, V., 1974. StudentsÕ Flora of Egypt, second ed. Cairo University Publications, Beirut, p. 888. Tomova, M.P., Botscheva, D.M., Zaikin, W.G., Wulfson, N.S., 1977. Steroid saponins and sapogenins V: Hecogenin from Tribulus terrestris. Planta Medica 32, 223–224. Tomova, M.P., Gjulemetova, R., Zarkova, S., Peeva, S., Pangarova, T., Simova, M., 1981. Steroidal spooning from Tribulus terrestris L. with a stimulating action on the sexual functions. In: First

2945

International Conference on Chemical, Biotechnological and Biologically Active Natural Products, Proceedings Varna, September 3, pp. 299–303. Tomova, M.P., Panova, D., Wulfson, N.S., 1974. Steroid saponins and sapogenins IV: Saponins from Tribulus terrestris. Planta Medica 25, 231–237. Wang, Y., Ohtani, K., Kasai, D., Yamasaki, K., 1996. Steroidal saponins from fruit of Tribulus terrestris. Phytochemistry 42, 1417– 1422. Wang, Y., Ohtani, K., Kasai, D., Yamasaki, K., 1997. Steroidal saponins from fruit of Tribulus terrestris. Phytochemistry 45, 811– 817. Wo, T.S., Shi, L.S., Kuo, S.C., 1999. Alkaloids and other constituents from Tribulus terrestris. Phytochemistry 50, 1411–1415. Wu, G., Jiang, S., Jiang, F., Zhu, D., Wu, H., Jiang, S., 1996. Steroidal glycosides from Tribulus terrestris. Phytochemistry 42, 1677–1681. Xu, Y.X., Chen, H.S., Liu, W.Y., Gu, Z.B., Liang, H.Q., 1998. Two sapogenins from Tribulus terrestris. Phytochemistry 49, 199–201. Zhu, H.Z., Tsumagari, H., Honbu, T., Ikeda, T., Ono, M., Nohara, T., 2001. Peculiar steroidal saponins with opened E-ring from Solanum genera plants. Tetrahedron Letters 42, 8043–8046.