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Author's personal copy Biochemical Systematics and Ecology 39 (2011) 902–905
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Acylated iridoid glycosides from Scrophularia saharae Batt. & Trab. Rachid Chebaki a, Hamada Haba a, Christophe Long b, Laurence Marcourt b, Mohammed Benkhaled a, * a b
Laboratoire de Chimie et Chimie de l’Environnement (L.C.C.E), Département de Chimie, Faculté des Sciences, Université de Batna, Batna 05000, Algeria Centre de Recherche sur les Substances Naturelles, UMS CNRS 2597, 3 rue des Satellites, BP 94244, 31432 Toulouse, France
a r t i c l e i n f o Article history: Received 23 March 2011 Accepted 19 July 2011 Available online 19 August 2011 Keywords: Scrophularia saharae Scrophulariaceae Iridoids
1. Subject and source Scrophularia saharae Batt. & Trab. (Scrophulariaceae) is a perennial plant, common throughout the desert region of Algeria (Battandier, 1889; Ozenda, 1991). If the North African traditional medicine does not mention a possible therapeutic use for S. saharae Batt. & Trab. (deserti Coss., not Del.) (Battandier, 1889), the species Scrophularia deserti Del. growing in Saudi Arabia, is used in local folk medicine as an antipyretic, hypoglycemic, cardiotonic, diuretic in thyphoid fever, a remedy for kidney diseases and lung cancer (Ahmed et al., 2003). A pharmacological study performed from the same species revealed that the two iridoid glycosides, scropolioside D and harpagoside B, were found to possess significant antidiabetic and antiinflammatory activities, respectively (Ahmed et al., 2003). The plant material was collected in May 2004 in the vicinity of Biskra (Algeria) and was identified by Pr Bachir Oudjehih, Agronomic Department of the University of Batna, where a voucher specimen has been desposited under N 533/LCCE. 2. Previous work The genus Scrophularia is known for the presence of variety of compounds. Previous works allowed isolation of iridoids (Giner et al., 1998; Li et al., 1999; Ahmed et al., 2003; Nguyen et al., 2005), saponins (Bhandari et al., 1997; Calis et al., 1993a; Giner et al., 2000), phenylethanoids (Miyase and Mimatsu, 1999; Díaz et al., 2004). No previous phytochemical or biological properties of S. saharae have been reported. 3. Present study In a continuation of our studies on the chemical constituents of Scrophulariaceae family (Arrif et al., 2006, 2008; Ferhat et al., 2010), we have investigated S. saharae Batt. & Trab. The present paper describes the isolation and characterization of
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[email protected] (M. Benkhaled). 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.07.017
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Fig. 1. Pertinent correlations observed in the HMBC spectrum for compound 3.
a new iridoid glycoside 3, together with four known iridoids: harpagoside, 8-O-acetylharpagide, scropolioside B 1 and scropolioside D. All the isolated compounds were identified by means of spectroscopic methods. 3.1. Extraction and isolation of constituents 200 g of powdered roots of S. saharae were extracted twice with 2 L of cyclohexane at room temperature during 4 days. The residue was extracted by CHCl3 (2 times 2 L) at room temperature during 4 days. The combined chloroformic extracts were
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R. Chebaki et al. / Biochemical Systematics and Ecology 39 (2011) 902–905
Table 1 NMR data of compounds 1 and 3 [500 MHz (1H), 125 MHz (13C), CDCl3 þ MeOD, d (ppm) (J ¼ Hz)]. Atom
1
2
1
H
1
97.3 141.0 101.7 35.7 83.5 58.0 65.1 41.8 59.9
4.92 6.36 5.13 2.55 4.02 3.65 – 2.66 3.98 4.00
d (9.6) d (5.8) dd (5.8–4.7) m dd (8.7–2.1) d (2.1)
C
5.03 6.39 5.13 2.55 4.06 3.67 – 2.64 3.89 4.11
d (9.7) dd (1.6–6) dd (4.7–6) m d (8.1) br s
10 20 30 40 50 60
4.81 3.33 3.46 3.31 3.35 3.71 3.92
d (7.9) dd (7.9–9) t (9) t (9) m dd (5.4–12.1) dd (1.3–12.1)
98.2 73.4 76.2 70.6 77.2 61.5
4.82 3.36 3.51 3.49 3.38 3.77 3.88
10 0 20 0 30 0 40 0 50 0 60 0
5.08 5.42 5.54 5.33 4.13 1.30
d (1.7) dd (1.7–3.5) dd (3.5–10.3) t (10.3) m d (6.2)
96.3 70.3 70.0 70.9 66.8 16.3
5.03 5.41 5.53 5.34 4.10 1.28
10 0 0 20 0 0 , 60 0 0 30 0 0 , 50 0 0 40 0 0
– 7.50 7.37 7.37 6.36 7.62 – –
134.0 127.9 128.5 130.3 116.4 145.9 165.8 –
– 7.49 7.36 7.35 6.33 7.63 – –
134.0 127.9 128.5 130.3 116.1 146.2 166.2 – 19.3 170.2
– 7.46 6.89 – 6.25 7.64 – 3.83 2.20 –
a b CO OCH3 100 00 200 00 , 600 00 300 00 , 500 00 400 00
a0 b0 CO OCH3 CH3CO CH3CO
– 7.53 7.37 7.37 6.44 7.71 – – 2.22 –
m m m d (16) d (16)
m m m d (16) d (16)
s
13
H
1 3 4 5 6 7 8 9 10
dd (7.6–9.7) d (13.2) d (13.2)
3
13
C
1
13
H
C
94.6 141.3 102.5 35.9 83.6 58.4 65.0 42.4 60.9
5.02 6.39 5.13 2.55 4.06 3.67 – 2.64 3.89 4.11
d (9.7) dd (1.6–6.1) dd (4.8–6.1) m d (7.9) br s
d (7.8) dd (7.2–7.8) dd (7.2–7.8) dd (7.2–7.8) m dd (2–11.4) dd (5.5–11.4)
99.0 73.2 76.2 69.6 76.7 61.4
4.80 3.31 3.45 3.37 3.34 3.71 3.92
d (7.9) dd (7.9–8.7) t (8.7) t (8.7) m dd (5.3–12) dd (2–12)
98.2 73.4 76.2 70.3 77.2 61.5
d (1.2) dd (1.2–3) dd (3–9.6) t (9.6) m d (6.6)
96.6 70.4 69.1 71.7 67.3 17.5
5.07 5.42 5.54 5.33 4.13 1.31
d (1.7) dd (1.7–3.5) dd (3.5–10) t (10) m d (6.2)
96.3 70.0 69.4 70.8 66.8 16.3
134.1 128.3 128.9 130.6 117.0 146.2 166.1 –
– 7.63 6.79 – 6.36 7.62 – 3.76
s
134.0 128.0 113.0 161.0 116.4 146.0 166.2 54.3
126.8 130.2 114.4 161.7 114.3 146.1 166.9 55.5 21.0 170.5
– 7.53 m 7.37 m 7.37 m 6.44 d (16) 7.71 d (16) – – 2.1 s –
134.0 127.9 128.5 130.3 116.4 146.2 165.9 – 19.2 170.2
dd (7.8–9.6) dd (2.0–11) dd (5.5–11)
dd (2.5–8.5) t (8.5) t (8.5) d (16) d (16)
d (9) d (9) d (16.2) d (16.2) s s
dd (7.6–9.7) d (13.1) d (13.1)
d (8.9) d (8.9) d (16) d (16)
93.7 141.1 101.7 35.7 83.3 58.0 65.1 41.8 59.9
concentrated under vacuum to dryness to yield 4.5 g. 4 g of this extract were chromatographed over a silica gel column eluted with petroleum ether, petroleum ether-ethyl acetate 90:10, 85:15, 80:20, 60:40, 50:50 and 20:80, to afford 30 fractions. Fraction 22 (723 mg) was subjected to a silica gel column chromatography using a gradient of hexane-ethyl acetate 80:20, 60:40, 50:50, 30:70, 20:80 and ethyl acetate, to afford 15 fractions. Purification of fractions (91 mg) eluted with ethyl acetate by semi-preparative HPLC using a gradient of H2O/MeCN (60:40 to 40:60) yielded three compounds: scropolioside B 1 (14 mg), scropolioside D (20 mg) and the new iridoid glycoside 3 (10 mg). Original fraction 25 (189 mg) was applied to silica gel chromatography column eluting with petroleum ether-ethyl acetate 80:20, 50:50, 20:80,10:90, ethyl acetate, ethyl acetate-methanol 99:1 and 98:2, to give seven fractions. Purification of fractions (38 mg) eluted with ethyl acetate-methanol 98:2 by preparative TLC on silica gel (eluent: CHCl3-MeOH 70:30) yielded 8-O-trans-cinnamoyl harpagide named harpagoside. Original fraction 29 (218 mg) was submitted to a silica gel column chromatography using a gradient of hexane-ethyl acetate 50:50, 20:80, ethyl acetate, ethyl acetate-methanol 99:1 and 98:2, to provide six fractions. Filtration of fractions (25 mg) eluted with ethyl acetate-methanol 98:2 on Sephadex LH-20 column in methanol, yielded 8-O-acetylharpagide. 3.2. Identification of constituents Isolated compounds were identified by UV (Beckman DU-600), IR (KBr, Shimadzu IR-470 and Jasco FT/IR-4100), positive and negative ESI-MS (ion trap Bruker Esquire) and extensive 1D and 2D NMR analysis (COSY, HSQC, HMBC, NOESY, ROESY,
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Bruker Avance Spectrometer, 1H 500 MHz, 13C 125 MHz). CC was carried out on Kieselgel 60 (320–400 mesh) and Sephadex LH-20. HPLC was performed with a Waters apparatus equiped with an ASI-717, a 600E pump, a diode array detector UVD 996 and a millenium software, on a column Lichrospher 100 RP-18 (250 25 mm, 5 mm). Analytical and preparative (1 mm thickness) TLCs were carried on silica gel (Kieselgel 60 F254, Merck) and RP-18 (Kieselgel 60 F254S) plates. We isolated a new iridoid glycoside 3 along with four known iridoids. The known iridoids were identified as harpagoside (Li et al., 1999), 8-O-acetylharpagide (Akdemir et al., 2004), scropolioside D (Calis et al., 1993b) and scropolioside B 1 (Calis et al., 1988; Bhandari et al., 1992), by comparison of their NMR spectra with published data. Compound 3 was obtained as a white powder. The ESI mass spectrum recorded in the positive ionization mode showed a molecular ion at m/z 863 [M þ Na]þ and m/z 875 [M þ Cl] in the negative mode suggesting a molecular formula of C42H48O18. Its UV spectrum showed bands at 201, 217 and 277 nm characteristic of an iridoid enol ether system and cinnamoyl chromophore (Calis et al., 1988). The chemical shifts of protons and carbons, established mainly by COSY H–H, HSQC and HMBC, are similar to those reported for the glycoside iridoid 2 named scrophuloside B4 (6-O-(2"-O-acetyl-3"-O-trans-cinnamoyl-4"-O-p-methoxy-trans-cinnamoyl)-a-L-rhamnopyranosyl catalpol), isolated from the roots of Scrophularia ningpoensis (Nguyen et al., 2005). However, the HMBC correlations (Fig. 1) indicate unambiguously the p-methoxy-transcinnamoyl at C-300 position of the rhamnopyranosyl moiety instead of C-400 position in the case of compound 2 (scrophuloside B4). Indeed, the proton H-300 at dH 5.54 ppm of the rhamnopyranosyl identified by COSY H–H showed a long range coupling in the HMBC spectrum with the carbonyl carbone at dC 165.9 ppm, which correlates with H-a and H-b at dH 6.36 and 7.62 ppm respectively. Carbone C-b (dC 146 ppm) identified by HSQC correlates in HMBC with two equivalents protons H-2"’ and H-6"’ at dH 7.63 ppm (2H, d, J ¼ 8.9 Hz). These two protons correlate with the carbon C-4"’ (dC 161 ppm), indicating that the methoxyl group is located at this position (Fig. 1). Compound 3 is thus (6-O-(2"-O-acetyl-3"-O-p-methoxy-trans-cinnamoyl4"-O-trans-cinnamoyl)-a-L-rhamnopyranosyl) catalpol) for which we propose the trivial name of scrophuloside B5 (Table 1). Compound 3: white powder, ½a22 D 23.6 (c 0.21, MeOH); UV lmax(MeOH): 201, 217, 277 nm. IR bands (CHCl3): 3408, 2912, 1740, 1715, 1596 cm1. ESIMS m/z: positive mode 863 ([M þ Na]þ 100%), negative mode 875 ([M þ Cl] 100%). 4. Chemotaxonomic significance Iridoids and their glycoside derivatives are chemotaxonomic markers of the family Scrophulariaceae, to which the genus Scrophularia belongs (Kooiman, 1970; Albach et al., 2007; Jensen et al., 2008). Iridoid glycosides isolated from Scrophularia species are most frequently derived from 6-O-a-L-rhamnopyranosyl catalpol where the most common substitutions occur throughout ester bonds to the rhamnopyranosyl moiety (Kajimoto et al., 1989; Giner et al., 1998; Miyase and Mimatsu, 1999; Stevenson et al., 2002). Harpagide and their acylated derivatives such as harpagoside (Kajimoto et al., 1989; Fernandez et al., 1995; Li et al., 1999; Ahmed et al., 2003) are also widely represented in the Scrophularia genus. According to the previous studies on the chemical composition of Scrophularia species, S. saharae Batt. & Trab. also contains harpagide derivatives and iridoid glycosides derived from 6-O-a-L-rhamnopyranosyl catalpol as chemotaxonomic markers. 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