Iridoids and flavonoid from Linaria aegyptiaca (L.) Dum. subsp. fruticosa

Biochemical Systematics and Ecology 38 (2010) 833–835

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Iridoids and flavonoid from Linaria aegyptiaca (L.) Dum. subsp. fruticosa Maria Ferhat a, Hassina Harkat a, Catherine Lavaud b, Hamada Haba a, Christophe Long c, Mohammed Benkhaled a, * a

Laboratoire de Chimie et Chimie de l’Environnement (L.C.C.E), Département de Chimie, Faculté des Sciences, Université de Batna, Batna 05000, Algeria UMR CNRS 6229, Institut de Chimie Moléculaire de Reims, BP 1039, 51097 Reims Cedex 2, France c Centre de Recherche sur les Substances Naturelles, UMS CNRS 2597, 3 rue des Satellites, BP 94244, 31432 Toulouse, France b

a r t i c l e i n f o Article history: Received 4 March 2010 Accepted 24 June 2010 Keywords: Linaria aegyptiaca Scrophulariaceae Iridoids Flavonoid

1. Subject and source Linaria aegyptiaca (L.) Dum. subsp. fruticosa is a woody perennial plant belonging to the Scrophulariaceae family (Quezel and Santa, 1963). It grows in the southern part of Algeria, especially in arid land and desert. Linaria reflexa Desf. is used in the North African folk medicine for the treatment of certain skin diseases (Boukef, 1986). 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 deposited under No. 401. 2. Previous work The genus Linaria is known for the presence of variety of compounds. Previous investigations led to isolation of alkaloids (Hua et al., 2002), iridoids (Otsuka, 1995; Ahmad et al., 2006; Tundis et al., 2008), flavonoids (Otsuka, 1992) and diterpenoids (Gordaliza et al., 1995). The neo-clerodane diterpenoids and flavonoids previously isolated respectively from Linaria saxatilis var. saxatilis and L. reflexa Desf. showed cytotoxic activity (Gordaliza et al., 1997; Tundis et al., 2005). No previous phytochemical or pharmacological properties of L. aegyptiaca have been reported. 3. Present study In a continuation of phytochemical studies on Algerian Saharan plants particularly of Scrophulariaceae family (Arrif et al., 2006, 2008), the aerial parts of L. aegyptiaca (L.) Dum. subsp. fruticosa were investigated. In the present study three iridoids 1, 2, 3 and a flavonoid 4, were isolated from the methanol extract. * Corresponding author. Tel./fax: þ213 33 86 89 46. E-mail address: [email protected] (M. Benkhaled). 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.06.006

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3.1. Extraction and isolation of constituents Powdered aerial parts (1000 g) of L. aegyptiaca were successively extracted twice with 10 L of cyclohexane, ethyl acetate and methanol at room temperature. The combined methanol extracts were concentrated under vacuum to dryness to yield 16 g. This extract (10 g) was chromatographed over a polyamide column eluted with water, water–methanol 80:20, 50:50, 20:80 and 10:90 successively, to afford 9 fractions. Fraction 1 (534 mg) was subjected to a silicagel column chromatography (eluent: CHCl3–MeOH–H2O 90:10:1, 80:20:1, 80:20:2, 70:30:2, 60:40:2 and methanol) and preparative TLC on RP-18 (eluent: H2O–MeOH 70:30) which led to compound 2 (12 mg). Fraction 2 (500 mg) was submitted again to a silicagel column chromatography (eluent: CHCl3–MeOH–H2O 95:5:0.5, 93:7:0.5, 90:10:1, 85:15:1.5, 80:20:1.5 and 50:50:0) and preparative TLC on RP-18 eluted with water–methanol 70:30 and 60:40, to allow isolation of compound 1 (16 mg) and 3 (8 mg) respectively. Fraction 8 (308 mg) contained a fluorescent compound coloured in yellow after sulphuric acid-vanillin spray. It was submitted to a silicagel column chromatography (eluent: CHCl3 and CHCl3–MeOH–H2O 95:5:0.5, 93:7:0.5, 90:10:1 and 85:15:1.5). Recrystallisation in methanol of grouped fractions eluted with CHCl3–MeOH–H2O 90:10:1 furnished the compound 4 (20 mg). 3.2. Identification of constituents Isolated compounds were determined by UV (Shimadzu UV-3101 spectrophotometer), IR (KBr, Shimadzu model IR-470 spectrometer), 1D and 2D NMR experiments (COSY, HSQC, HMBC, NOESY, Bruker Avance Spectrometer, 1H 500 MHz, 13C 125 MHz), positive and negative ESI-MS (ion trap Bruker Esquire), HR-ESI-MS (Bruker Micromass Q-TOF), X-ray crystallography (Kappa CCD diffractometer) and optical rotations (Perkin–Elmer 241 polarimeter) compared with literature data. They were identified as antirrhinoside (1) (Sticher, 1971; Kitagawa et al., 1973; Handjieva et al., 1993), linarioside (2) (Kitagawa et al., 1972; Kapoor et al., 1974), mussaenosidic acid (3) (Damtoft et al., 1984) and ladanein (4) (Yang et al., 1996; Horie et al., 1998) (Fig. 1). 4. Chemotaxonomic significance Iridoids and their glycoside derivatives are chemotaxonomic markers of the family Scrophulariaceae (Albach et al., 2007; Jensen et al., 2008), to which the genus Linaria belongs. Antirrhinoside and related iridoid constituents are the chemical characters for the genera of the Scrophularioideae–Antirrhineae tribe of the Scrophulariaceae family, including Linaria, Antirrhinum, Asarina, Kickxia and Maurandia (Kooiman, 1970; Nicoletti et al., 1988). Iridoids isolated from Linaria

HO

OH

HO

O

CH3

OH

Cl

O

O HO

H O

OH

O

HO

CH3

H O

OH

O

HO

OH

OH

HO

HO

1

2

COOH H

OCH3

O HO CH3

O

H3CO

H O

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O

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OH

HO

HO

OH

3

O

4 Fig. 1.

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species are most frequently anthirrinoside, 6-acylanthirrhinoside and 5-deoxyanthirrhinoside derivatives, as in the case of iridoids identified from Linaria clementei Boiss. (Marco, 1985), Linaria multicalis (L.) Miller subsp. multicalis (Tundis et al., 2008), Linaria vulgaris (Ilieva et al., 1992), L. Arcusangeli Atzei et Camada (Bianco et al., 1996a), Linaria flava subsp. sardoa Arrigoni (Bianco et al., 1996b) and Linaria japonica Miq. (Otsuka, 1994). Two antirrhinoside derivatives acylated at C-60 of the glucoside unit were isolated from L. flava subsp. sardoa Arrigoni: 60 -O-senecioylanthirrhinoside and 60 -O-angeoylanthirrhinoside (Bianco et al., 1996b). Two anthirrhinoside derivatives substituted at C-5 were also identified from Linaria dalmatica (L.) Mill.: 5-O-glucosylanthirrhinoside and 5-O-alloylanthirrhinoside (Handjieva et al., 1993). Linarioside, a rare naturally chlorinated iridoid glucoside, found in many Linaria species such as L. japonica Miq. (Kitagawa et al., 1972), L. clementei Boiss. (Marco, 1985), Linaria arcusangeli and L. flava subsp. sardoa Arrigoni (Bianco et al., 1996a), L. dalmatica, L. genistifolia, Linaria simplex, L. vulgaris and Linaria pelisseriana (Ilieva et al., 1993), seems to be a good chemotaxonomical marker of Linaria species. According to the previous studies on the composition of the Linaria genus which belongs to the Scrophularioideae–Antirrhineae tribe, L. aegyptiaca (L.) Dum. susbsp. fruticosa also shows antirrhinoside as specific chemotaxonomic marker. In accordance with the previous isolations from Linaria species, the chemical study of L. aegyptiaca subsp. fruticosa confirms the occurrence of antirrhinoside and linarioside. Pectolinarin and linariin isolated from Linaria species as L. japonica Miq. (Morita et al., 1974; Otsuka, 1992) and L. reflexa Desf. (Tundis et al., 2005), are the most common flavonoid derivatives of the Linaria genus. Previous phytochemical studies showed that ladanein is a flavonoid present in several species such as Colebrookea oppositifolia (Labiatae) (Yang et al., 1996), Marrubium lu and Aksit, 2002) and Centaurea clementei (Asteraceae) (Gonzalez Collado et al., 1985), trachyticum (Lamiaceae) (Çitog and was identified, to our knowledge for the first time in the Scrophulariaceae family. Mussaenosidic acid which was previously isolated from Globularia vulgaris (Plantaginaceae) (Taskova et al., 2006) and Melapyrum cristatum (Scrophulariaceae) (Damtoft et al., 1984) is described for the first time in the Linaria genus. Acknowledgments Dr G. Massiot is gratefully acknowledged for the MS and NMR technical support from his Group (Pierre Fabre Research Institute). The authors thank D. Patigny, service de spectrométrie de masse, UMR CNRS 6229, Institut de Chimie Moléculaire de Reims (France) for performing the MS spectra. 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