Biochemical Systematics and Ecology 38 (2010) 833–835

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Biochemical Systematics and Ecology

journal homepage: www.elsevier .com/locate/biochemsyseco

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, AlgeriabUMR CNRS 6229, Institut de Chimie Moléculaire de Reims, BP 1039, 51097 Reims Cedex 2, FrancecCentre 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 4 March 2010Accepted 24 June 2010

Keywords:Linaria aegyptiacaScrophulariaceaeIridoidsFlavonoid

* Corresponding author. Tel./fax: þ213 33 86 89 4E-mail address: mbenkhaled@yahoo.fr (M. Benk

0305-1978/$ – see front matter � 2010 Elsevier Ltddoi:10.1016/j.bse.2010.06.006

1. Subject and source

Linaria aegyptiaca (L.) Dum. subsp. fruticosa is a woody perennial plant belonging to the Scrophulariaceae family (Quezeland Santa, 1963). It grows in the southern part of Algeria, especially in arid land and desert. Linaria reflexa Desf. is used in theNorth African folk medicine for the treatment of certain skin diseases (Boukef, 1986). The plant material was collected in May2004 in the vicinity of Biskra (Algeria) and was identified by Pr Bachir Oudjehih, Agronomic Department of the University ofBatna, 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 saxatilisvar. saxatilis and L. reflexa Desf. showed cytotoxic activity (Gordaliza et al., 1997; Tundis et al., 2005). No previous phyto-chemical 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. fruticosawere investigated. In the present study three iridoids 1,2, 3 and a flavonoid 4, were isolated from the methanol extract.

6.haled).

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M. Ferhat et al. / Biochemical Systematics and Ecology 38 (2010) 833–835834

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 acetateand methanol at room temperature. The combined methanol extracts were concentrated under vacuum to dryness to yield16 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 columnchromatography (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 preparativeTLC 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. Itwas submitted to a silicagel column chromatography (eluent: CHCl3 and CHCl3–MeOH–H2O 95:5:0.5, 93:7:0.5, 90:10:1 and85:15:1.5). Recrystallisation in methanol of grouped fractions eluted with CHCl3–MeOH–H2O 90:10:1 furnished thecompound 4 (20 mg).

3.2. Identification of constituents

Isolated compounds were determined by UV (Shimadzu UV-3101 spectrophotometer), IR (KBr, Shimadzu model IR-470spectrometer), 1D and 2D NMR experiments (COSY, HSQC, HMBC, NOESY, Bruker Avance Spectrometer, 1H 500 MHz, 13C125 MHz), positive and negative ESI-MS (ion trap Bruker Esquire), HR-ESI-MS (Bruker Micromass Q-TOF), X-ray crystallog-raphy (Kappa CCD diffractometer) and optical rotations (Perkin–Elmer 241 polarimeter) compared with literature data. Theywere identified as antirrhinoside (1) (Sticher, 1971; Kitagawa et al., 1973; Handjieva et al., 1993), linarioside (2) (Kitagawaet al., 1972; Kapoor et al., 1974), mussaenosidic acid (3) (Damtoft et al., 1984) and ladanein (4) (Yang et al., 1996; Horieet 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 thechemical characters for the genera of the Scrophularioideae–Antirrhineae tribe of the Scrophulariaceae family, includingLinaria, Antirrhinum, Asarina, Kickxia and Maurandia (Kooiman, 1970; Nicoletti et al., 1988). Iridoids isolated from Linaria

O

OH

HO O OH

OHHO

HO

HO

OO

OH

HO O OH

OHHO

HO

HO

CH3

Cl

HO

1 2

O

H

HO O OH

OHHO

HO

CH3HO

3

COOH

O

O

H3CO

HO

OH

OCH3

4

CH3

Fig. 1.

M. Ferhat et al. / Biochemical Systematics and Ecology 38 (2010) 833–835 835

species are most frequently anthirrinoside, 6-acylanthirrhinoside and 5-deoxyanthirrhinoside derivatives, as in the caseof iridoids identified from Linaria clementei Boiss. (Marco, 1985), Linaria multicalis (L.) Miller subsp. multicalis (Tundiset al., 2008), Linaria vulgaris (Ilieva et al., 1992), L. Arcusangeli Atzei et Camada (Bianco et al., 1996a), Linaria flavasubsp. sardoa Arrigoni (Bianco et al., 1996b) and Linaria japonica Miq. (Otsuka, 1994). Two antirrhinoside derivativesacylated at C-60 of the glucoside unit were isolated from L. flava subsp. sardoa Arrigoni: 60-O-senecioylanthirrhinoside and60-O-angeoylanthirrhinoside (Bianco et al., 1996b). Two anthirrhinoside derivatives substituted at C-5 were also identifiedfrom 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. (Kitagawaet 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 goodchemotaxonomical marker of Linaria species. According to the previous studies on the composition of the Linaria genuswhich belongs to the Scrophularioideae–Antirrhineae tribe, L. aegyptiaca (L.) Dum. susbsp. fruticosa also shows anti-rrhinoside as specific chemotaxonomic marker. In accordance with the previous isolations from Linaria species, thechemical study of L. aegyptiaca subsp. fruticosa confirms the occurrence of antirrhinoside and linarioside. Pectolinarin andlinariin 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 thatladanein is a flavonoid present in several species such as Colebrookea oppositifolia (Labiatae) (Yang et al., 1996), Marrubiumtrachyticum (Lamiaceae) (Çito�glu and Aksit, 2002) and Centaurea clementei (Asteraceae) (Gonzalez Collado et al., 1985),and was identified, to our knowledge for the first time in the Scrophulariaceae family. Mussaenosidic acid which waspreviously isolated from Globularia vulgaris (Plantaginaceae) (Taskova et al., 2006) and Melapyrum cristatum (Scrophu-lariaceae) (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 ResearchInstitute). The authors thank D. Patigny, service de spectrométrie de masse, UMR CNRS 6229, Institut de Chimie Moléculairede Reims (France) for performing the MS spectra.

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