CHEMICAL AND STRUCTURAL STUDIES OF TUNISIAN Anacyclus Cyrtolepidioïdes BUTANOL EXTRACT S. BCHIR, S. HAMMAMI, A. BERGAOUI, Z.

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1 J.Soc.Alger.Chim., 0, (), Journal de la Société Algérienne de Chimie CEMICAL AND STRUCTURAL STUDIES F TUNISIAN Anacyclus Cyrtolepidioïdes BUTANL EXTRACT S. BCIR, S. AMMAMI, A. BERGAUI, Z. MIGRI UR 0-Chimie Appliquée-Environnement, Département de Chimie, Faculté des Sciences de Monastir. (Reçu le 9//0, accepté le 0/09/) ملخص خلال هذا البحث قمنا بعزل نارنجينين دي هيدروآسي بنزويك و البيتا سيتوستيرول -قليكوزيد D-β-- من مستخرج البيتانول لنبتة أناسيكليس سيرتوليبيدويدس. ولقد قمنا بتحديد بنية هذه المواد الطبيعية بالاعتماد على تقنيات الرنين المغناطيسي النووي ذات البعد الواحد و ذات البعدين و مدعومة بمطيافية الكتلة ABSTRACT Naringenin,.-dihydroxybenzoic acid and β-sitosterol---β-d-glycoside were isolated from the butanol extract of A. cyrtolepidioides fresh flowers. Their structures were established using ( and C) and D NMR spectroscopic methods as well as ES Mass Spectrometry. Key words: A. Cyrtolepidioides, Asteraceae, Naringenin,.-dihydroxybenzoic acid, β- sitosterol---β-d-glycoside. RESUME La naringénine, l acide,-dihydroxybenzoïque at le β-sitostérol---β-dglucoside ont été isolés de l extrait butanolique des fleurs fraîches de l espèce A. cyrtolepidioïdes poussant en Tunisie. Leurs structures ont été établies par RMN et D ( et C) et par spectrométrie de masse en mode Electro-Spray. Mots clés: A. Cyrtolepidioides, Asteraceae, Naringénine, l acide,-dihydroxybenzoïque, β-sitostérol---β-d-glycoside. 59

2 S. BCIR, INTRDUCTIN Anacyclus cyrtolepidioïdes (Asteraceae) is one from three species of the same genus and being widely distributed in Tunisia []. Chemical investigation of Egyptian Anacyclus species revealed the presence of sesquiterpene lactones reported from their cytotoxic, phytotoxic and antineoplastic properties [], these components have never been isolated from Tunisian Anacyclus specie. Moreover, Anacycline has been reported as a natural insecticide in Anacyclus clavatus [-5]. In continuation to our attention paid to the chemical and biological study of Medicinal plants [6-], we have extensively examined the butanol extract of Tunisian A. Cyrtolepidioïdes flowers. Thus Naringenin,.-dihydroxybenzoic acid and β-sitosterol---β-d-glycoside were isolated and have been characterised for the first time as secondary metabolites from the indicated plant Compound ' ' ' 6' ' 5' ' 5' ' 6' ' ' 5 6 Compound Compound EXPERIMENTAL Plant material: the plant was collected in March 005 in Gabes city (south of Tunisia). Samples were identified by Dr. F. arzallah-skhiri (Institut Supérieur de Biothechnologie de Monastir) and voucher specimens were deposited in the herbarium of the Institut Supérieur agronomique de Chott-Mariem, Université de Sousse, Tunisie. Flower heads were separated from stems and leaves prior to extraction. 60

3 Journal de la Société Algérienne de Chimie Extraction and isolation of the chemical constituents: Compound : A sample of A. cyrtolepidioïdes fresh flowers (000g) was subjected to hydrodistillation in a steam distiller apparatus during hours to obtain a volatile fraction and a water residue which was exhausted successively with ethylacetate and butanol. Fractionation of the butanol extract from fresh flowers was pursued on a silica gel open column using a mixture of (EP/AcEt) (8:) gradually increased with ethylacetate and methanol, 5 fractions of 00mL were thus collected and regrouped into 8 main fractions (G -G 8 ) according to their TLC profiles. Fraction G was further purified successively over three silica gel columns eluted with C Cl /Me gradient (9:, 8:, and :) to afford mg of compound. Compound : fraction G deriving from separation of butanol crude extract was subjected to a silica gel column chromatography eluted with a mixture of CCl /Me (0:0, 9:, 8:) to yield 8 subfractions (F -F 8 ). The third one was successively rechromatographed over two silica gel columns to afford.5mg of compound as a white solid. Compound : fraction G was purified through three silica gel columns using as eluent a mixture of CCl /Me (8.5:.5) gradually increased by Me. Which efforded mg of compound as a white solid. RESULTS AND DISCUSSIN Compound The NMR spectrum displayed three double doublets appearing at δ.0ppm (J =.z, J =.0z), δ.ppm (J =.z, J =.9z) and δ 5.ppm (J =.9z, J =.0z) assigned to protons α, β and of characteristic pyron ring, this suggested that compound was a flavanone. Aromatic protons at δ 5.8 (, d, J meta =.z) and δ 5.89 (, d, J meta =.z) were assigned to 6 and 8 in flavanon A ring. Two characteristic doublets appearing at δ 6.8ppm (, J ortho =8.z,, 5 ) and δ.ppm (, J ortho =8.z,, 6 ) indicated the presence of a C monosubstitution on the B-ring moiety. The C NMR spectrum of compound showed carbon atoms: from which one methylene carbon at δ C.5 (C ), six quaternary carbons appearing at δ C 68.8, 65.9, 65., 59.5,., 0. and a characteristic flavanone carbonyl carbon appearing at δ C 98.ppm. Comparison of and C NMR data with those of the literature [-], suggested that compound is the 5,-dihydroxy--(-hydroxyphenyl)-,-dihydrochromen--one named Naringenin isolated for the first time from Anacyclus cyrtolepidioïdes. This was reinforced by the significant heteronuclear long range correlations: 6 -C 8, -C, 8 -C 9, 8 -C 0,, 5 -C and -C (figure ). 6 8 A C ' B ' ' ' ' ' 5 6 Fig.: eteronuclear multiple-bond correlations for compound 6

4 S. BCIR, Arrows point from proton to carbon Compound was isolated as a white solid from the butanol extract of A. cyrtolepidioïdes fresh flowers. Its Electro Spray Mass spectrum exhibited a pseudomolecular ion peak at [M+] + at m/z 55, revealing a molecular formula of C 6 (spectrum ). The identification of its structure was deduced from and D NMR data (, C, MQC and MBC spectra) (Table I). Significant - C long range correlations -C ; 5 -C ; 5 -C ; 5 -C and 6 -C allowed to confirm the proposed skeleton structure as.-dihydroxybenzoic acid (figure ) signalled for the first time in Anacyclus cyrtolepidioïdes. 5 6 Fig. : eteronuclear multiple-bond correlations for compound Arrows point from proton to carbon Spectrum : ES Mass spectrumof Compound [M+] + Compound Structure of β-sitosterol---β-d-glycoside was characterized by comparison of its NMR spectral data with those reported previously [5]. 6

5 Journal de la Société Algérienne de Chimie Table I. C NMR and NMR spectral data for compounds and Compound (CD D, 00Mz) Position C ; dd ;.0 ;.9 α.5. ; dd ;. ;.0 β.5.0 ; dd ;. ; ; d; ; d; ; d; ; d; ; d; ; d; 8. Compound (CD D, 00Mz) Position C ; d ; ; d ; ; dd ; 8 ; AKNWLEDGEMENTS The authors wish to thank Pr. S. Jegham (Sanofi-aventis-R et D-, Rue du Professeur Josef Bayac- 8 Montpellier Cedex 0, France) for elucidation of Mass Spectra. REFERENCES. G. Potier-Alapetite, Flore de la Tunisie, Publications Scientifiques Tunisiennes, Imprimerie fficielle de la République Tunisienne, Tunis, 98.. M. A. Koheil, Bull. Fac. Pharm. Cairo Univ 000, 8(),.. A. Bergaoui, S. ammami, M. Ben alima Kamel, L. Sakka Rouis,. Boussada, D. ouas, Z. Mighri, J. Entomol. 008, 5,.. M. J. Pascual-Villalobos, A. Robledo, Ind. Crops Prod. 998, 8, A. Ahmed, M.A. Aboul-ELA, A.A. El-Din, Pharmazie 990, 5,. 6. A. Bergaoui, S. ammami,. Ben Jannet, Z. Mighri, Nat. Prod. Res. 00, (8), 88.. A. Z. Bergaoui, F.. Skhiri, S. ammami, I. Cheraif, J. Cheriaa-Issa,. Ben Jannet, Z. Mighri, JEBP 008, (6), A.T. Karoui, S. ammami,. Ben Jannet, M.A. amza, P.Abreu, Z. Mighri, J. Soc. Alg. Chim. 008, 8(), 9. 6

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