International Conference on Chemical, Environmental and Biological Sciences (ICCEBS'2012) Penang, Malaysia

Phytochemical Study of the Arborescens

Ammar.Dibi, Fatma.Bitam

We report here the chemical investigation of petroleum Abstract—The chloroform extract of the aerial parts of the ether and methanol extracts of the aerial part of Launaea was separated by repeated silica gel wcich lead to the isolation of one triterpene and one lighane column chromatography to give two naturals pure compounds: lactone; not previously reported from the genus Launaea and lignane lactone and the taraxast-20-ene-3β,30-diol 2. The structures the tribe Lactuceae. elucidation are followed by exploitation of the 1HNMR, 13CNMR, and 2D NMR (COSY , HMBC , and HSQC) spectroscopic methods as well as by the mass spectroscopy. These two compounds are II. RESULTS AND DISCUSSION detected here for the first time in this genus. The dried aerial parts of the Launaea arborescens were extracted with petroleum ether, chloroform and methanol Keywords—, Launaea, Lignans, Triterpenes. solvents. The petroleum ether extract was found to be dominated by triterpenes. The extract was submitted to I. INTRODUCTION subsequent chromatographic steps to obtain the pur compound: taraxast-20-ene-3β,30-diol (I). In the same way, The genus Launaea belongs to the tribe Lactuceae of the the purification of the ethyl extract gave an arylnaaphtalene lignan (II), named previously chinensin. Asteraceae family and contains about 40 species. In the flora of , five of the nine Launaea species present are The molecular formula of compound I was deduced to be endemic of north Africa and include Launaea arborescens C30H50O2 by both the EIMS (electronic impact mass spectrum) displaying a molecular ion peak at m/z 442 and the (Batt.) Murb, synonym Zollikoferia spinosa DC[1]- [2]. 13 In Algeria, it grows in the area from the South to Beni- C NMR spectrum exhibiting 30 signals (seven methyls, ten methylenes, seven methines, and six quaternary carbons). Abbés and Tademait [2]. 1 The plant is appreciated by livestock, mainly by camel. It’s The H NMR spectrum of compound (I) disclosed the known as a medicinal plant being used as an antidiarrhoic and presence of six tertiary methyl groups [δ 0.75 (3H, s, H3-28), antispasmodic. Its milk is used in the treatment of the skin 0.76 (3H, s, H3-24), 0.85 (3H, s, H3-25), 0.95 (3H, s, H3-27), diseases. 0.96 (3H, s, H3-23), and 1.04 (3H, s, H3-26)], a secondary In the frame of a study by a Spanish group of three species methyl [δ 1.01 (3H, d, J = 6.5 Hz, H-29), and a vinyl proton at of the genus Launaea, including L. arborescens, the aerial δ 5.58 (1H, dd, J = 6.5 and 1.8 Hz, H-21). The 1H NMR parts of this species was found to contain eight common spectrum contained also an AB system δ 4.13 (1H, d, J = 12.3 phenolic compounds including three flavonoids, luteolin, Hz, H-30α) and δ 4.02 (1H, d, J = 12.3 Hz, H-30β), which was luteolin-7-O-glucoside, luteolin-7-O-rhamnoside, two attributed to an isolated hydroxyl methylene group, along with coumarins, aesculetin and its glycoside cichoriin, and ethyl- the typical carbinol signal at δ 3.21 (1H, dd, J = 11.2 and 4.9 caffeoate and ferulic acid [3]. It was also found that the other Hz, H-3). These spectral data suggested that compound (I) is two species L. acanthoclada and L. residifolia were an unsaturated pentacyclic triterpene with two hydroxyl characterized by the same phenolic content and that cichoriin groups. was the most abundant compound in all samples [3]. The comparison of the chemical data with those of In addition, very interesting antifungal, antibacterial and pseudotaraxasterol [6], showed that the chemical shift values insecticidal activities have been reported for the methanol were almost identical strongly supporting the location of the extract of the plant [4 ]-[5]. double bond and the hydroxymethylene group in the ring E, the same as pseudotaraxasterol. Thus, compound (1) should be a derivative of ψ-taraxasterol. In the 1H-1H COSY spectrum, the vinyl signal H-21 showed Ammar. Dibi :Laboratoire LCCE, Faculté des Sciences, Departement de cross-peak correlations with two non-equivalent methylene Chimie, Université de Batna , Batna 05000 , Algerie. (corresponding author proton signals at δ 1.64 (1H, m, H-22a) and δ 1.78 (1H, m, H- to provide phone: +21333868980 fax:+21333868980 ; e-mail: [email protected]). 22β), and with the hydroxyl methylene H2-30 by allylic Fatma .Bitam : LCCE, Departement de Chimie, Faculté des Sciences coupling. The methyl doublet at δ 1.01 (H3-29) was correlated Université de Batna , Batna, Algerie; (e-mail: [email protected] ). with a methine proton signal at δ 1.88 (H-18), which in turn

51 International Conference on Chemical, Environmental and Biological Sciences (ICCEBS'2012) Penang, Malaysia

+ showed a coupling with the hydroxyl methylene H2-30. and 403.1, which correspond respectively to [M + Na] ,[2M + Significant HMBC correlationswere observed between the Na]+, and [M + K]+. quaternary sp2 carbon at δ 143.7 (C-20) and the isolated TABLE I hydroxyl methylene H2-30 thus confirming the presence of the 1H and 13C chemical shifts of the compound I in CDCl fragment -CH(CH )-C(CH OH)=CH-CH - in the ring E. 3 3 2 2 These data led us to identify compound (I) as taraxast-20-ene- Position δ 13C δ 1H m, J (Hz) HMBC ( C to H ) 3β,30-diol (Fig. 1). 1 38.7 0.91 m H-2 The interpretation of 1H-1H COSY, HSQC and HMBC 1.73 m - experiments allowed all proton and carbon assignments (Table 2 27.6 1.54 m - 1.55 m I). 3 79.1 3.23 dd (11.2, 4.9) H-23, H-24, H-1 Derivatives of ψ-taraxasterol oxygenated at C-30 are rare in 4 38.9 - - H-3, H-5, H-23, H-24 nature. To date, only three compounds including compound (I) 5 55.3 0.76 m H-1, H-7, H-23, H-24, H- 6 18.3 1.54 m 25 have been reported from the [6] (Shiojima et al., 1.45 - - 1996). In particular, taraxast-20-ene-3β,30-diol (I) was isolated 7 34.2 1.38 m - from Ixeris chinensis from the Lactuceae tribe (Shiojima et al., - - H-6, H-11, H-26 8 41.1 - - - 1996), from the whole plant of Saussurea petrovii belonging to 9 50.4 1.36 dd (11.7, 4.7) H-7, H-9, H-26 the Asteraceae family [7], and from the aerial parts of Picris 10 37.1 - - H-7, H-12, H-25, H-26 evae of the Lactuceae tribe[8]. 11 21.6 1.27 m H-1, H-5, H-9, H-25 1.59 m H-11 Our findings confirmed the typical occurrence of this 12 27.1 0.92 - compound in the Lactuceae tribe. H-11, H-27 13 39.2 1.65 m H-9, H-12, H-27 OH 14 42.4 - - H-13, H15, H-27 30 15 27.4 1.11 m - 29 20 1.68 m - 16 36.7 1.30 m H-28 19 17 34.5 1.35 - H-16, H-18, H-22, H-28 11 22 18 48.5 1.25 m H-28 25 26 19 32.1 1.88 m H-21 1 14 27 - m - 16 10 20 143.7 - - H-19 ,H-21, H-29, H-30 3 21 120.7 5.58 br d (6.2) H-30 HO 22 41.7 1.64 m H-21 6 1.78 m - 24 23 23 15.4 0.96 s H-24 24 27.9 0.76 s H-23 25 16.3 0.85 s - Fig. 1 Structure of Compound I 26 16.0 1.04 s H-7 27 14.8 0.95 s H-15 The second compound was isolated as a crystal. It had the 28 17.7 0.75 s H-18 29 22.5 1.01 d (6.5) H-18 molecular formula as deduced by both ESIMS and 13C NMR 30a 65.5 4.02 d (12.3) - spectra. 30b - 4.13 d (12.3) H-21 Compound (II) was identified as 6,7-methylenedioxv-]-

(3′,4′-dimethoxyphenyl)-3-hydroxymethylnaphthalene-2- These data are consistent to the molecular formula C21H28O8 carboxylic acid lactone by spectroscopic data that were (molecular weight 364). collected by recording the 1H NMR, HSQC, HMBC and the From the above data, we strongly suggested a lignan mass spectra. structure exhibited an aryl naphthalene skeleton. The structure In the NMR spectrum of compound (II), we observe two was fully confirmed by some 2D experiments (HSQC, and signals of two protons which resonated at δ 5.35 and HMBC). characterised of the presence of 1-aryl-2,3-naphtalene [9], and A survey of the literature on the structures of some lignans another signal integrated for two protons and resonating at those exhibited an arylnaphtalene skeleton indicates that δ6.09 indicated the presence of a methylenedioxy group [9]. compound (II) had strong similarities with chinensin, a lignan The spectrum present some signals in the range of δ [6.86- which was isolated from Polygala chinensis [10] and some 7.69] which were attributed to the presence of a series of some species of publeurum belonging to the umbelliferea family aromatic protons. Finally, the spectrum showed the presence of [11]. two strong signals resonated at δ3.85 and δ3.95 indicated the Thus the structure of compound (II) is chinensin (Fig. 2). presence of two methoxyl group linked to the aromatic ring. In the 13C NMR spectrum in modulated J, we deduce easily eleven quaternary carbons, six methines, two methylene and two methoxyl groups. The ESIMS spectrum of compound (II) recorded in positive mode showed three peaks at 387.1, 751.1,

52 International Conference on Chemical, Environmental and Biological Sciences (ICCEBS'2012) Penang, Malaysia

5 4 3a O 6 10 3 fine powder .The masse of 900 grammes was extracted with O different organic solvents, first with petroleum ether, then 1 2a O 7 9 2 with ethyl acetate, and finally with n-Butanol. 8 1' O Those wich exhibited the same compounds, as judged from 2' 6' TLC, were combined into five fractions. 3' The treatment of the petroleum ether extract (10 g) on a 5' OCH 4' 3 silica gel column with petroleum ether and gradient petroleum OCH3 ether–ethyl acetate allowed 132 fractions. Fraction 90-104 Fig. 2 Structure of Chinensin presents on TLC an invisible spot and which appears by sulphuric vanillin in mauve. This fraction is subjected to TABLE II 1 13 repeat chromatography purifications on silica gel column H and C chemical shifts of the compound II in CDCl3 using pure CH2Cl2 as eluant, to afford the pure compound I

Position δ 13C δ 1H m, J (Hz) HMBC ( C to H) (10 mg). 1 140.4 - - - In the same way, the ethyl acetate extract was 2 118ff.7 - - H-4 prefractionated on silica gel colunm chromatography Merck 2a 169.8 - - H3a 60 (230-400 mesh), using gradient of dichloro methane in 3a 67.9 5.35 s - 3 79.1 3.23 - H-3a methanol to afford twelve main fractions (F1-F12); the 4 118.9 7.69 s H-5, H-3 fraction F6 which present some uv visible spots under the 5 103.6 7.20 s H-4 wave length 254 was subjected to a series of purification in 6 148.9 - - CH O , H-8, H-5 2 2 silica gel column chromatograph. The fraction eluted with the 7 149.9 - - CH2O2, H-8, H-5 8 103.7 7.12 s - gradient CH2Cl2/CH3OH (95:5) gave the pur compounds II 9 130.5 - - H-4 (50 mg). 10 134.6 - - H-4, H-8 1’ 127.1 - - H-5’ 2’ 113.3 6.86 d (1.9) OCH3(3’) C. Compound I 3’ 148.5 - - OCH3(3’) 4’ 148.6 - - OCH3(4’) Taraxast-20-ene-3β,30-diol: white crystals ; [α]D = -10 5’ 110.7 7.03 d (8.2) OCH3(4’) IR KBr ν max cm -1: 3424, 2929, 2856, 1456, 1426. 6’ 122.4 6.91 dd (8.2,1.9) H-2’ OCH3(3’) 55.9 3.85 s - EI-MS m/z ( rel. Int.): 442 (100%), 1H NMR ( CDCl3); see OCH3(4’) 55.8 3.95 s - table I CH2O2(C6, C7) 101.7 6.09 s - D. Compound II 6,7-methylenedioxy-(3′,4′-dimethoxyphenyl)-3-hydroxymethy lnaphthalene-2-carboxylic acid lactone : white crystals A. General experimental procedures UV MeOH λ max nm: 220, 255, 310 and 350. IR KBr ν max cm -1: 1762, 1615, 1467, and 934. Silica-gel chromatography was performed using pre-coated 1H NMR (CDCl ); see table II. Merck F254 plates and Merck Kieselgel 60 powder. 1H and 3 13C NMR spectra were obtained on a Bruker Advance IV. CONCLUSION spectrometer and Bruker Advance 2 spectrometer equiped The chemical investigation of the areal part of the Algerian with dual cryosonde in CDCl3(500 MHz and 125MHz, respectively). 2D NMR experiments were performed using plant Launaea arboresens lead to the isolation of a triterpene standard Bruker microprograms (XWIN-NMR version 2.6 and a lignan lactone, these finding were consistent with the software and TOPSPIN 1.3). Positive and negative mass previous studies on different species of the same tribe spectra were performed using a Bruker Esquire Ion trap. reporting related terpenoid compounds.

III. EXPERIMENTAL PART ACKNOWLEDGMENT We thanks the research centre on natural products, UMS, A. Plant material CNRS 2597 (Toulouse, France), and the staff of NMR of the The plant L. arborescens was collected in April 2002 in institute of chemistry of the university of Strasbourg (France), Bechar (Laabadla, South of Algeria) and identified by Prof. for their help and availability by allowing us to achieve the Bachir Oudjehih, Institute of Agronomy of University of mass and NMR spectra. Batna (Algeria). A voucher specimen is deposited in the herbarium of the department of thesame University under the REFERENCES number code 423/HIAB mass). [1] P, Quezel, S. Santa, « In: Nouvelle Flore de l’Algérie et des Régions Désertiques Méridionales » , vol. 1–2, CNRS, Paris, 1963, p. 1162. B. Extraction and isolation [2] P, Ozenda, « Flore et Végétation du Sahara », CNRS, Paris, 2004, p. The aerials parts of launaeaa arboresens were dried in the 662.

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