International Conference on Emerging Trends in Computer and Image Processing (ICETCIP'2014) Dec. 15-16, 2014 Pattaya (Thailand) Secondary Metabolites from

A. Dibi, A. Jebara, F. Bitam, and M.C. Aberkane.

Abstract— This work is based on the phytochemical study Analysis of the 1H and 13C NMR data MeOD of compound of the species Pallenis spinosa, belonging to the 1and comparison with literature data of sterols derivatives family which grows in , with the aim of discovering strongly suggested a skeleton of a stigmasterol. new naturals therapeutic compounds. In particular, the 1H NMR spectrum indicated six signals The purification and the analysis spectroscopic resulted in the due to the presence of six methyl groups: δH 0.68 H-18 (s), δH structure determination of two sterols: Stigmasterol-3-O-β-D- 1.00 H-19 (s), δH 0.86 H-26 (d, J = 6.7 Hz), δH 0.81 H-27 (d, J glucopyranoside-6’-O-Ester I and β-Sitosterol-3-O-β-D- = 6.7 Hz), glucopyranosyl II. The structures of these compounds are δH 0.84 H-29 (t, J = 6 Hz) and δH 0.91 H-21(d, J = 7.5 Hz)], established by spectral data, including 1D, 2D NMR and mass two olefinic signals due to the presence of a double bond δH spectroscopy. 5.03 (H-23) et 5.17 (H-22). The spectrum showed also the Keywords—Asteraceae, Palenis, Sterols. appearance of signals in the range [3.1- 4.7] corresponding to protons of a glycopyranose moiey: δH 3.30 (H-2’), δH 3.57 (H-3’), δ 3.42 (H-4’), δ 3.44 (H-5’) et H-6’ (δ 4.48, H-6’a I. INTRODUCTION H H H and δH 4.27, H-6’b.² HE Palenis spinosa (L.) belongs to the The Cosy, HSQC and HMBC experiments allowed the TAsteracaea family, and tio the tribe . It is one assignment of carbon and proton resonances as reported in of the important medicinal Algerian which are table I. The down chemical shift of the proton H-3 (δH 3.55, δC commonly used for the treatment of gastralgia, circulatory 79.5), in comparison with non glucosylated compounds problems, contusion, injury, inflammation, mouth infections confirm well the attachment of the glucose part at the carbon and respiratory problems [1-2]. C-3. In the other hand, the down chemical shift of the two The chemical investigations on the genus Palenis have been protons H-6’ (δH 4.48 H-6’a , δH 4.27 H-6’b, δC = 63.5) and 13 reported in the literature data and sesquiterpene lactones are the carbonyl signal in CNMR spectrum at δH = 174.6 suggest the most isolated compounds [2-5]. that the OH group at the C-6’ position is esterified. This idea We report here our chemical study of the chloroform soluble is confirmed by analysing of other signals in the NMR spectra fraction of the aerial parts of Palenis spinosa which yielded to of compound I [δH 2.33 (t, J= 2.3 Hz, H-2’’) / δC 34.2 (C-2’’), the isolation of four sterols. We describe here the isolation and δH 1.61 (m, H-3’’), δC 24.9 (C-3’’), δH 1.19-1.25, δC 29.2-29.7 elucidation structure of the two main sterols of this plant: and 0.88 (s), δC14.1]. These signals are corresponding to the plant: Stigmasterol-3-O-β-D-glucopyranoside-6’-O-Ester I presence of a fatty acid alkyl chain. and β-Sitosterol-3-O-β-D-glucopyranosyl II. These two From the above data, and in comparison with literature data , compounds are isolated here for the first time from the genus compound (I) was deduced to be a stigmastéryl-3β- Palenis. glucopyranoside-6’-O-ester that had been previously reported along with related metabolites from Euphorbia soongarica II. RESULTS AND DISCUSSION Centaurea regia and Euphorbia guyoniana [6- 8]. A mass of 20 g of chloroform extract was subjected to a VLC chromatography in silica gel using petroleum ether and ethyl acetate as elution system to give three main fractions (A, B, and C). Fraction B ( 1.8 g) was subjected to silica gel column chromatography in normal and reversed phase to obtain the pure compound I (5 mg). The fraction C was further purified in the same manner as fraction B to give the pure compound II

(10 mg). Fig. 1 Structure of Compound I

Ammar.Dibi, Laboratoire LCCE, Faculté des Sciences, Departement de Chimie, Université de Batna , Batna 05000 , Algerie. (corresponding author Compound (II) was also isolated as white powder, the to provide phone: +21333868980 ,fax:+21333868980 ; e-mail: molecular formula C29H36O11was deduced by the ESIMS [email protected]). spectrum in positive and negative mode that showed the Fatma .Bitam , LCCE, Departement de Chimie, Faculté des Sciences sodiated molecular peaks at m/z 599.3 [M + Na]+ and 575 [M- Université de Batna , Batna, Algerie; (e-mail: [email protected] ). - Amira. Jebara, LCCE, Departement de Chimie, Faculté des Sciences H] . The NMR spectra of this compound present similar signals Université de Batna , Batna, Algerie; (e-mail : [email protected] in comparison of those of compound I, according to the Mohamed.Cherif. Aberkane: Departement de Chimie, Faculté des presence of the same sterol skeleton. Some differences are Sciences Université de Batna, Batna, Algerie; (e-mail: [email protected] observed in the NMR spectrum of compound II, it showed the

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International Conference on Emerging Trends in Computer and Image Processing (ICETCIP'2014) Dec. 15-16, 2014 Pattaya (Thailand) absence of the signals of the protons of the double bond at the 28 29 21 22 position C22-C23, and the absence of the esterification of the 26 24 18 20 25 OH group at the position C-6’ of the glucose linkage. 23 12 The signals of six methyls groups are resonated at [δH 0.71 H- 17 11 27 19 13 18 (s), δH 1.03 H-19 (s), δH 0.94 H-21 (d, J = 6.7 Hz), δH 0.86 16 1 9 H OH H-26 (d, J = 6.7 Hz), δ 0.84 H-27 (d, J = 6 Hz) and δ 0.87 H- 14 H H 2 10 8 15 6' H 29 (t, J = 7.5 Hz) and the prton of H-3 is resonated at δH 3.63. 4' O 5' 3 HO 7 these chemical data are in good agreement of a β-Sitostérol 5 HO 2' O 4 6 derivative. The presence of the glucopyranose part is H 1' 3' OH confirmed by the signals of the carobns resonated at δC 101.0 H H (C-1’), (δC 70.1 C-4’), (δC 73.5 C-2’), (δC 76.2 C-5’) (δC 76.5 Fig. 2 Structure of compound II C-3’), and (δC 61.4 C-6’). TABLE II In comparison with the chemical data reported in the literature 1 13 H AND C CHEMICAL SHIFTS OF THE COMPOUND II IN CDCL3 (Table II), compound II is determined to be Daucostérol[175] (β-sitostérol-3β-D-glucopyranosyl), a common compound in Position δ (ppm) δ (ppm) ; m, J (Hz) C H the Asteraceae family, in particular in Centaurea genus[9-10]. 1 CH2 37.1 1.89 m H-1α 1.08 m H-1β TABLE I 1 13 2 CH2 29.3 1.61 m H-2β ; 1.92m H-2α H AND C CHEMICAL SHIFTS OF THE COMPOUND II IN MEOD 3 CH 78.6 3.63 m 4 CH2 38.3 2.27 tl 13 H-4 β ; Position δC (ppm) δH (ppm) ; m, J (Hz) 2.43 dm 13; 2 H-4α

1 CH2 37.3 3.46 d 3.5 5 C 140.3 - 2 CH2 29.5 1.95 m 6 CH 121.5 5.38 dl 5 3 CH 79.5 3.55 m 7 CH2 31.7 1.45 -2.00 m 4 CH2 38.9 2.36 m 8 CH 31.6 1.97 m 5 C 140.8 - 9 CH 50.1 0.98 m 6 (=CH) 122.1 5.38 dl 10 C 36.4 - 7 CH2 31.9 1.46-1.90 m 11 CH2 20.8 1.50 m H-11α,1.02 mH-11β 8 CH 31.9 1.38 m 12 CH2 39.6 1.10 m H-12α -2.1 mH-12 β 9 CH 50.2 0.85 m 13 C 42.0 - 10 C 36.1 - 14 CH 56.6 1.06 m 11 CH2 21.1 1.36-1.42 m 15 CH2 25.7 1.52 m H-15α ;1.02m H-11β 12 CH2 39.8 1.08-1.94 m 16 CH2 28.0 1.87 m H-16α ;1.27m H-16β 13 C 42.3 - 17 CH 55.9 1.14 m 14 CH 56.1 0.91 m 18 CH3 12.2 0.71 s 15 CH2 24.3 0.93-1.33 m 19 CH3 19.1 1.03 s 16 CH2 28.2 1.80-2.00 m 20 CH 35.9 1.36 m 17 CH 56.9 1.03 m 21 CH3 18.3 0.94 d 6.7 18 CH3 11.8 0.68 s 22 CH2 33.7 1.34 m H-22α ;1.06 m H-22β 19 CH3 19.3 1.00 s 23 CH2 25.7 1.19 m 20 CH 36.7 1.28 m 24 CH 45.7 0.95 m 21 CH3 19.0 0.91 d 7.5 25 CH 29.3 1.68 m 22=CH2 138.2 5.17 m 26 CH3 18.7 0.86 d 6.7 23=CH2 129.3 5.03 m 27 CH3 19.1 0.84 d 6 24 CH 51.2 0.93 m 28 CH2 22.7 1.27 m 25 CH 31.9 1.59 m 29 CH3 12.2 0.87 t 7.5 26 CH3 19.0 0.86 d 6.7 27 CH3 19.8 0.81 d 6.7 Glucose 28 CH2 25.4 1.26 m 1’ CH 101.0 4.43 d 7.8 29 CH3 12.2 0.84 m 2’ CH 73.5 3.20 t 7.9 Glucose 3’ CH 76.5 3.35 m 1’ CH 101.2 4.37 d 7.4 4’ CH 70.1 3.39 m 2’ CH 73.6 3.30 t 8 5’ CH 76.2 3.28 m 3’ CH 76.0 3.57 t 8 6’ CH2 61.4 3.88 d H-6’α 4’ CH 70.2 3.42 t 8 3.71dd 11.9 ; 4.9 H-6’β 5’ CH 74.0 3.44 m

6’ CH2 63.1 4.48 dd 5-12.2 H-6’a 4.27 d 11.1 H-6’b Ester A. General experimental procedures 1’’ C 174.6 - Silica-gel chromatography was performed using pre-coated 2’’ CH2 34.2 2.33 t 7.9 3’’ CH2 24.9 1.61 m Merck F254 plates Merck Kieselgel 60 powder and n’’ CH2 29.2-29.7 1.19-1.26 m Lichroprep RP-8 Merck (40-63 μm). 1H and 13C NMR and (n+1)’’ CH3 14.1 0.88 t 7.1 2D NMR experiments spectra were obtained on a Bruker

Advance spectrometer DRX-500 in CDCl and MeOD (500 3 MHz and 125MHz, respectively). were performed using

standard Bruker microprograms (XWIN-NMR version 2.6

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International Conference on Emerging Trends in Computer and Image Processing (ICETCIP'2014) Dec. 15-16, 2014 Pattaya (Thailand) software and TOPSPIN 1.3). Positive and negative ESI mass [4] Ahmed A., Jakupovic J. Dihydroxypallenone, a sesquiterpene with a New carbon Skeleton from Pallenis spinosa .Phytochemistry, vol. 29, spectra were performed using a Micromasse Q-TOF. pp. 3355-3358, 1990. [5] Ahmed, A. A. Mark, S. A sesquiterpene alchool from Pallenis. Biochem. III. EXEPERIMENTAL PART System. Ecol, vol. 20, pp. 785-786, 1992. [6] Guo, D.A. Venkatramesh, M Developmental regulation of sterol A. Plant material biosynthesis in Zea mays. Lipids, vol. 30, pp. 203-219, 1995. [7] Volkman, J.K. Sterols in microorganism. Appl. Microbiol. Biotechnol, Pallenis spinosa was collected in June 2009 from the vol. 60, pp. 495-506, 2003. region of Batna (Eastern Algeria). It was identified by [8] Chappel, J. The genetics and molecular genetics of terpene and sterol Professor Bashir OUDJEHIH from the Institute of Agronomy origami. Curr. Opin. Plant Biol, vol. 5, pp. 151-157, 2002. of University of Batna (Algeria). The dried plant material was [9] Flamini, G., Pardini, M., Morelli, I. A flavonoid sulphate and othercompounds from the roots of Centaurea bracteata. Phytochemistry, pulverized and the powder vegetal material was subjected to vol. 58, pp. 1229-1233, 2001. extractions with different solvents. [10] Ramdane, S., Ouahiba, B., Ratiba, M., Flavonoid with cytotoxic activity and other constituents from Centaurea africana. Phytochemistry, vol. 2, B. Extraction and isolation pp. 114–118, 2009. Dried and powdered aerial parts (2 kg) of Pallenis spinosa [11] Panda, S.; Jafri, M.; Kar, A.; Meheta, B.K. Thyroid inhibitory, antiperoxidative and hypoglycemic effects of stigmasterol isolated from were macerated in chloroform (15l) to give a crude Buetamonosperm. Fitoterapia, vol. 80, pp. 123-1262, 2009. chloroform extract (40g). A part of this extract was subjected to VLC chromatography in silica gel (gradient light petroleum ether/EtOAc) to give three main fractions. Purification of two of these fractions using a series of chromatography in normal phase (silica gel) and reversed phase (RP-18) allowed us to obtain four sterols. The two main compound (I, II) (5mg and 10mg) are identified using NMR spectroscopic techniques), C. Compound I

Stigmastéryl-3β-glucopyranoside-6’-O-Ester

Amorphous powder; Rf = 0.5 (90 /10 : CHCl3/ MeOH ) 1H and 13C NMR ( MeOD) see Table I

D. Compound II β-Sitosterol-3-O-β-D-glucopyranosyl Amorphous powder; Rf = 0.5 (90 /10 : CHCl3/ MeOH ) ESI : m/z 599.3 [M+Na], m/z 575 [M-H]- 1H and 13C NMR (CDCl ) see Table II. 3

IV. CONCLUSION

This is the first chemical characterization of the phytosterols components of the Algerian plant Palenis spinosa. These molecules are identified here for the first time from the genus Palenis and the medicinal activities of this species is mostly related to these compounds since sterols have been reported for many biological activities [11].

ACKNOWLEDGMENT We thank the staff of NMR of the institute of chemistry of the University of Strasbourg (France), for their help and availability by allowing us to achieve NMR spectra.

REFERENCES [1] Benitez, G; Gonzalez, M.R. & Mallero, M. J. Pharmaceutical ethnobotany in the western part of Granada. Journal of Ethnopharmacology, vol. 129, pp. 87-105, 2010. [2] Giovanni A.; Jasmin J. ,Sven J.. Sesquiterpenoids from Pallenis spinosa. Phytochemistry. 46, 1039-1043, 1997. [3] Sanz,JI.; Marcoj. A.A Germacrane, Derivative from Pallenis Spinosa. Phytochemistry. 30, 2788-2790, 1991.

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