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The Chemical Constituents of CapparisSpinosa of Jordanian OriginMonther A. Khanfar a , Salim S. Sabri b , Musa H. Abu Zarga a &Klaus-Peter Zeller ca Chemistry Department , University of Jordan , Amman, 11943,Jordanb Faculty of Arts and Sciences , University of Sharjah , P.O. Box27272, UAEc Institut für Organische Chemie , Universität Tübingen , Auf derMorgenstelle 18, Tübingen, D-72076, GermanyPublished online: 27 Oct 2010.

To cite this article: Monther A. Khanfar , Salim S. Sabri , Musa H. Abu Zarga & Klaus-Peter Zeller(2003) The Chemical Constituents of Capparis Spinosa of Jordanian Origin, Natural Product Research:Formerly Natural Product Letters, 17:1, 9-14, DOI: 10.1080/10575630290034302

To link to this article: http://dx.doi.org/10.1080/10575630290034302

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Natural Product Research, Vol. 17, No. 1, pp. 9–14

THE CHEMICAL CONSTITUENTS OF CAPPARISSPINOSA OF JORDANIAN ORIGIN

MONTHER A. KHANFARa,z, SALIM S. SABRIb,y, MUSA H. ABU ZARGAa,*and KLAUS-PETER ZELLERc

aChemistry Department, University of Jordan, Amman, 11943, Jordan; bFaculty of Arts andSciences, University of Sharjah, P.O. Box 27272, UAE; cInstitut fur Organische Chemie,

Universitat Tubingen, Auf der Morgenstelle 18, D-72076 Tubingen, Germany

(Received 17 March 2002; In final form 14 April 2002)

Investigation of Capparis spinosa of Jordanian origin lead to isolation of two new compounds �-sitosterylglu-coside-60-octadecanoate (1) and 3-methyl-2-butenyl-�-glucoside (2). Linked Scan MS measurements were usedto propose a mass fragmentation pattern for the alkaloid Cadabicine isolated here for the second time fromnature.

Keywords: Capparis spinosa; Capparedaceae; 3-methyl-2-butenyl-�-glucoside; �-sitosterylglucoside-60-octa-decanoate; Cadabicine

INTRODUCTION

Root bark ofCapparis spinosa (L.) (Caper spinosa (L.)) (Capparedaceae orCappareaceae)is known in folk medicine to possess aperient, tonic, duratic, expectorant, antihelmintic,emmenagogue and analgesic effect [1,2]. Also it is reported that the juice of the freshplant kills the worms in the ear [2] and the dried leaves steeped in vinegar are usedfor treatment of ulcers [3]. Previous studies on this plant led to isolation of stachydrine[4], polyisoprenoid alcohols [5], flavonoids [6–9], and glucosinolates [10]. We reporthere the isolation and characterization of two new compounds from C. spinosa.These are �-sitosterylglucoside-60-octadecanoate (1) and 3-methyl-2-butenyl-�-gluco-side (2). They were identified and characterized by spectral data and chemical evidence.In addition fifteen known compounds were isolated from the plant during thisstudy. These are: isorhamnitine-3-O-rutinoside, 1-tetradecanol, p-hydroxybenzalde-hyde, 6,10,14-trimethyl-2-pentadecanone, ursolic acid, glycerol monotetracostanoate,4-coumaric acid, nicotinamide, methyl hexadecanoate, �-sitosterol, �-sitosterylgluco-side, cadabicine, octadecanoic acid, rutin, and stachydrine. The first seven compoundsare reported for the first time from the genus Capparis. This is the second report of the

zPresent address: Chemistry Department, Hashemite University, Zarqa, 13115, Jordan.yOn leave from the University of Jordan.*Corresponding author.

ISSN 1478-6419 print: ISSN 1478-6427 online � 2003 Taylor & Francis Ltd

DOI: 10.1080/10575630290034302

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sperimidine-type alkaloid, cadabicine (3), from a natural source [11]. The last threecompounds have been previously reported from C. spinosa [4,7,12].

RESULTS AND DISCUSSION

Compound 1 was isolated as pure solid. FAB-MS spectrum shows a quasi-molecularion peak at m/z 865 [MþNa]þ which corresponds to the molecular formula[C53H94O7þNa]þ. Also the spectrum shows a peak m/z 267, which is due to thefatty acyl residue.The 1H-NMR (CDCl3) and proton decoupled 13C-NMR (CDCl3) spectra show

great similarities with those of octadecanoate and �-sitosterylglucoside, which havebeen separated from the plant. 1H-NMR shows a huge peak between 1.2–1.3 ppmcorresponding to the fatty acid chain protons. A triplet at 2.34 ppm (J¼ 7.5Hz)corresponds to the �-methylene protons of the fatty acid. DEPT experiments confirmthe presence of the terminal methyl group of the fatty acid at 14.1 ppm in additionto the six methyls of the aglycone. A peak at 174 ppm in the 13C-NMR indicates thepresence of the ester carbonyl.The � values of the different carbons of the sugar moiety are comparable to those

in �-sitosterylglucoside, except for C60 signal, which is shifted downfield (63.9 ppm)relative to the corresponding signals in �-sitosterylglucoside (61.0 ppm). This confirmsthe location of the octadecanoate group at C60.Acidic hydrolysis of compound 1 produced �-sitosterol, glucose, and octadecanoic

acid as compared with authentic samples. Also the basic hydrolysis of this compoundproduced octadecanoic acid and �-sitosterylglucoside as indicated by TLC usingdifferent solvent systems. Confirmation of the structure of the acid was achieved byEIMS of the free acid (m/z 284 [M]þ) and its methyl ester (m/z 298 [M]þ).Acetylation of compound 1 afforded a triacetyl derivative as indicated by 1H and

13C-NMR spectra. It is noteworthy that the multiplet corresponding to C60-methylenein 1H-NMR (�¼ 4.25 ppm) was not shifted upon acetylation which further confirms thelocation of the octadecanoate group at C60.Compound 2 was obtained as amorphous solid. FAB-MS shows quasi-molecular ion

peak at m/z 249 corresponding to the ion [C11H20O6þH]þ. Also the spectrum showstwo peaks at m/z 271 and 519 which correspond to the [MþNa]þ and [2MþNa]þ,respectively.

1H-NMR (DMSO-d6) shows two sharp singlets each integrating to three protons at1.57 and 1.65 ppm which are assigned for the two methyl groups protons. The oneproton multiplet at 5.22 ppm corresponds to the olefinic proton while the peakat 4.64 ppm integrating for two protons is assigned to the allylic methylene group.

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A number of multiplets in the region 3–5 ppm was indicative of the presence of thesugar residue.Proton decoupled 13C-NMR and DEPT experiments (DMSO-d6) show two methyl

carbons at � 18.3 and 25.9 ppm corresponding to C4 and C4a. The peak at �64.8 ppm is assigned to the allylic carbon. The olefinic methine and aglycon quaternarycarbon resonate at � 121.5 and 136.0 ppm, respectively. In addition the 13C-NMRconfirms the presence of the hexose unit.Compound 2 gave upon acetylation a tetraacetyl derivative, as indicated by 1H- and

13C-NMR. Hydrolysis of 2 gave glucose as compared with an authentic sample by TLCusing different solvent systems. The above spectral and chemical transformation datawas consistent with the �-glucoside of 3,5-dimethylallyl alcohol 2.The �-glycosidic linkage was conferred from the coupling constant of the anomeric

proton (J¼ 7Hz).The [MþH]þ ion of cadabicine 3 in the ESI mass spectrum is found at m/z 436 (35%).

Its collision induced fragmentation produced a MS/MS spectrum exhibiting fragmentsat m/z 419 (22), 362 (7), 348 (92), 320 (12), 291 (100), 265 (49), 263 (43), 203 (61).The key step in this fragmentation pattern (see Scheme 1) is cleavage of the bonds

between the ammonium–N-atom and the adjacent carbon atoms by a �-eliminationprocess. The two isomeric ring-opened ions can then eliminate 1,3-diaminopropaneand 1,4-diaminobutane, respectively, thus forming the ions at m/z 362 and 348.Alternatively, NH3 can be eliminated with formation of the fragment at m/z 419.Loss of 3-aminopropane from the m/z 348 ion produces the fragment at m/z 291. Thefragment at m/z 203 results from cleavage of two bonds as indicated in Scheme 1.

SCHEME 1

CHEMICAL CONSTITUENTS OF CAPPARIS SPINOSA 11

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EXPERIMENTAL

General Experimental Procedures

Melting points were measured at an Electrothermal Mel-Temp apparatus andare reported without correction. MS spectra were obtained at 70 eV using a TSQ70-Triple Quadrapole Instrument. Electrospray-MS/MS data have been obtained ona API III triple quadrapole mass spectrometer (Sciex). The samples have beenintroduced by direct loop as solutions in methanol with a drop of trifluoroaceticacid. MS parameters: ion spray voltage, 4800V; orifice voltage, 70V. The data havebeen acquired and processed with a Sciex MassSpec data system. 1H-NMR spectrawere carried out on a Bruker DPX-300MHz in CDCl3, DMSO-d6 using TMS asinternal standard, 13C-NMR spectra were recorded at 75.4767, using same instrument.

Plant Material

C. spinosa was collected in the region of Mahes 20–25 km north of Amman-Jordan inJune 1993, and identified by Prof. Dawud Al-Eisawi (Department of BiologicalSciences, Faculty of Science, University of Jordan). A voucher specimen was depositedat the herbarium of the University of Jordan, Amman-Jordan.

Extraction and Isolation

The dried ground plant material (18 kg) was defatted in petroleum ether at roomtemperature and then extracted extensively with ethanol (4 times). After evaporationof the solvent, the concentrated ethanolic extract (ca. 1300 g) was partitioned betweenchloroform and water. The dried chloroform layer was further fractionated betweenn-hexane and 10% water/methanol. The aqueous methanol soluble fraction (A: 530 g)was separated into ten main fractions (AI–AX) using CC silica gel (70–230mesh) and agradient of methanol/chloroform as eluent. Further purifications of AI (17.9 g) by CCon silica gel (400mesh) using hexane/chloroform then chloroform/methanol mixturesas eluent and TLC afforded 1-tetradecanol (350mg), hexadecanoate methyl ester(550mg) and octadeanoic acid (250mg). Chromatography of AII (29.5 g) on a silicagel column, using the same solvent mixtures of AI gave 6,10,14-trimethyl-2-pentadeca-none (300mg), �-sitosterol (550mg) and glycerol monotetracostanoate ester (3.0 g). TheCC of AIV (83.3 g) over a silica gel column using a gradient of benzene/acetone andTLC afforded p-hydroxybenzaldehyde (30mg) and ursolic acid (350mg).Chromatography of Fraction AVI (63.3 g) on a silica gel column using a gradientof methanol/chloroform as eluent and TLC afforded �-sitosteryl-glucoside-60-octade-canoate (1) (200mg), whereas the chromatography of fraction AVII (126.9 g) gave�-sitosterylglucoside (9.0 g).The polar organic compounds were extracted from the water layer by n-butanol. The

n-butanol extract (250 g) was separated into nine main fractions (BI–BIX), using CCsilica gel (70–230mesh) and a gradient of methanol/chloroform as eluent. Fractionsof BI–BVIII were further purified by CC on silica gel (400mesh) using methanol/chloroform mixtures as eluent and TLC. Fraction BII (8.2 g) afforded coumaric acid(300mg), fraction BIII (5.8 g) gave nicotinamide (70mg), fraction BV (23.4 g) afforded3-methyl-2-butenylglucoside (2) (400mg), fraction BVII (22.5 g) gave cadabicine

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(80mg) and isorhamnitine-3-O-rutinoside (1.5 g), whereas fraction BVIII (31.5 g)afforded rutin (15 g) and cadabicine. Fraction BIX (77.7 g) was chromatographedusing the system chloroform/methanol/water (65/27/8) as eluent to give stachydrine(300mg).

�-Sitosterylglucoside-6 0-octadecanoate (1) Amorphous solid, FAB-MS: m/z 865[MþNa]þ, 575 (M-octadecanoate]þ, 429 [M-gluc-octadecanoateþCH5]

þ. 1H-NMR.(CDCl3, 300MHz): � 0.65 (3H, s, C18-H), 0.80–0.98 (overlapped doublet, methyls),1.25 (huge broad, fatty acid chain), 1.47–2.00 (complex CH2 and CH), 2.34 (2H, t,J¼ 7.5Hz, CH2COO), 3.36 (1H, m, C3-H), 3.51–4.37 (several multiplets, sugarmoiety), 5.34 (1H, m, C6-H). 13C-NMR (CDCl3, 75.5MHz): � 11.9, 12.0, 14.1, 18.8,19.0, 19.8, 21.1, 22.7, 23.1, 24.3, 25.0, 26.2, 28.3, 29.2, 29.4, 29.6, 29.7–29.8, 31.88,31.95, 34.0, 34.3, 36.2, 36.7, 37.3, 38.9, 39.8, 42.3, 45.8, 50.2, 56.2, 56.8, 63.9, 70.6,73.4, 73.7, 76.4, 79.8, 101.3, 122.0, 140.4, 174.1.

�-Sitosterylglucoside-6-octadecanoate triacetyl (1a) Compound (1) was acetylatedusing acetic anhydride in pyridine. The product was purified by TLC to give 1a;1H-NMR: (CDCl3, 300MHz): � 2.00 (3H, s, CH3COO), 2.02 (3H, s, CH3COO), 2.05(3H, s, CH3COO).

13C-NMR (CDCl3, 75.5MHz): � 68.7, 70.5, 71.5, 71.8, 72.9, 80.1,169.3, 169.4, 170.4.

3-Methyl-2-butenyl-�-glucoside (2) Amorphous, FAB-MS m/z [MþH]þ 249, 271[MþNa]þ, 519 [2MþNa]þ. 1H-NMR: (DMSO-d6, 300MHz): � 1.57 (3H, s, C4-H),1.65 (3H, s, C4a-H), 2.47–4.15 (sugar moiety), 4.64 (2H, br, m, OCH2), 5.22 (1H, br,m, C2-H). 13C-NMR (DMSO-d6, 75.5MHz): � 18.3 (C4), 25.9 (C4a), 61.6 (C60), 63.8(C1), 70.6 (C40), 73.9 (C20), 77.1 (C50), 77.3 (C30), 102.0 (C10), 121.5 (C2), 136.0 (C3).

3-Methyl-2-butenyl-�-glucoside tetraacetate (2a) Compound 2 was acetylated usingacetic anhydride in pyridine. The product was purified by TLC to give 2a. 1H-NMR:(DMSO-d6, 300MHz): � 2.01, 2.03, 2.04, 2.09 (3H, s, 4CH3COO).

13C-NMR(DMSO-d6, 75.5MHz): � 20.6–20.7 (4CH3COO), 169.3, 169.4, 170.3, 170.7(4CH3COO).

Acknowledgments

We are grateful to Dr. Jalal Zahra and Miss Kyan Barghouti of our University forcollection of the NMR spectra.

References

[1] J.S. Mosa, M.A. Al-Yahya and I.A. Al-Meshal (1987). Medicinal Plants of Saudi Arabia, p. 1. King SaudUniversity Press, Riyadh.

[2] H.M. Said (1972). Pharmacographia Indica, XV. Hamdard National Foundation, Pakistan.[3] R.A. Blakelock and C.C. Townsend (1980). Capparidaceae. In: C.C. Townsend and E. Guest (Eds.), Flora

of Iraq. Ministry of Agriculture, Iraq.[4] M.M. Mansurov (1972). Farmakol. Toksikol., 35, 715; (1973). Chem. Abstr., 78, 164948.[5] M.S. Al-Said, S.I. Abdel Sattar, S.I. Khalifa and F.S. El-Feraly (1988). Pharmazie, 43, 640.

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[6] F. Tomas and F. Ferreres (1978). Rev. Agroquim. Tecnol. Aliment., 18, 232; (1979). Chem. Abstr., 90,100117; (1976). Rev. Agroquim. Tecnol. Aliment., 16, 252; (1976). Chem. Abstr., 85, 190887; (1976).Rev. Agroquim. Tecnol. Aliment., 16, 568; (1977). Chem. Abstr., 86, 185906.

[7] C. Charaux (1924). Bull. Soc. Chim. Biol., 6, 641.[8] M.V. Artem’eva, M.O. Karryev, A.A. Meshcheryakov and V.P. Gordienko (1981). Izv. Akad. Nauk

Turkm. SSR, Ser Fiz-Tekh., Kim. Geol. Nauk, 123; (1981). Chem. Abstr., 95, 129307.[9] M. Rodrigo, M.J. Lazaro, A. Alvarruiz and V. Giner (1992). J. Food Sci., 57, 1152.[10] H. Schraudolf (1988). Phytochemistry, 28, 259.[11] V.U. Ahmad, A.-R. Amber, S. Arif, M.H.M. Chen and J. Clardy (1985). Phytochemistry, 24, 2709.[12] R. Hegnauer (1961). Planta Med., 9, 37.

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