Chemical Constituents from Stem Bark and Roots of Clausena anisata

Phytochemical investigations on the stem bark and roots of the tropical shrub Clausena anisata led to the isolation and characterization three carbazole alkaloids: girinimbine, murrayamine-A and ekeberginine; two peptide derivatives: aurantiamide acetate and N-benzoyl-l-phenylalaninyl-N-benzoyl-l-phenylalaninate; and a mixture of two phytosterols: sitosterol and stigmasterol. The structures of these compounds were established by nuclear magnetic resonance (1H-NMR, 13C-NMR, COSY, HSQC, HMQC, HMBC and NOESY) spectroscopy and electrospray ionization mass spectrometry (MS).


Introduction
Clausena anisata (Will). Hook.f. ex .benth., is a tropical shrub or tree growing up to ten meters in height in and on the margins of evergreen forests [1]. Different parts (stem bark, roots, and leaves) of this plant are widely used in traditional medicine to treat many diseases. Traditional healers in Tanzania use Clausena anisata against oral candidiasis and fungal infections of the skin [2], whereas in the Temeke district (Daressalam, Tanzania), Clausena anisata is used against epilepsy and as an anticonvulsant [3]. In some parts of Africa and in the Philippines, the burning of fresh leaves is utilized to repel mosquitoes [4]. Previous phytochemical investigations on this taxon yielded mostly carbazole alkaloids [5][6][7][8][9][10][11][12], coumarins [13][14][15][16] and limonoids [17]. In continuation of our investigation on the Rutaceae plants [18], we report the identification of seven compounds isolated from Clausena anisata.
The sodium adducts of aurantiamide acetate (4) and N-benzoylphenylalaninyl-Nbenzoylphenylalaninate (5)  Product ions 5b and 5c at m/z 292 (major) and m/z 260 (minor), respectively, were observed upon CID of the m/z 529 ion 5a, formed by ionization of N-benzoylphenylalaninyl-N-benzoylphenylalaninate (5) (Scheme 3). The sum of the masses of the two product ions (5b and 5c) equals the mass of the precursor ion 5a plus that of a sodium ion, indicating that both product ions are sodium ion adducts. Cleavage of the C-1-O bond accompanied by transfer of H-2 to the C-1′ carbonyl oxygen via a six-membered cyclic transition state accounts for the formation of both product ions from the m/z 529 ion 5a; the product ion observed depends on which half of ion 5a retains the sodium ion.

General
Melting points (uncorrected) were determined on a Gallenkamp melting point apparatus in open capillary tubes. Mass spectra were obtained by electrospray ionization on a Bruker microTOF (accurate mass measurements) and Thermo-Finningan LCQ Duo (tandem mass spectra) mass spectrometers using flow injection in methanol [48]. Collision-induced dissociation (CID) energies are given in parentheses in the arbitrary units (%) supplied by the software. NMR spectra (both 1D and 2D) were acquired on a Bruker AVANCE 500 MHz spectrometer (500.13 MHz for 1 H and 125.76 MHz for 13 C) and a Bruker AV-III 700 MHz spectrometer (700.23 MHz for 1 H and 176.09 MHz for 13 C) equipped with a 5-mm TCI cryoprobe. Chemical shifts (δ, ppm) are reported relative to TMS as internal standard, and coupling constants (J) are given in Hz. Methyl, methylene and methine carbons were distinguished by DEPT experiments. UV spectra were collected in methanol on an Agilent 8345 spectrophotometer, and optical rotations were measured using a Rudolph Instruments Digipol 781 automatic polarimeter. Column chromatography was performed on silica gel (70-230 mesh, 60 Å) using hexane, hexane-ethyl acetate and ethyl acetate as eluents. PTLC was carried out using Merck Si gel 60GF 254 on glass plates (20 × 20 cm) at a thickness of 0.5 mm. TLC was carried out on Sigma-Aldrich TLC plates, Si gel matrix with fluorescent indicator. Spots on TLC and PTLC plates were visualized under UV light (254 and 366 nm) and by spraying with Dragendorff's reagent and/or aqueous sulfuric acid (10%).

Plant Material
The stem bark and roots of Clausena anisata were collected from Limbe, south west region, Cameroon, in August 2009. The plant was identified by M. Litonga Ndive Elias, taxonomist at Botanic Garden of Limbe, Cameroon, where the voucher specimen has been deposited.

Extraction and Isolation
Air dried plants (2.4 kg of stem bark and 3.1 kg of roots) were ground to a fine powder and extracted twice with methanol (2 × 10 L, 4 days) at ambient temperature. The stem bark and root extracts were concentrated under reduced pressure to yield dark brown viscous syrups (39 and 35 g, respectively). Each crude extract was subjected to column vacuum chromatography over silica gel and eluted with mixtures of hexane, ethyl acetate and methanol, in order of increasing polarities to give about 110 fractions each. Work up procedures on the fractions afforded three carbazole alkaloids (compounds 1, 2 and 3), two peptide derivatives (compounds 4 and 5), and two phytosterols 6 and 7, which were identified using spectroscopic methods (1D and 2D-NMR, MS).