STEROIDAL AND PHENOLIC COMPOUNDS FROM Sidastrum Paniculatum (L.) FRYXELL AND EVALUATION OF CYTOTOXIC AND ANTI-INFLAMMATORY ACTIVITIES

Sidastrum paniculatum (L.) Fryxell belongs to the family Malvaceae and is popularly known as “malva roxa” or “malvavisco”. The phytochemical study of the hexane, CHCl3 and EtOAc phases from the crude ethanol extract of S. paniculatum led to the isolation of six compounds: a mixture of b-sitosterol and stigmasterol, 4-methoxy-3-hydroxybenzoic acid, 4-methoxy-3-hydroxybenzaldehyde, N-trans-feruloyltyramine and kaempferol-3-O-β-D-(6’’-E-p-coumaroyl) glucoside. The structural identification of the compounds was made on the basis of spectroscopic methods such as IR, 1H and 13C NMR with the aid of including two-dimensional techniques, besides comparison with literature data. The b-sitosterol and stigmasterol mixture showed a significant anti-inflammatory activity.

Keywords: Sidastrum paniculatum; Malvaceae; anti-inflammatory activity.

INTRODUCTION

Sidastrum paniculatum (L.) Fryxell is a shrub belonging to the family Malvaceae with wide distribution in the neotropics.1 In Northeast of Brazil this species is popularly known as “malva-roxa” or “malvavisco”. For long time, this species was belonging to the genus Sida, as Sida paniculata L., and was transferred to Sidastrum by Frixell.2

Sidastrum Baker f. is one of 70 genera informally included by Bayer and Kubitzki as a member of the tribe Malveae, subfamily Malvoideae (Malvaceae).3 The genus comprises about eight species with neotropical distribution, occurring from Mexico to West Indies to Argentina.1,3 In Brazil, the northeast region is probably the diversity core of the genus Sidastrum since a great number of species can be found there.1

Recent molecular studies of the Sida generic alliance4 strongly support the phylogenetic relationships between Sidastrum and the genus Meximalva as well as an Australian group of Sida species, which form a well-supported clade as suggested before by Fryxell1 based on morphological characters.

Previous phytochemical studies on species from Malvaceae, specially of the genus Sida have reported the presence of steroids,5-8 phenols,6,7,9 flavonoids,6-8,10,11 triterpenes,5,9 essential oils,12 alkaloids,13 sesquiterpene lactone,14 fatty acids,15 and phaeophorbide.6

Aiming at contributing to the chemotaxonomic study of the family Malvaceae, subfamily Malvoideae, and considering the absence of data in literature concerning the chemical constitution of the genus Sidastrum, the species Sidastrum paniculatum was submitted to a phytochemical study to isolate and identify its chemical constituents, through usual chromatographic and spectroscopic methods, besides comparison with literature data. In addition to that, an evaluation of the cytotoxic and anti-inflammatory activities with some of the isolated compounds will be herein described.

EXPERIMENTAL

General procedures

NMR spectra (HOMOCOSY, HETCOR, HMQC, HMBC and NOESY) were registered in CDCl3, CD3OD and recorded on a Mercury Varian instrument operating at 200 MHz and 50 MHz for 1H and 13C, respectively. The solvent signal was used as internal standard. IR spectra were measured on a Perkin-Elmer, FT-IR-1750 spectrometer in KBr pellets. Chromatography columns were carried out on silica gel (Merck) and Sephadex LH-20 (Merck). TLC were performed on silica gel PF254 plates and the spots were visualized under UV light (254 and 366 nm) and by exposure to the iodine vapor.

Plant material

The whole plant of S. paniculatum was collected in Pedra da Boca, in the municipality of Araruna, State of Paraíba, on February 2004, and a voucher specimen (M. F. Agra et al. 6051) was deposited at the Herbarium Prof. Lauro Pires Xavier (JPB), Universidade Federal da Paraíba.

Extraction and isolation

The plant material (10 kg) was subjected to dehydration in an oven in a temperature of 40 ºC for 72 h. After that, it was grounded in a mechanical mill, yielding 5.7 kg of a powder which was submitted to maceration with ethanol for three consecutive days. This process was repeated until the maximum extraction of the chemical constituents. The obtained ethanol extractive solution was concentrated in a rotatory evaporator, yielding 500 g of crude ethanol extract (CEE). The latter was suspended in ethanol:H2O (9:1) and successively partitioned with hexane, CHCl3, EtOAc and n-butanol. The hexane phase (6.0 g) was subjected to column chromatography packed with silica gel and eluted with hexane, CHCl3, EtOAc and methanol. 69 fractions of 20 mL each were collected, analysed and combined through analytical thin-layer chromatography (TLC). The sub-fraction 37/42 (1.36 g) was rechromatographed on

Steroidal and phenolic compounds from Sidastrum paniculatum

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silica gel column eluted with hexane, CHCl3 and methanol providing 15 sub-fractions which were analysed and joined through analytical TLC. The sub-fraction 11/15 showed itself as crystals, yielding 0.093 g of a b-sitosterol (1) and stigmasterol (2) mixture. The CHCl3 phase provided a precipitate (5.0 g) and a supernatant (13.0 g). 6.0 g of the latter were submitted to chromatography on a column packed with silica gel and eluted with hexane, EtOAc and methanol, giving 200 fractions that were analysed and joined through analytical TLC. Fraction 5/15 (0.81 g) was also chromatographed on silica gel column using the above chromatographic procedures, yielding 120 sub-fractions.

The sub-fraction 26/57 (0.05 g) showed itself as an amorphous solid, defined as 4-hydroxy-3-methoxybenzoic acid (3). Fraction 26/102 (0.45 g), on the other hand, was chromatographed following the same methodology, providing 96 sub-fractions of which the sub-fraction 43/56 was recrystallized in chloroform and methanol, yielding 0.025 g of the 4-hydroxy-3-methoxybenzaldehyde (3). 0.15 g of the precipitate was submitted to preparative TLC, using a mixture of hexane:ethyl acetate (1:1) as eluent, resulting on the isolation and purification of 0.021 g of N-trans-feruloyltyramine (4). The ethyl acetate phase (3.0 g) was subjected to chromatography column packed with Sephadex LH-20 and eluted with methanol, providing 23 fractions that were analysed and combined through analytical TLC. The sub-fraction 12/16 (0.087 g) was also chromatographed on Sephadex LH-20, yielding 14 fractions. The sub-fraction 5/8 (0.032 g) was defined as kaempferol-3-O-β-D-(6’’-E-p-coumaroyl) glucoside (5).

4 (N-trans-feruloyltyramine)1H NMR (d, CDCl3, 200 MHz): 7.37 (d, J=15.8 Hz, H-7), 6.96 (d, J=8.4 Hz, H-2’/6’), 6.93 (brd, J=8.4 Hz, H-6), 6.90 (brs, H-2), 6.74 (d, J=8.4 Hz, H-5), 6.68 (d, J=8.4 Hz, H-3’/5’), 6.16 (d, J=15.8 Hz,