screening for cytotoxic activity of extracts and isolated alkaloids
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Phytomedicine 15 (2008) 882885
Screening for cytotoxic activity of extracts and isolated alkaloids frombulbs of Hippeastrum vittatum
A.F.S. Silva a , J.P. de Andrade a , K.R.B. Machado b , A.B. Rocha b , M.A. Apel a ,M.E.G. Sobral a , A.T. Henriques a , J.A.S. Zuanazzi a,
a Programa de Po s-Graduacao em Cie ncias Farmace uticas, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752,90610-010 Porto Alegre, RS, Brazil b Universidade Luterana do Brasil, Av. Farroupilha, 8001 Canoas, RS, Brazil
Abstract
The dichloromethane and n-butanol extracts obtained from fresh bulbs of Hippeastrum vittatum (Amaryllidaceae),collected in Southern Brazil, were evaluated for their cytotoxic activity in vitro against ve human cell lines (HT29 colonadenocarcinoma, H460 non-small cell lung carcinoma, RXF393 renal cell carcinoma, MCF7 breast cancer, and OVCAR3epithelial ovarian cancer), using the sulphorhodamine B assay. Both extracts showed potential antiproliferative activity.From CH 2Cl2 fraction, three alkaloids were isolated: lycorine, vittatine and montanine. The two last compounds weresubmitted to the antiproliferative assay and the highest level of cytotoxicity was found for the alkaloid montanine.r 2007 Elsevier GmbH. All rights reserved.
Keywords: Hippeastrum vittatum ; Amaryllidaceae; Cytotoxic activity; Alkaloids; Montanine; Vittatine
Introduction
Alkaloids are widespread in the plant kingdom andalso present in plants used in medicines ( Pettit et al.,1986). Many plant-derived drugs to treat cancers arealkaloids, and pancratistatin from Pancratium littoraleJacq. (Amaryllidaceae) has been selected by the UnitedStates Cancer Institutes to be analyzed in preclinicaltrials ( Bibby et al., 2000 ). Others biological activities
have been established for the Amaryllidaceae alkaloids,including antimalarial, antiviral and anticholinesterase.H. vittatum was investigated previously ( Ali et al., 1984 )and several alkaloids have been identied from it.
The present paper is part of a search for alkaloids innative Amaryllidaceae species of Brazil ( Hoffmann et al.,
2003, 2004 ). It reports the evaluation of the cytotoxicactivity of two extracts, dichloromethane and n-butanol,from fresh bulbs of Hippeastrum vittatum against vehuman cell lines (HT29 colon adonocarcinoma, H460 non-small cell lung carcinoma, RXF393 renal cell carcinoma,MCF7 breast cancer and OVCAR3 epithelial ovariancancer), according to established protocols ( Skehan et al.,1990). The CH 2Cl2 fraction was submitted to isolation andits main compounds were identied ( Fig. 1 ), and in turn
evaluated for their antiproliferative activity.
Experimental
Plant material
Bulbs of H. vittatum (LHe r.) Herb. were collected inthe owering stage in South of Brazil, in January 2002,and was identied by Marcos Sobral (Faculty of
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Corresponding author. Tel.: +55 51 3308 5450;fax: +5551 33085437.
E-mail address: [email protected] (J.A.S. Zuanazzi).
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Pharmacy, UFRGS). Voucher specimen is deposited atthe Herbarium of the Universidade Federal do RioGrande do Sul (UFRGS, ICN) under Sobral et al., 8889.
Extraction and isolation
Fresh bulbs (2.29 kg) were triturated and maceratedwith EtOH. The procedure is repeated until negative testagainst Bertrand reagent. The EtOH extracts were driedunder vacuum, and the residue was partitioned in lightpetroleum and HCl (10%). The HCl phases werewashed with CH 2Cl 2 . The remaining acid phase wasbasied with NH 4OH (pH 9) and the extract withCH 2Cl 2 and subsequently with n-butanol. The residuesobtained by drying under vacuum yielded 3.73 and 8.0 gto CH 2Cl 2 and n-butanol extracts, respectively. InCH 2Cl 2 extract when re-suspended in the same solvent,80 mg of lycorine was precipitated. The supernatants of this fraction was chromatographed using circularcentrifuge technique on silica-gel, eluted with anincreasing gradient of CH 2Cl 2 MeOH. Fractions withsimilar TLC behavior were combined to yield two majorfractions 1 and 2. Fraction 1 resulted in 2.0g of puremontanine alkaloid (PM 301), and fraction 2 waspuried by preparative TLC eluted with CH 2Cl 2 MeOH(928) to afford vittatine (0.0067g).
Cell culture maintenance
The colon adenocarcinoma (HT29), non-small celllung carcinoma (NCI-H460), renal cell carcinoma
(RXF393), breast cancer (MCF7) and epithelial ovariancancer (OVCAR3) cell lines were maintained asexponentially growing cultures in RPMI 1640 cellculture medium, supplemented with 10% fetal bovineserum, pH 7.4. All cell lines were cultured at 37 1 C in air/carbon dioxide (95:5) atmosphere.
Cell growth inhibition assay
All the samples, including CH 2Cl2 and n-butanolextracts and the isolated compounds, were tested at 1, 5,10, 25 and 50 mg/ml concentrations; each experiment wasreplicated three times. The samples were dissolved onDMSO and further diluted with cell culture medium inthe different concentrations of DMSO. The assay wascarried out as described previously using sulphorhoda-mine B (SRB) test with an ELISA microplate reader, andthe absorbances were read at a wavelength of 515nm(Labsystems Multiscan EX plate reader) ( Monks et al.,2002). Those extracts and alkaloids which produce anSRB absorbance lower than 25% that of the time-zerocontrol value in the cell lines were considered to becytotoxic. From the doseresponse curves IC 50 values(concentration that induce 50% inhibition of cell growth)were calculated from the dose response curves.
Results and discussion
Among the three alkaloids identied in the CH 2Cl 2extract, montanine produced a remarkable yield pre-senting 0.09% of the fresh bulbs. This is the rst time
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Fig. 1. Structures of alkaloids.
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that this alkaloid has been isolated from H. vittatum .The structures of the compounds ( Fig. 1 ) were eluci-dated by physico-chemical and spectral analysis and arein agreement with those reported in the literature.Montanine [1] and lycorine [2]: Dufeld et al. (1965) andMu gge et al. (1994) ; vittatine [3]: Viladomat et al. (1995)and Frahm et al. (1985) .
( )-Montanine [1] (Fig. 1 ): white powder. C 17 H 19 NO 4.l Max,nm 240, 290. IR bands (KBr): 3430, 1900, 1550,1500, 1250, 1000 cm 1. [a ]D 71.4
1 (CHCl 3; c1.00).MS (70eV) m/z (rel. int.): 301 (M + , 18.5).( )-Lycorine [2] (Fig. 1 ): white powder. C 16 H 17 NO 4 .l Max,nm 240, 290. IR bands (KBr): 3300, 1900,1550, 1500, 1250, 1000 cm 1). [a ]D 54.4
1 (MeOH,c0.18). MS (70 eV) m/z (rel. int.): 287 (M + , 33), 286(24), 268 (29), 252 (18), 251 (22), 250 (54), 228 (12),227 (73), 226 (100), 147 (14), 119 (10).(+ )-Vittatine [3] (Fig. 1 ): yellow crystals. C 16 H 17 NO 3 .l Max,nm 240, 290. [a ]D +89,0
1 (CHCl 3 , c0,67).MS (70 eV) m/z (rel. int.): (M + , 74), 228 (73), 119(76), 187 (75), 173 (25), 55 (100).
The extracts and the isolated alkaloids were evaluatedfor their cytotoxic activity and the results obtainedrevealed that both extracts presented strong activityagainst the cell lines, inhibiting their growth by 4 95%in all of them ( Table 1 ). These values are in accordancewith the parameters recommended by NCI ( Pisha et al.,1995) and it can therefore be seen that these extractspossess such activity and that this activity makes themcandidates for further investigation.
Taking into account the results obtained, the CH 2Cl2was selected for isolation of the actives constituents. Thealkaloids montanine and vittatine were isolated andassayed for the cytotoxic activity. Due to the toxicity of lycorine described in the literature ( Ghosal et al., 1985 ;Lin et al., 1995 ), this substance was not analyzed.Montanine and vittatine displayed growth inhibitoryactivity ( o 10% of control SRB absorbance) in all thecell lines tested. Although these two alkaloids have beenisolated from the same species, they present distinctstructural fundamental nucleus. Vittatine has been
assayed by Lin et al. (1995) for cytotoxic activity, butno effect was reported. In our work, montanine showedstronger activity, and such response could be related to itsmontanine skeleton. The IC 50 values for these compoundsare reported in Table 1 . For all cell lines, the IC 50 valuesdemonstrated that all of them are more resistant tovittatine as compared to montanine. Montanines majoreffect seemed to be its high concentration in the extract.Our ndings suggest that montanine could be the maincompound responsible for the cytotoxic activity of CH 2Cl2 extract, since its concentration in this fraction ishigher than the other two alkaloids. The n-butanol extractalso showed cytotoxic activity ( Table 1 ). Further investi-gations will involve the isolation and elucidation of theconstituents responsible for its cytotoxic effects.
References
Ali, A.A., Mesbah, M.K., Frahm, A.W., 1984. Phytochemicalinvestigation of Hippeastrum vittatum . Part IV: Stereo-chemistry of pancracine, the rst 5,11-methanomorphan-thridine alkaloid from Hippeastrum structure of hipagine.Planta Med. 50, 188189.
Bibby, M.C., Holwell, S.E., Thompson, M.J., Pettit, G.R.,2000. Preclinical activity of the novel anti-vascular agent,pancratistatin phosphate. In: Symposium on New Drugs inCancer Therapy, p. 281.
Dufeld, A.M., Aplin, R.T., Budzikiewicz, H., Djerassi, C.,Murphy, C.F., Wildman, W.C., 1965. Mass spectrometry in
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Table 1. Cytotoxic activity of CH 2Cl 2 and n-butanol extracts and isolated alkaloids of Hippeastrum vittatum
Sample Cell lines a
HT29 H460 RXF393 MCF7 OVCAR3
CH 2Cl 2 0.687 0.21 0.62 7 0.06 0.79 7 0.52 1.60 7 0.34 0.84 7 0.29
n-Butanol 4.087
0.61 3.347
0.30 2.937
0.6 3.747
0.29 3.567
0.25Montanine 0.71 7 0.10 0.57 7 0.57 0.65 7 0.01 0.74 7 0.02 0.84 7 0.11Vittatine 21.91 7 1.61 15.88 7 3.28 29.57 7 12.66 NT NT
a Results are expressed as IC 50 values ( mg/ml), means of 3 determinations, measured by SRB; activity: o 5 strong, 520 moderate, 2050 weak, 4 50inactive. Key to cell lines employed: HT29 (colon adenocarcinoma), H460 (non-small cell lung carcinoma), RXF393 (renal cell carcinoma), MCF7(breast cancer), and OVCAR3 (epithelial ovarian cancer). NT: not tested.
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