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Screening for cytotoxic activity of extracts and isolated alkaloids from bulbs of Hippeastrum vittatum.


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 five human cell lines (HT29 colon adenocarcinoma, H460 non-small cell lung carcinoma, RXF393 renal cell carcinoma, MCF7 breast cancer, and OVCAR3 epithelial ovarian cancer), using the sulphorhodamine B assay. Both extracts showed potential antiproliferative activity. From [CH.sub.2][Cl.sub.2] fraction, three alkaloids were isolated: lycorine, vittatine and montanine. The two last compounds were submitted to the antiproliferative assay and the highest level of cytotoxicity was found for the alkaloid montanine.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Hippeastrum vittatum; Amaryllidaceae; Cytotoxic activity; Alkaloids; Montanine; Vittatine


Alkaloids are widespread in the plant kingdom and also present in plants used in medicines (Pettit et al., 1986). Many plant-derived drugs to treat cancers are alkaloids, and pancratistatin from Pancratium littorale Jacq. (Amaryllidaceae) has been selected by the United States Cancer Institutes to be analyzed in preclinical trials (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 identified from it.

The present paper is part of a search for alkaloids in native Amaryllidaceae species of Brazil (Hoffmann et al., 2003, 2004). It reports the evaluation of the cytotoxic activity of two extracts, dichloromethane and n-butanol, from fresh bulbs of Hippeastrum vittatum against five human cell lines (HT29 colon adonocarcinoma, H460 non-small cell lung carcinoma, RXF393 renal cell carcinoma, MCF7 breast cancer and OVCAR3 epithelial ovarian cancer), according to established protocols (Skehan et al., 1990). The [CH.sub.2][Cl.sub.2] fraction was submitted to isolation and its main compounds were identified (Fig. 1), and in turn evaluated for their antiproliferative activity.


Plant material

Bulbs of H. vittatum (L'Her.) Herb. were collected in the flowering stage in South of Brazil, in January 2002, and was identified by Marcos Sobral (Faculty of Pharmacy, UFRGS). Voucher specimen is deposited at the Herbarium of the Universidade Federal do Rio Grande do Sul (UFRGS, ICN) under Sobral et al., 8889.

Extraction and isolation

Fresh bulbs (2.29 kg) were triturated and macerated with EtOH. The procedure is repeated until negative test against Bertrand reagent. The EtOH extracts were dried under vacuum, and the residue was partitioned in light petroleum and HCl (10%). The HCl phases were washed with [CH.sub.2][Cl.sub.2]. The remaining acid phase was basified with [NH.sub.4]OH (pH 9) and the extract with [CH.sub.2][Cl.sub.2] and subsequently with n-butanol. The residues obtained by drying under vacuum yielded 3.73 and 8.0 g to [CH.sub.2][Cl.sub.2] and n-butanol extracts, respectively. In [CH.sub.2][Cl.sub.2] extract when re-suspended in the same solvent, 80 mg of lycorine was precipitated. The supernatants of this fraction was chromatographed using circular centrifuge technique on silica-gel, eluted with an increasing gradient of [CH.sub.2][Cl.sub.2]-MeOH. Fractions with similar TLC behavior were combined to yield two major fractions 1 and 2. Fraction 1 resulted in 2.0 g of pure montanine alkaloid (PM = 301), and fraction 2 was purified by preparative TLC eluted with [CH.sub.2][Cl.sub.2]-MeOH (92-8) to afford vittatine (0.0067 g).

Cell culture maintenance

The colon adenocarcinoma (HT29), non-small cell lung carcinoma (NCI-H460), renal cell carcinoma (RXF393), breast cancer (MCF7) and epithelial ovarian cancer (OVCAR3) cell lines were maintained as exponentially growing cultures in RPMI 1640 cell culture medium, supplemented with 10% fetal bovine serum, pH 7.4. All cell lines were cultured at 37 [degrees]C in air/carbon dioxide (95:5) atmosphere.


Cell growth inhibition assay

All the samples, including [CH.sub.2][Cl.sub.2] and n-butanol extracts and the isolated compounds, were tested at 1, 5, 10, 25 and 50 [micro]g/ml concentrations; each experiment was replicated three times. The samples were dissolved on DMSO and further diluted with cell culture medium in the different concentrations of DMSO. The assay was carried out as described previously using sulphorhodamine B (SRB) test with an ELISA microplate reader, and the absorbances were read at a wavelength of 515 nm (Labsystems Multiscan EX plate reader) (Monks et al., 2002). Those extracts and alkaloids which produce an SRB absorbance lower than 25% that of the time-zero control value in the cell lines were considered to be cytotoxic. From the dose-response curves [IC.sub.50] values (concentration that induce 50% inhibition of cell growth) were calculated from the dose response curves.

Results and discussion

Among the three alkaloids identified in the [CH.sub.2][Cl.sub.2] extract, montanine produced a remarkable yield presenting 0.09% of the fresh bulbs. This is the first time that this alkaloid has been isolated from H. vittatum. The structures of the compounds (Fig. 1) were elucidated by physico-chemical and spectral analysis and are in agreement with those reported in the literature. Montanine [1] and lycorine [2]: Duffield et al. (1965) and Mugge et al. (1994); vittatine [3]: Viladomat et al. (1995) and Frahm et al. (1985).
 (-)-Montanine [1] (Fig. 1): white powder. [C.sub.17][H.sub.19]
 [N0.sub.4]. [[lambda].sub.Max, nm] = 240, 290. IR bands (KBr): 3430,
 1900, 1550, 1500, 1250, 1000 [cm.sup.-1]. [[alpha].sub.D] = -71.4
 [degrees] (CH[Cl.sub.3]; c1.00). MS (70eV) m/z (rel. int.): 301
 ([M.sup.+ *], 18.5).
 (--)-Lycorine [2] (Fig. 1): white powder. [C.sub.16][H.sub.17]
 [NO.sub.4]. [[lambda].sub.Max, nm] = 240, 290. IR bands (KBr): 3300,
 1900, 1550, 1500, 1250, 1000 [cm.sup.-1]). [[alpha].sub.D] = -54.4
 [degrees] (MeOH, c0.18). MS (70eV) m/z (rel. int.): 287 ([M.sup.+*],
 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.sub.16][H.sub.17]
 [NO.sub.3]. [[lambda].sub.Max, nm] = 240, 290. [[alpha]].sub.D] = +89,
 0[degrees] ([CHCl.sub.3], c0, 67). MS (70eV) m/z (rel. int.):
 ([M.sup.+ *], 74), 228 (73), 119 (76), 187 (75), 173 (25), 55 (100).

The extracts and the isolated alkaloids were evaluated for their cytotoxic activity and the results obtained revealed that both extracts presented strong activity against the cell lines, inhibiting their growth by > 95% in all of them (Table 1). These values are in accordance with the parameters recommended by NCI (Pisha et al., 1995) and it can therefore be seen that these extracts possess such activity and that this activity makes them candidates for further investigation.
Table 1. Cytotoxic activity of [CH.sub.2][Cl.sub.2] and n-butanol
extracts and isolated alkaloids of Hippeastrum vittatum

 Sample Celll ines(a)

 HT29 H460 RXF393

[CH.sub.2][Cl.sub.2] 0.68 [+ or -] 0.62 [+ or -] 0.79 [+ or -]
 0.21 0.06 0.52

n-Butanol 4.08 [+ or -] 3.34 [+ or -] 2.93 [+ or -]
 0.61 0.30 0.6

Montanine 0.71 [+ or -] 0.57 [+ or -] 0.65 [+ or -]
 0.10 0.57 0.01

Vittatine 21.91 [+ or -] 15.88 [+ or -] 29.57 [+ or -]
 1.61 3.28 12.66


[CH.sub.2][Cl.sub.2] 1.60 [+ or -] 0.34 0.84 [+ or -] 0.29

n-Butanol 3.74 [+ or -] 0.29 3.56 [+ or -] 0.25

Montanine 0.74 [+ or -] 0.02 0.84 [+ or -] 0.11

Vittatine NT NT

(a) Results are expressed as [IC.sub.50] values ([micro]g/ml),
means of 3 determinations, measured by SRB; activity: < 5 strong, 5-20
moderate, 20-50 weak, > 50 inactive. 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.

Taking into account the results obtained, the [CH.sub.2][Cl.sub.2] was selected for isolation of the actives constituents. The alkaloids montanine and vittatine were isolated and assayed 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 inhibitory activity (< 10% of control SRB absorbance) in all the cell lines tested. Although these two alkaloids have been isolated from the same species, they present distinct structural fundamental nucleus. Vittatine has been assayed by Lin et al. (1995) for cytotoxic activity, but no effect was reported. In our work, montanine showed stronger activity, and such response could be related to its montanine skeleton. The [IC.sub.50] values for these compounds are reported in Table 1. For all cell lines, the I [C.sub.50] values demonstrated that all of them are more resistant to vittatine as compared to montanine. Montanine's major effect seemed to be its high concentration in the extract. Our findings suggest that montanine could be the main compound responsible for the cytotoxic activity of [CH.sub.2][Cl.sub.2] extract, since its concentration in this fraction is higher than the other two alkaloids. The n-butanol extract also showed cytotoxic activity (Table 1). Further investigations will involve the isolation and elucidation of the constituents responsible for its cytotoxic effects.


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* Corresponding author. Tel: + 55 51 3308 5450; fax: +55 51 3308 5437.

E-mail address: (J.A.S. Zuanazzi).

0944-7113/$-see front matter [c] 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2007.12.001

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 Pos-Graduacao em Ciencias Farmaceuticas, 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
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Author:Silva, A.F.S.; Andrade, J.P. de; Machado, K.R.B.; Rocha, A.B.; Apel, M.A.; Sobral, M.E.G.; A.T.; Hen
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Oct 1, 2008
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