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Boletín de la Sociedad Chilena de Química

Print version ISSN 0366-1644

Bol. Soc. Chil. Quím. vol.45 n.4 Concepción Dec. 2000

http://dx.doi.org/10.4067/S0366-16442000000400019 

A DNA BINDING COMPOUND FROM PROSOPIS
TAMARUGO PODS

GUILLERMO SCHMEDA-HIRSCHMANN1* AND JASMIN JAKUPOVIC2

1Laboratorio de Química de Productos Naturales, Instituto de Química de
Recursos Naturales, Universidad de Talca, Casilla 747, Talca, Chile.
e-mail: schmeda@pehuenche.utalca.cl
2 ANALYTICON. Gustav-Meyer-Allee 25,13355 Berlin, Germany.
(Received: July 24, 2000 - Accepted: August 1°, 2000)

ABSTRACT

The tricyclic compound maltoxazine was isolated as the DNA binding compound occurring in the basic extract of Prosopis tamarugo pods. The structure was elucidated by spectroscopic methods.

KEY WORDS: Prosopis tamarugo, Mimosaceae, maltoxazine, DNA binding.

RESUMEN

El compuesto tricíclico maltoxazina se identificó como el producto bioactivo con capacidad de unión al ADN presente en el extracto básico de frutos de Prosopis tamarugo. La estructura se determinó por métodos espectroscópicos.

PALABRAS CLAVES: Prosopis tamarugo, Mimosaceae, maltoxazina, unión al ADN.

Prosopis tamarugo Phil. (Mimosaceae) is a typical tree of the northern desert of Chile. The pods are used as feed for sheep, goats and cattle1). DNA binding activity in tamarugo pods was reported in a study dealing with the proximate composition and biological activity of food plants gathered by Chilean Amerindians2) . Following our studies on Chilean economic plants, we now report the isolation and identification of a DNA binding compound from P. tamarugo pods.

a. Plant material

Prosopis tamarugo pods were collected in the Pampa del Tamarugal, I Región, Chile in October, 1995. A voucher herbarium specimen (JIL 163) is kept at the Herbario de la Universidad de Talca.

b. DNA binding activity

The assay was performed according to previous work2-4). Calf thymus DNA was purchased from Sigma Chemical Co. The HPLC equipment used was a Merck-Hitachi apparatus with an L-4000 absorbance detector set at 254 nm and a D-2500 recorder/data module. Column: C 18 RP (Lichrospher 100, 5 mm particle size). Mobile phase: water:methanol (100:0 to 0:100). Under these conditions, free DNA eluted with Rt=0.5 min. Test samples and DNA solutions were pre-mixed and incubated at room temperature for 30 min. before injection. Extracts and pure compound were assayed at 0.50 mg/mL. Doxorubicine and vinblastine were used as reference compounds.

c. Isolation

The ripe, air-dried pods (20 kg) were ground and extracted with EtOH:H2O 7:3 under reflux and concentrated to afford a crude syrup. The syrup was made acidic with HCl and defatted with petrol (3x2 L). After filtration, the aqueous phase was basified to pH 11 and extracted with CHCl3 (3x1 L) to yield 1.6 g of a crude basic extract. The basic extract was chromatographed on silica gel (48 g) with a cyclohexane (200 ml); cyclohexane:EtOAc 9:1 (200 mL); cyclohexane:EtOAc 9:1 + 10 drops NH3 conc. (200 mL); cyclohexane:EtOAc 8:2 + 10 drops NH3 conc. (200 mL); cyclohexane:EtOAc 1:1 + 10 drops NH3 conc. (200 mL); EtOAc + 10 drops NH3 conc. (200 mL); EtOAc:MeOH 9:1 + 20 drops NH3 conc. (200 mL) gradient. A Dragendorff-reactive fraction was obtained (100 mg; Rf 0.45; silica gel, cyclohexane:EtOAc 1:1 + 5 drops NH3 conc. /100 mL). Preparative HPLC (LiChrogel PS 1; 2-propanol, 5 ml/min., isocratic) yielded 12 mg p-hydroxybenzoic acid (Rt 14 min) and 24 mg maltoxazine 1 (Rt 59 min).

Maltoxazine 1: colourless resin, MS (m/z): 179.0949 (calc. for C10H13NO2: 179.0949) (94); 150 (100)[C9H12NO]; 136 (10), 122 (12), 108 (8), 95 (16), 75 (33), 45 (78), 31 (48).
1H-NMR (400 MHz, CDCl3, d-values): 1.86-1.94 m (H-4); 2.56 m and 2.42-2.46 m (H-5);
4.51 ddd (17.5, 1.9, 1.4) and 4.38 d br (17.5) (H-6); 4.62 dd (6.2, 2.5) (H-7); 2.22 dddd (10.6, 7.8, 6.2) and 1.86 m (H-8); 1.86 m and 1.94 m (H-9); 3.73 dddd (9.0, 6.7, 6.0, 3.5) and 2.83 dddd (11.0, 9.0, 7.1, 5.6) (H-10). 13C-NMR (100 MHz, CDCl3, d-values): 203.11 s (C-1); 142.52 s (C-2); 149.32 s (C-3); 23.95 t (C-4); 33.76 t (C-5); 66.93 t (C-6); 89.55 d (C-7); 31.00 t (C-8); 24.17 t (C-9); 48.39 t (C-10). IR (KBr) cm-1: 3394 (OH), 2925, 1703 (C=O), 1642(-C=C-C=O), 1125, 1030.

From the basic extract of P. tamarugo pods, the Dragendorff-positive maltoxazine5) was isolated and identified as the DNA binding constituent of the pods. DNA binding of crude pod extracts from P. tamarugo collected in 1996 and 1997 showed DNA binding values of 34 and 18%, respectively2) while the basic extract displayed a stronger effect (78%) and maltoxazine (95%) was almost as active as vinblastine at 0.50 mg /mL. Under our assay conditions, the DNA binding of the standard inhibitors doxorubicine and vinblastine at 0.50 mg/ml were 100 and 70%, respectively.

DNA binding alkaloids have been identified from the aerial parts of Argentinian and Chilean Prosopis species3-4) with higher activities for piperidine derivatives. Piperidine alkaloids have also been isolated from Prosopis juliflora 6-10). The proximate composition and biological activity of Chilean Prosopis species has been reported11) .

The heterocycle maltoxazine was isolated as the DNA binding compound present in the basic extract of Prosopis tamarugo pods. The product, with a cereal flavor, contributes to the characteristic aroma of tamarugo pods. The compound has been reported as a reaction product of hexoses with a-aminoacids like proline under conditions of the Maillard reaction5).

ACKNOWLEDGEMENTS

We acknowledge the financial support of FONDECYT, Grant 1941165 and CONAF-II Región for allowing the collection of plant material in the National Park. GSH thanks the Alexander von Humboldt Foundation for a fellowship. We are indebted to Prof. T. Anke, Biotechnology Lab. and Prof. Kreiter, Department of Chemistry, University of Kaiserslautern, Germany, for laboratory and instrumental facilities and Mrs. H. Ruzek for the spectral measurements.

*To whom correspondence should be addressed.

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