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Boletín de la Sociedad Chilena de Química

versión impresa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.44 n.3 Concepción set. 1999

http://dx.doi.org/10.4067/S0366-16441999000300016 

DNA BINDING ALKALOIDS FROM LOBELIA BRIDGESII HOOK ET 
ARN AND LOBELIA TUPA L.

OSCAR MARAMBIO1, ESTER MONSALVE1 AND
GUILLERMO SCHMEDA-HIRSCHMANN2*

1Departamento de Ciencia y Tecnología de los Alimentos, Universidd de Los Lagos, Casilla
933, Osorno, Chile.
2Laboratorio de Química de Productos Naturales, Instituto de Química de Recursos Natu-
rales, Universidad de Talca, Casilla 747, Talca, Chile.
e-mail:schmeda@pehuenche.utalca.cl
(Received: May 27, 1999 - Accepted: July 15, 1999)

ABSTRACT

The methanolic extract of the aerial parts of Lobelia bridgesii and L. tupa (Lobeliaceae) showed activity in the DNA binding assay. Bioassay-guided isolation led to the alkaloids norlobelanidine and norlelobanidine as the main bioactive constituents of othe plant. At 0.50 mg/mL, the percentual DNA binding of both compounds were 79% and 71%, respectively.

KEY WORDS: Lobelia bridgesii, L. tupa, Lobelioideae, DNA binding, alkaloids, norlobelanidine, norlelobanidine.

RESUMEN

El extracto metanólico de las partes aéreas de Lobelia bridgesii y L tupa (Lobeliaceae) mostró actividad en el bioensayo de unión al ADN. Mediante aislamiento bioguiado se identificaron los alcaloides norlobelanidina y norlelobanidina como los componentes bioactivos mayoritarios de estas plantas. A la concentración de 0,50 mg/mL, el porcentaje de unión de estos compuestos al ADN fue de 79% y 71%, respectivamente.

PALABRAS CLAVES: Lobelia bridgesii, L. tupa, Lobelioideae, unión al ADN, alcaloides, norlobelanidina, norlelobanidina.

INTRODUCTION

Several Lobelia species have been used as ritual plants by the Mapuche Indians of southern Chile. Lobelia tupa L., known under the common name "tupa" or "tabaco del diablo" (devil's tobacco) is reputed as hallucinogenic and anaesthetic. The milky sap of the stem and roots is sometimes placed into cavities to aleviate tooth pain1). Mösbach2) states similar uses for L. tupa, L. excelsa Bonpl., L. polyphylla H. et A. and other Lobelia species.

Following our chemical and biological studies on Chilean native plants, we now report the isolation of DNA binding alkaloids from L. bridgesii and L. tupa.

EXPERIMENTAL

a. Plant material

Lobelia bridgesii Hook et Arn. (Campanulaceae, Lobelioideae), an endemism of the Valdivia Province in Chile and Lobelia tupa L. were collected at San José de la Mariquina, Provincia Valdivia, X Región, Chile on November 1994. Voucher herbarium specimens were identified at the Botany Department, Universidad Austral de Chile and have been kept at this institution.

b. DNA binding activity

The assay was performed according to Pezzutto3) with some modifications4). Calf thymus DNA was purchased from Sigma Chemical Co. The HPLC equipment used was a Merck-Hitachi apparatus with a L-4000 absorbance detector set at 254 nm and a D-2500 recorder/data module. Column: C18 RP (Lichrospher 100, 5 mm particle size). Mobile phase: water to methanol (100:0 to 0:100). Under these conditions, free DNA eluted with a Rt=0.5 min. Test samples and DNA solutions were pre-mixed and incubated at room temperature for 30 min before injection. Extracts were assayed at 0.50 mg/mL. Doxorubicine and vinblastine were used as reference compounds. Cytotoxicity as well as inhibition of DNA, RNA and protein biosynthesis was carried out as described in5).

c. Isolation

The air-dried aerial parts (3.2 kg) and roots (1.96 kg) of L. bridgesii were extracted with MeOH. The crude extracts were resuspended in water and partitioned with petroleum ether. After biological testing the activity was found mainly in the aqueous phase. The aqueous phase was basified with NaOH to pH 10 and extracted with CHCl3 (3x500 mL) to obtain the much active crude alkaloid fraction. The alkaloid extract was chromatographed on silica gel with a petroleum ether: CHCl3 1:1; CHCl3; CHCl3:MeOH + 0.5% NH3 9:1 to CHCl3:MeOH + 0.5% NH3 1:1 gradient. Some 100 fractions of 20 mL each were collected. After TLC comparison and detection with Dragendorff's reagent, those fractions showing similar TLC patterns were pooled and subsequently purified by preparative TLC or recrystallization. 110 mg of compound 1 (colorless crystals from EtOH; 0.0034% yield) and 60 mg of compound 2 (0.0019% yield) were obtained. Lobelia tupa roots (2.13 kg) and aerial parts (0.86 kg) yielded 20 mg of compound 2 as the main bioactive metabolite. Compounds assayed were identified spectroscopically. The 1H- and 13C-NMR data of 1 and 2 are presented in Tables I and II.

TABLE I. 1H-NMR data of norlobelanidine 1 and norlelobanidine 2 (400 MHz in MeOH-d4 for 1, 300 MHz in CDCl3 for 2).


H
1
2

2,6
4.95 m
3.13 m
3,5
2.93 ddd (13.8; 12.6; 3.6), 3.42 m
1.2-1.6 m
4
3.4 m
 
7,9
3.38 m; 3.33 m
2.01 ddd (14; 10; 10)
8
6.40 dd (9.7; 4.1)
4.81 dd (9.2; 4.0)
10
 
3.69 m
11
-
1.43 dq (7.5)
12
-
0.93 t (7.5)
2’,6’
8.87 brd
7.38 dd br
3’,5’
8.80 br dd
7.28 br dd
8.72 br dd
7.25 m


j 2',3' = 3',4' = 4',5' = 7.7; 2',4' = 3',5' = 1.8

TABLE II. 13C-NMR data of norlobelanidine 1 and norlelobanidine 2 (100 MHz, MeOH-d4 for 1; 75 MHz, CDCl3 for 2).


C
1
2

2
58.99 d
58.22 d
3
30.48 t
29.20 t
4
23.58 t
22.59 t
5
30.48 t
29.13 t
6
58.99 d
58.06 d
7
43.40 t
41.44 t
8
74.47 d
73.29 d
9
43.40 t
39.03 t
10
74.47 d
72.77 d
11
-
30.64 t
12
-
9.35 q
145.76 s
143.73 s
2’,6’
126.40 d
128.12 d
3’,5’
129.40 d
127.32 d
128.59 d
125.24 d

Norlobelanidine 1: colorless crystals, mp 254, from EtOH.

[a]D: + 0.31 (c = 6.36); MeOH. MS (m/z, rel int. %): C21H27NO2 325.204 (50) [M]+ (calc. for C21H27NO2: 325.204), 204 (86) [M - C6H5CH(OH)CH2] + [C13H18NO]+, 186 (75) [204 - H2O]+ [C13H16N]+, 82 (100) [C5H8N]+, 79 (59) [C5H5N]+. IR (KBr, cm-1): 3237 (secondary OH), 2920, 2850 (aliphatic side chains), 1496, 1448 (C6H5-), 1352, 1205 (tertiary amine), 1088, 1061, 934, 829, 736, 699 (C6H5-), 548.

Norlelobanidine 2: [a]D: + 2.15 (c = 0.93, MeOH).

MS (m/z, rel int. %): C17H27NO2 277.204 (calc. for C17H27NO2: 277.204) (32); 204 (51) [C13H18NO]+ [M - CH3CH2CH(OH)CH2] +, 186 (71) [204 - H2O]+ [C13H16N]+, 156 [M - C6H5CH(OH)CH2] + (100), 82 (85) [C5H8N]+. IR (KBr, cm-1): 3289 (secondary OH), 3088, 2943 (aliphatic side chains), 2564, 1616 (m), 1495, 1448, 1413, 1335, 1284, 1266 (tertiary amine), 1090, 1057, 1030, 746, 699 (C6H5-), 631, 540, 486, 461.

RESULTS AND DISCUSSION

Lobelia bridgesii and Lobelia tupa extracts were assessed for DNA binding activity "in vitro". Lobelia bridgesii leaf extract showed a 23% reduction of the DNA peak at 0.50 mg/mL. Bioassay-guided isolation led to the alkaloids norlobelanidine and norlelobanidine as the main bioactive constituents of the plant. At 0.50 mg/mL, the percentual DNA binding activity of both compounds was 79 and 71%, respectively. At the same concentration, the DNA binding effect of doxorubicine was 100% and for vinblastine the figure was close to 70%. Norlobelanidine did not modify the DNA, RNA or protein biosynthesis at concentrations up to 200 µg/mL using L 1210 cells. The DNA binding activity of Lobelia extracts and alkaloids is presented in Table III.

TABLE III. DNA binding activity of Lobelia bridgesii and L. tupa extracts and alkaloids.


Plant part and extract
% DNA binding at 0.50 mg/mL 
 
Lobelia bridgesii
Lobelia tupa

L, MeOH
L, CH2Cl2
L, EtOAc
L, Aqueous
R, MeOH
Alkaloids
Norlobelanidine
Norlelobanidine
Reference compounds
Vinblastine
Doxorubicine
23
25
38
80
-

79
71

69
100
15
0
0
0
0

0
70

69
100


L: leaf; R: root. - : not done

Two piperidine alkaloids were identified as the mainDNA binding compounds from L. polyphylla and L. tupa. Norlobelanidine 1 was previously isolated from the aerial parts of Lobelia polyphylla Hook et Arn6), while norlelobanidine 2 was reported as a constituent of L. inflata7). Piperidine alkaloids are widely distributed in Lobelia and Sedum species7,8). Biological activities reported for Lobelia species comprises the cardiovascular effect of Lobelia alkaloids9) and the antidepressant principle b-amyrin palmitate from Lobelia inflata L.10,11).

CONCLUSIONS

From the methanolic extracts of the ritual plants Lobelia bridgesii and Lobelia tupa, two alkaloids were isolated as their main constituents with DNA binding activity. At 0.50 mg/mL norlobelanidine and norlelobanidine showed a DNA binding effect of 79 and 71%, respectively. The figure was similar to that of vinblastine in our assay conditions. The compounds, however, did not show activity in the DNA, RNA or protein biosynthesis up to 200 µg/mL using L 1210 cell cultures.

ACKNOWLEDGEMENTS

We acknowledge the financial support from the Dirección de Investigación, Universidad de Los Lagos, Grant 304.11. G.S.-H. thanks the Alexander von Humboldt Foundation for a fellowship. We are indebted to Prof. T. Anke, Biotechnology Lab. and Prof. Kreiter, Department of Chemistry, Universität 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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