J. Chil. Chem. Soc., 48, N 3 (2003) ISSN 0717-9324

¹Departamento de Ciencias del Ambiente, Facultad de Química y Biología,
Universidad de Santiago de Chile, Chile, email : rtorres@lauca.usach.cl
²Centro Chimica dei Reccettori del CNR, Istituto di Chimica, Universitá Cattolica del Sacro Cuore,
Largo Francesco Vito1, Roma, Italia.

(Received: October 25, 2002 ­ Accepted: May 22, 2003)

ABSTRACT

From the resinous exudate of twigs end leaves of Larrea nitida, two lignans nor isoguaiacine 1 and meso-nor-dihydroguaiaretic acid 2 and ferulic acid 3 were isolated. The antioxidant activities of resin and pure compounds were assesed by bleaching of the ABTS derived radical-cation.

Key Words: Zygophyllaceae, Larrea, lignans, ferulic acid, antioxidant activities.

INTRODUCTION

Larrea nitida is a resinous bush that grows on the Andean hills in Aconcagua and Coquimbo provinces of Chile¹. The chemical composition of its resin has not been described yet. This genus is characterized by the presence of flavonoids and lignans with antioxidant properties^2-3. In the present communication we describe the main components of the resinous exudates from the leaves of L. nitida: nor-isoguaiacine 1, meso-nor-dihydroguaiaretic acid 2 and ferulic acid 3, a phenylpropane derivative and biogenetic precursor of the former. The antioxidant activities of the isolated compounds are also assessed by the bleaching of the ABTS-derived radical cation.

EXPERIMENTAL

¹H-NMR spectra were recorded on a Bruker spectrometer (300 MHz), ¹³C-NMR spectra were obtained on a Varian model Jupiter 400 spectrometer (75 MHz). IR spectra were recorded on a Perkin-Elmer 735-B spectrophotometer. Mass spectra were obtained with a Fisons Autospec-Q VG-Analytical equipment. The optical rotations were measured with Perkin-Elmer 241 polarimeter. The melting points were measured on an Electrothermal instrument and were not corrected.

Plants samples: Larrea nitida Cav. was collected in Cochiguas (3017'S, 7032'W) Valle del Elqui, 4^th Region, Chile, in September 1999. The sample was identified and deposited in the herbarium of Museo de Historia Natural de Santiago.

Extraction and isolation: The leaves and twigs of the fresh plant (1.0 kg) were dipped into dichloromethane for 30 s. The organic extract was concentrated in a rotary evaporator to give a residue of 48.4 g of a resin (4.8% yield, based on the dry plant).

Purification and identification of compounds: Part of the extract (30 g) was purified by column chromatography (silica gel) using hexane-ethyl acetate mixtures of increasing polarity. Six fractions were separated, weighing 0.28 g, 2.3 g, 7.0 g, 75 g, 6.6 g and 6.2 g. These fractions were screened by thin-layer chromatography and ¹H RMN spectroscopy and purified by column chromatography. In this way, the following compounds were isolated and identified:

Nor-isoguaiacine 1, obtained in the form of a resin (28.6 mg), [a]24°_D = -27 (c, 1, CHCl3 ), IR (cm^-1): 3385; 2953; 1610, .¹H RMN (300 MHz, CDCl₃,TMS, d (ppm): 0.87 (d, 3H, J = 6.9 Hz, 9-CH3); 0.88 (d, 3H, J = 6.9 Hz, 9´-CH3); 1.91 (dt, 1H, J = 6.9; J = 6.5; J = 3.0 Hz, H-8); 2.00 (dd, 1H,J = 6.6; J = 3.0 Hz, H- 8´); 2.41 (dd, 1H, J = 16.5; J = 6.9 Hz, Ha-7); 2.83 (dd, 1H, J = 16.5; J = 6.9 Hz, Hb-7); 3.56 (d, 1H, J=6.5 Hz, H-7´); 3.79 (s, 3H, OCH3); 6.29 (s, 1H, H-3); 6.4 (dd, 1H, J = 1.9; J= 8.1 Hz, H- 6´); 6.54 (s, 1H, H 2´ ); 6.58 (s 1H, H-6); 6.74 (d, 2H, J = 7.8 Hz, H-5´). ¹³C RMN (300 MHz ,CDCl₃, TM) d (ppm): 15.6 (CH3); 15.7 (CH3); 29.1 (C); 34.8 (CH2); 40.5 (C); 50.3 (CH); 55.7 (OCH3); 111.8 (CH); 113.8 (CH); 114.7 (CH); 116.6 (CH); 121.8 (CH); 128.3 (CH); 129.7 (C); 139.3 (C); 142.1 (C); 142.5 (C); 143.4 (C); 146.4 (C). EIMS low resolution: m/z 314 (82 %), 258 (13 %), 227(60 %), 55 (37 %), 40 (100 %)

The heteronuclear C-H correlation (HETCOR) and the INEPT experiments with H-7' confirmed the proposed structure. Identical with an authentic sample.

Meso-nor-dihydroguaiaretic acid 2, (100 mg), white solid, m.p. 184-185°, [a]24 _D = 0° (c, 1, CHCl₃ ) IR (cm^-1): 3461; 1605; 1520; 1455; 1390. ¹H RMN (300 MHz , C₃ D₆ O ,TMS), d (ppm): 0.82 (d, 6H, J= 6.65 Hz); 1.73 (m, 2H); 2.20 (dd, 2H, J = 9.1; J = 13.3 Hz); 2.69 (dd, 2H, J = 4.9; J = 13.3 Hz); 6.52 (dd, 2H, J = 2.8 Hz); 6.68 (d, 2H, J = 2.0 Hz); 6.73 (d, 2H, J = 8.0 Hz). ¹³C RMN (300 MHz ,C₃ D₆ O, TMS, d (ppm): 17.2 (C9 and C9´); 39.9 (C7 and C7´); 40.8 (C8 and C8´); 116.5 (C5 and C5´); 117.6 (C2 and C2´); 121.8 (C6 and C6´); 135.1 (C1 and C1´); 144.5 (C4 and C4´); 146.4 (C3 and C3´). EIMS : m/z 302 (12 %), 151 (3 %),124 (21%) 123 (100 %). Identical with an authentic sample

Ferulic acid 3: (5.6 mg), white solid m.p.170-171°. IR (cm^-1): 3427; 2921. 1620; 1516; 1432. ¹H RMN (300 MHz, C₃ D₆ O, TMS), d (ppm): 3.91 (s, 3H); 6.36 (d, 1H, J = 15.9 Hz); 6.87 (d, 1H, J =8.2 Hz); 7.31 (s, 1H); 7.32 (s, 1H); 7.59 (d, 1H, J = 15.9 Hz). Identical with an authentic sample.

Antioxidant activity determination: evaluated from the bleaching of ABTS derived radical cations: The radical cation derived from ABTS [2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] was prepared by reaction of ABTS (150 mM) with MnO2 (25 mg/mL) in 5 mL of an aqueous buffered solution ( pH 7). After shaking the solution for a few minutes, it was centrifuged and filtered. The filtrate presented the typical green-blue color of the radical solution with absorbance 734 nm (e = 0,0015 mM -1 cm-1). An estimate of the concentration of the remaining ABTS was obtained from the absorbance of the sample at 340 nm ( e = 0,0039 mM -1 cm-1). Aliquots of 5 mL of the ethanolic solution containing the antioxidant were added to 3 mL of the radical solution. The decrease in the absorbance at 734 nm, due to the consumption of the preformed radical, was followed as a function of time. The antioxidant activity of the assessed compounds was measured in terms of its rate of radical bleaching and of its stoichiometry in microequivalents of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) by the determination of its fast (FRE) and total (TRE) reacting equivalents, given by equations (1) -(3). ⁴

where DA is the decrease in absorbance (734 nm) measured 10 s after the sample addition, DA¹⁵ is the decrease in absorbance (734 nm) measured after 15 minutes addition and f is a dilution factor. The antioxidant activities of the extract and fractions were expressed by their FRE and TRE values (see table I) using equations (1) and (3). The values correspond to the concentrations in mM of Trolox that produce the same effect of the samples. Table II lists the antioxidant activities of the pure compounds in equivalents of Trolox from equations (1) and (2).

Table I. FRE and TRE values of total extract and fractions obtained from equations 1 and 3 in mM of Trolox

Tabla II. FRE and TRE values obtained from equations 1 and 1 in Trolox equivalents.

RESULTS AND DISCUSSION

From the resinous exudate of the leaves from Larrea nitida were isolated nor-isoguaicine 1 and meso-nor-dihydroguaiaretic acid 2, obtained before from L.tridentata ⁵ and L.divaricata⁶, and ferulic acid 3, reported for the first time in this genus.

It has been proposed that the resinous exudates in species growing in arid regions, such as Larrea nitida, may act as filters against UV radiation and also against predators^7-9. Many of their secondary metabolites, comprising terpenes, flavonoids and lignans^10-11, exhibit antioxidant properties, suggesting a possible role of these compounds in preventing the oxidative degradation of the resin. In the present communication, we describe the antioxidant activity of the resinous exudate of Larrea nitida and of some compounds isolated from it. Significant activities were obtained for these compounds, when compared with the standard antioxidant Trolox (Tables I and II).

A good correlation was obtained between the total number of phenolic groups in each structure and the corresponding FRE or TRE. The former can be associated with the number of the more reactive groups and the TRE with the total reactive centers in the structures. FRE is a good measure of the quick protection of a given compound against oxidants. TRE measures the degree of long-term protection of the antioxidant, or how effective it is against a strong oxidative stress.

Nor-isoguaiacine 1 showed a TRE of 2.8 (Table II), owing to the presence of three reactive phenolic hydroxyls. The index FRE was 2.1 (Table II). This result shows that 2 of the 3 reactive OH groups tend to act quickly. The third OH group, from a quinoidal compound, reacts more slowly with ABTS. Meso-nor-dihydroguaiaretic acid 2 presented a TRE of 3.6 (Table II), in spite of having four phenolic hydroxyl groups. This reflects the fact that the reaction with the fourth phenolic hydroxyl is very slow, and is not complete in 15 minutes. The value of the FRE index, 2,6 (table II), confirms that only 3 reactive centers react quickly.

Ferulic acid 3, is the only evaluated monophenolic structure. The FRE and the TRE values were 1.3 and 1.9 respectively (Table 2). Those results show the presence of one reactive phenolic hydroxyl, but suggests other important factors responsible for an increase of the antioxidant protection by this compound. Our results are very similar to those obtained by Plumb and col.¹², who obtained for Ferulic acid a TRE value of 1.96 ± 0,009 in Trolox equivalents.

There are also evidences of an increase of the antioxidant behavior in the monophenolic compound by the presence of 1 or 2 methoxyl groups ortho to the hydroxyl group. This substitution tends to increase the stability of the aryloxy radical, thereby increasing its effectiveness as an antioxidant agent.¹³

The antioxidant activity of the total extract is higher than that of the isolated fractions, being 30% more active than the main fraction (Table I). This may be the result of some kind of synergism or of unidentified active compounds absent in the individual fractions. This relation is valid if one considers either the FRE or the TRE values.

The above results are in agreement with the hypothesis that the lignans and their precursors play an important role in the defense of the Larrea nitida against adverse conditions. Further in vivo studies about oxidant effects on surface leaves and on seasonal variations in the concentration of the isolated antioxidant agents are in progress.

ACKNOWLEDGEMENTS

This work was supported by DICYT(USACH) and Programa de Colaboración Internacional CONCICYT/CNR and in part FONDECYT, Proyecto 1030813. Also we thank CYTED Proyect (Prof. Alejandro Fernández Barrero, University of Granada) by helping in spectroscopical facilities.

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