Marino B. Arnao

The hydrophilic and lipophilic contribution to total antioxidant activity

Abstract The ABTS/H2O2/HRP decoloration method permits the evaluation of the antioxidant activity of complex food samples. This method, with slight modifications, is capable of determining both hydrophilic and lipophilic antioxidant properties, thus, it is possible estimate the antioxidant activity of both antioxidant types in the same sample. The method is easy, accurate and rapid to apply. Its application to three vegetable soups provided data on hydrophilic and lipophilic antioxidant activity, and the values reflect the contribution of the particular antioxidants (ascorbic acid and carotenoids) to the total antioxidant activity of the samples.

Some methodological problems in the determination of antioxidant activity using chromogen radicals: a practical case.

The two most widely used chromogen compounds to measure the antioxidant activity of biological material are the ABTS+· and the DPPH· radicals. Both present excellent stability in certain assay conditions but also show several important differences in their response to antioxidants and in their manipulation. In this study, we present the interferences, at different wavelengths, caused by plant-derived materials (citrus juices and wines) using the two above chromogens to measure their antioxidant activity.

An end-point method for estimation of the total antioxidant activity in plant material

The 2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase. This ABTS radical (ABTS ∞a ), a chromogen, is stable at room temperature but is unstable above 35°C and/or at pH values of above 7.5. Nevertheless, the most important factor in its stability is the ABTS/ABTS ∞a concentration ratio in the medium. The radical reacts with the antioxidant, L-ascorbic acid, with a high rate constant, the stoichiometry of the reaction being 1 mol of L-ascorbic acid per 2 mol of ABTS ∞a reduced. Based on these considerations, a spectrophotometric end-point method has been developed to evaluate L-ascorbic acid in aqueous media, and this represents an improvement over the lag-method previously reported. Under optimal conditions of temperature, pH and reagent concentration, the end-point method was capable of determining L-ascorbic acid with a limit of quantification of 0.38 nmol. In the assay described here, this ability is used to evaluate the total antioxidant activity of commercial citrus juices, in which ascorbic acid is a principal component. In our opinion this procedure can quickly provide useful information on the antioxidant content of foods and plant extracts. # 1998 John Wiley & Sons, Ltd. Phytochem. Anal. 9, 196‐202, 1998

A kinetic study on the suicide inactivation of peroxidase by hydrogen peroxide

In the absence of reductant substrates, and with excess H2O2, peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) shows the kinetic behaviour of a suicide inactivation, H2O2 being the suicide substrate. From the complex (compound I-H2O2), a competition is established between two catalytic pathways (the catalase pathway and the compound III-forming pathway), and the suicide inactivation pathway (formation of inactive enzyme). A kinetic analysis of this system allows us to obtain a value for the inactivation constant, ki = (3.92 +/- 0.06) x 10(-3) x s-1. Two partition ratios (r), defined as the number of turnovers given by one mol of enzyme before its inactivation, can be calculated: (a) one for the catalase pathway, rc = 449 +/- 47; (b) the other for the compound III-forming pathway, rCoIII = 2.00 +/- 0.07. Thus, the catalase activity of the enzyme and, also, the protective role of compound III against an H2O2-dependent peroxidase inactivation are both shown to be important.

Melatonin acts as a growth-stimulating compound in some monocot species

Abstract:  In a recent study melatonin (N‐acetyl‐5‐methoxytryptamine), a well‐investigated animal molecule but minimally studied in plants, was seen to have a physiological role as growth‐promoting molecule in lupin hypocotyls. In the present study, the role of melatonin as a growth promoter is extended to coleoptiles of canary grass, wheat, barley and oat, in which it shows a relative auxinic activity [with respect to indole‐3‐acetic acid (IAA), the main auxin in plants] of between 10 and 55%. In addition, melatonin is seen to have an important inhibitory growth effect on roots similar to that played by auxin. The quantitation by liquid chromatography with electrochemical detection and identification by tandem mass spectrometry of melatonin and IAA in etiolated coleoptiles of the monocots assayed showed that both compounds are present in similar levels in these tissues. These results point to the co‐existence of auxin and melatonin in tissues and raises the possibility of their co‐participation in some physiological actions as auxinic hormones in plants.

Melatonin: a growth-stimulating compound present in lupin tissues

Melatonin (N-acetyl-5-methoxi-tryptamine), a well-known animal hormone synthetised by the pineal gland, plays a key role in the circadian rhythm of vertebrates. An exhaustive bibliographical revision of studies on melatonin in plants published since 1990 points to very few studies (around 20), of which only 8 have a clear plant physiological focus. The data presented in this study demonstrate that melatonin plays a physiological role in plant tissues. Melatonin is seen to be a molecule that promotes vegetative growth in etiolated Lupinus albus L. hypocotyls, in a similar way to IAA. The measurements of melatonin and IAA in lupin hypocotyls by high-performance liquid chromatography with electrochemical detection, and their identification by tandem mass spectrometry, point to a different distribution of these molecules in etiolated hypocotyls.

Functions of melatonin in plants: a review

The number of studies on melatonin in plants has increased significantly in recent years. This molecule, with a large set of functions in animals, has also shown great potential in plant physiology. This review outlines the main functions of melatonin in the physiology of higher plants. Its role as antistress agent against abiotic stressors, such as drought, salinity, low and high ambient temperatures, UV radiation and toxic chemicals, is analyzed. The latest data on their role in plant–pathogen interactions are also discussed. Both abiotic and biotic stresses produce a significant increase in endogenous melatonin levels, indicating its possible role as effector in these situations. The existence of endogenous circadian rhythms in melatonin levels has been demonstrated in some species, and the data, although limited, suggest a central role of this molecule in the day/night cycles in plants. Finally, another aspect that has led to a large volume of research is the involvement of melatonin in aspects of plant development regulation. Although its role as a plant hormone is still far of from being fully established, its involvement in processes such as growth, rhizogenesis, and photosynthesis seems evident. The multiple changes in gene expression caused by melatonin point to its role as a multiregulatory molecule capable of coordinating many aspects of plant development. This last aspect, together with its role as an alleviating‐stressor agent, suggests that melatonin is an excellent prospect for crop improvement.

Melatonin: plant growth regulator and/or biostimulator during stress?

Melatonin regulates the growth of roots, shoots, and explants, to activate seed germination and rhizogenesis and to delay induced leaf senescence. The antioxidant properties of melatonin would seem to explain, at least partially, its ability to fortify plants subjected to abiotic stress. In this Review we examine recent data on the gene-regulation capacity of melatonin that point to many interesting features, such as the upregulation of anti-stress genes and recent aspects of the auxin-independent effects of melatonin as a plant growth regulator. This, together with the recent data on endogenous melatonin biosynthesis induction by environmental factors, makes melatonin an interesting candidate for use as a natural biostimulating treatment for field crops.

Protective effect of melatonin against chlorophyll degradation during the senescence of barley leaves

Abstract:  Melatonin (N‐acetyl‐5‐methoxytryptamine) is a highly conserved molecule whose presence is not exclusive to the animal kingdom. Indeed, numerous studies have demonstrated its presence in plants, where the possible role(s) of this indoleamine is (are) under active investigation. The present work aims to further our knowledge in this respect and presents the results of a study of the effect that melatonin has on foliar senescence. Barley leaves treated with melatonin solutions clearly slowed down the senescence process, as estimated from the chlorophyll lost in leaves. This effect of melatonin was concentration dependent, with an optimal response being obtained at 1 mm melatonin, after 48 hr of incubation in darkness. The already known effects of the phytohormones, kinetin, and abscisic acid, were also assayed. Of the phytohormone and melatonin combinations assayed, 1 mm melatonin presented the best protection against senescence. The levels of endogenous melatonin in control leaves were measured by liquid chromatography with fluorescence detection and in leaves treated with different exogenous melatonin concentrations (to demonstrate the absorption capacity of leaves). The possible physiological implications of this newly revealed action of melatonin in foliar senescence are discussed.

Melatonin promotes adventitious‐ and lateral root regeneration in etiolated hypocotyls of Lupinus albus L.

Abstract:  Melatonin is a well‐known animal substance, which has recently been detected in plant tissues. However, there are only a few studies concerning its possible physiological role in plants. In this paper, we investigate the possible effect of melatonin on the regeneration of lateral and adventious roots in etiolated hypocotyls of Lupinus albus L. compared with the effect of indole‐3‐acetic acid. We performed this study by measuring both molecules in roots. Six‐day‐old derooted lupin hypocotyls immersed in several melatonin or indole‐3‐acetic acid concentrations were used to induce roots. A macro‐ and microscopic study of the histological origin of the adventitious and lateral roots was made, while melatonin and indole‐3‐acetic acid in the roots were quantified using liquid chromatography with fluorescence detection. The data show that both melatonin and indole‐3‐acetic acid induced the appearance of root primordia from pericicle cells, modifying the pattern of distribution of adventitious or lateral roots, the time‐course, the number and length of adventitious roots, and the number of lateral roots. Melatonin and indole‐3‐acetic acid were detected and quantified in lupin primary roots, where both molecules were present in similar concentrations. The physiological effect of exogenous melatonin as root promoter was demonstrated, its action being similar to that of indole‐3‐acetic acid.