Influence of phenological stage on the antioxidant activity of Thymus zygis s . l . essential oil

When a species has proven antioxidant capacity and possible commercial application it is important to know whether or not this activity varies with the phenological stage of the raw material commodity. The present work aims to assess this topic for the case of Thymus zygis s.l. We analyzed the yield and the chemical composition (gas chromatography-mass spectrometry) of its essential oil in five wild populations from the Southwest of Iberian Peninsula at flowering and fruiting stage. We measured their TEAC (Trolox equivalent antioxidant activity) values. The main components were p-cymene (flowering: 33.6-16.2%; fruiting: 43.2-30.3%), γ-terpinene (flowering: 13.5-5.78%; fruiting: 4.67-1.96%) and thymol (flowering: 46.45-22.2%; fruiting: 54.24-13.9%); the latest, especially high (flowering: 46.45%; fruiting: 54.24%) in one of the populations, identified as Thymus zygis ssp. gracilis (Boiss.) R. Morales, which showed the highest antioxidant activity as well. In the set of samples TEAC oscillated between 16.12-30.52 mmol Trolox L–1 (flowering) and 10.96-40.24 mmol Trolox L–1 (fruiting). No significant differences between antioxidant activity of both stages were found, but yields were significantly higher at flowering time. These levels of antioxidant activity are close to those achieved as highly antioxidant food products, as coffee or wine. This species can be a good raw material for agribusiness use, specially the population of Thymus zygis ssp. gracilis. Additional key words: antioxidant capacity; chemical composition; Iberian Peninsula; Lamiaceae; phytogenetic resources; thyme.

in the fruiting stage.It was dried in an airy room in darkness, and conserved for two months in paper bags.Two extractions of the essential oil were carried out for each sample (hydrodistillation; European Pharmacopoeia, Council of Europe, 1996); one with the whole plant (WP) and another with flowers and leaves (FL).Essential oil yield and its percentage composition were calculated.
We found that the yields of the populations studied oscillated between 0.5 and 1.73% for the whole plant (WP) and between 0.96 and 3.42% for the flowers and leaves (FL) (Table 2).The high yields of this plant have made it one of the most comercial Spanish thymes (Jordán et al., 2009).Our results may suggest that it is much more advantageous the extraction in flowers and leaves than in the whole plant, because yields are significantly higher in the flowering (WP = 0.9-1.73%;FL = 1.43-3.42%)than fruiting (WP = 0.35-0.93%;FL = 0.96-1.92)[Wilcoxon test, (WP), n = 4, *, p = 0.043; (FL), n = 4, *, p = 0.043].

Introduction
Thymus zygis s. l., is a plant endemic to the Iberian Peninsula and northern Morocco, which is a pioneer scrub on all types of soils.Its traditional use is well known in its native range (Vázquez, 2008;Morales, 2010) and its essential oil has been chemically characterized using material from North Africa, Spain and Portugal (Blanco, 2005;Figueiredo et al., 2008).Some authors (Jiménez et al., 1993;Jordán et al., 2009) have pointed out its antioxidant power.This capacity makes it a plant of possible use in the food industry, important to inhibit oxidation, one of the major causes of food spoilage.It prevents rancidity and/or deterioration of the nutritional quality, colour, flavour and texture (Antolovich et al., 2002).
When a species has proven antioxidant capacity and possible commercial application it is important to know whether or not this activity varies with the phenological stage of the raw material commodity.Studies addressing this question have not been made and it is a highly interesting topic from the applied point of view.Thus, the present work aims to assess the influence of the phenological stage on the antioxidant activity of Th. zygis.
Table 2 summarizes chemical composition of essential oils studied.Chemical composition of thyme essential oil may vary throughout the growing season (Blanco et al., 2010).Our results offered only significant differences between the flowering and fruiting season in three of the components identified (Kruskal-Wallis test, p-cymene: n = 10, *, p = 0.028; limonene, n = 10, *, p = 0.015; γ-terpinene: n = 10, **, p = 0.009: see Table 2), without impact on the final antioxidant activity, although two of them did not represent residual amounts in the total mixture.It is evident that the total antioxidant activity is due to the sum of the activities of each component, many of them with demonstrated antioxidant capacity have been tested individually by Dorman et al. (2000a).
Finally, it is also noteworthy that this essential oil has a high proportion of thymol.This fact gives a remarkable quality of the product, given the particular organoleptic characteristics of this phenolic derivative, top rated by industry (Sotomayor, 1998), and much more competitive from a business perspective than others as carvacrol, present in related species but with unpleasant smelling, irritating properties, and a clearly lower value.56, 24.6, 17.21, 22.97, and 24.4 mmol Trolox L -1 .Fruiting stage: 37.69, 15.35, 11.98, 14.49, and 22.81 mmol Trolox L -1 .

Table 1 .
Provenance of the study material, indicating locality (all in Badajoz province), altitude, UTM coordinates, collection date, voucher specimens in the HSS Herbarium (Herbario del Suroeste de España, Badajoz, Spain), and population taxon (subspecies) sylvestris Short communication.Antioxidant activity of Thymus zygis essential oil Even though, for an industrial extraction process, it seems more logical to use the whole plant, because the separation of flowers and leaves consumes many hours of factory work.

Table 2 .
Percentage composition (whole plant, WP) and yield (WP and flowers and leaves, FL) of the essential oil of five Thymus zygis populations (P) 3 RTM = Retention time in GLC-MS (min).