Polyphenolic extract and essential oil quality of Thymus zygis ssp. gracilis shrubs cultivated under different watering levels

Abstract

Thymus zygis ssp. gracilis shrubs were cultivated as an experimental crop under different watering level, in order to achieve 81, 63, 44 and 30% of the local potential evapotranspiration (ETo). After 4 years of cultivation, thyme leaves were analyzed on the basis of their essential oil (yield and quality), total phenolic content, free radical-scavenging activity and polyphenolic profile.

Essential oil yield values ranged between (2.3 ± 0.7) and (3.6 ± 0.7)% for 81 and 30% ETo equivalent, respectively. The comparison of essential oil production at the 2nd and 4th years of cultivation showed that using watering levels higher than 30% ETo equivalents reduced significantly (P < 0.05) the essential oil yielded by these shrubs with time.

Analysis of total phenolic content, polyphenolic profile, and radical scavenging activity were performed using post-distillation dry leaves. Total phenolic content values ranged from (122.2 ± 19.3) to (108.5 ± 19.2) mg of gallic acid equivalents (GAEs)/g of dry plant for the highest and lowest watering level treatment, respectively. Regarding the polyphenolic profile, rosmarinic acid, followed by apigenin, ferulic, carnosic and caffeic acids, was the phenolic component quantified at the highest concentrations. Radical-scavenging activities (IC₅₀) concentrations varied from (3.7 ± 1.6) mg/mL for 81% ETo to (7.4 ± 2.3) mg/mL 30% ETo.

In spite of the intra-specific variability detected, the individual analysis of shrubs has allowed the selection of plants which are characterised by having adequate levels of essential oil and polyphenolic extract (yield and quality), almost all of them being cultivated under a 60% ETo watering level. These selected shrubs will allow us to make further vegetative propagations in order to obtain homogeneous field crops with plants of contrasted quality cultivated under a 60% ETo watering level.

Introduction

Thymus zygis ssp. gracilis (Boiss.) R. Morales (also known as red thyme) is a widespread endemic plant in the Iberian Peninsula. It is normally located from sea level to 2000 m above sea level and has its flowering period between May and July (Morales, 1986). This aromatic shrub has become one of the most commercial Spanish thymes because of the economical importance of its essential oil and its use as a condiment by herb-shops and the food industries.

The importance of aromatic plants as natural antioxidants has been well established (Cuvelier et al., 1996, Soares et al., 1997, Dapkevicious et al., 1998, Kähkönen et al., 1999, Bicchi et al., 2000, Parejo et al., 2002, Miura et al., 2002, Wellwood and Cole, 2004, Proestos et al., 2005, Smelcerovic et al., 2006). Their main secondary metabolites, (poly)phenolic compounds, are characterised by having redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers. They also have metal chelation properties (Rice-Evans and Miller, 1997, Kähkönen et al., 1999).

The antifungal and antibacterial activities present in essential oil of plants of the genus Thymus have been demonstrated by several researchers (Cruz et al., 1989, Crespo et al., 1990, Bhaskara Reddy et al., 1998, Marino et al., 1999, Consentino et al., 1999, Dafarera et al., 2000, Kulenova et al., 2000, Karaman et al., 2001). Its antioxidant activity against the thermal autooxidation of lard has been demonstrated (Kulevanova and Panovska, 2002), and it has been shown that a dietary supplementation of thyme essential oil could address the unfavourable antioxidant-pro-oxidant balance that occurs with age (Youdim and Deans, 1999).

Some of the aromatic plants which are widespread in the Mediterranean area have already been studied for their antioxidant activity, including sage, rosemary and thyme (Cuvelier et al., 1996, Haraguchi et al., 1996, Soares et al., 1997, Hidalgo et al., 1998, Wang et al., 1999, Bicchi et al., 2000, Parejo et al., 2002, Miura et al., 2002, Del Baño et al., 2003, Wellwood and Cole, 2004, Proestos et al., 2005). The search for natural antioxidants in aromatic plants by-products has become an alternative to synthetic antioxidants in the food and pharmaceutical industries (Soriano et al., 1996, Parejo et al., 2002). These authors considered the study of the remaining distillation material potentially interesting as a result of the water-soluble properties of phenolic compounds that rarely form part of essential oils. Phenolic compounds tend to be water soluble, because they frequently occur combined as glycosides, and they are usually located in the cell vacuole (Harborne, 1998).

Thymus zygis ssp. gracilis, also known as red thyme, is one of the most commercial Spanish thymes because of the economic importance that the presence of thymol has for thyme essential oil quality. For this species, the average yield of essential oil per plant is around 3% (w/v) (Sotomayor, 1998). Currently, in Spain, more than 1500 ton/year of dry leaves and 26 ton of thyme essential oil are exported to foreign countries (Sotomayor, 1999). Considering these data, an amount of 8 ton of dry red thyme leaves would be required for this oil production. This involves an overproduction of apparently useless distillate leaves. Different extracts from thyme leaves have shown the presence of a large number of flavonoids and vitamin E, compounds of great interest in the food industry due to their antioxidant activities (Guillén and Manzanos, 1998). However, as cultivation practices are still being developed for this species, there is not enough production to satisfy the external market demands. This necessitates the harvesting of wild plants, which imposes a great commercial and ecological inconvenience, due to the great heterogeneity of the chemical compositions of the final products and the lack of production control. As plants are not available throughout the year, production of extracts is interesting, since the industry demand is not met by this commercial thyme.

Thymus zygis ssp. gracilis has only been cultivated previously by Sotomayor (1998), in dry-land conditions, and by Sotomayor et al., 2001, Sotomayor et al., 2004, under an experimental irrigation design.

Sotomayor et al. (2004) evaluated the possibility of cultivating red thyme under drip-irrigation conditions and determined how irrigation affects the yield and quality of essential oil and dry material production. Nevertheless, so far, to our knowledge, there is no information about the variation of phenolic composition in cultivated thyme species under different watering-level conditions.

Generally, the contents of phenolic substances are influenced by a large number of external factors such as agro-technical processes, climatic conditions, harvest and post-harvest manipulations, and time of consummation (Waterman and Mole, 1994). However, according to Parejo et al. (2002), in general, the distilled plant material has a higher phenolic content as well as higher antioxidant and radical scavenging activities than the non-distilled material.

So far, this thyme species has only been cultivated considering the economical importance of its essential oil. However, interest is now also focused on the search for natural antioxidants from waste materials of aromatic plants. For these reasons, the main goal of the present study is to determine how irrigation conditions affect the polyphenolic extract quality from the distillate of red thyme. This result, along with those obtained for essential oil yield and quality, will allow us to define the best irrigation conditions for this thyme species. On the other hand, one of the biggest problems that the cultivation of this species exhibits is the great intra-specific variability observed among shrubs. As a consequence of the source of the seeds (wild populations), these shrubs are not adapted to the cultivation practices. So, a previous pre-selection is needed in order to choose those plants that are resistant to the cultivation practices and, at the same time, exhibit adequate yield and quality regarding the essential oil and polyphenolic extracts.