Leaf mass per area (LMA) is a morphological trait widely used as a good indicator of plant functioning (i.e. photosynthetic and respiratory rates, chemical composition, resistance to herbivory, etc.). The LMA can be broken down into the leaf density (LD) and leaf volume to area ratio (LVA or thickness), which in turn are determined by anatomical tissues and chemical composition. The aim of this study is to understand the anatomical and chemical characteristics related to LMA variation in species growing in the field along a water availability gradient. We determined LMA and its components (LD, LVA and anatomical tissues) for 34 Mediterranean (20 evergreen and 14 deciduous) woody species. Variation in LMA was due to variation in both LD and LVA. For both deciduous and evergreen species LVA variation was strongly and positively related with mesophyll volume per area (VA or thickness), but for evergreen species positive relationships of LVA with the VA of epidermis, vascular plus sclerenchyma tissues and air spaces were found as well. The leaf carbon concentration was positively related with mesophyll VA in deciduous species, and with VA of vascular plus sclerenchymatic tissues in evergreens. Species occurring at the sites with lower water availability were generally characterised by a high LMA and LD.

The anatomical basis for variation in LMA in woody species depends on the contrast made. Higher LMA in evergreens is mainly due to a greater leaf thickness, caused by a larger volume of mesophyll and air spaces. Within deciduous species and evergreens, higher LMA is caused by a higher density, due to higher volumetric fractions of mesophyll and lower fractions of air spaces and epidermis.

Supercritical fluid extraction (SFE) and liquid solvent sonication, in combination with two different sample treatments, were compared for the extraction of natural antioxidants from rosemary leaves. Dried, ground, and sieved rosemary leaves (20 mg) were subjected to SFE with CO(2) at 355 bar at 100 °C (CO(2) density 0.72 g/mL) for 20 min at a liquid flow rate of 4 mL/min. The analytes were concentrated on an ODS trap and subsequently eluted with acetone. Antioxidants in the SF and liquid solvent extract were analyzed by HPLC. Compounds of known antioxidant activity such as carnosol, carnosic acid, and methyl carnosate were identified by mass spectrometry of the HPLC fractions collected. Freezing and grinding the samples in liquid nitrogen resulted in decreased carnosic acid recoveries. Supercritical CO(2) extraction provided the highest recovery of carnosic acid from rosemary leaves (35.7 mg/g), the lowest relative standard deviation (4.4%), and the cleanest extract [Formula: see text] no cleanup prior to HPLC was required. Among the liquid solvents studies, only acetone provided comparable results (73% recovery relative to SC-CO(2) extraction); however, it required decoloration with active carbon prior to HPLC analysis.

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The concentration of carnosic acid in a representative selection of leaves from wild and cultivated populations of Rosmarinus officinalis was determined by reversed-phase HPLC following extraction with supercritical carbon dioxide. Different sources of variability including season, genetics, leaf age, and growing origin (wild or cultivated) were considered. Variability in the carnosic acid content among rosemary leaves appears to be largely due to seasonal and environmental factors, in addition to their individual origin (genetics). The results reveal excellent correlation (r = 0.93) between the carnosic acid concentration and photoperiod. The results presented can be used to improve the selection of raw materials for the extraction of carnosic acid from rosemary. Keywords: Rosmarinus officinalis; carnosic acid; antioxidant; supercritical fluid extraction