Bio-morphological and Chemical Characterization of Rosemary (Rosmarinus officinalis L.) Biotypes

The use of herbs in the food industry has recently increased significantly because of their antioxidant action on lipid degradation, besides their traditional role in food aroma. In particular, rosemary (Rosmarinus officinalis L.) extracts have proved to possess such useful antioxidant properties. The present research that reports on a study on the morphological and chemical characteristics, conducted on rosemary clones obtained from wild material collected in different environments of southern Italy, was aimed at better characterizing and evaluating their potential for present and possible new food technological uses. In addition to morphological characterizations aimed to detect differences among clones, a technological qualitative oil component “fingerprint” was conducted by means of GC-MS, comparing two different extraction techniques of the essential oil fraction (traditional steam distillation and dichloromethane solvent extraction) for the quantitative and qualitative differences. INTRODUCTION The genus Rosmarinus Linnaeus is by now represented by a single species, Rosmarinus officinalis L. (rosemary). However, the systematics of varieties, subspecies and biotypes appears uncertain and confused, as there are numerous cultivars that grow wild in the Mediterranean countries (Giugnolinini, 1985). Even though there are subspecies and varieties of R. officinalis L., some infraspecific differences are likely to occur within the species, in that morphologically identical plants may have a different essential oil composition. R. officinalis L., traditionally used fresh, dried, or as essential oil, actually contains a great quantity of essential oil (more than 1 %) that is largely used in medicine. The chemical composition of rosemary oil has been the subject of an extensive study, reviewed by Lawrence (1976–1977,1979–1980,1981–1987,1988–1991,1992–1994, 1995). Two major types of rosemary oil can be distinguished with respect to their main constituents: oils with over 40 % of 1,8-cineole (oils from Morocco, Tunisia, Turkey, Greece, Yugoslavia, Italy, France) and oils with approximately equal ratios (20–30 %) of 1,8-cineole, α-pinene and camphor (oils from France, Spain, Italy, Greece, Bulgaria) (Mastelic, 1997, Tomei et al., 1995). The literature also revealed some unusual chemical compositions for rosemary oils. The more recent interest in this species concerns the biological (antioxidant, antimicrobial and insecticidal) action of specific components of the essential oil of rosemary. The objective of this research was to characterise the morphological and chemical traits of some rosemary clones, obtained from wild material collected in different environments of southern Italy, to better assess their potentials for present and possible new food technological uses. Recently, essential oils deriving from medicinal plants such as Rosmarinus gained importance as to their quantitative and qualitative composition (Adams, 1995.). In addition to this, oil composition differences proved to be helpful for integrated ecotypechemiotype characterization approaches, helpful for practical issues such as definition of Proc. XXVI IHC – Future for Medicinal and Aromatic Plants Eds. L.E. Craker et al. Acta Hort. 629, ISHS 2004 Publication supported by Can. Int. Dev. Agency (CIDA) 472 optimum harvesting time or the evaluation of field-plant interactions (European Union Commission 2000, Falchi Delitali et al., 1980). This research is aimed to characterise the morphological and chemical traits of some clones obtained from wild material collected in different environments of southern Italy, with a view to better assess their potential for present and possible new food technological uses. MATERIALS AND METHODS Plant Material Seven biotypes belonging to the genus Rosmarinus, selected from wild populations in different environments of southern Italy (Apulia, Basilicata and Calabria), that were collected since 1999. Experimental Design The collection of rosemary clones obtained from wild mother plants was maintained in a collection field at the Agricultural Faculty of Bari (Italy). The field had been fertilized with 120 kg ha N, 100 kg ha P2O5 and 100 K2O kg ha. The experimental design was a randomised complete-block design with 3 replications. The planting distance was 0.75 m between rows and 0.35 m within rows. Harvest and Processing Spring shoots used for the determination of morphological parameters were collected for each biotype from three-year-old plants, during the period of slow growth (15th July, 2002). Measurements were made on the total fresh weight, number and weight of early shoots; their weight, length, stem diameter and the number of internodes were measured on defoliated shoots; the number and total weight of leaves of detectable size (>0.5 mm length) occurring on the whole shoot and on side branches was determined. The length, width and surface area of 30 leaves per biotype were measured, using an image analysis software (Scion image program), after acquisition by the videocamera. Keeping stems and leaves separate, samples were oven-dried at 38°C and re-weighed for the dry matter determination. Entire shoots, once oven-dried, were used for the distillation of essential oils, by dispersing 10 g of dry matter in 400 ml of water, by a 3-hour treatment at a temperature of 90°C. Content of Essential Oil For hydrodistillation extraction of oil, vegetal leaf material was air-dried on wooden benches away from direct sunlight for a fifteen days period. Dried leaves were then finely minced with an electric mill and placed in a 3-liter Clevenger-type assembly distillation apparatus. The plant material 150 g in 1500 ml of deionised H2O; was distilled for 3 hours. The essential oil was collected about 1 h after heating and dehydrated with the addition of anhydrous sodium sulphate (Merck). For the solvent extraction of oil, five grams of oven-dried (35°C for 72 hours) leaf-stem deriving from each single plant were submitted to solvent extraction in the plant material, 0.2 g was placed in 1.5 ml of dichloromethane for 15 min. in ultrasound bath. The obtained essential oil was collected in 2.5 ml borosilicate glass vials sealed by PTFE film, and stored at –18°C up to analysis. Composition of Essential Oil Essential oil samples were analyzed by means of gas chromatography mass spectrometry (GC-MS), by a Hewlett Packard 5973 mass selective detector connected with a 6890 Hewlett Packard gas chromatograph. Separation was achieved by a Hewlett Packard HP-5MS fused-silica capillary column (30 m x 0.2 mm I.D., film thickness 0.33 micron).The GC used helium as a carrier gas at a flow rate of 1 ml/min. The temperature ramp was from 50°C (5 min) to 270°C (20 min) at 4°C/min. The injection temperature