INTRODUCTION Thymus L. Is One of the Most Important Genera
Total Page：16
File Type：pdf, Size：1020Kb
Arch. Biol. Sci., Belgrade, 60 (3), 475-483, 2008 DOI:10.2298/ABS0803475D POPULATION VARIABILITY IN THYMUS GLABRESCENS WILLD. FROM SERBIA: MORPHOLOGY, ANATOMY AND EssENTIAL OIL COMPOSITION Zora Dajić-STEvanović1, I. Šoštarić1, P. D. Marin2, D. Stojanović3, and M. Ristić4 1Faculty of Agriculture, University of Belgrade, 11080 Belgrade, Serbia 2Institute of Botany, Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia 3Faculty of Pharmacy, University of Belgrade, 11000 Belgrade, Serbia 4Dr. Josif Pančić Institute for Medicinal Plant Research, 11000 Belgrade, Serbia Abstract — In five indigenous populations of Thymus glabrescens Willd. collected in the region of Banat (Serbia), the variability on leaf morphological traits, leaf and stem anatomy, and composition of the essential oil was studied. The major component in the studied populations was either thymol or γ-terpinene. Distinct differentiation of populations with respect to chemical composition of essential oils might be related to spatial distribution of the studied populations. No correlations between morphology, anatomy, and essential oil yield and composition were determined. Both capitate and peltate glandular trichomes were found on calyces, whereas the latter were noticed on the abaxial and adaxial leaf surface. Key words: Thymus glabrescens, essential oils, thymol, γ-terpinene, glandular trichomes, cluster analysis Udc 582.929.4:575.17:581.4:615 INTRODUCTION Hyphodromi, and Serpyllum. In Flora of Serbia 31 species of the genus Thymus are listed, with more Thymus L. is one of the most important genera as than 60 varieties, most of which grow in vari- regards the number of species (more than 200) with- ous meadow and pasture communities and in dry, in the family Lamiaceae. This genus belongs to the sunny, rocky habitats, on both limestone and ser- tribe Mentheae, subfamily Nepetoideae (M o r a l e s , pentine (D i k l i ć , 1974). 1986). That there are serious difficulties in the taxonomical interpretation of the taxa belonging Lamiaceae genera and species are known for sig- to the genus Thymus owing to the high variability nificant variability of the secretory structures respon- of populations with respect to many morphologi- sible for biosynthesis and accumulation of essential cal and micromorphological traits, as well as the oils [with respect to both their type (A s c e n s a o et composition of secondary compounds (Dajić- al., 1998) and their number and distribution within S t e v a n o v i ć and Š o š t a r i ć , 2006). This vari- the plant organs (Ve n k a t a c h a l a m et al., 1984, abily is caused both by environmental factors and Tu r n e r et al., 2000)]. Chemical polymorphism is genetic variation due to frequent hybridization lead- characteristic of the species of Thymus; numerous ing to variable chromosome number and expressed chemotypes have been defined, such as carvacrol gynodioecy, a sexual polymorphism in which natu- and thymol, a-terpineol, thujone, geraniol, linalool, ral populations contain two type of plants – females and others (Thompson et al., 1998). Thyme and hermaphrodites (T h o m p s o n , 2002). essential oils have been reported to possess antimi- crobial activities (B h a s k a r a et al., 1998; L o ž i e n é According to J a l a s (1971), the genus Thymus et al., 2007), most of which are mediated by thymol is divided into eight sections: Micantes, Mastichina, and carvacrol, as the phenolic components of the Piperella, Teucrioides, Pseudothymbra, Thymus, oil. Due to the hydrophobic nature of Lamiaceae 475 476 ZORA DAJIć-STEVANOVIć ET al. volatiles, the bacterial cell membrane has been pro- In analysis of leaf (N = 45) and stem (N = 30) posed as the primary target of their antimicrobial anatomical traits, plant material was fixed in FAA, action (M i t i ć –ć u l a f i ć et al., 2005). Spasmolytic subjected to the standard paraffin procedure, and and antioxidant activities (M i g u l et al., 2004; S a c - microtome sectioned using a LEICA SM 2000 R c h e t t i et al., 2005) have also been reported for the microtome, after which sections 7-10 µm thick phenolic oil extract of the plants. were stained with safranine and aniline blue. Image analysis was done with LEICA IM1000 software. Thymus glabrescens Willd. belongs to the section Serpyllum, subsection Isolepides (J a l a s , 1972). It is In order to describe the number of glandular a perennial herbaceous plant, distributed in Central trichomes on leaves, 30 leaves were taken from indi- and Eastern Europe as well as in Asia, inhabiting vidual plants and bright-field microscopy was con- open dry meadows, grasslands, and rocks with sunny ducted using a LEICA XTL-3400 D stereo-micro- exposure (J a l a s , 1972). In Serbia, the species has a scope. For scanning electron microscopy (SEM), scattered distribution being found on the slopes of small pieces of dry leaves and calyces of 10 plants Mt. Fruška Gora, in the Deliblato Sands, through- were sputter-coated with gold for 180 sec at 30 mA out Šumadija, and in Southwest Serbia (D a j i ć - using a BAL-TEC SCD 005 instrument and viewed S t e v a n o v i ć and Š o š t a r i ć , unpublished data). with a JEOL JSM-6460L Velectron microscope at an acceleration voltage of 20 kV. Despite broad scientific interest in the biology, taxonomy, chemotypes, and related biological activ- Isolation and analysis of essential oils ity of secondary metabolites of Thymus species, Air dried aerial parts were subjected to hydro- there is a general lack of information regarding Th. distillation for 3 h using a modified Clevenger-type glabrescens, with the exception of two reports from apparatus with a water-cooled oil receiver to reduce the Western Balkans (K a r u z a –S t o j a k o v i ć et hydrodistillation artifacts. The oil obtained was al., 1989; Kustrak, 1990) and one from Romania dried over anhydrous sodium sulfate and stored at (K i s g y ö r g y et al., 1983). We therefore studied 4-6°C until analyzed. the morphological, anatomical, and essential oils of autochthonous populations of Th. glabrescens. The GC-FID analysis was carried out in a Hewlett results are here present as a first report on compara- Packard 5890 II gas chromatograph equipped with tive morphology, anatomy, and essential oil compo- FID, a split-splitless injection system (split ratio of sition of this species from Serbia. 1:30), and a 25 m x 0.32 mm HP-5 fused silica capil- lary column (film thickness: 0.52 μm). The carrier MATERIAL AND METHODS gas was H2, the flow rate 1 ml/min. Oven tempera- ture was programmed from 40oC to 260oC at a 4oC/ Collection of plant material and study site min linear rate; injector and detector temperatures o o Th. glabrescens was collected during the flow- were maintained at 250 C and 300 C, respectively. ering period of June 2004 in the Banat region of The injection volume was 1 μl of 1 % (w/v) essential Northeast Serbia (Table 1). Voucher specimens were oil in ethanol. determined in accordance with the Flora of Serbia GC-MS analyses were performed with a Hewlett (D i k l i ć , 1974) and Flora Europaea (J a l a s , 1972) Packard G 1800C GCD Series II (GC-EID) instru- and deposited at the Department of Botany, Faculty ment fitted to a 30 mm x 0.25 mm HP-5 MS capillary of Agriculture, University of Belgrade, Belgrade. column (film thickness: 0.25 μm) using He (1 ml/min) Morphology and anatomy as the carrier gas, temperature of the transfer line being maintained at 260oC. Essential oil components were The following morphological features of leaves were identified by matching their mass spectra with pub- analyzed: length (mm), width (mm), and the length/ lished data (A d a m s , 1989) and by consulting librar- width ratio (N = 30) in all of the collected populations. ies of mass spectra (Wiley and NIST/NBS). population variabilitY of thYmus GLabrEscENS in SErbia 477 Table 1. Geographic position and habitat description for studied populations of Th. glabrescens Herbarium voucher Population code Locality Habitat GPS data N 45o16´53.6˝ P1 100604-1 Belo Blato Ruderal habitat E 020o22´33.2˝ Slightly salt-affected N 45o17´25.0˝ P2 100604-2 Lukino Selo meadow E 020o25´53.3˝ Slightly salt-affected N 45o28´41.6˝ P3 100604-3 Zrenjanin Melenci meadow E 020o20´17.7˝ N 45o00´05.1˝ P4 180604-1 Devojački Bunar Meadow on the sand E 020o56´19.5˝ N 45o04´20.6˝ P5 180604-4 Jablanka Steppe meadow E 021o23´04.2˝ Data processing other populations, probably due to the significantly lowest average number of glandular trichomes on Analysis of variance (ANOVA and Duncan both the upper and lower leaf epidermis (Fig. 1). multiple range test) and cluster analysis (based upon Euclidean distances) of all surveyed populations of Leaf and stem anatomy Th. glabrecens were performed using STATISTICA software, version 7.0. Leaf anatomical features measured in two differ- ent regions (at the central midrib and near the apex) RESULTS AND DISCUSSION showed that all characters were significant for dis- crimination of populations (ANOVA, Table 3). The Leaf morphology highest and lowest leaf depths were found for P4 and P1, respectively. The height of upper and lower epi- The population variability of leaf morphology dermal cells was greatest in P4 and P3, respectively.