Glycosidically Bound Volatile Compounds of Satureja montana L

In this paper, the glycoconjugated volatile compounds of four Croatian Satureja species (Satureja montana L., S. cuneifolia Ten., S. subspicata Vis. and endemic S. visianii Šilić) were investigated. Content and composition of these compounds were examined depending on the stage of plant development. GC and GC–MS analysis of volatile aglycones revealed twenty-one compounds. Thymoquinone, geraniol and carvacrol were detected in all vegetative phases of the investigated plants. Other quantitatively important aglycones were eugenol and thymol of S. montana, phenyl ethyl alcohol, benzene acetaldehyde, borneol, α-terpineol, thymol and eugenol of S. cuneifolia, phenyl ethyl alcohol, benzene acetaldehyde, terpinen-4-ol, α-terpineol and β-ionone of S. subspicata and camphor, thymol and 8a-acetoxylemolol of S. visianii. Moderate similarity in the chemical composition of essential oils and volatile aglycones of investigated plant species indicate that many biologically active compounds are glycosylated and accumulate as non-volatile glycosides.


Introduction
The plants of the genus Satureja (Lamiaceae) are often aromatic herbs and shrubs widely distributed in the Mediterranean area, Asia and boreal America.In the Mediterranean region of Croatia four Satureja species are reported: Satureja montana L., S. cuneifolia Ten., S. subspicata Vis. and an endemic S. visianii Šilić [1].These annual or perennial semi-bushy plants inhabit arid, sunny, stony and rocky habitats along the Adriatic coast.Among these plants, winter savory (S. montana) and wild savory (S. cuneifolia) are the most common in this part of Croatia [2].
In numerous Lamiaceae species, many secondary metabolites, including important biologically active compounds, are glycosylated and accumulate as non-volatile glycosides.These compounds are soluble in water, nonvolatile and odourless.Connected with the fact that non-volatile glycosides can release volatile flavour compounds (aglycones) by acid or enzymatic hydrolysis, these plant metabolites are a possible source of aroma.Free and glycosidically bound volatiles are secondary metabolites important for the plant survival, reproduction and chemotaxonomy since they occur in adaptations as strong phylogenetic and ecological components.Chemical composition, antimicrobial activity and other possible applications in phytotherapy of Satureja essential oils were previously widely investigated [2]- [8].Despite that, we observed a relative paucity of data on the composition of glycosidically bound volatile compounds in this group.Analyses of glycosidically bound volatiles and antioxidant activity of free volatile compounds were previously reported for S. montana L. [9] [10].Monoterpenes are usually present as main constituents of the aglycones as well as the main components in essential oil composition of aerial parts of Satureja plants [5] [11] [12].In the literature data, we found no other published results dealing with the glycosidically bound volatiles of other Satureja species.Considering all mentioned above, our goal was to study the composition of glycosidically bound volatile compounds of four Croatian Satureja species (S. montana, S. cuneifolia, S. subspicata and endemic species S. visianii) and compare composition of the obtained free and glycosidically bound volatiles with the composition of the essential oil.

Plant Material
Three Satureja species (S. montana, S. cuneifolia and S. subspicata) were collected during 2014 at the Kozjak Mountain (near the city of Split, Croatia), prior to flowering (in July, leaves and stalks) in the course of flowering (in September, flowering tops, leaves and stalks) and after flowering (in November, leaves and stalks).Satureja visianii was collected in the same period on the peninsula of Pelješac, Croatia.Voucher specimens are deposited in the herbarium at the Department of Biology, Faculty of Science, University of Split, Croatia [No.

Isolation of Glycosidically Bound Volatile Compounds
The glycosides of volatile compounds were isolated from 100 g of fresh plant material by extraction at room temperature (24 h).Extraction was effected by percolation with 500 ml ethyl acetate.As internal standard, 500 μg of octyl-β-D-glucoside was added to ethyl acetate.20 mg β-glucosidase from almonds (Fluka) was added to the glycosidic solution, along with 3 ml pentane for trapping liberated aglycones.Hydrolysis was carried out at 37˚C for 72 hours.

Gas Chromatography-Mass Spectrometry
Gas chromatography (GC) analyses were performed on a gas chromatograph (model 3900; Varian Inc., Lake Forest, CA, USA) equipped with flame ionization detector (FID), mass spectrometer (MS) (model 2100T; Varian Inc.), capillary column VF-5ms (30 m × 0.25 mm i.d., coating thickness 0.25 μm).The individual peaks were fixed by comparison of their retention indices, and/or authentic samples, as well as by comparing their mass spectra with literature data [13].

Results and Discussion
The content of aglycones obtained from fresh plant material varied from 1.8 to 7.6 mg•kg −1 according to the stage of plant development.The content of these compounds was the highest during the flowering period of plant, and decreased in the period after flowering.GC-MS analysis of the aglycones revealed twenty-one compound, representing 82.6% -97.2% of the total aglycone fraction (Table 1).
Based on the incidence throughout all the vegetative stages of plant development, the main aglycones in investigated Satureja species are thymoquinone (2.4% -56.9%), geraniol (2.2% -16.9%) and carvacrol (3.4% - 42.6%) (Table 1).Thymoquinine was also reported as a dominant aglycone (20.7%) of S. montana in the study by Radonić and Miloš [10].Other quantitatively important aglycones of S. montana were eugenol (17.8%) and thymol (11.3%) as a dominant compounds in the period after flowering (Table 1).Other quantitatively important aglycones in S. cuneifolia were phenyl ethyl alcohol, benzene acetaldehyde, borneol, α-terpineol, thymol and eugenol (Table 1).These compounds were detected in all vegetative phases.Aside from thymoquinone, geraniol and carvacrol, quantitatively important aglycones detected in all vegetative phases of S. subspicata were phenyl ethyl alcohol, benzene acetaldehyde, terpinen-4-ol, α-terpineol and β-ionone.In addition to above listed dominant aglycones, other quantitatively important components in all vegetative phases of the S. visianii were camphor, 8a-acetoxylemolol and thymol (Table 1).Aliphatic alcohols, 2-phenylethanol, benzyl alcohol, eugenol, linalool, geraniol, nerol and α-terpineol can, more or less, be considered common in aglycone fraction of Lamiaceae family [14] and the eugenol was found to be the main aglycone in most plants of this family [10] [14] [15].Eugenol was the main aglycone of S. montana in the period after flowering (Table 1).S. subspicata and S. cuneifolia contained significant concentration of eugenol during and after flowering, but among aglycones of endemic S. visianii eugenol was detected in a low percentage in the period after flowering (Table 1).Composition of essential oils of above listed Satureja species was reported in Table 1 in our previous paper [2].Comparison of the chemical composition of volatile aglycones (Table 1) with those of essential oils [2] reveals ten common compounds (1-octene-3-ol, linalool, camphor, borneol, terpinen-4-ol, α-terpineol, myrtenol, geraniol, thymol and carvacrol) (Table 1).Among two major constituent of the aglycone fraction of Satureja species, thymoquinone and carvacrol, only the latter has been detected among the free compounds in the essential oil [2].These results are consistent with the fact that the free and glycosidically bound plant secondary metabolites have different biosynthetic pathways which establish differences in their chemical composition [14].The results of our study show moderate similarity in the chemical composition of essential oil and free volatile aglycones of investigated plant species.Regarding the substantial interest for aromatic plants and their possible applications, this paper reveals the presence of compounds whose effect may been overseen due to the fact that they are present in glycosidic form.