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Study explored high voltage electrical discharge (HVED) for recovery of bioactive compounds from Dalmatian sage. HVED parameters were: treatment time (3, 9 min), ethanol content (0, 25, 50%, v/v), voltage for argon (15, 20 kV) and nitrogen (20, 25 kV). Findings were compared to conventional extraction (CE) with assessment of total phenolic content (TPC), antioxidant activity (EPR), NIR spectroscopy, colorimetric parameters, Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). Raw sage was analyzed for pesticide residues and heavy metals. The optimal HVED extraction expressed as TPC 57.21 mg GAE/g was obtained with nitrogen/9 min/25 kV/33% of ethanol. Sage is safe for extraction in terms of pesticide residues and heavy metals. HVED electroporation facilitated the extraction of bioactive compounds and produced extracts with high antioxidant potential, thus confirmed HVED as “green” extraction for sustainable functional food production.

Kadulja (Salvia officinalis L.) je samonikla ljekovita i aromatična biljka rasprostranjena u mediteranskog dijelu Hrvatske, a posebno u Dalmaciji. Cilj ovog rada bio je odrediti profil hlapljivih spojeva dalmatinske kadulje, i to odvojeno listova, a odvojeno cvjetova, odnosno odrediti sastav i sadržaj ovih spojeva. Radi što potpunijeg uvida u sastav i sadržaj hlapljivih spojeva pripremljena su tri uzorka hlapljivih spojeva, eterično ulje, vršne pare i apsolut. Analiza uzoraka provedena je plinskom kromatografijom-masenom spektrometrijom (GC/MS). Terpenski spojevi su kvalitativno i kvantitativno najvažniji sastojci eteričnog ulja, vršnih para i apsoluta listova i cvjetova kadulje. Glavni sastojci eteričnog ulja listova kadulje su terpenski alkoholi 1,8-cineol i borneol te keton kamfor, a eteričnog ulja cvjetova alkoholi viridiflorol i izoborneol, ugljikovodik -humulen te keton kamfor. U vršnim parama listova i cvjetova kadulje identificirani su svi spojevi. Svi sastojci vršnih para listova i cvjetova kadulje su terpeni, sa -humulenom kao glavnim sastojkom u vršnim parama listova i -pinenom u vršnim parama cvjetova. Za razliku od eteričnih ulja i vršnih para, apsolut listova kadulje karakterizira visoki udio masnih kiselina, 30,4 % u apsolutu cvjetova i 21,1 % u apsolutu listova. Ipak, kvalitativno i kvantitativno, i u apsolutima kadulje dominiraju terpenski spojevi kao što su epimanool, borneol i - gurjunen u apsolutu listova te epimanool, feruginol, pisiferol, -humulen i -gurjunen u apsolutu cvjetova kadulje. Sage (Salvia officinalis L.) is a wild-growing medicinal and aromatic plant which is widespread in the Mediterranean part of Croatia, especially in Dalmatia. The aim of this study was to determine the chemical profile of Dalmatian sage volatile compounds, separately of leaves and flowers, and determine the composition and content of these compounds. For more complete insight into the composition and content of volatile compounds, three samples of volatile compounds were prepared, namely essential oil, headspace and absolute. Analysis of all samples was carried out by gas chromatography-mass spectrometry (GC/MS). Terpenes are qualitatively and quantitatively the most important ingredients of essential oils, headspaces and absolutes of sage leaves and flowers. The major constituents of leaves essential oil were terpene alcohols 1,8-cineol and borneol as well as ketone camphor, while the main constituents of flowers essential oil were alcohols viridiflorol and isoberneol, hydrocarbon -humulene as well as ketone camphor. All the compounds of leaves and flowers headspace were identified. All constituents of leaves and flowers headspace are terpenes, with -humulene as the main constituent of the leaves headspace and -pinene as the main constituent of the flowers headspace. Opposite to the essential oils and headspaces, sage absolutes were characterized by a high content of fatty acids, 30,4 % in flowers absolute and 21,1 % leaves absolute. However, qualitatively and quantitatively, terpenes were dominating compounds in sage absolutes, such as epimanool, borneol i -gurjunene in leaves absolute and epimanool, ferruginol, pisiferol, -humulene i -gurjunene in sage flowers absolute.

Nine new microsatellite markers (SSR) were isolated from Salvia officinalis L. A total of 125 alleles, with 8 to 21 alleles per locus, were detected in a natural population from the east Adriatic coast. The observed heterozygosity, expected heterozygosity, and polymorphic information content ranged from 0.46 to 0.83, 0.73 to 0.93 and 0.70 to 0.92, respectively. New microsatellite markers, as well as previously published markers, were tested for cross-amplification in Salvia brachyodon Vandas, a narrow endemic species known to be present in only two localities on the Balkan Peninsula. Out of 30 microsatellite markers tested on the natural S. brachyodon population, 15 were successfully amplified. To obtain evidence of recent bottleneck events in the populations of both species, observed genetic diversity (HE) was compared to the expected genetic diversity at mutation-drift equilibrium (HEQ) and calculated from the observed number of alleles using a two-phased mutation model (TPM). Recent bottleneck events were detected only in the S. brachyodon population. This result suggests the need to reconsider the current threat category of this endemic species.

The essential oil composition of wild-growing Salvia officinalis L. (Lamiaceae) from Montenegro was analysed by GC-FID and GC-MS and its antimicrobial activity tested at different oil concentrations, Twenty-five compounds were identified in the essential oil, with major components: alpha-thujone (29.50%), camphor (22.52%) and 1,8-cineole (12.19%). The amount of several minor compounds in the essential oil was also significant: camphene (5.35%), borneol (4.43%) and limonene (4.21 %). Sixteen different microorganisms were used in this study as test organisms, including four reference ATCC strains and twelve clinically isolated strains. These included human pathogens, food poisoning and spoilage bacteria and the blastomycete opportunistic yeast Candida albicans. Results showed that the sage essential oil exhibited a significant antibacterial and antifungal activity. The most sensitive strain was Staphyloccocus aureus, and of Gram-negative species even at the lowest concentration of oil, Providencia stuartii was the most sensitive. The origin of the bacterial strain and the fact of being Gram-positive or Gram-negative appeared to have little influence on the sensitivity of the oil; in general all the bacteria, except Pseudomonas aeruginosa and Salmonella enteridis, were susceptible in various degrees,to the different concentration of sage essential oil. Noteworthy is significant activity of sage essential oil against clinically isolated bacterial strains and yeast C albicans.

Dalmatian sage (Salvia officinalis L.) represents one of the most significant medicinal autochthonous species in flora of eastern Adriatic coast and islands. It is evergreen outcrossing perennial subshrub with short woody stems that branch extensively and violet flowers. Apart from being native to Mediterranean karst of the east side of Adriatic it is cultivated in numerous countries worldwide with Mediterranean and temperate continental climate. From the earliest times it has been used in traditional medicine in healing gingiva, mouth cavity and the sore throat, against bacterial and fungal infections, for wound treatment, memory enhancement, for treating common cold, against sweating, stomach inflammation and ulcer formation etc. The essential oil has also been used in preservation of food and as spice as it gives both specific aroma and promotes digestion of food. The essential oil is extremely complex mixture of different active ingredients ; however, the thujones and camphor are the dominant compounds and are the parameter by which Salvia officinalis L. is distinguished from other Salvia species. The great variability of essential oil composition and yield has been detected depending on various factors such as genotype, environmental conditions, phonological stage, plant parts used for the extraction of essential oil and drying procedure. Molecular genetic analysis of S. officinalis is still limited and comprises the use of RAPD markers, AFLP and SSR markers in assessing mostly the genetic variability and structure of wild S. officinalis populations.

Nine new microsatellite markers (SSR) were isolated from Salvia officinalis L. A total of 125 alleles, with 8 to 21 alleles per locus, were detected in a natural population from the east Adriatic coast. The observed heterozygosity, expected heterozygosity, and polymorphic information content ranged from 0.46 to 0.83, 0.73 to 0.93 and 0.70 to 0.92, respectively. New microsatellite markers, as well as previously published markers, were tested for cross-amplification in Salvia brachyodon Vandas, a narrow endemic species known to be present in only two localities on the Balkan Peninsula. Out of 30 microsatellite markers tested on the natural S. brachyodon population, 15 were successfully amplified. To obtain evidence of recent bottleneck events in the populations of both species, observed genetic diversity (H$_E$) was compared to the expected genetic diversity at mutation-drift equilibrium (H$_{EQ}$) and calculated from the observed number of alleles using a two-phased mutation model (TPM). Recent bottleneck events were detected only in the S. brachyodon population. This result suggests the need to reconsider the current threat category of this endemic species.

Consumer demands for safe, stable and naturally preserved food products have intensified research of different plant phenolic extracts which are known to possess antioxidant activity and offering a potential alternative to synthetic preservatives. This study aimed to research the influence of phenophase on the phenolic profile and related antioxidant properties of sage, one of the most characteristic plants of Dalmatian karst. Sage leaves were picked up in different phenophases (February, May, August and November) at the area of the "Vrana Lake" Nature Park (Middle Dalmatia, Croatia). Total phenols, flavonoids, non-flavonoids, flavons and flavanols in sage extract were determined using spectrophotometric methods. The antioxidant potential of extracts was evaluated, in vitro, using five different antioxidant methods (FRAP, DPPH, ABTS, BR, β-carotene bleaching method). Significant differences in phenolic composition and antioxidant properties of analyzed sage extracts were confirmed, as expected. The total phenolic contents ranged from 4.34 to 5.16 g GAE/L. The best results were obtained for May extract. Relative content of flavonoids was around 17 % in May extract, while during rest of the year it ranged from 4 to 9 %. The content of flavones and flavonoles was also the highest in May extract. All plant extracts exhibited excellent antioxidant properties. The best antioxidant results using all methods (free radical scavenging ability, reducing power, the efficiency in protecting the oxidation of linoleic acid and inhibition of oscillations) were obtained for May extract. We can conclude that good antioxidant properties of Dalmatian sage leaves are a result of very high content of phenolic compounds, especially flavonoid fraction. Determined differences in phenolic composition and biological activity of sage extracts show greatest phenolic potential and best antioxidant properties for May leaves extracts, making it the best choice for further use as functional food ingredient.

The common sage (Salvia officinalis L.) is a native plant of northern and central Spain, southern France and western part of the Balkan Peninsula. Because of its medicinal and aromatic characteristics it is often gathered from the wild. The assessment of genetic diversity using molecular markers is a starting point for its introduction into plant breeding programs and agricultural production. Eleven new microsatellite markers were developed from genomic DNA libraries enriched for GA and GT repeats. 25 common sage plants from a natural population from the eastern Adriatic coast were examined for polymorphism using the developed microsatellites markers. A total of 142 alleles were observed across eleven loci, the number of alleles per locus ranged from eight to 26. The observed heterozygosity ranged from 0, 300 to 0, 833, and the expected heterozygosity from 0, 666 to 0, 934. 7 out of eleven microsatellites showed high polymorphic information content (PIC) of more than 0, 75 indicating that these microsatellite markers are very useful in assessing genetic diversity and population structure. All eleven microsatellite markers were tested for cross-amplification in four different Salvia species and Rosmarinus officinalis L. The species with most successful amplification was Salvia fruticosa Mill.. (60%), while in other tested species, amplification rate was: 25 % for S. pratensis L., 20 % for S. sclarea L., 20% for S. verticillata L. and 20% for R. officinalis L..

Dalmatian sage is a perennial shrub native of the northern coastal region of the Mediterranean. In Croatia, Dalmatian sage is often gathered from the wild and commercialized as medicinal and aromatic plant. The assessment of genetic diversity is a starting point for its introduction into plant breeding programmes and agricultural production since commercial gathering could have a negative impact on biodiversity conservation. The aim of this study was to assess the amount and structure of population genetic diversity, the geographic differentiation, the occurrence of demographic bottlenecks and the ecotypic divergence. For this purpose five microsatellite markers were developed from enriched genomic DNA libraries and successfully applied for the assessment of genetic diversity in ten Dalmatian sage populations collected in Croatia.

Dalmatian sage (Salvia officinalis L.) is a perennial subshrub belonging to Lamiaceae family. It is a native of the northern shore region of the Mediterranean, and grows wild in large masses in the calcereous, karstic mountains of Dalmatia, Herzegovina, Montenegro and Albania. It is used as an herb with beneficial healing properties and its dried leaf (Salviae folium) is an authorized drug in most pharmacopoeias. Genetic diversity and structure of ten Dalmatian sage populations in Croatia has been analysed using AFLP markers. Five populations were collected in Istria and Kvarner (Šušnjevica, Kamenjak, Stara Baška, Cres, Lošinj) and five in Dalmatia (Hvar, Vis, Pelješac, Mljet, Konavle). Each population consisted of 25 plants. The four AFLP primer combinations yielded 1389 polymorphic fragments. Shannon's information index as a measure of genetic diversity within populations ranged from 0.327 (Vis) to 0.370 (Lošinj). The analysis of molecular variance was used to partition the total genetic variance into among and within populations. The most of the genetic diversity was attributable to differences among individuals within populations (93%) in concordance with the patterns expected for an outcrossing and moderately long-lived species. Allelic frequencies at AFLP marker loci in each population were calculated from the observed frequencies of fragments assuming Hardy-Weinberg equilibrium, using the Bayesian approach. A low level of genetic differentiation among populations (FST = 0.039) was observed. Neighbor-joining tree based on Nei's genetic distance matrix among populations revealed two clades supported by a high bootstrap value in accordance with geographical origin of the populations (Istra and Kvarner vs. Dalmatia). Regression analysis of geographic distances and pairwise FST values revealed a typical pattern of isolation-by-distance.

This study included aromatic plants that grow wild and/or are often planted in Croatia: Lavandula latifolia, Salvia officinalis and Rosmarinus officinalis. Composition of the essential oils of plants collected on Korčula, Croatia, and commercially available oils of same plants were analysed, and their antibacterial activity was compared.