Trends in biological activity research of wild-growing aromatic plants from Central Balkans BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 61 Review Article Received: 10 October 2016 Revised: 02 November 2016 Accepted: 07 November 2016 Trends in biological activity research of wild-growing aromatic plants from Central Balkans Ana M. Džamić1*, Jelena S. Matejić2, Petar D. Marin1 1University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, 11.000 Belgrade, Serbia 2University of Niš, Faculty of Medicine, 18.000 Niš, Serbia * E-mail: simicana@bio.bg.ac.rs Abstract: Džamić, A.M., Matejić, J.S., Marin, P.D.: Trends in biological activity research of wild-growing aromatic plants from Central Balkans. Biologica Nyssana, 7 (2), December 2016: 61-73. Flowering plants consists of more than 300.000 species around the world, out of which a small percentage has been sufficiently investigated from phytochemical and biological activity aspects. Plant diversity of the Balkans is very rich, but still poorly investigated. The aim of this paper is survey of current status and trends in research of wild-growing aromatic plants from Central Balkans. Many aromatic plants are investigated from morphological, physiological, ecological, systematic and phytochemical aspects. However, traditionally used medicinal and aromatic plants can also be considered from applicative aspects, concerning their health effects, and from wide range of usage in cosmetics, and as food, agrochemical and pharmaceutical products. In order to achieve all planned objectives, following methodology has been applied: field research, taxonomic authentication and, comparative biologically assayed phytochemical investigations. The total herbal extracts, postdistillation waste (deodorized) extracts, essential oils and individual compounds of some autochthonous plants have been considered as potential source of antibacterial, antifungal, anti-biofilm, antioxidant and cytotoxic agents. In this manuscript, composition of essential oils and extracts were evaluated in a number of species, from the Apiaceae, Lamiaceae, Rosaceae and Asteraceae families. Extracts which were rich in phenols mostly of flavonoids, often showed high antioxidant potential. Also, phenolic compounds identified in essential oils and extracts were mostly responsible for expected antimicrobial activity. Current worldwide demand is to reduce or, if possible, eliminate chemically synthesized food additives. Plant-produced compounds are becoming of interest as a source of more effective and safe substances than synthetically produced antimicrobial agents (as inhibitors, growth reducers or even inactivators) that control growth of microorganisms. Many different pathogens have developed resistance toward synthetic antibiotics and mycotics, so, there is a need for discovering a new antimicrobials. It is worth noting that synergistic effect of components found in essential oils or in various extracts may pay key role in its biological activities. Key words: Antibacterial, antifungal, antioxidant, cytotoxic activity, plant extracts, essential oil 7 (2) • December 2016: 61-73 12th SFSES • 16-19 June 2016, Kopaonik Mt DOI: 10.5281/zenodo.200401 BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 62 Apstrakt: Džamić, A.M., Matejić, J.S., Marin, P.D.: Trendovi u istraživanjima biološke aktivnosti divljerastućih aromatičnih biljaka centralnog Balkana. Biologica Nyssana, 7 (2), Decembar 2016: 61-73. Broj cvetnica u svetu se procenjuje na više od 300.000 vrsta, od kojih je mali procenat u potpunosti proučen sa fitohemijskog i sa aspekta biološke aktivnosti. Biljni diverzitet Balkana je bogat, ali je i dalje slabo proučen. Cilj ovog rada je pregled trenutnog stanja i trendova u proučavanje samoniklih aromatičnih biljaka sa područja Centralnog Balkana. Mnoge aromatične biljke su proučavane sa morfološkog, fiziološkog, ekološkog, sistematskog i fitohemijskog aspekta. Međutim, lekovite i aromatične biljke koje se koriste u narodnoj medicini mogu se razmatrati i sa stanovišta njihove primenljivosti, imajući u vidu njihov efekat na zdravlje i široku upotrebu u kozmetici, kao hrane, i u agrohemijskim i farmaceutskim proizvodima. U cilju postizanja planiranih zadataka, primenjena je sledeća metodologija istraživanja: terenski rad, taksonomska autentifikacija i komparativna biološka i fitohemijska istraživanja. Ukupni biljni ekstrakti, ekstrakti postdestilacionih ostataka (deodorisani), etarska ulja i pojedinačne komponente autohtonih biljka su razmatrani kao potencijalni izvori antibakterijskih, antifungalnih, anti-biofilm, antioksidativnih i citotoksičnih agensa. U ovom radu prikazan je hemijski sastav etraskih ulja i ekstrakata brojnih vrsta iz familija Apiaceae, Lamiaceae, Rosaceae and Asteraceae. Ekstrakti bogati fenolima, naročito flavonoidima često poseduju visok antioksidativni potencijal. Takođe, fenolne komponente idetifikovane u etarskim uljima i ekstraktima se smatraju odgovornim za antimikrobnu aktivnost. Širom sveta se trenutno radi na smanjenju ili potpunom eliminisanju hemijski sintetisanih aditiva u hrani. Biljni proizvodi su postali značajni kao izvor efikasnijih i bezbednih supstanci nego što su sintetički antimikrobni agensi koji kontrolišu rast mikroorganizama (inhibitori, regulatori rastenja ili inaktivatori rasta). Mnogi različiti patogeni su postali rezistentni na sintetičke antibiotike i mikotike, pa je potrebno pronaći nove efikasne antimikrobne supstance. Potrebno je istaći da sinergistički efekat između komponenti u etarskom ulji ili različitim ekstraktima može imati ključni efekat za njegovu biološku aktivnost. Ključne reči: antibakterijska, antifungalna, antioksidativna, citotoksična aktivnost, biljni ekstrakti, etarska ulja Introduction Plant kingdom, with over 300.000 higher species, represents a source of new chemical agents for active pharmaceutical ingredients and lead compounds. Only a small percent 5% to15% of these plants have been chemically and pharmacologically investigated. Approximately 10.000 to 15.000 of the world’s plants have documented medicinal uses and roughly 150-200 have been incorporated in western medicine (K r a u s e & T o b i n , 2013). The Balkan flora is not only the richest in Europe, but comprises also many endemics. According to contemporary assessments, vascular flora of the Balkans comprises almost 8.000 taxa (S t e v a n o v i ć et al., 2007). In flora of Sebia 3.562 species and subspecies are recorded and among them 32.12% are protected (I g i ć et al., 2010). Plants which contain bioactive compounds are often categorised as medicinal or poison plants, and their consumation should have adverse or beneficial result which often depends on the dose of taking. Secondary metabolites are small organic molecules and specific biologically active compouds that are produced by plants. They pay important role in the function of plant organisms and are important for long-term survival and defence. Nowadays, it is known that production of secondary metabolites are the rule rather than exception. Secondary metabolites represent essential source for discovery of a novel compounds because of great diversity of their structures (B e r n h o f t , 2010). Modern approach adopted to explore plant and its bioactive constituents involve interdisciplinary work in botany, pharmacognosy, chemistry and toxicology (H o s t e t t m a n n et al., 1995) and should take following steps: 1. selection, collection, botanical identification, preparation of plant material 2. extraction with suitable solvents and preliminary analysis 3. biological and pharmacological screening of crude extracts 4. chromatographic separation of pure bioactive constituents guided by bioassay 5. structure determination 6. analyses and pharmacological profile of pure compounds 7. toxicological testing If plants show no signs of being attacked by pests and neither has pieces eaten out of the leaves, there is a good chance that some metabolites are present which act as insecticides or antimicrobial agents. The evaluation of plant extracts to ensure efficacy and safety is followed by identification of active principles, dosage formulations, efficacy and pharmaco-kinetic profile of the new drug. Many plants have been used because of their antibacterial, antifungal, anti-biofilm, antioxidant, cytotoxic, anti- inflammatory, antidiabetic, anti-cholinesterase, anti- nociceptive, anti-proliferative, genotoxic, anti- genotoxic, neuroprotective, herbicidal, enzyme BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 63 inhibition, anti-Alzheimer properties and have been investigated by a number of researchers worldwide. Ethnopharmacologists, botanists, microbiologists, natural product chemists, and many others are searching for phytochemicals which could be developed for some applications (D a s et al., 2010). It has to be pointed out that biological activity data for majority of chemically investigated wild growing plants are still missing (Č a v a r Z e l j k o v i ć & M a k s i m o v i ć , 2014). This study gives an overview of current status and trends in research of potential of wild-growing aromatic plants of different biological aspects and methods in vitro. Extracts and Essential oils Plants are complex matrices, which produce a wide range of secondary metabolites with different functional groups and polarities. Categories of natural products commonly encountered include waxes and fatty acids, polyacetylenes, terpenoids (e.g. monoterpenoids, iridoids, sesquiterpenoids, diterpenoids, triterpenoids), steroids, essential oils (lower terpenoids and phenylpropanoids), phenolics (simple phenolics, phenylpropanoids, flavonoids, tannins, anthocyanins, quinones, coumarins, lignans), alkaloids and glycosidic derivatives (e.g. saponins, cardiac glycosides, flavonoid glycosides). For preliminary investigation of biological properties the most important is to test complex mixture as total plant extracts. Several approaches can be employed to extract the plant material. Although water is used as an extractant in many traditional protocols, organic solvents of varying polarities are generally selected in modern methods of extraction to exploit a solubility of plant constituents. Solvent extraction procedures applied for the initial extraction plant natural products include maceration, percolation, Soxhlet extraction, pressurized solvent extraction, ultrasound assisted solvent extraction, and steam distillation. Terpenoids and essential oils are secondary metabolites that are highly enriched in compounds based on an isoprene structure. The most abundant terpenes in essential oils are monoterpenes (C10) and sesquiterpenes (C15) while diterpenes (C20), as well as aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters or lactones (S e i d e l , 2006; B o h l i n et al., 2012) are often present. Antimicrobial activity Current standard antimicrobial methods approved by various organizations such as Clinical and Laboratory Standards Institute (CLSI), and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) exist, for guidelines of antimicrobial susceptibility testing of convenient drugs, these might not be exactly applicable to plant extracts and modifications have to be made. In general, antimicrobial methods are broadly classified into diffusion and dilution methods. Diffusion tests include agar well diffusion, agar disk diffusion, poison food technique, and bioautography, while dilution methods include agar dilution, broth microdilution and broth macrodilution technique (D a s et al., 2010). Agar well diffusion and disk diffusion have been applied in the first screening, where extract or test substance are placed into agar or applied on the filter disc. Zone of inhibition have been measured. Bioautography is also employed as a preliminary phytochemical screening technique, by bioassay guided fractionation, to detect active components. Bioautography is a very convenient way of testing plant extracts and pure phytochemical compounds for their effect on both human pathogenic and plant pathogenic microorganisms. It can be employed in the target directed isolation of active constituents (C h o m a & G r z e l a k , 2011). However, the most preferred method is broth microdilution assay (or micro-well dilution assay), using microtitration plates, by making the serial two- fold dilutions of test substances in corresponding medium. Using this method, minimum inhibitory concentrations (MIC) and minimum bactericidal/fungicidal concentrations (MBC/MFC) are recorded. Minimal inhibitory concentration (MIC) is defined as the lowest concentration of the samples inhibiting visible growth. Minimal bactericidal/fungicidal concentration (MBC/MFC) is defined as the lowest sample’s concentration that kills 99.9% of bacterial/fungal cells. The main advantages of broth methods, especially microdilution which is carried out in microtiter trays are lower workloads for a larger number of replicates and the use of small volumes of the test substance and growth medium. In this method dilution of the oil is better and there is no agar in the medium, which both enable better diffusion through the liquid medium (S o k o v i ć et al., 2011). Antimicrobial activity of wild growing Apiaceae species The in vitro antimicrobial activity of essential oils and various extracts of wild growing Apiaceae has been investigated by the microdilution method, where a panel of pathogenic bacteria and microfungi was tested (M i l o s a v l j a v i ć et al., 2007; S o k o v i ć et al., 2009; S t o j k o v i ć et al., 2009; BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 64 G l a m o č l i j a et al., 2011; M a t e j i ć et al., 2012, 2013, 2014, 2015; M a r č e t i ć et al., 2014a; 2014b; M i l e s k i et al., 2014, 2015; P o p o v i ć et al., 2015a, 2015b) (Tab. 1). Essential oil analyses of inflorescence and aerial parts of endemic species, Ferulago macedonica showed α-pinene as the main compound (43% and 23%, respectively). Both oils demonstrated strong antibacterial activity. The essential oil of Echinophora spinosa, containing δ-3- carene (60.86%) as the dominant component, was the most prominent against Escherichia coli and Pseudomonas aeruginosa while the most resistant bacterial species against the oil was the Staphylococcus aureus (G l a m o č l i j a et al., 2011). Essential oil of Echinophora sibtorpiana (characterized by presence of methyl eugenol (60.40%), followed by p-cymene (11.18%) and α- phellandrene (10.23%) demonstrated considerable antimicrobial activity. The lowest antimicrobial activity was detected for aqueous extracts. The most resistant were fungus Aspergillus niger and bacteria Micrococcus flavus and Escherichia coli (M i l e s k i et al., 2014). Essential oil of Seseli rigidum, with dominance of α-pinene (48.5%), showed antimicrobial activity. The most resistant bacterial strains were Micrococcus flavus and Staphylococcus epidermidis, and the most resistant micromycete was Aspergillus niger (S t o j k o v i ć et al., 2009). Methanol extracts of various Seseli taxa revealed activity against different bacteria and yeast Candida albicans (with MICs of 0.78-12.5 mg/ml) (Matejić et al., 2012). Methanol and ethyl acetate extracts of Cacrys cristata - rare and critically endangered species in the flora of Serbia, exhibited similar antibacterial activity. Methanol extract from aerial parts, inflorescence and fruits of Opopanax hispidus showed significant antimicrobial effect (M a t e j i ć et al., 2014, 2015). Antimicrobial activity of wild growing Lamiaceae species The in vitro preliminary antimicrobial tests were carried out by disc diffusion method. The results of the antimicrobial activity obtained by disc-diffusion assay showed that the diethyl ether extracts of Stachys inhibited the growth of all the tested bacteria, and S. plumosa exhibited significant antimicrobial activity against pathogens E. coli, P. aeruginosa and S. aureus (comparable to antibiotics used as the positive controls (L a z a r e v i ć et al., 2010). In last decade majority of antimicrobial experiments used microdilution method. It is well known, that essential oils from Lamiacae family showed high antimicrobial potential. Antibacterial and antifungal activity of essential oils of wild growing Thymus, Mentha, Mellisa, Hyssopus, Salvia, Satureja, Sideritis and Stachys species were analyzed (Tab. 1). Essential oils of Salvia sclarea (linalyl acetate and linalool) and Mentha longifolia with dominance of cis- and trans-dihydrocarveol and piperitone, were analyzed for their antifungal potential. The most susceptible was phytopathogen Phomopsis helianthi, while the most resistant was Trichoderma viride (D ž a m i ć et al., 2008, 2010). Essential oil of Satureja kitaibelii characterized by dominance of p- cymene showed strong antimicrobial activity (MIC values of 0.10-25 mg/ml) (K u n d a k o v i ć et al., 2011; M i h a j i l o v -K r s t e v et al., 2011), while essential oil of Hyssopus officinalis subsp. pilifer (1,8-cineole, β-pinene and isopinocamphone) showed lower antimicrobial potential (D ž a m i ć et al., 2013). Methanolic extract of Satureja kitaibelii and its major compound rosmarinic acid possessed strong antimicrobial activity against tested bacteria and microfungi (S t a n o j k o v i ć et al., 2013). The components with phenolic structure are known to be highly active antimicrobial agents. Thymol, a main constituent of various Thymus essential oils, was responsible for wide spectrum of antibacterial and antifungal activity (S o k o v i ć et al., 2009; V l a d i m i r -K n e ž e v i ć et al., 2012; P e t r o v i ć et al., 2016). Carvacrol and thymol are recognized as strong antimicrobial agents able to disintegrate the outer membrane of Gram-negative bacteria, releasing lipopolysaccharides and increasing the permeability of the cytoplasmic membrane. Antimicrobial activity is associated with chemical structure of compounds. The most important is lipophilic character of hydrocarbon skeleton and hydrophilic character of functional group. Antimicrobial activity should be arranged in this order: phenols > ketones > alcohols > ethers > aldehydes > hydrocarbons (Dorman & Deans, 2000). Antimicrobial activity of wild growing Rosaceae species Antibacterial activity of three wild fruits, red wild berry fruit (Cornus mas), blackthorn (Prunus spinosa) and wild blackberry (Rubus fruticosus) extracts was analyzed using disc-diffusion and microdilution methods against 12 bacterial strains (Tab. 1). The antimicrobial activity of wild berry fruits extracts was high against almost all the tested bacterial strains (R a d o v a n o v i ć et al., 2013). Antimicrobial activity of wild growing Asteraceae species Essential oil of Helichrysum arenarium in bioautography assay showed clear inhibition zones which were indication of strong antimicrobial activity Gram (+) (Micrococcus luteus, BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 65 Staphylococcus aureus, Staphylococcus epidermidis) and Gram (-) bacteria (Escherichia coli, Pseudomonas tolaasii, Salmonella enteritidis, Salmonella typhimurium) and yeast Candida albicans (R a n č i ć et al., 2005). The most rarely used was micro-atmosphere assay. The essential oil evaporated and the activity by vapor contact has been measured. Due to the high lipophilic nature of mycelia coupled with a large surface area relative to the volume of a fungus, vapours of essential oils may act mainly by accumulation on mycelia than in the agar (I n o u y e et al., 2000). S t u p a r et al. (2014) investigated antifungal activity of the essential oil of Helichrysum italicum using micro-atmosphere method. The main components of the oil were γ- curcumene, α-pinene, and neryl acetate. The essential oil of H. italicum showed moderate antifungal activity against fungi isolated from cultural heritage objects. The most susceptible fungi to oil treatment were Epicoccum nigrum and Penicillium sp., while the most resistant was Trichoderma viride. Pathogenic strain Pseudomonas aeruginosa causes pathological changes on vegetables, and it is considered as conditional parasite of plants, but also as a human parasite because it causes infections of urinary and respiratory tract. The effect of the water and ethanol plant extract was tested against pathogenic strains Pseudomonas aeruginosa and P. fluorescens using disc diffusion method. Water and ethanol extract of Achillea millefolium and Salvia officinalis showed the greatest inhibitory effect on Pseudomonas aeruginosa, (the water extract of Achillea). Great inhibitory effect of the ethanol extract had Helichrysum arenarium, Rosmarinus officinalis, Satureja montana (P e t r o v i ć et al., 2003). In general, microdilution assay is the most rewarding method for different analyses. The several strains of microorganisms should be tested on one plate, and small amount of tested sample could be used. Nowadays this technique is the most frequently used. The chemical composition and the potential to have antimicrobial activity depend highly on the plant species, habitat and ontogeny phase as well as of methods of processing, extraction solvent and extract dose. Many times the values of the inhibition zones were proportional to the concentration of the extracts and essential oils. Sometimes dominant compounds are crucial for the biological activity. Syinergistic effect of the compounds are also important. Antioxidant activity Compounds with antioxidant activity are mainly phenolic acids, flavonoids and polyphenols (D i l l a r d & G e r m a n , 2000). Phenols provide the plants with defense mechanisms to neutralize reactive oxygen species (ROS) in order to survive and prevent molecular damage and damage by microorganisms, insects, and herbivores. Flavonoids show a wide range of biological activities such as inhibition of cell-proliferation, induction of apoptosis, inhibition of enzymes and other antibacterial and antioxidant effects (P a n d e y & G u p t a , 2014). Antioxidants may directly react with the reactive radicals to destroy them by accepting or donating electron(s) or they may indirectly decrease the formation of free radicals by inhibiting the activities or expressions of free radical generating enzymes or by enhancing the activities and expressions of other antioxidant enzymes. Many research models have been established in chemical and/or biological systems for studying the mechanisms of action of antioxidants and for identifying new antioxidants (Lu et al., 2010) and it is suggested to test potential antioxidants by different methods. Important step for determination of the antioxidant capacity is quantification of phenolic compounds in samples by using Total phenol content (TPC) assay with Folin–Ciocalteu reagent, as well as quantification of total flavonoids (TFC). The most frequently used in vitro antioxidant methods are: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azino-bis(3-ethylbenz- thiazoline-6-sulfonic acid (ABTS) radical scavenging activity, Ferry reducing power (FRAP) assay, Total reducing power (TRP) assay, β-carotene- linoleic acid assay etc. Available literature data of in vitro antioxidant activity of wild growing species from Central Balkans are given in Tab. 1. Antioxidant activity of wild growing Apiaceae species Methanolic extract of Seseli taxa tested for its antioxidant potential (total phenols, total flavonoids DPPH and ABTS assays) revealed Seseli libanotis ssp. libanotis for its highest reducing power (M a t e j i ć et al., 2012). Essential oil of Echinophora sibthorpiana possessed high radical scavenging capacity in DPPH and ABTS assays. Aqueous extract of aerial parts was the strongest tested extract. Extracts of aerial parts expressed higher radical scavenging activity in comparison to the root extracts. All extracts had lower antiradical activity compared to BHA and vitamin C (M i l e s k i et al., 2014). In the case of various extracts of Ferulago macedonica, it was found that there is no strong relationship between total phenolic and flavonoid contents and radical scavenging activities BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 66 (DPPH, ABTS). These results can be explained since the antioxidant property of a plant extract is generally considered as the result of the combined activity of a wide range of compounds including, not only phenols, but also peptides, organic acids and other components. Both, phenols and flavonoids were present in higher amounts in F. macedonica extracts of inflorescence in comparison to the aerial parts (M i l e s k i et al, 2015). Antioxidant activity of wild growing Lamiaceae species Extracts of waste water after hydrodistillation of Satureja montana and S. cuneifolia, showed better ability to reduce stable DPPH radical than the standards essential oils or thymol, carvacrol, and thymoquinone. This is probably due to the high concentration of other active compounds, such as (E)-coniferyl alcohol. To compare these results, total antioxidant activity of these two Satureja species was also tested using the phosphomolybdenum method. This test is based on the reduction of Mo(VI) to Mo(V) by the extract and subsequent formation of a green phosphate/Mo(V) complex at acid pH. Total antioxidant activity of the phosphomolybdenum model evaluates both water-soluble and fat-soluble antioxidants, i. e. total antioxidant capacity. The highest activity in this method was found for essential oil of S. cuneifolia (Č a v a r et al., 2013). For scavenging ability on DPPH radicals, various extracts of Hissopus officinalis subsp. pilifer were effective in the order of: deodorized aqueous extract > deodorised metanol extract > deodorised ethyl acetate extract. The total phenol content, determined by Folin–Ciocalteau reagent was in the same order. Essential oil of H. officinalis subsp. pilifer possessed the lowest activities compared to other extracts and control substances (D ž a m i ć et al., 2013). Antioxidant potential of Melittis melissophyllum various hydro alcoholic, ethanol and methanol extracts revealed a high scavenging activity by DPPH and ABTS methods. Reducing power of extracts were evaluated through the FRAP assay. The highest phenol and flavonoid content were identified in the 96% ethanol extract and the lowest were in the ethanol 10% solution. Also, the highest correlation was observed between total phenol and flavonoid contents. Statistically significant correlations were registered for DPPH and ABTS assays which is logical due to their similar mechanism of action as radical scavengers. A hierarchical cluster analysis (HCA) was performed on the total phenols and total flavonoids content DPPH, ABTS, and FRAP assay. The dendrogram (Fig. 1) shows that total phenols and total flavonoids content are quite homogeneous, while DPPH was placed in separate cluster (G r u j i ć et al., 2014). Tree Diagram for 5 Variables Ward`s method Euclidean distances 0 10 20 30 40 50 60 70 80 90 100 110 (Dlink/Dmax)*100 IC50 DPPH, (mg/mL) FRAP, (µmolFe/mg) ABTS, mg Vit C/g Total flavonoids (QuE/g) Total phenols (GAE/g) Fig. 1. Dendrogram of applied assays for antioxidant characteristics evaluation for Melittis melissophyllum extracts (G r u j i ć et al., 2014). BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 67 Table 1. Antimicrobial and in vitro antioxidant activity of extracts and essential oils of wild-growing species from Central Balkans Fam Species Extracts EO composition Antimicrobial methods Antioxidant methods References A p ia c e a e Cachrys cristata DC. Methanol, ethyl acetate, acetone, aqueous Aerial parts, fruits Antibacterial Antifungal Microdilution Total phenols Total flavonoids DPPH ABTS Matejić et al.,2012, 2014 Daucus carota L. Fruits, flowers, root, leaves and stem Antibacterial Antifungal Microdilution Soković et al., 2009 Echinophora sibthorpiana Guss. Echinophora spinosa L. Methanol, ethanol, aqueous Inflorescence aerial parts Antibacterial Antifungal Microdilution Total phenols Total flavonoids DPPH ABTS Mileski et al., 2014 Glamočlija et al., 2011 Eryngium palmatum Pančić and Vis. Methanol chloroform Root Microdilution Antibacterial Anticandidal Total phenols DPPH FRAP Marčetić et al., 2014a; 2014b Ferulago macedonica Micevski and E. Mayer Methanol, ethanol, aqueous Antibacterial Antifungal Microdilution Total phenols Total flavonoids DPPH ABTS Mileski et al., 2015 Laserpitium latifolium L. L. zernyi Hayek L. ochridanum Micevski Chlorophorm Underground parts aerial parts fruits Antibacterial Antifungal microdilution antibiofilm Popović et al, 2015 a,b Opopanax hispidus (Friv.) Griseb. Methanol, ethyl acetate Antibacterial Anticandidal Microdilution Total phenols Total flavonoids DPPH ABTS Matejić et al., 2015 Seseli pallasii Besser, Seseli libanotis (L.) Koch ssp. libanotis Seseli libanotis ssp. inter- medium (Rupr.) P. W. Ball Seseli rigidum Waldst. & Kit. Seseli globiferum Vis. Seseli annuum L. Methanol Aerial parts fruits Antibacterial Antifungal Microdilution Total phenols Total flavonoids DPPH ABTS FRAP Matejić et al., 2012 Ilić et al., 2014; 2015 Janacković et al, 2011 Stojković et al, 2009 Milosavljević et al., 2007 Tordylium maximum L. Methanol, aqueous Antibacterial Anticandidal Microdilution Total phenols Total flavonoids DPPH ABTS Matejić et al., 2013 L a m ia c e a e Hyssopus officinalis ssp. pilifer Deodorized aqueous, methanol and ethyl acetate Aerial parts Antifungal Microdilution Total phenols DPPH Džamić et al., 2013 Mentha longifolia L. Aerial parts Antifungal Microdilution DPPH Džamić et al., 2010 Mellisa officinalis L. Aerial parts Antibacterial Antifungal Microdilution DPPH OH radical Mimica-Dukić et al., 2004 Melittis melissophyllum L. Methanol, Ethanol (hydroalcoholic) Total phenols Total flavonoids DPPH ABTS FRAP Grujić et al., 2014 Salvia nemorosa L. ssp. nemorosa Salvia sclarea L. Aerial parts Antibacterial Antifungal Microdilution DPPH Phosphomolybd enum Božin et al., 2011 Džamić et al., 2013 Satureja montana L. Satureja cuneifolia Ten Satureja montana ssp. pisidica (Wettst.) Šilić Satureja montana L. ssp. montana Aerial parts Antibacterial Antifungal Microdilution DPPH ORAC TRP APF Čavar et al., 2013 Kundaković et al., 2014 Mihajilov-Krstev et al., 2014; 2011 BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 68 Table 1. continuation of the table Fam Species Extracts EO composition Antimicrobial methods Antioxidant methods References L a m ia c e a e Satureja kitaibelii Wierzb. ex Heuff. Satureja horvatii Šilić Bukvički et al., 2014 Lakušić et al., 2008 Sideritis montana L. Aerial parts Antibacterial Microdilution Miladinović et al., 2012 Stachys germanica ssp. heldreichii (Boiss) Hayek Stachys iva Griseb. Stachys plumosa Griseb. Stachys scardica Griseb. Diethyl ether, ethyl acetate Antibacterial Antifungal Disk-diffusion Phosphomolybd enum Lazarević et al., 2010 Thymus praecox Opiz ssp. polytrichus Thymus longicaulis C. Presl Thymus serpillum L. Thymus tosevii Velen Supercritical extracts Methanol, ethanol, aqueous Aerial parts Antibacterial Antifungal Microdilution Macrodilution Disk-diffusion DPPH Petrović et al., 2016 a, b Vladimir-Knežević et al., 2012 Soković et al., 2009 R o sa c e a e Crataegus oxyacantha L. Antibacterial Disk-diffusion Total phenols Anthocyan content Flavonoid content DPPH Kostić et al., 2012 Cornus mas L. Prunus spinosa L. Acidified methanol solution Antibacterial Disk-diffusion Microdilution DPPH Radovanović et al., 2013 Rosa canina L. Methanol Antibacterial Antibiofilm Microdilution Živković et al., 2015 Rubus caesius L. var. aquaticus Weihe and Nees Rubus fruticosus L. Methanol, ethanol, acetone, aqueous Acidified methanol solution Antibacterial Disk-diffusion Microdilution Total phenols Total flavonoids DPPH ABTS FRAP TRC Veličković, et al., 2015; Radovanović et al., 2013 A st e r a c e a e Achillea collina Becker ex Heimerl s.l. Achillea pannonica Scheele Aerial parts Disk-diffusion DPPH Božin et al., 2008 Ambrosia artemisiifolia L. 70% aqueous acetone extracts Total phenols Total flavonoids DPPH FRAP Maksimović, 2008 Artemisia alba Turra Artemisia absinthium L. Ethanol Aerial parts Antibacterial Anticandidal Microdilution Total phenols Total flavonoids DPPH FRAP ABTS Đorđević et al., 2013 Mihajilov-Krstev et al., 2014 Helichrysum arenarium (L.) Moench Helichrysum italicum (Roth) G. Don Aerial parts Antibacterial Antifungal Microdilution Bioautography Rančić et al., 2005 Stupar et al., 2014 Hieracium pillosela L. Methanol, dychlorometane, ethyl acetate, dychlorometane:me thanol Antibacterial Disk-diffusion Stanojević et al., 2008 Xeranthemum annuum L Methanolic acetone ethyl acetate Antibacterial Antifungal Microdilution Total phenols Total flavonoids DPPH Stanković et al., 2011 BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 69 Antioxidant activity of wild growing Rosaceae species R a d o v a n o v i ć et al. (2013) found that in polyphenolic extracts of fruits from tree Rosaceae species main compounds were gallic acid, caffeic acid, p-coumaric acid, ferulic acid, (+)-catechin, procyanidin B2, (-)-epicatechin, quercetin, rutin and quercetin-3-glucoside. All extracts showed high scavenging effect on DPPH radical. Extracts obtained from Hieracium pilosella have significant free radical scavenging activity on stable DPPH• and high reactive hydroxyl radical. The data suggest that aqueous, ethanolic and methanolic extracts of H. pilosella from Southeastern Serbia are a potential source of natural antioxidants. Chlorogenic acid was detected in the highest quantities in all investigated extracts (S t a n k o v i ć et al., 2009). Antioxidant activity of wild growing Asteraceae species According to B o ž i n et al. (2008) the main detected compounds in essential oil of Achillea callina were β-pinene, chamazulene and E-caryophyllene, while in essential oil of A. pannonica 1,8-cineole and camphor were the dominant components. Authors represented that both oils possess strong antioxidant effects in vitro and in vivo assays. Antioxidant capacities are influenced by many factors, which cannot be fully described with a single method. The most commonly used method for assessment of antioxidant properties of natural products is DPPH radical assay. The DPPH radical assay overcomes the limitations of monitoring the activity of the numerous samples over a specified period of time. It is reproducible and strongly correlated with phenolic compounds (M i l i a u s k a s , 2004). Cytotoxic activity Brine shrimp, Artemia salina, is the test organism that is used for evaluation of possible cytotoxic activity of plant extracts or essential oils. Brine shrimp assay is simple, inexpensive method and results are obtained quickly. In the study represented by J a n a ć k o v i ć et al. (2008) a brine shrimp assay was used to examine potential cytotoxic activity of different Centaurea species, measuring lethal concentration (LC). Cytotoxic activity of methanol and ether extracts was tested. The methanol extract of C. arenaria showed very significant activity while the lowest activity was found with C. chrysolepis methanol extract. Among ether extracts, the most active was C. splendens (and the lowest activity was recorded for the ether extract of C. scabiosa. Toxicity test on Drosophila melanogaster showed that the essential oil of A. absinthium is toxic for developing insect larvae. Starting with the concentration of 0.38 % of essential oil in medium, significant mortality of larvae exposed to the oil was noted when compared to the control. The essential oil also affected the development of D. melanogaster larvae and significantly delayed achievement of the pupa stadium (M i h a j i l o v -K r s t e v et al., 2014). Many plant extracts and natural products, especially phenols, with high antioxidant activity have shown cytotoxic effects in different cell lines. Flavonoid anticancer activities include inhibition of cell growth, inhibition of protein kinase activities, and induction of apoptosis. Popular and broadly used for cytotoxity examination is the MTT (3-[4,5- dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay which is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity. Methanolic extract of Origanum vulgare showed significant antiproliferative activity in both colon cancer (HCT- 116) and breast cancer (MDA-MB-231) cell lines. Inhibitory activity appeared to be particularly conspicuous in treated HCT-116 cell lines, whereas at the highest concentration (500 µg/mL), only 10% of the cells remained viable. The results indicate that O. vulgare is considered to be a particularly valuable source of effective anti-proliferative and cytotoxic substances (G r b o v i ć et al., 2013). The methanol extract of Satureja kitaibelii exhibited strong activity against Fem-x human malignant melanoma cells, and moderate activity against breast cancer cell lines (S t a n o j k o v i ć et al., 2013). The cytotoxic effect of the essential oils from Satureja montana ssp. pisidica from two localities (mountains Korab and Galičica) was tested against cancer cell lines MDA- MB-361, MDA-MB-453, HeLa, LS174 and MRC5 cells. The essential oil from Korab demonstrated significantly better results than the oil from Galičica, particularly against HeLa and MDA-MB-453 cell lines, while the oil from Galičica was the most active on the human epithelial cervical cancer HeLa cells (K u n d a k o v i ć et al., 2014). Other activities Most studies describe the action of essential oil and extracts for other biological activities. Strong inhibition of human serum cholinesterase by essential oil of Satureja montana, suggests this as a natural source of compounds that can be used in the treatment of foodborne and neurological diseases, wound and other infections, as well as for general health improvement (M i h a j i l o v -K r s t e v et al., 2014). Considering human and horse serum BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 70 cholinesterase inhibition, both essential oils acted as strong inhibitors, even higher than effect of their dominant constituent α-pinene, leading to the conclusion that the other constituents of the essential oil manifested their action toward cholinesterase (Ilić et al., 2015). Thyme essential oil, as well as its major phenolic constituent’s thymol and carvacrol were researched for application in Alzheimer’s disease treatment or as remedies for cognitive disorders. It was noted that phenolic chemotypes possess higher antioxidant activity than non-phenolic ones, as well as acetylholinesterase inhibitory activity (Č a v a r Z e l j k o v i ć & M a k s i m o v i ć , 2014). Conclusion The reported results from in vitro studies hint the potential antimicrobial and antioxidant value of the wild growing plants from Central Balkan. It was prepared based on plenty literature search of biological activity of extracts and essential oils. The microdilution method was found as the mostly used in antimicrobial assay. DPPH method is the most frequently used in antioxidant assay. The traditional use of many aromatic species leads to discovering the unknown biological potential of related species. The most important approach is combination of phytochemical investigation and bioassay. Essential oil and extracts without identifying of compounds responsible for this activity are also insufficient for preclinical trials. In spite the fact that a lot of studies have been reported so far, many of wild-growing plants are still waiting to be explored. Acknowledgements. This work was founded by the Ministry of Education, Science and Technological Development of the Republic of Serbia for financial support (Grant No. 173029). References Bernhoft, A. 2010. A brief review on bioactive compounds in plants. In: Bernhoft, A. (ed.), Bioactive compounds in plants – benefits and risks for man and animals 11-17, The Norwegian Academy of Science and Letters, Oslo. Bohlin, L., Alsmark, C., Göransson, U., Klum, M., Wedén, C., Backlund, A. 2012: Strategies and methods for a sustainable search for bioactive compounds. Planta Medica, 78-OP7. Božin, B., Lakić, N., Srđenović Čonić, B., Kladar, N., Orčić, D., Mimica-Dukić, N. 2012: Antioxidant and antimicrobial properties of a new chemotype of woodland sage (Salvia nemorosa L. subsp. nemorosa, Lamiaceae) essential oil. Biologica Serbica, 34: 51-60. Božin, B., Mimica-Dukić, N., Bogavac, M., Suvajdžić, Lj., Simin, N., Samojlik, I., Couladis, M. 2008: Chemical composition, antioxidant and antibacterial properties of Achillea collina Becker ex Heimerl s.l. and A. pannonica Scheele essential oils. Molecules, 13: 2058-2068. Bukvički, D.R., Stojković, D.S., Soković, M.D., Vannini, L., Montanari, C., Pejin, B, Savić, A., Veljić, M. M, Grujić, S.M., Marin P.D 2014: Satureja horvatii essential oil: In vitro antimicrobial and antiradical properties and in situ control of Listeria monocytogenes in pork meat. Meat Science, 96(3): 1355-1360. Čavar, S., Edita Šolić, M., Maksimović, M. 2013: Chemical composition and antioxidant activity of two Satureja species from Mt. Biokovo. Botanica Serbica, 37(2): 159-165. Čavar Zeljković, S., Maksimović, M. 2015: Chemical composition and bioactivity of essential oil from Thymus species in Balkan Peninsula. Phytochem Review, 14: 335–352. Choma, I.M., Grzelak, E.M. 2011: Bioautography detection in thin-layer chromatography. Journal of Chromatogrphy A, 1218(19): 2684-91. Das, K., Tiwari, R.K.S., Shrivastava, D.K. 2010: Techniques for evaluation of medicinal plant products as antimicrobial agent: Current methods and future trends. Journal of Medicinal Plants Research, 4(2): 104-111. Dillard, C.J., German, J.B. 2000: Review phytochemicals: nutraceuticals and human helth. Journal of the Science of Food and Agriculture, 80: 1744–1756. Đorđević, S., Stanisavljević, D., Ristića, M., Milenković, M., Veličković, D., Stojičević, S., Zlatković, B. 2013: Chemical, antioxidant and antimicrobial analysis of the essential oil and extract of Artemisia alba Tura. Digest Journal of Nanomaterials and Biostructures, 8(4): 1377– 1388. Doroman, H.J.D., Deans S.G. 2000: Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88: 308-316. Džamić, A., Soković, M.D., Ristić, M.S., Novaković, M., Grujić-Jovanović, S., Tešević, V., Marin P.D. 2010: Antifungal and antioxidant activity of Mentha longifolia (L.) Hudson (Lamiaceae) essential oil. Botanica Serbica, 34(1): 57-61. Džamić, A., Soković, M., Ristić, M., Grujić- Jovanović, S., Vukojević, J., Marin, P.D. 2008: Chemical composition and antifungal activity of Salvia sclarea (Lamiaceae) essential oil. Archives of Biological Sciences, 60: 233-237. Džamić, A.M., Soković, M.D., Novaković, M., Jadranin, M., Ristić, M.S., Tešević V., Marin, BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 71 P.D. 2013: Composition, antifungal and antioxidant properties of Hyssopus officinalis L. subsp. pilifer (Pant.) Murb.essential oil and deodorized extracts. Industrial Crops and Products, 51: 401–407. Glamočlija, J.M., Soković, M.D., Šiljegovic, J.D., Ristić, M.S., Ćiric, A.D., Grubišić, D.V. 2011: Chemical composition and antimicrobial activity of Echinophora spinosa L. (Apiaceae) essential oil. Records of Natural Products, 5(4): 319-323. Grbović, F., Stanković, M. S., Ćurčić, M., Đorđević, N., Šeklić, D., Topuzović, M., Marković, S. 2013: In vitro cytotoxic activity of Origanum vulgare L. on HCT-116 and MDA-MB-231 cell lines. Plants, 2(3): 371-378. Grujić, S., Stojanović, G., Mitić, V., Stankov- Jovanović, V., Džamić, A., Alimpić, A., Marin P. 2014: Evaluation of antioxidant activity of Melittis melissophyllum L. extracts. Archives of Biological Sciences, 66(4): 1401-1410. Hostettmann, K., Marston, A., Wolfender, J.-L. 1995. Strategy in the search for new biologically active plant constituents. In: Hostettmann, K., Marston A., Millard M. and Hamburger M. (eds.), Phytochemistry of Plants used in Traditional Medicine 17-45, Oxford science publications. Oxford University press Inc., New York. Igić, R., Vukov, D., Božin, B., Orlović, S. 2010: Lekovite biljke. Prirodni resursi Vojvodine. Društvo za zaštitu životne sredine “Vrelo”, Novi Sad, Srbija. Ilić, M.D., Stankov Jovanović, V.P., Mitić, V.D., Jovanović, O.P., Mihajilov-Krstev, T.M., Marković, M.S., Stojanović, G.S. 2015: Comparison of chemical composition and biological activities of Seseli rigidum fruit essential oils from Serbia. Open Chemistry, 13: 42–51. Inouye, S., Tsuruoka, M., Watanabe, M., Takeo, K., Akao, M., Nishiyama, Y., Yamaguchi H. 2000: Inhibitory effect of essential oils on apical growth of Aspergillus fumigatus by vapour contact. Mycoses, 43: 17–23. Janaćković, P., Tešević, V., Marin, P., Milosavljević, S., Duletić-Laušević, S., Janaćković, S., Veljić, M. 2008: Brine shrimp lethality bioassay of selected Centaurea L. species (Asteraceae). Archives of Biological Sciences, 60(4): 681-685. Janaćković, P.T., Soković, M.D., Vujisić, Lj.V., Vajs, V.E., Vućkovic, I.M., Krivošej, Z.Dj., Marin, P.D. (2011): Composition and antimicrobial activity of Seseli globiferum essential oil. Natural Product Communications, 6(8): 1163-1166. Kostić, D.A., Velicković, J.M., Mitić, SS., Mitić, M.N., Randelović, S.S 2012: Phenolic content, and antioxidant and antimicrobial activities of Crataegus oxyacantha L. (Rosaceae) fruit extract from Southeast Serbia. Tropical Journal of Pharmaceutical Research, 11(1): 117-124. Krause, J., Tobin G. 2013: Discovery, Development, and Regulation of Natural Products. In: Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century. 3-35. Kundaković, T., Milenković, M., Zlatković, S., Kovacević, N., Goran, N. 2011: Composition of Satureja kitaibelii essential oil and its antimicrobial activity. Natural Product Communications, 6(9): 1353-1356. Kundaković, T., Stanojković, T., Kolundzija, B., Marković, S., Sukilović, B., Milenković, M., Lakušić, B. 2014: Cytotoxicity and antimicrobial activity of the essential oil from Satureja montana subsp. pisidica (Lamiceae). Natural Product Communications, 9(4): 569-572. Lakušić, B., Ristić, M., Slavkovska, V., Antić Stanković, J., Milenković, M. 2008: Chemical composition and antimicrobial activity of the essential oil from Satureja horvatii Šilić (Lamiaceae). Journal of Serbian Chemical Society, 73(7) 703–711. Lazarević, J.S., Palić, R.M., Radulović, N.S., Ristić, N.R., Stojanović, G.S. 2010: Chemical composition and screening of the antimicrobial and antioxidative activity of extracts of Stachys species. Journal of Serbian Chemical Society, 75(10): 1347–1359. Lu, J.-M., Lin, P.H., Yao, Q., Chen, C. 2010: Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. Journal of Cellular and Molecular Medicine, 14(4): 840-860. Maksimović, Z. 2008: In vitro antioxidant activity of ragweed (Ambrosia artemisiifolia L., Asteraceae) herb. Industrial Crops and Products, 28(3): 356– 360. Marčetić, M., Petrović, S., Milenković, M., Niketić, M.S. 2014a: Composition, antimicrobial and antioxidant activity of the extracts of Eryngium palmatum Pančić and Vis. (Apiaceae). Central European Journal of Biology, 9(2): 149-155. Marčetić, M., Petrović, S., Milenković, M., Vujisić, Lj. V., Tešević, V.V., Niketić, M.S 2014b: Composition and antimicrobial activity of root essential oil of Balkan endemic species Eryngium palmatum. Chemistry of Natural Compounds, 49(6): 1140-1142. Matejić, J., Džamić, A., Mihajilov-Krstev, T., Ranđelović, V., Krivošej, Z., Marin, P. 2012: Total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 72 extracts from three Seseli L. taxa. Central European Journal of Biology, 7(6): 1116-1122. Matejić, J.S., Džamić, A.M., Mihajilov-Krstev, T.M., Ranđelović, V.N., Mileski, K.S., Marin, P.D. 2014: Total phenolic and flavonoid contents and biological activities of Cachrys cristata Dc. extracts. Archives of Biological Science, 66(3): 1117-1123. Matejić, J., Džamić, A., Mihajilov-Krstev, T., Ranđelović, V., Krivošej, Z., Marin, P. 2013: Total phenolic and flavonoid content, antioxidant and antimicrobial activity of extracts from Tordylium maximum. Journal of Applied Pharmaceutical Science, 3(1): 055-059. Matejić, J.S., Džamić, A.M., Mihajilov-Krstev, T.M., Ranđelović, V.N., Marin, P.D. 2015: Antioxidant and antimicrobial potential of Opopanax hispidus (Apiaceae) extracts. Lekovite sirovine, 35: 141- 150. Mihajilov-Krstev, T.M., Kitić, D.V., Radnović, D.V., Ristić, M.S., Mihajlović-Ukropina, M., Zlatković, B.K. 2011: Chemical composition and antimicrobial activity of Satureja kitaibelii essential oil against pathogenic microbial strains. Natural Product Communications, 6(8): 1167- 1172. Mihajilov-Krstev T.M., Jovanović B., Jović J. L., Ilić B.S., Miladinović D.L., Matejić J.S., Rajković J.S., Djordjević Lj.B., Cvetković V.J., Zlatković B.K. 2014: Antimicrobial, antioxidative, and insect repellent effects of Artemisia absinthium essential oil. Planta Medica, 80(18): 1698-1705. Mihajilov-Krstev, T., Radnović, D., Kitić, D., Stankov-Jovanović, V., Mitić, V., Stojanović- Radić, Z., Zlatković, B. 2014: Chemical composition, antimicrobial, antioxidative and anticholinesterase activity of Satureja montana L. ssp montana essential oil. Central European Journal of Biology, 9(7): 668-677. Miladinović, D.L., Ilić, B.S., Mihajilov-Krstev, T.M., Jović, J.L., Marković, M.S. 2014: In vitro antibacterial activity of Libanotis montana essential oil in combination with conventional antibiotics. Natural Product Communications, 9(2): 281-286. Miladinović, D.L., Ilić, B.S., Mihajilov-Krstev, T.M., Nikolić, N.D., Milosavljević, V.N., Nikolić, D.M. 2012: Antibacterial potential of the essential oil from Sideritis montana L. (Lamiaceae). Hemijska industrija, 66(4): 541–545. Mileski, K., Džamić, A., Ćirić, A., Grujić, S., Ristić, М., Matevski, V., Marin, P.D. 2014: Radical scavenging and antimicrobial activity of essential oil and extracts of Echinophora sibthorpiana Guss. from Macedonia. Archives of Biological Sciences, 66(1): 401-413. Mileski, К., Džamić, А., Ćirić, А., Ristić, М., Grujić, S., Matevski, V., Marin, P.D. 2015: Composition, antimicrobial and antioxidant properties of endemic species Ferulago macedonica Micevski and E. Mayer. Records of Natural Products, 9(2): 208-223. Miliauskas, G., Venskutonis, P.R., van Beek, T.A. 2004: Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chemistry, 85: 231-237. Milosavljević, S., Tešević, V., Vučković, I., Jadranin, M., Vajs, V., Soković, M., Janaćković, P., Jovanović, A. 2007: Composition and antifungal activity of the essential oil of Seseli annuum wild- growing in Serbia. Fitoterapia, 78(4): 319–322. Mimica-Dukić, N.M., Božin, B.N., Soković, M.D., Simin, N.D. 2004: Antimicrobial and antioxidant activities of Melissa officinalis L. (Lamiaceae) essential oil. Journal of Agricultural and Food Chemistry, 52(9): 2485-2489. Pandey S., Gupta R.K. 2014: Screening of nutritional, phytochemical, antioxidant and antibacterial activity of Chenopodium album (Bathua). Journal of Pharmacognosy and Phytochemistry, 3(3): 1-9. Petrović, J., Stanojković, A., Čomic, Lj., Ćurčić, S. 2003: Biotic activity of some aromatic plants on Pseudomonas aeruginosa and Pseudomonas fluorescens. Lekovite sirovine, 23: 31-36. Petrović, N.V., Petrović, S.S., Džamić, А.M., Ćirić, А.D., Ristić, М.S., Milovanović, S.L., Petrović, S.D. 2016a: Chemical composition, antioxidant and antimicrobial activity of Thymus praecox supercritical extracts. Journal of Supercritical Fluids, 110: 117–125. Petrović, N.V., Petrović, S.S., Ristić, М.S., Džamić, А.M., Ćirić, А.D. 2016b: Chemical composition and antimicrobial activity of Thymus praecox Opiz ssp. plytrychus essential oil from Serbia. Records of Natural Products, 10(5): 633-638. Radovanović, B., Milenković-Anđelković, A.S., Radovanović, A.B., Anđelković, M.Z. 2013: Antioxidant and antimicrobial activity of polyphenol extracts from wild berry fruits grown in Southeast Serbia. Tropical Journal of Pharmaceutical Research, 12(5): 813-819. Seidel, V. 2006. Initial and Bulk Extraction. In: Sarker, S.D., Latif, Z., Gray, A.I. (ed.), Methods in Biotechnology Vol. 20, Natural Products Isolation, 2nd ed. Humana Press Inc., Totowa, NJ. Soković, M.D., Stojković, D.S., Glamočlija, J.M., Ćirić, A.D., Ristić, M.S., Grubišić, D.V. 2009: Susceptibility of pathogenic bacteria and fungi to essential oils of wild Daucus carota. Pharmaceutical Biology, 47(1): 38-43. BIOLOGICA NYSSANA 7 (2)  December 2016: 61-73 Džamić, A. et al.  Trends in biological activity research of wild… 73 Soković, M.D., Vukojević, J., Marin, P.D., Brkić, D.D., Vajs, V., van Griensven, L.J. 2009: Chemical composition of essential oils of Thymus and Mentha species and their antifungal activities. Molecules, 14: 238-249. Stanković, M.S., Radojević, I.D., Stefanović, O.D., Topuzović, M.D., Čomić, Lj.R., Branković, S.R. 2011: Immortelle (Xeranthemum annuum L.) as a natural source of biologically active substances. Excli Journal, 10: 230-239. Stanojević, Lj.P., Stanković, M.Z., Nikolić, V.D., Nikolić, Lj.B. 2008: Anti-oxidative and antimicrobial activites of Hieracium pillosela L. extracts. Journal of Serbian Chemical Society, 73(5): 531-540. Stanojević, Lj., Stanković, M., Nikolić, V., Nikolić, Lj., Ristić, D., Čanadanović-Brunet, J., Tumbas V. 2009: Antioxidant activity and total phenolic and flavonoid contents of Hieracium pilosella L. extracts. Sensors, 9(7): 5702–5714. Stanojković, A., Ceković, J., Pivić, R., Stanojković, A. 2008: In Vitro Antibacterial activity of Thymus serpyllum extracts. Proceedings 43rd Croatian and 3rd International Symposium on Agriculture, 516-519. Stanojković, T., Kolundžija, B., Ćirić, A., Soković, M., Nikolić, M., Kundakovića, T. 2013: Cytotoxicity and antimicrobial activity of Satureja kitaibelii Wierzb. ex Heuff (Lamiaceae). Digest Journal of Nanomaterials and Biostructures, 8(2): 845 – 854. Stevanović, V, Tan, K, Petrova, A. 2007: Mapping the endemic flora of the Balkans—a progress report. Bocconea, 21: 131–137. Stojković, S., Petrović, S., Kukić, J., Džamić, A., Ristić, M., Milenković, M., Glamočlija, J., Soković, M., Stojković, D. 2009: Chemical composition and antimicrobial and antioxidant actyvity of essential oil from the flowers of Sesili rigidum. Chemistry of Natural Compounds, 4: 253-256. Stupar, M., Ljaljević Grbić, M., Džamić, A., Unković, N., Ristić, M., Vukojević, J. 2014: Antifungal activity of Helichrysum italicum (Roth) G. Don (Asteraceae) essential oil against fungi isolated from cultural heritage objects. Archives of Biological Science, 66(4): 1539-1545. Vladimir-Knežević, S., Kosalec, I., Babac, M., Petrović, M., Ralić, J., Matica, B., Blažeković B. 2012: Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory pathogens. Central European Journal of Biology, 7(6): 1109-1115. Živković, J., Stojković, D., Petrović, J., Zdunić, G., Glamočlija, J., Soković, M. 2015: Rosa canina L. – new possibilities for an old medicinal herb. Food and Function, 6(12): 3687-3692.