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Wild edible Mediterranean herbs have always played an important role in human diet and traditional medicine, and recently special attention has been paid to halophytic species since about 1% of the total world flora consists of these plants. They are native to saline habitats because of their special adaptive mechanisms to the harsh conditions to which they are exposed. While the total area of saline soils is increasing (1-2% per year) due to rapid climate change, agricultural productivity is decreasing, and along with population growth, it is estimated that the 21st century will be the century of halophyte agriculture expansion. Although halophytes are often underestimated and underutilised, their innate resilience to these changes could play a key role in ensuring food security. The aim of this study is to gain insight into the most widespread halophyte species (Crithmum maritimum ; Salicornia europea, Inula crithmoides, Arthrocnemum fructicosum, Limonium cancellatum, Cakile maritima Scop., Pancriatium maritimum, ...) growing along the Adriatic coast, their main characteristics, potential for use in different sectors, and environmental importance.

Medicinal plants are potential sources of bioactive compounds. One of the medicinal plants used in the traditional medicine of Bosnia and Herzegovina is endemic Satureja subspicata L. In this work, we examined the ability of Satureja subspicata L. essential oil and hot water and methanol extracts to inhibit the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) using Ellman’s method. The ability of Satureja subspicata L. essential oil in concentration of 1 mg/mL and 2 mg/mL to inhibit enzymes was moderate: 72.82%, and 76.89% for AChE, and 51.51%, and 27.15% for BChE, respectively. Analyzed hot water and methanol extracts in concentration of 1 mg/mL showed weak ability of cholinesterase inhibition. Extracts were additionally analyzed regarding to ability to protect proteins from oxidation, during 1 h and 24 h incubation periods. After incubation for 1 h hot water extract showed a very good protective effect (10.61%), while the methanolic extract showed prooxidative activity. After incubation for 24 h, both extracts showed prooxidative activity. The obtained results show that the examined essential oil and extracts of S. subspicata L. contain compounds with cholinesterase inhibition and antioxidant potential, and therefore can be useful in treatment of Alzheimer's disease.

Based on field research, an analysis of the ecology of the species Dalmatocytisus dalmaticum (Vis.) Trinajstić was conducted, and the collected plant material was subjected to phytochemical analysis. As a stenoendemic species, this plant is found only near the town of Sinj on the hills Poljakova greda, Šušnjevača and Medinjak. It grows in the composition of sub-Mediterranean dry grasslands of the class Festuco-Brometea Br.-Bl. & R.Tx. 1943, on which it forms carpet-like coverings. On Mount Greda it creates larger stands on shallow carbonate mud soils and it reproduces by creeping underground stems and to a lesser extent by seeds. Recently, the number of habitats of D. dalmaticum has been decreasing, due to afforestation with black pines and the succession from rocky grasslands to thickets. Isolation of essential oil from fresh plant material collected in 2021, was performed by water distillation in a modified Clevenger apparatus. Obtained oil sample was analyzed by GC/MS analysis. A total of 60 compounds were identified, accounting for 88.06% of the total oil. The major groups of compounds were monoterpene hydrocarbons (3.22%), oxygenated monoterpenes (5.12%), sesquiterpene hydrocarbons (4.54%), oxygenated sesquiterpenes (9.89%), diterpene alcohols (9.09%), carbonyl compounds (22.93%), and other compounds (29.84%). The most abundant compounds of each group in the extract are α-pinene, (E)-damascenone, (Z)-caryophyllene, hexahydrofarnesyl acetone, 4-vinylphenol, pentacosane, phytol, and palmitic acid. Some of the compounds mentioned are known to have cytotoxic, anticancer, antibacterial, and antiviral properties, suggesting the possibility of further research on this species.

Teucrium montanum L. - Chemical Composition and Biological Activity of Aqueous and Methanolic Extract

Study of Phytochemical Composition and Biological Potential of Teucrium montanum L. Essential oil

Medicinal plant Mentha pulegium L. can be found in the area of Europe and Mediterranean area, where it is used in as traditional medicine and in nutrition of local population. Because of the mint-like odour M. pulegium essential oil has wide application as food component of food and as a fragrance. In this work the chemical composition, antioxidant activity as well as and anticholinesterase potential of the essential oil of M. pulegium growing in Bosnia and Herzegovina (BiH) were was assessed. Monoterpenoids pulegone (54.4%), p-menthone (14.0%) and piperitenone (12.8%), identified by GC-MS and GC-FID, were dominant essential oil chemical components. Analysezed essential oil showed good antioxidant activity, tested wby ith DPPH and FRAP assay, and moderate cholinesterase inhibition potential, tested by Elman’s assay. The promising biological activity of tested essential oil indicates to the possibility of its use in food as protecting antioxidant, in prevention of free radical linked diseases and diseases associated with low content of acetylcholine.

Artemisia genus is one of the largest of the Asteraceae family. This genus is consists of large number of species which are widespread throughout Europe, Asia, Africa, and North America. Many Artemisia plants is often used in traditional medicine as antimalarial, antiviral, antitumor, spasmolytic, and others agents (Tan et al, 1998). In this work the volatile oils of six different Artemisia species (A. absinthium, A. abrotanum, A. annua, A. arborescens A. verlotiorum and A. vulgaris) from Dalmatia (Croatia) and Bosnia and Herzegovina were tested on chemical composition and anticholinesterase power. Artemisia volatile oils were isolated by hydrodistillation and their chemical compositions were determined by GC-MS/FID. Volatile oil of A. absinthium was characterized by high amounts of Z-sabinyl acetate and Z-epoxy-ocimene ; A. abrotanum oil was characterized by Z-davanone and piperitone ; A. annua oil was characterized by camphor and artemisia ketone ; A. arborescens oil was characterized by camphor and chamazulene ; A. verlotiorum oil was characterized by Z-thujone ; A. vulgaris oil was characterized by E-thujone. Anticholinesterase (antiAChE and antiBuChE) power was tested by Ellman method (Ellman, 1961). All of the tested volatile oils showed moderate anticholinesterase power comparing with huperzin A, as good anticholinesterase agent. AChE inhibition power of tested Artemisia volatile oils (1 mg/mL) were in the range of 29.5-55.6 %, while BuChE inhibition power of tested oils were in the range of 11.4-49.0 %.

Inhibition of important enzymes is one of the fundamental therapies for treating diseases such as Alzheimer's dementia and Diabetes mellitus. Genus Centaurea is one of the most important genuses of the Asteraceae family, with a large number of species. In Croatia there are around 80 species of this genus, of which 27 are endemic.In this study, the chemical profile and enzyme inhibition potential of the water extracts of five plants of the genus Centaurea was determined. The chemical profile of the extracts was determined by quantitative determination of the total phenol content and the total flavonoid content. Enzyme inhibition potential of the extracts were investigated in terms of the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase BuChE) by Ellman method as well as a-glucosidase enzymes by method described from Brueggeman and Hollingsworth.The results showed that all Centaurea water extracts contain a relatively small content of phenolic and flavonoid compounds. C. scabiosa and C. rhenana extracts show low inhibition of AChE, C. triumfetti and C. ragusina show very low inhibition of AChE, whereas C. alba does not inhibit AChE. C. alba, C. rhenana and C. scabiosa extracts poorly inhibiting BuChE, while C. triumfetti and C. ragusina do not inhibit this enzyme. C. scabiosa extract show moderate inhibition on a-glucosidase. C. rhenana extract show low inhibition on a-glucosidase enzyme, while the other extracts do not inhibit this enzyme.

Fruits such as tangerine, grapefruit, sweet orange and lemon (genus Citrus, Rutaceae) are one of the important horticultural crops. Peel, as a by-product of the Citrus processing, represents the source of the most widely used essential oils in the world. They have gained acceptance in the food industry primarily as an aroma flavour in food products such as: marmalades, gelatines, sweets, soft drinks, ice creams, dairy products, candies, cakes even alcoholic and non-alcoholic beverages etc. Citrus essential oils are also used in pharmaceutical industry, perfumery and cosmetic industry, and even as green solvent for Soxhlet extraction. For this reason, it is crucial to have a fast and reliable analytical method for detection of its components. The aim of this study was development and validation of the method for qualitative and quantitative determination of limonene as the main Citrus essential oil component using gas chromatography. Commercial essential oils of tangerine, grapefruit, sweet orange and lemon were used for this purpose. The method had good separation of components and reproducible limonene retention time, while LOQ, LOD and R2 were 1.6×10-3, 4.7×10-4, 0, 9949, respectively.

The medicinal plant Mentha pulegium L. (Lamiaceae) is used in traditional medicine of Bosnia and Herzegovina to treat neurological and gastrointestinal disorders [1]. Healing properties of M. pulegium are attributed to monoterpenoids present in its essential oil and polyphenol derivatives [2]. These bioactive components have an important role in the prevention and treatment of chronic diseases related to oxidative stress. One of them is Alzheimer's disease, a neurological brain disorder and the most common form of dementia, affecting the older population. Inhibitors of cholinesterases have an important role in the treatment of Alzheimer’s disease. The aim of this work was to analyze the chemical composition as well as the antioxidant activity and cholinesterase inhibition potential of M. pulegium essential oil from Bosnia and Herzegovina. The chemical composition of the essential oil was determined by GC/MS and GC/FID techniques. The antioxidant potential was tested using DPPH and FRAP methods [3, 4]. Inhibition of acetylcholinesterase and butyrylcholinesterase was determined using the Ellman’s method [5]. The major components found in M. pulegium essential oil where: pulegone (54.4%), p-menthone (14.0%), piperitenone (12.8%) and piperitone (3.7%). A solution of the essential oil (1 g/L) showed a low antioxidant potential and a good inhibition of both cholinesterases.

Genome size and chromosome number are characteristic species trait important in systematics, taxonomy and evaluation of biodiversity. The Adriatic coast of Croatia presents a unique area for biological and chemical research of plant material. Genus Centaurea (Asteraceae family) is already known for many medicinal plants that present a valuable source of potential medicines. In Croatia 79 Centaurea taxa have been listed, comprising 27 endemics. Previous chemical and biological observations of extracts and volatiles isolated from Centaurea species, showed differences in chemical composition and biological activity of volatile substances between different species. Some Centaurea species have many similarities in their morphological appearance, therefore the criteria of genome size and number of chromosomes contribute to plant material identification. Due to a high demand for a clear and authentic plant material identification in chemical research, genome size and number of chromosomes for six Centaurea species have made prior to chemical and biochemical research of isolated plant extracts. The genome size was estimated using flow cytometry. The chromosome number was determined using the standard karyological methods. The plant samples, seeds and leaves, were collected from wild growing natural populations of Croatia. The studied species present three basic chromosome numbers, x= 9, 10 and 11. The ploidy level of investigated species was diploid and tetraploid. Centaurea cristata and C. rhenana were tetraploid, whilst other species were diploid. The nuclear DNA amounts (2C DNA) range from 1.70 to 3.57 pg. Using the Leitch's et al. criteria all these taxa belong to the group of very small genome size. The results present the novel values for C. cristata, and C. rhenana. The studied species will be the object of our future studies concerning the chemical composition of volatile and non-volatile plant extracts and their biological activity and differences in chemical composition due to a level of ploidy.

The Croatian Adriatic coast and islands form a unique and distinctive area as a habitat for 80 Centaurea (Asteracea) taxa out of which 27 are endemics ; out of total 400- 700 species, distributed in 27 sections in this genus (1). Section Acrocentron, where both studied Centaurea salonitana L. and Centaurea rupestris L. belong, is one of the largest sections of Centaurea genus (2). There are some studies connecting the cell nuclei ploidy and variation in volatile oil composition implicating possible connection (3). The aim of the present study was to investigate the involvement of cell nuclei ploidy, determined using flow cytometry, in variation of essential oil composition in two species of Acrocentron section: C. salonitana and C. rupestris, from two different localities in Croatia, Adriatic region. The essential oil composition was determined using GC and GC-MS analysis of C. salonitana diploid and tetraploid and C. rupestris two diploid population’s essential oils. The diploid and tetraploid C. salonitana showed very similar composition of essential oils, number of determined components and yield of essential oil. There were more differences among two different species than the same species with different ploidy level. However when comparing two populations of the same species from different ecological conditions, such as continental and Mediterranean climate we have noticed there are differences among the essential oils of the same species from two different localities. The study presents the first results on C. salonitana essential oil composition, as well as first comparison of diploid and tetraploid population’s volatile oils.

Centaurea scabiosa is wild edible plant used in food and nutrition in Mediterranean area. A whole juvenile plant is used in local cuisine of Dalmatian region, Croatia. Volatile oils may comprise variety of biologically active compounds. Aim of this work was to investigate the volatile oil chemistry of C. scabiosa species and its biological activity: antioxidant potential, inhibition of cholinesterases and toxicity on human cells. The chemical composition of essential oil was assessed using GC/MS technique. Antioxidant capacity of essential oil was determined using DPPH and FRAP methods. Inhibition of both acetylcholinesterase and butyrylcholinesterase was determined using Ellman method. Toxicity of C. scabiosa volatile oil was measured on primary fibroblasts through the MTT assay. [11] The major compounds found in essential oil were heptacosane, caryophyllene oxide, alloaromadendrene epoxide, α-cyperone and α-bisabolol. Essential oil in stock concentration of 1g/L showed moderate inhibition of both cholinesterases while antioxidant potential was low on both tested methods. C. scabiosa volatile oil showed no toxicity on primary fibroblasts at the highest tested dose of 0, 125g/L in 0, 5% DMSO.

The Centaurea genus species herbal remedies have been used in ethnomedicines in Mediterranean area for treatment of various diseases as they possess a valuable source of bioactive compounds. Sesquiterpene lactones, phenols, flavonoids and lignans are usually regarded as a group of compounds responsible for diverse biological activity of Centaurea plant extracts The chemical constituents were obtained and tentatively identified using high performance liquid chromatography HPLC-DAD-ESI-TOF-MS in negative ionisation mode. The aqueous extracts of C. ragusina were investigated for correlation of their effects on human bladder (T24) and human glioblastomma (A1235) cancer cell lines using the MTT assay. Cells were exposed subsequently to both extracts in order to determine type of cell death induced by treatment. The main compounds of herba extract were sesquiterpene lactones: solstitialin A-3, 13 diacetate and epoxyrepdiolide ; quinic acid as organic acid. The main compounds of flower extract were organic acids: quinic acid, citric acid, malic acid ; sesquiterpene lactone cynaropicrin ; phenolic compounds: chlorogenic acid and phenylpropanoid syringin. Although both extracts showed significant dose- and time-dependent cytotoxicity against both cancer cell lines, the flower extract exhibited slightly better activity. The majority of the cells died by treatment induced apoptosis. Flower extract (26.25%) compared to a leaf extract (22.15%) showed slightly higher percentage of an apoptosis in T24 cells compared to non-treated cells (3.27 %). Centaurea ragusina L., an endemic Croatian plant species, revealed a good anti- proliferative activity of aqueous extracts on human bladder (T24) and human glioblastomma (A1235) cancer cell lines. Centaurea ragusina aqueous extract represents a valuable source of sesquiterpene lactones and flavonoids with anticancer properties reported in numerous studies.

Due to an antibiotic resistance around 700, 000 people each year worldwide is life threatened and killed by drug-resistant microbes (1). Therefore the new antimicrobial agents are needed and search for them presents a top priority in this area of science (1). Recent interest in natural compounds has even risen due to a rapid development of resistant bacterial strains. This forced natural product re-evaluation as a source of novel chemical skeletons with antibacterial activity for elaboration in drug development (2). Centaurea genus, Asteraceae family, is widespread in worldwide area comprising around 400-700 species (3). They are well known, on medicinal purposes, in eastern Mediterranean ethno-pharmacology ; due to a variety of bioactive compounds they comprise (4). Good antimicrobial activity was previously reported for more than 20 Centaurea species essential oils, with more than 200 different compounds present. This presents a good model for development of mathematical tool in order to characterise the chemical compounds responsible for a good antimicrobial activity. Biological activity of essential oils is usually connected with the dominant components, but some compounds in minor percentage may also have a strong impact (3). Therefore, we have used mathematical tool to detect the specific combination of compounds responsible for antimicrobial activity. For the theoretical base for multidimensional characterisation we have developed variance-covariance matrix and calculated its eigenvalues and eigenvectors. This tool may be used for a focused research of compounds that have good antimicrobial activity, but also for detection of any other essential oil characteristics that can be utilized in cosmetics, pharmaceutical, food or any other industry.

Satureja subspicata Vis. (mountain savory) is a rare, endemic species distributed in the eastern Mediterranean area [1]. In traditional medicine of Bosnia and Herzegovina (BiH) S. subspicata is used to treat leukemia and syndromes of lymphatic nodes since long ago. In this area preparations of S. montana and S. subspicata have been used with excellent results for different cardiovascular disorders, especially arrhythmia, heart fibrillation and the diseases of blood vessels [1]. There are only a few studies about the chemical composition and biological activity of non-volatile extracts of S. subspicata growing in Croatia and none report on the extracts fom BiH. In this work we investigated the phytochemical composition of methanolic and hot water extracts of S. subspicata collected in BiH, total phenols and flavonoids. In addition, the aim of this work was to determine the antioxidant potential and inhibition of cholinesterases. Chemical composition of extracts was assessed using HPLC/DAD technique. Antioxidant capacity was determined using DPPH, FRAP methods [3-4] and by determining activity of extracts in linoleic acid emulsion [5]. Using Ellman method inhibition of acetilcholinesterase and butyrylcholinesterase was determined [6]. The major compounds found in methanolic extract where: rosmarinic acid, eriodictyol and kaempferol, while in hot water extract major compounds where: rosmarinic acid, myricetin and ferulic acid. Obtained results for content of total phenols and total flavonoids indicate considerable amount of these compounds. In concentration of 1 g/L in stock solution extracts showed low inhibition of both cholinesterases and good antioxidant potential.

Centaurea genus, Asteracea family, is widespread in worldwide area comprising around 400 up to 700 species, of which around 80 with 27 endemics are present in Croatia. Centaurea species are well known for their medicinal properties as result of many bioactive compounds they comprise. In eastern Mediterranean ethnopharmacology they are used as medical plants in treatment of diseases like peptic ulcer, herpes labialis, malaria, common cold, abscesses, hemorrhoids, dyspepsia and some gastrointestinal disorders. Good biological activity of Centaurea essential oils is usually connected with the dominant compounds present in essential oils. Aim of this work was to characterize the essential oil composition as well as antioxidant and anticholinesterase activity of Centaurea rupestris from Croatia. Essential oils were hydrodistilled from aerial parts of the plants in Clevenger-like apparatus while chemical composition of oil was assessed using gas chromatography/mass spectrometry technique (GC/MS). The antioxidant potential was tested by DPPH and FRAP method while anticholinesterase potential was tested by Ellman method. The main constituents present in isolated oil were germacrene D (24.3%), heptacosane (14.4%), phytol (6.7%) and β-caryophyllene (5.0%). All listed compound are common constituents of Centaurea essential oils. Antioxidant potential was no showed promising effect unlike anticholinesterase potential.

The aim of this study was to determine the phenolic composition and antiproliferative activity of five water extracts of plants which belong to Lamiaceae family. The aerial parts extracts of the plants, obtained from Teucrium montanum, Calamintha nepetoides, Satureja subspicata, Salvia sclarea and Melissa officinalis, were tested on human prostate cancer (T24) and human breast cancer (MDA-MB-231) cell lines by using the MTT assay. The phenolic content of the plants were determined by high performance liquid chromatography (HPLC). Among the tested extracts, the extract of Teucrium montanum showed the best antiproliferative activity against T24 and MDA-MBA-231 cell lines. Furthermore, the extract of Satureja subspicata and Melissa officinalis showed significant antiproliferative activity against T24 cell line, too. Generally, all extracts showed better antiproliferative activity against T24 cell line in comparison to MDA-MBA-231 cell line. The HPLC data of the plant extracts showed the presence of the following phenolic compounds: protocatechuic acid, caffeic acid, p-coumaric acid, cinnamic acid and predominatly rosmaric acid. The findings of this study indicate that there is some justification for the use of the tested plants as traditional anticancer medicinal herbs.