The alphabet of sea fennel: Comprehensive phytochemical characterisation of Croatian populations of Crithmum maritimum L.

Extreme environmental conditions affect the synthesis and accumulation of bioactive metabolites in halophytic plants. The aim of this study was to investigate the presence and quantity of key health-promoting phytochemicals in Croatian sea fennel, one of the most popular Mediterranean halophytes with a wide range of uses. The EOs were characterised by a high content of limonene (up to 93%), while the fatty acid profile shows a low content of oleic acid and the presence of valuable linoleic acid (ω-6) and linolenic acid (ω-3) in high percentages. The dominances of lutein and α-tocopherol were also confirmed in all samples. The results confirm the great variability in the chemistry of sea fennel populations in the Mediterranean region, with significant differences in the composition of the Croatian samples compared to the others, as well as the presence and high concentrations of the analysed bioactive compounds that contribute to the plant's health-promoting attributes.

Phenolic, Headspace and Sensory Profile, and Antioxidant Capacity of Fruit Juice Enriched with Salvia officinalis L. and Thymus serpyllum L. Extract: A Potential for a Novel Herbal-Based Functional Beverages.

Since certain constituents are not naturally present in pure fruit juices, incorporating herbal extracts can provide specific sensory properties to the beverages and improve their biopotential. In our previous research, it was found that sage (Salvia officinalis L.), wild thyme (Thymus serpyllum L.), and combinations of their extracts had the highest total phenolic content and a unique composition of volatile compounds, which can contribute to the aromatic and antioxidant qualities of functional products. Therefore, this research aimed to investigate the potential of sage and wild thyme extracts, as well as their mixture (wild thyme:sage at 3:1, v/v), to enrich fruit juices (apple, pineapple, and orange). Obtained beverages were evaluated for sensory properties as well as phenolic and headspace composition (UPLC-MS/MS and HS-SPME/GC-MS analysis) and antioxidant capacity (ORAC assay). The incorporation of wild thyme extract in pineapple juice provided the most harmonious flavor and the highest content of volatile compounds (on PDMS/DVB fiber). The orange juice formulations were the most enriched with phenolic and volatile compounds (on DVB/CAR/PDMS fibers). The highest antioxidant capacity was observed in the formulation with orange juice and sage extract (22,925.39 ± 358.43 µM TE). This study demonstrated that enriching fruit juices with sage and wild thyme extracts could create functional beverages with improved sensory and health-promoting properties, providing valuable insights for the food and beverage industry to meet the growing demand of health-conscious consumers for natural and functional products.

In the Beginning Was the Bud: Phytochemicals from Olive (Olea europaea L.) Vegetative Buds and Their Biological Properties.

Even though Olea europaea L. is one of the most important and well-studied crops in the world, embryonic parts of the plants remain largely understudied. In this study, comprehensive phytochemical profiling of olive vegetative buds of two Croatian cultivars, Lastovka and Oblica, was performed with an analysis of essential oils and methanol extracts as well as biological activities (antioxidant, antimicrobial, and cytotoxic activities). A total of 113 different volatiles were identified in essential oils with hydrocarbons accounting for up to 60.30% and (Z)-3-heptadecene being the most abundant compound. Oleacein, oleuropein, and 3-hydroxytyrosol had the highest concentrations of all phenolics in the bud extracts. Other major compounds belong to the chemical classes of sugars, fatty acids, and triterpenoid acids. Antioxidant, antimicrobial, and cytotoxic activities were determined for both cultivars. Apart from antioxidant activity, essential oils had a weak overall biological effect. The extract from cultivar Lastovka showed much better antioxidant activity than both isolates with both methods (with an oxygen radical absorbance capacity value of 1835.42 µM TE/g and DPPH IC50 of 0.274 mg/mL), as well as antimicrobial activity with the best results against Listeria monocytogenes. The human breast adenocarcinoma MDA-MB-231 cell line showed the best response for cultivar Lastovka bud extract (IC50 = 150 µg/mL) among three human cancer cell lines tested. These results demonstrate great chemical and biological potential that is hidden in olive buds and the need to increase research in the area of embryonic parts of plants.

Antioxidant and Antimicrobial Activity of Hydroethanolic Leaf Extracts from Six Mediterranean Olive Cultivars.

Phenolic profiles, antioxidant, and antimicrobial activities of hydroethanolic olive leaf extracts from six Mediterranean olive cultivars (Croatian: Lastovka, Levantinka, Oblica; Italian: Moraiolo, Frantoio, Nostrana di Brisighella) were investigated. As expected, various distributions of phenolic levels were observed for each cultivar and the total phenolic content showed high variability (ranging from 4 to 22 mg GAE/g of dry extract), with the highest amount of phenolics found in the Oblica sample, which also provided the highest antiradical (ORAC) and reducing activity (FRAP). The screening of individual compounds was performed by HPLC-PDA-ESI-QTOF-MS and the main detected compounds were oleuropein, hydroxytyrosol, oleoside/secologanoside, verbascoside, rutin, luteolin glucoside, hydroxyoleuropein, and ligstroside. While the antioxidant activity of the samples was relatively high, they showed no bactericidal and bacteriostatic activity against E. coli and S. Typhimurium; weak activity against Staphylococcus aureus, Bacillus cereus, and Listeria innocua; and inhibitory effects against Campylobacter jejuni at 0.5 mg dry extract/mL. The obtained results support the fact that olive leaf extracts, and especially those from the Oblica cultivar, could potentially be applied in various industries as natural preservatives and effective and inexpensive sources of valuable antioxidants.

Investigation of Antioxidant Synergisms and Antagonisms among Phenolic Acids in the Model Matrices Using FRAP and ORAC Methods.

The total antioxidant potential of a sample cannot be predicted from the antioxidant activity of its compounds; thus, scientists usually explain the overall activity through their combined effects (synergistic, antagonistic, or additive). Phenolic compounds are one of the most powerful and widely investigated antioxidants, but there is a lack of information about their molecular interactions. This study aimed to investigate the individual and combined antioxidant activity of equimolar mixtures (binary, ternary, quaternary, and quinary) of 10 phenolic acids (protocatechuic, gentisic, gallic, vanillic, syringic, p-coumaric, caffeic, ferulic, sinapic, and rosmarinic acid) at different concentrations using ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Gallic acid showed the highest antioxidant activity, determined using the FRAP assay (494-5033 µM Fe2+) and rosmarinic acid with the ORAC assay (50-92 µM Trolox Equivalents (TE)), while the lowest antioxidant potential was observed for p-coumaric acid (FRAP 24-113 µM Fe2+ and ORAC 20-33 µM TE). The synergistic effect (by FRAP) in the equimolar mixtures of hydroxybenzoic acids was confirmed for a large number of tested mixtures, especially at low concentrations. All mixtures containing gentisic acid showed a synergistic effect (28-89% difference). Using the ORAC method, only two mixtures of hydroxybenzoic acids showed an antagonistic effect, namely a mixture of gentisic + syringic acids (-24% difference) and gallic + vanillic acids (-30% difference), while all other mixtures showed a synergistic effect in a range of 26-236% difference. Among mixtures of hydroxycinnamic acids, the highest synergistic effect was observed for the mixtures of p-coumaric + ferulic acids and caffeic + sinapic acids with differences of 311% and 211%, respectively. The overall antioxidant activity of phenolic acids could be explained by the number or position of hydroxyl and/or methoxy functional groups as well as the compound concentration, but the influence of other parameters such as dissociation, intramolecular hydrogen bonds, and electron donating or withdrawing effect should not be neglected.

Effects of Rubus fruticosus and Juniperus oxycedrus derivatives on culturability and viability of Listeria monocytogenes.

The consumers' demand for safe foods without chemical additives increased the research for green solutions, based on natural antimicrobials. Plants can be an important source of bioactive compounds able to prevent the development of foodborne pathogens and spoilage microflora. This paper aimed to characterize phenolic extracts (PEs) and essential oils (EOs) obtained from Mediterranean Rubus fruticosus leaves and Juniperus oxycedrus needles and to evaluate their antimicrobial effects against Listeria monocytogenes Scott A. The growth dynamics with sub-lethal concentrations of plant derivatives were modeled and flow cytometry was used to better evidence the effect on cell viability and culturability. The results showed that these plant derivatives affected the growth of L. monocytogenes, increasing lag phase (about 40 h in the presence of PEs vs. 8 h in the control) and decreasing the final cell load of at least 1 log cycle with respect to the control. R. fruticosus EO was the most effective, determining an initial decrease of cell counts of about 6 log cycles, followed by a restart of growth after 10 h, with rate similar to the control (0.08 with R. fruticosus EO vs. 0.09 ((log CFU/ml)/h in the control) but significantly lower final cell load (7.33 vs. 8.92 log CFU/ml). According to flow cytometry, only R. fruticosus EO induced a relevant increase of dead cells, while the other plant derivatives determined different extent of sub-lethal cell injury. The discrepancy observed in some cases between viability and culturability could indicate the presence of cells not able to grow in culture media, whose fate needs to be further investigated to assess their potential recovery, thus bringing to an overestimation of the antimicrobial effect of these substances. This research contributed to increase the knowledge of these underused raw materials such as blackberry leaves and juniper needles that can be exploited in food and other industries.

Variations in the Composition, Antioxidant and Antimicrobial Activities of Cystoseira compressa during Seasonal Growth.

The underexplored biodiversity of seaweeds has recently drawn great attention from researchers to find the bioactive compounds that might contribute to the growth of the blue economy. In this study, we aimed to explore the effect of seasonal growth (from May to September) on the in vitro antioxidant (FRAP, DPPH, and ORAC) and antimicrobial effects (MIC and MBC) of Cystoseira compressa collected in the Central Adriatic Sea. Algal compounds were analyzed by UPLC-PDA-ESI-QTOF, and TPC and TTC were determined. Fatty acids, among which oleic acid, palmitoleic acid, and palmitic acid were the dominant compounds in samples. The highest TPC, TTC and FRAP were obtained for June extract, 83.4 ± 4.0 mg GAE/g, 8.8 ± 0.8 mg CE/g and 2.7 ± 0.1 mM TE, respectively. The highest ORAC value of 72.1 ± 1.2 µM TE was obtained for the August samples, and all samples showed extremely high free radical scavenging activity and DPPH inhibition (>80%). The MIC and MBC results showed the best antibacterial activity for the June, July and August samples, when sea temperature was the highest, against Listeria monocytogenes, Staphylococcus aureus, and Salmonella enteritidis. The results show C. compressa as a potential species for the industrial production of nutraceuticals or functional food ingredients.

Seasonal Changes in Essential Oil Constituents of Cystoseira compressa: First Report.

Marine macroalgae are well known to release a wide spectrum of volatile organic components, the release of which is affected by environmental factors. This paper aimed to identify the essential oil (EO) compounds of the brown algae Cystoseira compressa collected in the Adriatic Sea monthly, from May until August. EOs were isolated by hydrodistillation using a Clavenger-type apparatus and analyzed by gas chromatography coupled with mass spectrometry (GC-MS). One hundred four compounds were identified in the volatile fraction of C. compressa, accounting for 84.37-89.43% of the total oil. Samples from May, June, and July were characterized by a high share of fatty acids (56, 69, and 34% respectively) with palmitic acid being the dominant one, while in the August sample, a high content of alcohols (mainly phytol and oleyl alcohol) was found. Changes in the other minor components, which could be important for the overall aroma and biological activities of the algal samples, have also been noted during the vegetation periods. The results of this paper contribute to studies of algal EOs and present the first report on C. compressa EOs.

Sustainable sources for antioxidant and antimicrobial compounds used in meat and seafood products.

The contribution of food in promotion of health has become of most importance. The challenges that lie before the global food supply chain, such as climate changes, food contamination, and antimicrobial resistance may compromise food safety at international scale. Compounds with strong antimicrobial and antioxidant activity can be extracted from different natural and sustainable sources and may contribute to extend the shelf life of meat and seafood products, enhance food safety and enrich foods with additional biologically active and functional ingredients. This chapter describes the use of bioprotective cultures, essential oils, plant extracts, seaweed extracts and grape pomace compounds in production of value-added meat and seafood products with improved shelf life and safety, following the requests from the market and consumers.

Bioactive Phenolic Metabolites from Adriatic Brown Algae Dictyota dichotoma and Padina pavonica (Dictyotaceae).

In this study, the influences of temperature (20, 40 and 60 °C) and extraction solvents (water, ethanol) on the ultrasound-assisted extraction of phenolics from the Adriatic macroalgae Dictyota dichotoma and Padina pavonica were studied. The extracts were analysed for major phenolic sub-groups (total phenolics, flavonoids and tannins) using spectrometric methods, while the individual phenolics were detected by HPLC. The antioxidant activities were evaluated using three methods: Ferric Reducing/Antioxidant Power (FRAP), scavenging of the stabile 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and Oxygen Radical Antioxidant Capacity (ORAC). The aim of the study was also to find the connection between the chemical composition of the extracts and their biological activity. Therefore, principal component analysis (PCA), which permits simple representation of different sample data and better visualisation of their correlations, was used. Higher extraction yields of the total phenolics, flavonoids and tannins were obtained using an alcoholic solvent, while a general conclusion about the applied temperature was not established. These extracts also showed good antioxidant activity, especially D. dichotoma extracts, with high reducing capacity (690-792 mM TE) and ORAC values (38.7-40.8 mM TE in 400-fold diluted extracts). The PCA pointed out the significant influence of flavonoids and tannins on the investigated properties. The results of this investigation could be interesting for future studies dealing with the application of these two algae in foods, cosmetics and pharmaceuticals.

Antimicrobial Activity of Selected Red and White Wines against Escherichia coli: In Vitro Inhibition Using Fish as Food Matrix.

Five different wines (standard Grasevina, macerated Grasevina with and without sulfur, rosé, and standard Plavac Mali), all typical Croatian wines, were tested to determine the antimicrobial activity against two Escherichia coli bacterial strains (ATCC® 25922 and ATCC® 8739) in vitro and using sea bass (Dicentrarchus labrax) fillets as food matrix. The chemical composition of wines (pH, acidity, alcohol, total phenolics, anthocyanins, tannins, and sulfur content) and antimicrobial activity (minimal inhibitory concentration (MIC), agar-well diffusion method) were determined. The total phenolic content of the wines ranged from 305-3210 mg gallic acid equivalents per liter (GAE/L), and did not correlate to antimicrobial activity. The two wines with the lowest phenolic content (standard Grasevina and rosé) had the lowest MIC values (122 and 429 mg GAE/L). A specific relation between the winemaking process and a particular MIC value was not established. There was also no relation found between the pH value, ethanol content, sulfur, or phenolics in regards to the antimicrobial effect. In fish fillets marinated in wine + water mixture (v/v = 1:1) and inoculated with 7 log colony forming units (CFU)/25 g the growth of bacteria was reduced after three days of storage at 4 °C. Subsequent storage resulted in the growth of bacteria in all samples, with the lowest growth of E. coli ATCC® 25922 in macerated Grasevina and E. coli ATCC® 8739 in standard Grasevina. All wines showed the capacity to reduce the number and growth of heavily infected sea bass filets, but correlation with specific wine constituents was not found. Taking into account the numerous reactive mechanisms between food and wine, all in vitro studies in controlled laboratory conditions should be further verified in the relevant environment, and additional research is needed to clarify the role of individual wine components in the mechanism of antimicrobial activity.

Antioxidant and Antimicrobial Potential of Phenolic Metabolites from Traditionally Used Mediterranean Herbs and Spices.

The phenolic extracts of fifteen Mediterranean medicinal plants, as well as their antioxidant and antimicrobial activities were investigated to grade their potential as additives in the food industry. Phenolic profiles of plant extracts were determined spectrophotometrically (total phenolics and phenolic subgroups) while individual compounds were identified using chromatographic assays. The biological activity of samples was determined using five antioxidant assays, while the antibacterial potential was determined against six foodborne pathogens (Camplyobacter coli, Escherichia coli, Salmonela Infantis, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus). The results showed significant variations in phenolic profile of plants and consequently their biological activity. Bearberry contained the highest concentration of phenolics, was extremely rich in non-flavonoids and also had the highest amount of catechins that resulted with good reducing and free radical scavenging properties and low chelating activity. All extracts were not effective against tested microorganisms with Gram-positive bacteria being more sensitive (especially S. aureus). The most effective extracts were St. Johns wort against S. aureus with minimal inhibitory concentration (MIC) of 1.00 mg/mL), bay laurel and nettle against B. cereus (MICs of 1.67 mg/mL), and woodland strawberry against L. monocytogenes (MIC of 3.33 mg/mL).

Interactions of resveratrol with other phenolics and activity against food-borne pathogens.

The aim of this study was to investigate the antibacterial activity of individual phenolics and their binary mixtures with resveratrol against selected food-borne pathogens. The antibacterial activity was quantified using the broth microdilution method by the determination of minimal inhibitory concentrations (MICs). Interactions between compounds in the binary phenolic mixtures were determined by calculating the fractional inhibitory concentration index (FICI). The influence of the number of OH groups in the phenols' structure on their antibacterial activity was assessed by principal component analysis (PCA). The most effective compounds were flavone luteolin and flavonol rutin, while the weakest antimicrobial activity was observed for phenolic acid and flavan-3-ols (catechin and epicatechin). The synergistic effect (FICI ≤0.5) of equimolar mixture of resveratrol with kaempferol was confirmed against Staphylococcus aureus, Bacillus cereus, and Escherichia coli, while the mixture of rutin with resveratrol proved synergistic only against S. aureus. The increasing concentrations of resveratrol in the mixtures with kaempferol and rutin resulted in a loss of synergism which indicates that only selected phenolic mixtures, with optimal concentrations of their individual components, result in synergistic antibacterial activity. We did not find an association between total number of OH groups and antibacterial activity of either individual phenolics or their mixtures.

Phenolic Content of Brown Algae (Pheophyceae) Species: Extraction, Identification, and Quantification.

Over the last few decades, isolations and chemical characterizations of secondary metabolites with proved biological activities have been of interest for numerous research groups across the world. Phenolics, as one of the largest and most widely distributed group of phytochemicals, have gained special attention due to their pharmacological activity and array of health-promoting benefits. Reports on phenolic potentials of marine algae, especially brown algae (Pheophyceae) that are characterized by the presence of phlorotannins, are still scarce. The aim of this review paper is to provide an overview of current knowledge about phenolic potential of different brown algae species (74 species from 7 different orders). Studies on brown algae phenolics usually involve few species, thus the focus of this review is to provide information about the phenolic potential of reported algae species and to get an insight into some issues related to the applied extraction procedures and determination/quantification methods to facilitate the comparison of results from different studies. The information provided through this review should be useful for the design and interpretation of studies investigating the brown algae as a source of valuable phytochemicals.

Production and Refinement of Omega-3 Rich Oils from Processing By-Products of Farmed Fish Species.

In this study, the effect of a four-stage chemical refining process (degumming, neutralization, bleaching, deodorization) on the quality parameters, fatty acid composition and volatile compounds of crude oils produced from processing by-products of farmed fish species (tuna, seabass and gilthead seabream) was evaluated. The quality of the oils was compared to commercially available cod liver oil on the basis of free fatty acid, peroxide value, p-anisidine, total oxidation (TOTOX), thiobarbituric acid reactive species (TBARS), oxidative stability at 80, 100 and 120 °C, tocopherol content, and volatile components, while the fatty acid profile and the proportion of polyunsaturated fatty acids (PUFAs) were used as an indicator of the nutritional values of fish oils. Quality parameters of the studied oils and oil oxidative stability were enhanced with refining and were within the limits recommended for fish oils without the loss of PUFAs. In tuna by-product refined oils, the proportion of PUFAs was over 40%, with 30% of eicosapentaenoic and docosahexaenoic fatty acids. The volatile compounds of the oils were quantified (in mg/kg) and major components were 2,4-heptadienal, pentadecane, 2,4-decadienal, 2,4-nonadienal and dodecane. The use of aquaculture by-products as an alternative source for fish oil production could contribute to a more sustainable and profitable aquaculture production, providing economic benefits for the producers and setting new standards for a fish by-product disposal strategy.

Effect of winemaking on phenolic profile, colour components and antioxidants in Crljenak kastelanski (sin. Zinfandel, Primitivo, Tribidrag) wine.

Crljenak kastelanski or Tribidrag is a Croatian autochthonous grape variety which is a parent of the America's most popular variety Zinfandel or Italian Primitivo. The aim of this study was to investigate the dynamics of extraction of phenolics and changes in antioxidant activity of the samples collected during maceration of C. kastelanski grapes processed using two different macerating enzymes. According to the obtained results it can be concluded that wines from C. kastelanski grapes are great source of bioactive phenolics although differences in phenolic profiles between control and enzyme-treated wines were detected. The highest content of total phenolics was detected in control wine (2691 mg GAE/L). Use of pectolitic enzyme Vinozym Vintage improved anthocyanin extraction, while higher colour parameters were observed for wine samples produced using Sihazym Extro. The statistical analysis confirmed great influence of total phenolics and concentrations of some individual phenolic compounds (e.g. catechin, gallic acid, epicatechin) on reducing and free radical scavenging activity of wine samples while the impact of anthocyanins was not detected. According to the obtained results it can be concluded that use of enzymes has slightly negative effect on total phenolics and wine antioxidant properties, but it increases the extraction yield of wine colour components what enables shorter maceration and prevents colour losses during the aging process.

Sea fennel (Crithmum maritimum L.): phytochemical profile, antioxidative, cholinesterase inhibitory and vasodilatory activity.

Sea fennel, a rediscovered star of the coastal cuisine, has been investigated for its phytochemical profile and biological potential. Sea fennel flowers, stems and leaves were analyzed for essential oils (EOs) isolated by hydrodistillation, as well as non-volatiles obtained by ethanolic extraction. Limonene were found to be a dominant compound in EOs and ethanolic extracts; ranging from 57.5-74.2 % and 0.7-8.1 mg/g dry plant material, respectively. In addition total phenolic content was determined for ethanolic extracts. All samples and their main phytochemicals were tested for various methods. EO and extract obtained from flowers were tested for vasodilatory activity on rat aortic rings. Antioxidant activity of EOs was extremely low in comparison to extracts, on the contrary to cholinesterase inhibition where EOs showed better activity than extracts. Flower extract and chlorogenic acid showed stronger vasodilators in comparison to EO and limonene. The obtained results point out the potential impact of the dominant compounds from EO and extract on the biological properties of the sea fennel.

Insight into the Presence of Stilbenes in Medicinal Plants Traditionally Used in Croatian Folk Medicine.

Over the last years, great interest has arisen concerning plant stilbenes, especially resveratrol, which has a whole spectrum of positive biological activities. In this study, we investigated the presence of resveratrol monomers (trans- and cis- form) and naturally occurring derivatives of trans-resveratrol (piceid, astringin and isorhapontin) in phenolic extracts of twenty medicinal plants traditionally used in Croatian folk medicine. The investigated compounds were present in the samples, in free form or as glucosides, and the highest share was found in immortelle, common yarrow and Lamiaceae plants. The obtained results indicate that biological activity of selected medicinal plants can be related to the presence of this valuable group of phytochemicals.

Phenolic acids profile, antioxidant and antibacterial activity of chamomile, common yarrow and immortelle (Asteraceae).

Chamomile, common yarrow and immortelle ethanolic extracts were chemically analysed with respect to phenolics. Twelve phenolic acids were separated and identified by HPLC-DAD and the presence of rosmarinic acid was additionally confirmed by LC-MS. Five methods were applied for the evaluation of extracts' antioxidant properties (FRAP, DPPH, ABTS, chelating activity, Briggs-Rauscher reaction), while the antibacterial activity was tested against some of the major food-borne pathogens (Campylobacter coli, Escherichia coli, Salmonella Infantis, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus). Immortelle extract was the richest in phenolics with a dominant flavonoid fraction, while the other two extracts contained higher amount of non-flavonoids. The phenolic acid profile also varied; high concentration of rosmarinic acid was found in chamomile and common yarrow, while caffeic acid was dominant in immortelle. The best antioxidant properties were obtained for chamomile extract, while good antimicrobial activity, especially against Gram-positive bacterial species, was detected for immortelle. The obtained results could be used as a tool for chemotaxonomic classification of the investigated plants or for their potential application as natural antioxidants/antimicrobials.

Antioxidative/acetylcholinesterase inhibitory activity of some Asteraceae plants.

The extracts obtained by 80% EtOH from some Asteraceae plants (Calendula officinalis, Inula helenium, Arctium lappa, Artemisia absinthium and Achillea millefolium) were studied. Rosmarinic acid, one of the main compounds identified in all extracts, was determined quantitatively by using HPLC. In addition, spectrophotometric methods were evaluated as an alternative for rosmarinic acid content determination. Total phenolic content was also established for all extracts. A. millefolium extract was found to have the highest content of rosmarinic acid as well as total phenols. All extracts were tested for antioxidant and acetylcholinesterase inhibitory activity. A. millefolium was shown to possess the best antioxidant activity (for all tested methods) as well as acetylcholinesterase inhibitory activity. Highly positive linear relationships were obtained between antioxidant/acetylcholinesterase inhibitory activity and the determined rosmarinic acid content indicating its significance for the observed activities.