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Essential oils from Mediterranean wild plants are widely used, but the hydrodistillation residues produced in parallel with these essential oils are significantly understudied and underexploited. Since there are only fragmentary data in the literature, we have, for the first time, systematically analyzed the chemical composition of the by-products obtained after hydrodistillation of sage, bay laurel, and rosemary leaves, i.e., hydrolates, water residues, and solid residues. The chemical composition of the hydrolates changed compared to their respective essential oils towards the dominance of more hydrophilic, oxygenated compounds, such as camphor in sage, 1, 8-cineole in bay laurel, and berbenone in rosemary. However, some compounds, mostly sesquiterpenes, which were present in considerable amounts in essential oils, were absent or only present in very small amounts in the hydrolates. Furthermore, both the water and the solid residues were rich in polyphenols, such as procyanidins in bay laurel and rosmarinic acid in rosemary and sage. In conclusion, we demonstrate the valuable chemical composition of sage, rosemary, and bay laurel hydrodistillation by-products and discuss a wide range of their possible applications.

The influence of ultrasonic pretreatments prior hydrodistillation of basil (Ocimum basilicum L.) was investigated to increasing essential oils yield. The hydrodistillation was preceded by ultrasonic pretreatment (ultrasonic output power of 200 W, a probe diameter of 14 mm) for 5, 10, 20, 30 and 40 minutes of extraction time. In order to determine the impact of pretreatments prior hydro distillation on the yields of basil essential oil, yields were compared to no pretreatment control. Ultrasonic pretreatments did not increase the yield of basil essential oil compared to the no pretreatment control, while with a longer time of ultrasonic pretreatment there was no increase in the yield of basil essential oil. After ultrasonic pretreatment of basil, enzymatic pretreatment with the addition of cell-degrading enzymes xylanase-pectinase-cellulase (1:1:1) was performed at 40 ˚C for 4 h. However, enzymatic pretreatment did not increase oil yield compared to ultrasonic pretreatment and no pretreatment control. However, the results showed that despite ultrasonic and enzymatic pretreatments prior hydro distillation of basil, the yields of essential oils did not significantly increase. In the production of essential oils, hydrolate is formed as a by-product, which has a strong antioxidant capacity determined by the FRAP method. In conclusion, the results of this study showed that ultrasonic and enzymatic pretreatment prior hydrodistillation did not increase the yield of basil essential oils.

Sage (Salvia officinalis L.) is rich in biologically active compounds, among which the most important are polyphenolic compounds. It is a growing interest in efficient and environmentally acceptable extraction methods with low solvent consumption, short extraction time and high extraction yield. The ultrasound-assisted extraction employing an ultrasonic device with direct cavitation using lower solvent consumption and short extraction time can result in higher recovery of polyphenolic compounds. Enzyme- assisted extraction has been intensively studied in the last years, enzymes can degrade and disrupt plant cell wall components, thus enabling better release and more efficient extraction of bioactive compounds from plants. We hypothesized that enzymatic pretreatment prior ultrasonic extraction can improve the recovery of polyphenols from sage. The aim of this study was to optimized extraction time of ultrasound-assisted extraction (ultrasonic output power of 200 W, a probe diameter of 14 mm). Furthermore, enzymatic pretreatment with addition of 5 mg and 50 mg of each enzyme (celulase, pectinase, xylanase and mixture of celulase- pectinase-xylanase) was performed at 40 ˚C for 1 h prior ultrasound-assisted extraction of polyphenols. Ultrasound-assisted extraction of polyphenols from sage was performed after enzymatic pretreatment with ultrasonic device for 5 minutes with aqueous methanol (1:1, v/v). The mass fraction of polyphenols (35.5 mg/g) extracted with ultrasound-assisted extraction under optimal extraction time of 5 minutes using methanol (1:1, v/v) was higher than mass fraction of polyphenols after enzymatic pretreatment (6.2-12.9 mg/g). However, enzymatic pretreatment prior ultrasonic extraction did not increase the mass fraction of total polyphenols compared to ultrasonic pretreatment. This suggests that the enzymatic pretreatment prior ultrasonic extraction has to be carefully re-evaluated. We have demonstrated the valuable effects of ultrasound-assisted extraction for only 5 minutes on the recovery of polyphenols from sage. In our case, ultrasound-assisted extraction of sage was enough to disrupt the cells and addition of enzymes was redundant.

To enhance the essential oils yields for hydrodistillation of bay laurel and sage, the impact of ultrasonic and enzymatic pretreatment (under optimized xylanase activity) was investigated. Response surface methodology (RSM) was used to optimize the reaction conditions of pH, temperature, and reaction time to obtain maximum xylanase activity. The impact of optimized conditions during enzymatic pretreatment induced variations in the essential oil yields. After the pretreatments, bay laurel and sage essential oils were isolated using Clavenger type apparatus and their yields were measured and compared with the essential oils obtained from non-treated plant materials (no pretreatment control). In order to determine the impact of pretreatments on the chemical composition, isolated essential oils were analyzed by gas chromatography and mass spectrometry (GC-MS), and afterward, the correlations were tested using Spearman’s test. The main constitutes of the sage essential oils are oxygenated monoterpens (α- and β-thujone, 1, 8-cineole and camphor). 1, 8-cineole is the major component of bay laurel essential oils, followed by α-terpinyl acetate, and several monoterpene hydrocarbons and phenylpropane derivates with dominant components eugenol and methyleugenol. Several chemical compounds (mostly oxygenated monoterpenes) of bay laurel and sage essential oils were detected in greater quantity after the ultrasonic and enzymatic pretreatments in comparison to untreated plant material.However, the results showed that despite optimization, the activity of xylanase did not significantly affect the quantity and quality of bay laurel and sage essential oils. The main advantage of ultrasonic extraction is shortening of the pretreatment time and only 10 minutes of ultrasonic pretreatment before the enzymatic pretreatment caused 50 % enhancement of sage and 40 % of bay laurel essential oil yields. Due to their chemical composition (oxygenated monoterpenes, monoterpene and sesquiterpene hydrocarbons), the essential oils obtained by the ultrasonic and enzymatic pretreatment prior hydrodistillation possess potential in the food industry applications.

In recent years, new pre-treatments prior hydrodistillation of plant material have been intensively investigated, with the aim of increasing their yield. The aim of this study was to investigate whether ultrasonic pretreatment prior Clavenger hydrodistillation could increase the yield of rosemary (Rosmarinus officinalis L.) essential oil. The hydrodistillation was preceded by ultrasonic pretreatment (ultrasonic output power of 200 W, a probe diameter of 14 mm) for 5, 10, 20, 30 and 40 minutes of extraction time. Ultrasonic pretreatments increased the yield of rosemary essential oil for 50 % compared to the negative control after 5 and 10 minutes, while with a longer time of ultrasonic pretreatment there was no increase in the yield of rosemary essential oil. After ultrasonic pretreatment of rosemary, enzymatic pretreatment with the addition of cell-degrading enzymes xylanase-pectinase- cellulase (1:1:1) was performed at 40 ˚C for 4 h. However, enzymatic pretreatment did not increase oil yield compared to ultrasonic pretreatment and negative control. This suggests that the increase in oil yield is caused by ultrasonic pretreatment after only 5 and 10 minutes, and not the enzymatic activity that did not show an increase in the yield of essential oil. In the production of essential oils, hydrolate is formed as a by- product, which has a strong antioxidant capacity determined by the FRAP method. In conclusion, the results of this study showed that ultrasonic pretreatment prior hydrodistillation can be useful in the production of rosemary essential oil, the main advantage is an increase in oil content by 50% after only 5 and 10 minutes of ultrasonic pretreatment.

Water hydrodistillation residues produced in parallel with bay laurel, rosemary and sage essential oils contain a remarkable source of bioactive molecules. Despite rich bioactive content, these fractions considered as waste and are often underexploited. To date, there are no studies reporting on the chemical composition of water residues remaining after hydrodistillation of bay laurel and sage leaves, while there are only few reports demonstrating the valuable composition of other hydrodistillation co-products of these plants as a valuable secondary source of polyphenol components. We have used different pretreatments prior to hydrodistillation, i.e. reflux extraction, ultrasound and enzyme assisted extraction. It was described the effect of these pretreatments on the composition of polyphenolic compounds in the water hydrodisillation residues. HPLC analysis showed that the water hydrodistillation residues of bay laurel, rosemary and sage were richest in polyphenols. In bay laurel water hydodistillation residues, procyanidins represented the dominant components, with procyanidin dimer I and II (up to 4.65 mg/g), tetramer II (up to 5.43 mg/g), trimer II, III and IV (up to 4.37, 4.92, 13.15 mg/g, respectively) as major compounds. Among other components, epicatechin-hexoside, epicatechin and epicatechin- 3-O-gallate (up to 3.56, 1.60, 1.07 mg/g, respectively) were also detected in significant quantities. In sage hydrodistillation water residues, rosmarinic acid (up to 5.39 mg/g) was major compounds, followed by caffeic acid methyl ester (up to mg/g) and epicatechin (up to 2.06 mg/g). Finally, in rosemary hydrodistillation water residues rosmarinic acid, galocatehin, p- coumaric acid and syringic acid (up to 7.57, 4.83, 2.19, 3.25 mg/g, respectively) were most represented components. In general, the pretreatments didn’t significantly affect the phenolic composition of water hydrodistillation residues (p

The essential oil manufacture produces a significant quantity of co-products. These potentially valuable hydrodistillation residues include hydrolates contain a remarkable source of bioactive molecules. In this study, we have focused on aromatic Mediterranean plants rich in volatile compounds: bay laurel, rosemary and sage. Their essential oils are frequently used, but hydrodistillation residues produced in parallel with these essential oils are much less explored. Despite rich bioactive content, these fractions are often underexploited or even considered waste. Increasing market demand for essential oils resulted in the development of many innovative methods for improvement of their yield and composition. Traditional extraction technologies prior hydrodistillation mainly related to the high temperature mainted for long extraction periods and the consumption of hazardous organic solvents as well as low extraction efficiency of desired compounds. So, we have analysed different pretreatments prior to hydrodistillation i.e. reflux extraction, ultrasound and enzyme assisted extraction on chemical composition of hydrolates in bay laurel, rosemary and sage. GC-MS analysis showed that the hydrolates of all plants were richest in oxygenated monoterpenes: up to 46%, 42% and 30% of total peak area in bay laurel, rosemary and sage hydrolates, respectively. Sesquiterpenes were also well represented: up to 42%, and 10% of total peak area in rosemary and bay laurel hydrolates, respectively. Dominant compounds were confirmed: 1, 8-cineole, camphor, borneol and berbenone in rosemary hydrolates, 1, 8-cineole, linalool, camphor, α- and β-terpineol, methyleugenol and eugenol in bay laurel hydrolates and 1, 8-cineole, camphor, borneol and berbenone in sage hydrolates. In general, the pretreatments didn’t significantly affect the volatile compounds of hydrolates (p

Saprolegnia parasitica, uzročnik saprolegnioze salmonidnih riba i Aphanomyces astaci, uzročnik račje kuge, oomicetni su patogeni koji uzrokuju značajne ekonomske štete u akvakulturi. Budući da su kemikalije koje se koriste za njihovu kontrolu štetne za ljude i okoliš, cilj ovog istraživanja je istražiti ekološki prihvatljive metode njihove inhibicije. Testiran je inhibicijski potencijal eteričnih ulja odabranog samoniklog mediteranskog bilja, kadulje (Salvia officinalis L.), ružmarina (Rosmarinus officinalis L.) i lovora (Laurus nobilis L.) prema rastu micelija i klijavosti zoospora patogena te su određene EC50 vrijednosti. Testirana eterična ulja bila su bogata hlapivim bioaktivnim komponentama, pretežno monoterpenima, te su pokazala inhibicijsku aktivnost prema rastu micelija i klijavosti zoospora, naročito eterično ulje kadulje koje je imalo najsnažnije djelovanje. To se dijelom može pripisati dominantnim komponentama, primjerice kamforu, ali još vjerojatnije sinergističkom djelovanju velikog broja komponenti. Micelij i zoospore patogena A. astaci bile su osjetljivije od istih razvojnih stadija patogena S. parasitica, gdje je samo eterično ulje kadulje u potpunosti inhibiralo rast micelija. Zaključno, dobiveni rezultati mogu poslužiti kao osnova budućih in vivo istraživanja i razvoja ekološki prihvatljivih metoda kontrole patogenih oomiceta u akvakulturi.

Introduction Saprolegnia parasitica and Aphanomyces astaci (Oomycetes) are pathogens with negative impact in freshwater aquaculture. Saprolegnia parasitica causes saprolegniosis, a disease affecting mostly salmonid fishes, while A. astaci causes crayfish plague. Chemicals harmful to humans and the environment are being used globally in aquaculture facilities to prevent the spread of these pathogens. Thus, the development of new, ecologically acceptable methods for their control is urgently needed. The aim of this study was to examine whether propolis, known for its antimicrobial properties and stimulatory effect on the host immune system, can inhibit the life stages of A. astaci and S. parasitica in vitro. Materials and methods Two propolis formulations were used: P1 (pure propolis in ethanol), and P2 (propolis in ethanol with the addition of sage and pepper mint). Their chemical composition was determined by ultra- performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) and gas chromatography/mass spectrometry (GC-MS). Two main life stages of the oomycete pathogens, namely mycelium and zoospores, were treated with propolis formulations and their main components, pinocembrin and chrysin. The effect of these compounds on mycelial growth was assessed using the disk diffusion assay and radial growth inhibition test, followed by determination of EC50 values. To test the inhibition of zoospore germination, sporulation was induced by washing the mycelium grown in liquid PG1 with natural water. Test compounds were added to the resulting zoospore suspension in a range of concentrations, and their effect was assessed by comparing the percentage of germinated zoospores with the results of control experiments. Malachite green, with known toxicity towards A. astaci and S. parasitica, was used as a positive control. Results Both propolis samples were rich in volatile and polyphenol compounds. As shown by UPLC-MS/MS analysis, chrysin was most abundant in both propolis samples (up to app. 50 µg/mL), followed by pinocembrin in P2 (4 µg/mL). Significantly higher number of different volatile components was found by GC-MS in P2 than in P1 propolis sample, probably because of addition of sage and pepper mint. In case of S. parasitica, both propolis formulations moderately inhibited the mycelial growth: EC50 (P1) = 206.2 µg/mL, EC50 (P2) = 206.6 µg/mL for P2, when compared with a known inhibitory compound malachite green (EC50 = 0.1 µg/mL). In contrast, EC50 values for P1, P2, and malachite green for mycelial growth inhibition of A. astaci were up to 36 times lower (5.6, 8.6, and 0.02 µg/mL, respectively). Considering zoospore germination, the minimal concentration that caused complete inhibition of S. parasitica germination (minimum inhibitory concentration, MIC) was 62.0 and 39.0 µg/mL for P1 and P2 propolis samples, respectively, while for malachite green it was 0.08 µg/mL. Similar results were obtained for Aphanomyces astaci zoospores: MIC was 31.3 and 39.0 µg/mL for P1 and P2, respectively, while malachite green inhibited zoospore germination at a concentration of 0.04 µg/mL. The inhibitory potential of main propolis components, chrysin and pinocembrin, was also tested but these compounds didn´t show significant anti-oomycete activity when applied in concentrations determined in propolis samples. This suggests that the observed anti-oomycete activity of propolis formulations was probably due to synergistic activity of a number of minor bioactive components. Conclusions Our results demonstrate the inhibitory activity of propolis towards life stages of pathogenic oomycetes S. parasitica and A. astaci. Interestingly, S. parasitica mycelium was relatively resistant to propolis in comparison to mycelium of A. astaci, but zoospores of both species (as main infection agents) were highly susceptible. Future in vivo testing is needed to explore the suggested host- protective effects of propolis during the infection process and to demonstrate its applicability as a feed additive in the fish farms.

Solid hydrodistillation residues produced in parallel with essential oils of bay laurel, rosemary and sage are much less explored or even considered waste despite rich polyphenolic content. Prior to hydrodistillation, plant material is often treated with ultrasound, microwaves or enzymes, in order to disrupt the cell wall and improve the access of solvent to the cell content, thus enhancing the release of bioactive compounds. When comparing different extraction procedures used to process solid hydrodistillation residues like enzyme assisted or reflux extraction, ultrasound application is considered gentler in comparison with other extraction procedures, working at lower temperature and for shorter time, which makes it more suitable to preserve polyphenols from thermal degradation. HPLC analysis showed that the solid hydrodistillation residues of all plants were richest in polyphenols. In bay laurel solid hydrodistillation residues extract, procyanidins represented the dominant components, with procyanidin trimer, dimer and tetramer as major compounds. Among other components, epicatechin, epicatechin-hexoside and epicatechingallate were also detected in significant quantities. In sage solid hydrodistillation residues extract, salvianolic acid I and K were major compounds, followed by luteolin-3-O-glucuronide. Finally, in rosemary solid hydrodistillation residues extract, rosmarinic acid was the most represented component. Applied solvents didn’t show significant variations (p

Uvod: Posljednjih se godina intenzivno istražuju nove metode izolacije eteričnih ulja, s ciljem povećanja njihovog prinosa. Ciljevi: Cilj ovog istraživanja bio je istražiti da li različiti predtretmani vodene destilacije mogu povećati prinos eteričnog ulja kadulje, ružmarina i lovora te utvrditi eventualne promjene u kemijskom sastavu eteričnih ulja. Metode: Vodenoj destilaciji predhodili su različiti predtretmani: klasična ekstrakcija refluksiranjem, ekstrakcija refluksiranjem uz dodatak enzima pektinaze, celulaze i ksilanaze te ultrazvučna ekstrakcija. Kemijski sastav dobivenih ulja analiziran je GC- MS i NMR analizom. Rezultati: Svi predtretmani povećali su prinos eteričnih ulja u usporedbi s negativnom kontrolom, pri čemu je kvaliteta ulja ostala nepromijenjena. No, predtretman ekstrakcijom refluksiranjem s dodatkom enzima nije povećao prinos ulja u odnosu na sami predtretman ekstrakcijom refluksiranjem (kontrola bez enzima). Ovo upućuje na to da je povećanje prinosa ulja uzrokovala ekstrakcija refluksiranjem, a ne enzimska aktivnost, i suprotno je navodima mnogih ranijih istraživanja. Međutim, u tim istraživanjima prinos ulja nakon predtretmana enzimima uspoređen s negativnom kontrolom (bez predtretmana), umjesto s kontrolom bez enzima. Zaključno, rezultati ovog istraživanja pokazali su da predtretmani ultrazvukom i ekstrakcijom refluksiranjem mogu biti korisni u proizvodnji eteričnih ulja navedenih biljaka, ali da primjena enzima u ovu svrhu treba biti dodatno istražena.

Background: Oomycetes are globally important pathogens of plants and animals, causing significant economic losses in agriculture and aquaculture. Since current methods for their treatment include chemicals harmful to humans and the environment, new environmentally friendly methods for their control should be developed. Objectives: The aim of this study was to examine anti-oomycete effects of propolis, a natural substance with known antimicrobial activity but poorly investigated anti-oomycete potential. Methods: Two propolis formulations were used: P1 and P2, and their chemical composition was determined spectrophotometrically. Their anti- oomycete activity was determined using an in vitro plate assay and a range of pathogenic oomycetes that parasitize on animals (Aphanomyces astaci, crayfish pathogen, and Saprolegnia parasitica, parasite of salmonid fish) and plants (Phytophora cactorum and P. plurivora). Results: Both propolis samples contained high, mg / mL concentrations of bioactive compounds: flavonoids, total phenols, hydroxyacetic acids and flavanols. Accordingly, high inhibitory activity of propolis was demonstrated against all tested Oomycetes in vitro: mycelium growth was slowed down from 30 % to 75 % in the presence of propolis formulations and the degree of inhibition was positively correlated with the concentration of flavonoids in the samples. In conclusion, this is one of the first studies that demonstrates strong inhibitory effect of propolis towards pathogenic Oomycetes. In the future, the effect of propolis should be tested in vivo, e.g. as food additive in freshwater aquaculture facilities.

Mikroorganizmi iz skupine Oomycetes, poznatiji kao „vodene plijesni“, uzročnici su bolesti mnogih slatkovodnih organizama te uzrokuju značajne štete u akvakulturi. Postojeće metode suzbijanja ovih patogena uključuju korištenje kemikalija štetnih za čovjeka i okoliš, stoga ću se u okviru svog doktorskog rada baviti razvojem ekološki prihvaljivih metoda njihove kontrole. Kao modelne organizme koristit ću oomicete iz roda Saprolegnia, uzročnike saprolegnioze - bolesti pastrve i drugih salmonidnih riba, te vrstu Aphanomyces astaci, uzročnika smrtonosne bolesti slatkovodnih deseteronožnih rakova. Prvi cilj mog doktorskog rada je istražiti da li odabrani biljni ekstrakti imaju sposobnost inhibicije rasta micelija i/ili inhibicije sporulacije modelnih oomiceta. U tu ću svrhu, koristeći ekološki prihvatljive načine ekstrakcije, pripremiti ekstrakte mediteranskog samoniklog bilja te analizirani njihov sastav spektrofotometrijski, tekućinskom kromatografijom visoke djelotvornosti (HPLC), plinsko kromatografsko masenom spektrometrijom (GC-MS) i nuklearnom magnetskom rezonancijom (NMR). Potom ću ispitati njihov inhibicijski potencijal prema modelnim oomicetima in vitro i in vivo. U drugom dijelu doktorskog rada istražiti ću mogućnost biokontrole patogenih oomiceta uz pomoć komenzalnih nepatogenih mikroorganizama prirodno prisutnih na/u domaćinu. Selekcionirati ću, izolirati i molekularno karakterizirati sakupljene bakterijske izolate te testirati njihovu potencijalnu inhibicijsku aktivnost prema patogenim oomicetima. Rezultati ovog istraživanja biti će temelj za razvoj modernih metoda kontrole bolesti uzrokovanih oomicetima u slatkovodnoj akvakulturi.

The use of sage (Salvia officinalis, L.) as a corrosion inhibitor for tinplate in 3 % sodium chloride solution has been evaluated by using electrochemical impedance spectroscopy techniques (EIS) at 25 °C. Inhibitor concentrations of sage extract were included within the range between 0 and 500 ppm. EIS techniques have shown that sage extract could serve as an effective inhibitor (90.2 %) for the corrosion of tinplate in 3 % in concentration of 200 ppm, at temperature 25 °C. These results confirm that sage extract exhibits good inhibitive performance for tinplate in a 3 % NaCl solution, and that it is more efficient at inhibiting the corrosion of tinplate at 200 ppm concentration than in higher concentrations of sage extract. The passivating film properties of the tinplate were improved by the presence of polyphenols in the sage extracts.

High hydrostatic pressure extraction (HHPE) was used to obtain polyphenols from sage (Salvia officinalis L.) leaves. In order to determine the optimal conditions for the HHPE extraction of sage polyphenols, the influence of ethanol concentrations (30, 96 %), pressure (300, 450, 600 MPa), extraction time (5, 10, 15 min) and extraction temperature (25, 60 °C) on the composition and mass fraction of polyphenols of wild sage were investigated. Based on the mass fraction of total phenols were selected optimal extraction conditions as follows: 30 % ethanol concentration, 600 MPa of high hydrostatic pressure, extraction time of 5 min and extraction temperature at 60 °C. HHPE reduce the processing time, enhance mass transport phenomena and greater solvent penetration into plant material that provide optimal extraction yields. Using HPLC coupled with UV/PDA, polyphenols isolated from sage including phenolic acid (e.g. rosmarinic acid, rosmarinic acid hexoside, salvianolic acid K, salvianolic acid I, methyl rosmarinate), flavones (e.g. apigenin and luteolin glycosides) and catehines (e.g. catechin, epicatechine). Compared to apigenin and luteolin glycosides and catechines, rosmarinic acid is present at the highest level. The results showed that HHPE can reduce the processing time and provide high extraction yields of polyphenols from plant. The results of this research may lead to finding the possible ecological alternatives for the recovery of polyphenols from sage, enhancing the use of environmental friendly solvents and energy-efficient technologies and promoting the advantages of HHPE for extraction purposes.

Oomycetes from the genus Phytophthora are plant pathogens that cause enormous economic and environmental damage. Extracts of various plants are considered promising and environmentally – friendly alternative to synthetic fungicides and are increasingly tested as anti-oomycete agents. Thus, we have tested the anti-Phytophthora activity of Bay Laurel (Laurus nobilis L.) leaves extracts, using two Phytophthora species with wide host range as models - P. plurivora and P. cactorum. Bay Laurel (Laurus nobilis L.) is a valuable medicinal plant from Lauraceae family, which has been widely used as spice and flavouring agent. Furthermore, due to the numerous bioactive compounds, essential oils and plant extracts show a wide range of biological activities such as antimicrobial, preservative, antioxidant and antifungal properties. The aim of this research was to determine the yields of total phenols extracted from the Bay Laurel leafs using different solvents and to test the anti—oomycete potential of phenolic compounds. In order to investigate the effect of different solvents on the extraction efficiency of polyphenols, ethanol (ethanol-water), acetone (acetone-water), methanol (methanol-water), ethanol-acetone-methanol (ethanol-acetone-methanol-water) and water were used. Mass fraction of total phenols of Bay Laurel leaves varied considerably as function of solvent nature and ranged from 18.4 to 59.9 mg GAE/g dry leaves, for pure acetone and methanol-water extracts, respectively. The highest mass fraction of polyphenols was obtained by using methanol-water. These results clearly demonstrate the influence of the solvent on the extractability of phenolic compounds, and water addition to the solvent improved the total phenols extraction for ethanol, methanol and acetone. Thus, binary solvent system containing hydro-organic solvents was superior to mono-component solvent systems regarding the extraction of total phenols. Next, the prepared plant extracts were tested for anti-Phytophthora activity using the modified well diffusion method. Results, expressed as inhibition of oomycete radial growth, have showed that both organic and hydro-organic solvent extracts of Bay Laurel leaves exhibited high contents of polyphenols and good anti–Phytophtora activity. In conclusion, L. nobilis extracts contain bioactive natural substances and may be considered as promising anti-oomycete agents in the future.

Crithmum maritimum L., commonly known as sea fennel or rock samphire, is an aromatic edible halophyte plant growing on maritime cliffs and sometimes in sands in the European and Atlantic coast. This halophyte plant has traditionally used in Mediteranean countries as food ingredient and in folk medicine. Sea fennel represents various economical interests, due to its high contents in minerals, vitamin C and polyphenols with antioxidant properties. The aim of this research was to determine the yields of total polyphenols and hydroxycinnamic acids of the sea fennel leaf depending on the solvent extraction applied for their isolation. Results showed that phenolic contents varied considerably as function of solvent polarity. The addition of 50 % water to methanol, acetone or ethanol enhance the extraction yields of polyphenols from sea fennel. Therefore, ethanol/water (1:1) seems to be the most recommended solvent for polyphenolic extraction from sea fennel. Sea fennel leaf extract using ethanol/water mixture show the highest mass fraction of total polyphenols (77.05 mg GAE/g) and mass fraction of hydroxycinnamic acids (50.94 mg CAE/g). These results indicate that sea fennel can be considered as a valuable source of polyphenols, especially of hydroxycinnamic acids that is known to have strong antioxidant capacity. Our findings identified the appropriate solvent for extracting polyphenols that might provide a rich and novel source of natural antioxidants. Sea fennel might find a number of industrial applications as food additives replacing synthetic ones in food industry.

Aphanomyces astaci (Oomycetes) is the causative agent of crayfish plague disease. It exerts significant negative impacts on European crayfish species, both in natural settings and in freshwater aquaculture. However, there are no specific anti-oomycete treatments, and current therapies use harmful chemicals such as formaldehyde and malachite green. Research of specific anti-oomycetal treatments using plant extracts present a new trend in aquaculture. Thus, the aim of this study was to test the anti-Aphanomyces activity of the sage leaves extracts. Sage (Salvia officinalis, Lamiaceae) is an aromatic Mediterranean plant rich in bioactive secondary metabolites. Such substances, including polyphenols and simple phenolic acids (i.e. phenolic acids, flavonoids, lignans, stilbenes), have known antimicrobial and antifungal properties, but their activity against freshwater Oomycetes wasn’t tested to date. In this study, the extraction of polyphenols from sage leaves was performed using extracting solvent under magnetic stirring at boiling temperature for 1 h under reflux in a condensation system. In order to investigate the effect of different solvents on the extraction efficiency of polyphenols, ethanol (ethanol-water), acetone (acetone-water), methanol (methanol-water), ethanol-acetone-methanol (ethanol-acetone-methanol-water) and water were used. While all sage extracts exhibited high content of polyphenols, extraction yields nevertheless varied considerably as function of solvent nature. The antiAphanomyces activity of sage extracts was assessed using modified well diffusion method and the results were quantified by measuring the retardation of oomycete radial growth. As will be presented, both organic and hydro-organic solvent extracts of sage leaves exhibited good anti–Aphanomyces activity. In conclusion, this research demonstrates the potential of Mediterranean plant extracts in control/treatment of oomycete-caused diseases in freshwater aquaculture.

Tinplate is one of the most common materials in food packaging. Acidic or alkaline effects of such products cause corrosion, but also loss of their organoleptic characteristics. In recent years, due to the growing interest and attention of the world towards environmental problems and towards the protection of the environment, therefore synthetic and toxic inhibitors that are widely used in industrial processes should be replaced with new environment-friendly inhibitors. The sage (Salvia officinalis L.) is rich in biologically active compounds, especially polyphenols, and represent a potential source of, so-called, green inhibitors. Sage leaves were extracted with 30 % ethanol and high hydrostatic pressure (HHP) under operating pressure of 600 MPa and temperature 60 °C for 300 s. The inhibition efficiencies of sage extract calculated from electrochemical impedance spectroscopy (EIS). The measurement was carried out in 3 % sodium chloride solution at 25 °C in presence and absence in different concentrations (0.2 – 2.0 g/L) of sage extract. EIS techniques have shown that sage extract acts as a good inhibitor (93 %), at temperature 25 °C, and concentration of 0.6 g/L. This results confirm that sage extract exhibits good inhibitive performance for tinplate in 3 % NaCl solution, and it is more efficient to inhibit the corrosion of tinplate at the addition of 0.6 g/L than higher concentrations of sage extract. The results of ATR – FTIR analysis of the hydrophobic film modified on the tinplate surface after exposure to the 3 % NaCl solution with additions of 0.6 g/L sage extract was utilised to confirm the presence of some functional groups with O, N and S atoms adsorbed on tinplate surface. Sage extracts present good inhibition properties for tinplate corrosion because of their chemical composition and their high share of phenolic compounds whose attachment to adsorption sites on tinplate surface is responsible for the inhibition process. Modern green extraction techniques, HHP tend to be eco - friendly in terms of polyphenols extraction efficiencies from plants. Thinking long-term, the results of this research could facilitate the development of new products, by implementing plant extracts in the alkaline medium during tinplate food packaging or tinplate coating, which would reduce or completely replace the use of synthetic solutions.

In order to determine the optimal conditions for the extraction of sage polyphenols, the influence of extraction solvent (water, 30 % (by volume) aqueous ethanol and 30 % (by volume) aqueous acetone), extraction time (3, 5, 7, 9 and 11 min) and microwave power (500, 600 and 700 W) on the composition and concentration of phenolic compounds of dry wild sage (Salvia officinalis L.) during microwave-assisted extraction (MAE) was studied. Optimized MAE method was compared with conventional extraction (CE). Based on the amount of total phenols, microwave power of 500 W and extraction time of 9 min were selected as optimal extraction conditions, resulting in higher content of polyphenols when compared with CE. Ethanol and acetone solutions were equally effective extraction solvents, both producing higher extraction capacity than water. Using HPLC coupled with UV/PDA, fourteen polyphenols were identified (caffeic and rosmarinic acid derivatives, luteolin- and apigenin-glycosides) with rosmarinic acid and luteolin glycosides at the highest concentrations. The mass fractions of all individual polyphenols were higher in the MAE extracts than in the CE ones.

The industrial processing of tomato results in the accumulation of large amounts of by-products composed of peel, seed and small amount of pulp. This material is disposed of as a solid waste or used as animal feed, but the abundance of bioactive compounds in by-product fractions suggests the possibility of utilizing it as a cheap source of different biopolymers. One of them is pectin. Although pectin occurs in the plant tissues, the number of sources that may be used for the commercial manufacture of pectin is limited (citrus fruits and apples). The alternative sources for pectin extraction were described by several authors, but to date, no research has been done on the extraction of pectin from dried tomato peels. This work reports methodology of pectin isolation from two types of tomato peels obtained as waste from canning factory. Pectin was extracted under reflux at 90°C, using ammonium oxalate/oxalic acid as extracting agent. The extraction was carried out during 24 h in the first step, and then again with a fresh volume of extracting agent during 12 h in the second step. The isolated pectins were evaluated in terms of methoxyl (MeO) and anhydrouronic acid (AUA) contents and degree of esterification (DE). Total pectin substance estimated as calcium pectate was determined gravimetrically. The structure of pectin was evaluated using FTIR spectroscopy. The results indicate that extraction parameters such as peel to extractant ratio, number of extractions and extraction time have a different effect on the yield and quality of pectin. Amongst them, number of extractions as well as time of extraction has shown the strongest effect on all measured parameters. The highest pectin yield but the lowest pectin quality was obtained at extraction time of 12 h which points out that higher pectin yields are not necessarily connected with higher pectin quality. The highest pectin yield (15.0 and 14.9 %), MeO content (5.8 and 4.0 %), AUA content (40.0 and 27.4 %) and DE (81.5 and 82.7 %) were obtained after 24 h of extraction of two different batches of dried tomato peels, respectively. The results of FTIR analyses revealed that there are no differences in structure between extracted pectins. This preliminary investigation point out that utilization of dried tomato peel for pectin production will not only solve the problems of waste disposal but also meet the requirements of pectin in the market to some extent.

Tomato (Lycopersicum esculentum) is one of the most consumed vegetable in the world, either as a raw or as a processed product. During tomato processing a considerable amount of waste is generated. Tomato peel, a by-product of tomato processing industry, contains significant levels of dietary fiber and bioactive compounds such as phenolics. The objective of this study was to determine the total phenols and flavonoids in the pectin biopolymer extracted from tomato peel as well as in peel residue after removal of pectin. The dried tomato peel was firstly treated with aqueous acid solution (ammonium oxalate/oxalic acid) under reflux at 90 C for 24 h, and then again after 12 h, to remove the pectin. The resulting two-phase system, e.g. extract of isolated pectin and tomato peel residue after extraction, were used for further analyses. Pectin was precipitated with addition of pure ethanol, while the peels were Soxhlet extracted with two solvents (pure and 70 % ethanol, by volume), for 6 and 12 h. Total phenols in extracts were determined with Folin-Ciocalteu assay, while flavonoids were measured with aluminium chloride colorimetric assay. The results have shown that total phenols are present in both pectin extracts obtained from two different batches of dried tomato peels, while flavonoids were found only in traces. Better extraction yield of total phenols from tomato peel residue was obtained by Soxhlet extraction, using pure instead of 70 % (by volume) ethanol (10.94 mg GAE/g and 9.17 mg GAE/g, respectively) and 6 h of extraction. The best extraction yields of flavonoids (2.80 mg RE/g) were obtained by using pure ethanol as solvent after 12 h of extraction, although extraction with 70 % ethanol provided acceptable recovery. In further work the use of mixed water/ethanol solvents for the extraction of tomato peel, as environmentally acceptable systems, will be investigated. As tomato peel contains considerable amount of phenolics should be regarded as a beneficial source of bioactive compounds. Moreover, the phenolics isolated from tomato peel could be potentially used as a food supplement to improve different physical, chemical, and nutritional properties of foods.

Sage (Salvia officinalis L.) is one of the most appreciated natural sources of bioactive compounds, among which polyphenols have attracted more attention due to antioxidative activity. Phenolic compounds sage differ significantly based on chemical structure, therefore different extraction techniques have been applied to obtain polyphenols from sage. The most frequently used methods for extracting phenolic compounds from their plant sources include either solvent extraction or supercritical fluid extraction. Solvent extraction technique (Soxhlet, conventional, microwave and ultrasound assisted extraction) were reported in literature to obtain sage extracts. Even if these techniques are able to provide with bioactive extracts, more environmentally friendly and selective extraction technique are nowadays preferred. The aim of this study was to analyse the effect an acid treatment (10, 20 and 25% HCl in 30% ethanol and aceton) and extraction time (30, 60 min) on extraction yield of polyphenols. The total phenolics and flavonoid content of sage extracts were evaluated using Folin-Ciocalteu method and aluminum chloride (AlCl3) method, respectively. The individual phenolic compounds were quantified using HPLC UV-PDA. In addition, the antioxidant capacity was evaluated by using DPPH (2, 2'-diphenyl-1-picrylhydrazyl) free radical scavenging method. Comparing the solvents used for extraction, the highest extraction yield of tolal phenols and flavonoids was observed using 30% ethanol through 30 minutes. The addition of 10% HCl was influenced the increase extraction capacity of phenolic compounds, while higher percentage of HCl (20 and 25%) negatively affecting the isolation of sage phenolics. The total phenolic content ranged from 3253.33 to 6278.12 mg/100g of dry sage, expressed as rosmarinic acid equivalents. The flavonoid content ranged from 3022.08 to 5649.60 mg/100g of dry sage, expressed as rutin equivalents. The main phenolic compounds determined in sage extract were: rosmarinic acid, folowed by luteolin and apigenin glycosides, and caffeic acid derivates. The antioxidant capacity of obtained extract correlated with concentration od total phenolics and flavonoids, and among individual phenolics the rosmarinic acid contributed the most to the AOC. The best extraction capacity of phenolics from sage was achieved by applying 30% ethanol through 30 minutes, and the addition of acid did not significantly affect the increase the extraction yield of phenols. Application of acid hydrolysis in extraction of sage phenols decreased their amounts, but at the same time increase the amount of aglycones.

Processing of tomato fruits into various products (tomato juice, paste, purée, ketchup, sauce and salsa) by food industries generate significant quantity of biodegradable material. Recycling the tomato peels to complex colloidal polymeric carbohydrate pectin was the main target of this study. Pectins are widely used in food industry for a various purpose such as a gelling agent, thickener, texturiser, emulsifier and stabilizer. In order to get better yields, purity and functional properties in shorter time, two different extraction methods, e.g. classical (refluxing) and ultrasonic bath were used in the study. The extraction of dried tomato peels, under reflux in a condensation system, was performed at 60 and 80 °C using ammonium oxalate/oxalic acid as extracting solvent, at time of 24 h in the first step and then subsequently with a fresh volume of extracting solvent during 12 h in the second step. Ultrasonic bath extraction with the same solvent was carried out at frequency of 30 kHz, temperature of 60 and 80 °C, and sonication time of 15, 30, 45, 60 and 90 min. A preliminary characterization of pectins was performed by titrimetric (methoxyl and anhydrouronic acid content, and degree of esterification), gravimetric (calcium pectate) and spectrophotometric (D-galacturonic acid) methods in order to investigate the influence of different extraction methods as well as extraction conditions on the chemical composition of the extracts. The colour of sample was determined as Hunter a, b and L values using Hunter colorimeter. The results obtained for ultrasonic bath extraction showed that yield, methoxyl (MeO) and anhydrouronic acid (AUA) contents and degree of esterification (DE) varied significantly (p

The effect of extraction solvents (30, 50 and 70 % aqueous solutions of ethanol and acetone, and 100 % distilled water), extraction temperature (60 and 90 °C) and extraction time (30, 60 and 90 min) on the composition and mass fraction of polyphenolic compounds in Dalmatian wild sage (Salvia officinalis L.) extracts has been investigated. The total polyphenolic content of sage extracts was determined spectrophotometrically using Folin-Ciocalteu method, whereas the individual polyphenols were determined by HPLC UV/PDA method. Results indicated that the main polyphenols in sage extracts were vanillic, caffeic, syringic, salvianolic K and salvianolic I acids, methyl rosmarinate, 6-hydroxyluteolin-7-glucoside, luteolin-7-glucuronide, luteolin-7-glucoside, apigenin-7-glucunoride, apigenin-7-glucoside, with rosmarinic acid and luteolin-3-glucuronide as predominant compounds. The mass fractions of total and individual polyphenols significantly depend on the type of extraction solvent, solvent composition and extraction temperature. The results showed that binary solvent systems are more efficient than mono-solvent systems in the extraction of polyphenolic compounds in regard to their relative polarity. The aqueous solutions of ethanol or acetone (30 %), extraction temperature of 60 °C and extraction time of 30 min were the most efficient for the extraction of polyphenols from dry sage leaves.

Istraživan je utjecaj klasične ekstrakcije te ekstrakcije potpomognute ultrazvukom na sastav i udio fenolnih spojeva kadulje u ovisnosti o vrsti i polarnosti ekstrakcijskog otapala (voda, 30, 50 i 70 % vodene otopine etanola i acetona), temperaturi (60 i 90 °C), vremenu ekstrakcije (30, 60 i 90 min) kod klasične ekstrakcije te snazi ultrazvuka (100 i 400 W), vremenu ekstrakcije (1 do 40 min), amplitudi (20, 60 i 100 %), temperaturi (40 do 60 C) kod ekstrakcije potpomognute ultrazvukom. Optimalni uvjeti ekstrakcije definirani su na temelju masenog udjela fenolnih spojeva. Najbolji ekstrakcijski kapacitet kod klasične ekstrakcije i ekstrakcije potpomognute ultrazvukom primjenom ultrazvučne kupelji postignut je uz upotrebu 30 % vodene otopine etanola pri temperaturi od 60 °C kroz 30 minuta, a primjenom ultrazvučne sonde izlazne snage 400 W, promjera sonde 22 mm, amplitudi 100 % kroz 11 minuta također uz upotrebu 30 % vodene otopine etanola. U svim ekstraktima kadulje neovisno o uvjetima ekstrakcije ili lokaciji uzgoja u najvećem udjelu zastupljena je ružmarinska kiselina i luteolin-3-glukuronid dok su ostali fenolni spojevi zastupljeni u značajno nižim udjelima. U usporedbi s klasičnom ekstrakcijom, ekstrakcija potpomognuta ultrazvukom se pokazala značajno učinkovitijom (ekstrakcijski kapacitet povećao se oko 20 %, a vrijeme ekstrakcije skratilo tri puta). U drugom djelu rada istraživan je utjecaj lokacije uzgoja (Pag, Pirovac, Brač, Lošinj) na sastav i udio fenolnih spojeva kadulje, a ekstrakcija je provedena pri optimalnim uvjetima definiranim u prvom dijelu istraživanja. Najveći udio fenolnih spojeva oderđen je u kadulji s otoka Paga, te je utvrđeno da lokacija uzgoja ima značajan utjecaj na količinu fenolnih spojeva kadulje.

Phenolic compounds were investigated in wild sage (Salvia officinalis L.). Conventional extraction (CE) in water bath shaking, extraction in an ultrasonic water bath (US) 35 kHz at 60 °C, 30 minutes and microwave assisted extraction (MAE) power 500 W at 60 °C, 10 minutes were compared. Extractions were performed using 30% ethanol and acetone with the use of acid hydrolysis with 10% and 20% HCl. Sage was harvested on the Mediterranean part of Croatia, dried in the shade and stored in paper bags. Total phenols (TP) were determined spectrophotometrically with Folin-Ciocalteu method. The flavonoids (FL) assay is based on the spectrophotometric evaluation of a complex with AlCl3. The influence of solvents on the amount of extracted TP and FL, and the application of acid hydrolysis with 10% and 20% HCl depending on the applied extraction were examined. Comparing the solvents used for extraction, 30% ethanol extracted higher amount of TP and FL than 30% acetone. Combined 30% ethanol as solvent with acid hydrolysis (10 % HCl) and MAE obtained the highest amount of TP (90.07 mg GAE / g dm), followed by US extraction (78.22 mg GAE/g dm), while CE produced lower amount of TP (62.78 mg GAE/g dm). Conclusions: Using MAE with acid hydrolysis the amount of TP and FL increased and was higher then the one obtained with CE. Application of acid hydrolysis with US extraction decreased the amounts of TP and FL. Extracts obtained with 30% ethanol and acetone without addition of HCl obtained approximately equal value of TP and FL. Acid hydrolysis with 10% HCl increased TP and FL, while the amounts decreased with application of 20% HCl .

Saprolegnia parasitica and Aphanomyces astaci are oomycete pathogens that cause significant economic losses in salmonid and crayfish aquaculture. In this dissertation, inhibition of their growth and reproduction by sage, rosemary and bay laurel essential oils and fluid propolis formulations was investigated, as environmentally friendly alternatives to the toxic chemicals currently used. Considering the high cost of essential oil production, the isolation of essential oil from bay laurel, sage and rosemary leaves was optimised to increase the yield. Ultrasonic and reflux extraction hydrodistillation pretreatments increased the essential oil yield by up to 60%, while the overall quality of the oil remained largely unchanged. In contrast, the use of cell wal degrading enzymes as hydrodistillation pretreatments did not affect the yield. Next, it was shown that the tested essential oils and propolis formulations strongly inhibited the motility and germination of zoospores and the growth of the mycelium of both pathogens, albeit with differences in efficacy depending on the substance, oomycete species and life cycle stage. The fluid propolis formulations most potently inhibited mycelial growth of A. astaci, while the sage essential oil most strongly inhibited zoospore germination and mycelium growth of S. parasitica and zoospore germination of A. astaci. Although the results point to several compounds as the basis of the inhibitory effect, such as camphor in sage and chrysin in propolis, the observed anti-oomycetic activity is probably due to the synergistic effect of many different bioactive components present at low concentrations. Overall, the results of this doctoral thesis provide a starting point for further in vivo testing of the use of essential oils and propolis formulations in freshwater aquaculture as an environmentally acceptable control of diseases caused by oomycetes. Saprolegnia parasitica i Aphanomyces astaci su patogeni iz skupine vodenih plijesni (Oomycota) koji uzrokuju značajne ekonomske štete u akvakulturi salmonida i rakova. U ovoj disertaciji istražena je njihova inhibicija eteričnim uljima kadulje, ružmarina i lovora te tekućim pripravcima propolisa, kao ekološki prihvatljivim alternativama toksičnim kemikalijama koje se trenutno koriste. Obzirom na visoku cijenu proizvodnje eteričnih ulja, s ciljem povećanja prinosa optimizirani su uvjeti izolacije eteričnog ulja iz listova kadulje, lovora i ružmarina. Predtretmani ultrazvukom i klasičnom ekstrakcijom uz refluks povećali su prinos eteričnog ulja do 60 %, pri čemu je kvaliteta eteričnog ulja ostala uglavnom nepromijenjena. Nasuprot tomu, primjena enzima koji razgrađuju staničnu stijenku kao predtretman hidrodestilaciji nije povećala prinos ulja. Nadalje, pokazano je da su testirani uzorci eteričnih ulja i propolisa snažno inhibirali pokretljivost i klijavost zoospora i rast micelija oba patogena, ali s razlikama u djelotvornosti ovisno o uzorku, vrsti patogena te fazi životnog ciklusa. Tekuće formulacije propolisa najsnažnije su inhibirale rast micelija vrste A. astaci, dok je eterično ulje kadulje najjače inhibiralo klijavost zoospora i rast micelija vrste S. parasitica te zoospore vrste A. astaci. Iako rezultati ukazuju na nekoliko komponenti kao osnovu inhibicijskog učinka, primjerice kamfor u kadulji i krizin u propolisu, demonstrirano inhibicijsko djelovanje je vjerojatno posljedica sinergističkog učinka većeg broja različitih bioaktivnih komponenti prisutnih u niskim koncentracijama. Zaključno, rezultati ovog doktorskog rada predstavljaju polazište za daljnja in vivo istraživanja primjene eteričnih ulja i propolisa u slatkovodnoj akvakulturi u svrhu ekološki prihvatljive kontrole bolesti uzrokovanih vodenim plijesnima.

Cilj ovog diplomskog rada bio je istražiti postupke ekstrakcije i predtretmane koji prethode ekstrakciji eteričnih ulja samoniklog ljekovitog bilja porodice Lamiaceae (Rosmarinus officinalis, Thymus vulgaris, Thymus serphyllum, Lavandula, Salvia officinalis) i Lauraceae (Laurus nobilis) na temelju dostupne literature. Kod proizvodnje eteričnog ulja zaostaju nusprodukti kao što su hidrolat, vodeni i biljni ostatak. Eterično ulje i navedeni nusprodukti zbog svog kemijskog sastava predstavljaju vrijedan izvor biološki aktivnih spojeva koji pozitivno utječu na ljudsko zdravlje. Eterična ulja i hidrolati sadrže visoke udjele monoterpena, dok vodeni i biljni ostatak polifenole. Primjena eteričnih ulja, hidrolata i biljnog ekstrakta ovih biljaka u prehrambenoj industriji je označena kao sigurna za korištenje, te je istražena njihova potencijalna primjena u prehrambenoj industriji kao prirodnog konzervansa. Također, istražena je njihova primjena u proizvodnji jestivih filmova i premaza kao i načini kvalitete eteričnih ulja i mogućih patvorenja. The aim of this study was to investigate the extraction methods and pretreatments that precede the extraction of essential oils of wild medical plants of the family Lamiaceae (Rosmarinus officinalis, Thymus vulgaris, Thymus serphyllum, Lavandula, Salvia officinalis) and Lauraceae (Laurus nobilis) based on available literature. In the production of essential oil, by-products such as hydrolate, aqueous and plant residues are left behind. Due to its chemical composition, the essential oil and the mentioned by-products represent a valuable source of biologically active compounds that have a positive effect on human health. Essential oils and hydrolats contain high amounts of monoterpenes, while aqueous and plant residues contain polyphenols. The use of essential oils, hydrolats and plant extracts of these plants in the food industry is marked as safe for use, so their potential application in the food industry as a natural preservative has been investigated. Also, their use in the production of edible films and coatings and ways to control essential oils quality as well as and possible forgeries have been explored.

Cilj ovog rada bio je odrediti udio eteričnog ulja i fenolni sastav ekstrakta lista ružmarina nakon vodene destilacije po Clavengeru. Vodenoj destilaciji po Clavengeru prethodili su predtretmani: ekstrakcija refluksiranjem, ekstrakcija refluksiranjem potpomognuta enzimima te ultrazvučna ekstrakcija s ili bez dodatka enzima. Uz eterično ulje dobiveni su nusprodukti (vodeni ekstrakt i hidrolat) koji u svom sastavu sadrže fenolne spojeve. Biljni ostaci podvrgnuti su ekstrakciji potpomognutoj ultrazvukom primjenom otapala različite polarnosti (etanol-voda (1:1), metanol-voda (1:1) i etanol-metanol-voda (1:1:1)). Najveći udio eteričnog ulja (1 mL/100 g) dobiven je uz predtretman ekstrakcije potpomognute ultrazvukom i dodatak enzima ksilanaze. Najveći maseni udio ukupnih fenolnih spojeva određen je u vodenim ekstraktima ružmarina (44,02 ± 5,38 mg GAE g-1), dok je najveći udio ukupnih fenolnih spojeva u hidrolatu (0,2 mg GAE g-1) određen ekstrakcijom potpomognutom ultrazvukom. Pri ekstrakciji fenolnih spojeva iz biljnog ostatka važan je izbor otapala te primijenjeni predtretmani, a najveći prinosi fenolnih spojeva dobivaju se primjenom otapala etanol-voda (1:1) i prethodnim tretmanom enzimom ksilanazom. The aim of this study was to determine the yield of essential oil and phenolic composition of rosemary leaves extract after Clavenger hydro-distillation. Clavenger hydro-distillation was preceded by refluxing or ultrasound-assisted extraction with or without the addition of an enzyme. By-products (aqueous extract and hydrolate) containe phenolic compounds in their composition. Plant residues were subjected to ultrasound-assisted extraction using solvents of different polarity (ethanol-water (1:1), methanol-water (1:1) and ethanol-methanol-water (1:1:1)). The highest yield of essential oil (1 mL/100g) was obtained by ultrasound pre-treatment and addition of xylanase enzyme. The highest mass fraction of total phenolic compounds in aqueous extracts (44,02 ± 5,38 mg GAE g-1) was obtained by refluxing, while the highest fraction of total phenolic compounds in the hydrolate (0,2 mg GAE g-1) was obtained with ultrasound-assisted extraction. In the extraction of phenolic compounds the choice of solvent and the applied pretreatments are shown to be important factors. The highest proportion of phenolic compounds was extracted using the ethanol-water solvent (1:1) and the xylanase enzyme.

Cilj ovog rada bio je odrediti optimalne parametre za provođenje predtretmana enzimima (ksilanaza) te njegov utjecaj na udio eteričnog ulja kadulje i lovora nakon vodene destilacije po Clavengeru. U prvom dijelu rada ispitivao se utjecaj pH-vrijednosti otapala, koncentracije enzima i vremena trajanja reakcije na aktivnost ksilanaze pri 40 °C. Utvrđeno je da optimalni uvjeti podrazumijevaju upotrebu pufera 2 (pH = 6,5), masenu koncentraciju enzima od 0,20 mg mL-1 te vrijeme provođenja enzimske reakcije u trajanju od 4 sata. U drugom dijelu rada istraživan je utjecaj ekstrakcije potpomognute enzimima te ekstrakcije potpomognute ultrazvukom (50 W, 10 minuta) uz dodatak enzima kao predtretmana na udio eteričnog ulja nakon vodene destilacije. Kod obje biljke je najveći ekstrakcijski kapacitet postignut primjenom ekstrakcije potpomognute enzimima kao predtretmana, dok ekstrakcija potpomognuta ultrazvukom uz dodatak enzima nije značajno doprinijela povećanju udjela ekstrahiranog eteričnog ulja. Zaključno, ekstrakcija potpomognuta enzimima kao predtretmana vodenoj destilaciji po Clavengeru bi se u budućnosti mogla koristiti kao ekološki prihvatljiva metoda za izdvajanje eteričnog ulja iz listova lovora i kadulje. The aim of this study was to determine optimal parameters for enzyme-assisted extraction (xylanase) and its impact on the content of sage and bay laurel essential oils after Clavenger hydrodistillation. Firstly, influence of solvent type, enzyme concentration and time of reaction on the xylanase activity at 40 °C was tested. It was found that the optimal parameters include the usage of buffer 2 (pH = 6,5), mass concentration of 0,20 mg mL-1 and reaction time of 4 hours. Secondly, influence of enzyme-assisted extraction and ultrasound-assisted extraction (50 W, 10 minutes) with the addition of enzymes on the essential oil yield after Clavenger hydrodistillation was tested. The highest extraction capacity was observed using enzyme-assisted extraction as pretreatment, while ultrasound-assisted extraction with the addition of the enzymes hasn't significantly contributed to the increase of the essential oil yield. In conclusion, pretreatment with enzyme-assisted extraction prior to the Clavenger hydrodistillation could be used as a green extraction method for bay laurel and sage essential oils.

Cilj ovog rada bio je odrediti udio eteričnog ulja i fenolni sastav lista lovora nakon provedene vodene destilacije. Predtretman klasičnom ekstrakcijom refluksiranjem i ekstrakcijom potpomognutom ultrazvukom uz dodatak enzima (celulaza, pektinaza, ksilanaza) neposredno prije provedene Clavenger vodene destilacije nije pridonio povećanju udjela ekstrahiranog eteričnog ulja i udjela fenolnih spojeva iz lista lovora. Spektrofotometrijski su određeni visoki maseni udjeli ukupnih fenola u vodenom ekstraktu (27,27 mg GAE g-1) i ekstraktu biljnog ostatka lista lovora (20,01 mg GAE g-1) koji zaostaju nakon provedene vodene destilacije po Clavengeru. U biljnim ostacima lovora nakon provedene vodene destilacije je ispitivan utjecaj smjese otapala, te su primjenom smjese etanol-metanol-voda (1:1:1) ekstrahirani veći maseni udjeli fenolnih spojeva nego primjenom smjese etanol-voda (1:1) i metanol-voda (1:1).Vodeni ekstrakt, ekstrakt biljnog ostatka, eterično ulje i hidrolat lovora predstavljaju vrijedan izvor biološki aktivnih spojeva, te će se u daljnjim analizama ispitati njihovo djelovanje na patogene oomicete. The aim of this study was to the determine the oil content and phenolic composition of laurel bay. Pretreatment with classical extraction by refluxing and ultrasound-assisted extraction with the addition of enzymes (cellulase, pectinase, xylanase) just prior to the Clevenger hydro-distillation did not contribute to the increase of the extracted essential oil from the laurel bay leaves. In the aqueous extract and the hydrolate that are left behind after Clevenger hydro-distillation are spectrophotometrically determined mass fraction of total phenols, hydroxycinnamic acids, flavonoids and flavonols. After the hydro-distillation the influence of solvent mixture was investigated in the plant residues of the laurel bay leaves and the highest mass fraction of phenolic compounds was extracted using ethanol-methanol-water (1:1:1), while using ethanol-water (1:1) and methanol.water (1:1) significantly less. Aqueous extract, hydrolate and extract of laurel bay leaves are valuable sources of phenolic compounds and in further analysis will be determined their oomicete activity.

Motar je aromatska halofilna biljka koja se zbog svog bogatog sastava biološki aktivnih spojeva koristi u prehrani. U ovom radu istraživan je utjecaj različitih vrsta otapala (voda, etanol (96%), etanol-voda (1:1), aceton (100%), aceton-voda (1:1), metanol (100%), metanol-voda (1:1), etanol-metanol-aceton (1:1:1) i etanol-aceton-metanol-voda (1:1:1:1)) na ekstrakciju fenolnih spojeva iz lista motar. Ekstrakcija je provedena refluksiranjem pri temperaturi ključanja otapala kroz 1 h. U dobivenim ekstraktima provedeno je spektrofotometrijsko (UV/VIS) određivanje masenih udjela ukupnih fenola, flavonoida, hidroksicimetnih kiselina i flavonola. Dobiveni rezultati su pokazali da primjena ekstrakcijskog otapala ima utjecaj na izolaciju fenolnih spojeva te da se bolji učinak ekstrakcije fenolnih spojeva postiže dodatkom vode u otapalo, nego primjenom čistog otapala, a vodena otopina etanola ima najbolji ekstrakcijski učinak kod izolacije ukupnih fenola (77,05 mg/g), hidroksicimetnih kiselina (53,49 mg/g) i flavonola (38,24 mg/g), dok je primjena čistog otapala, odnosno metanola, pokazala najbolji učinak kod ukupnih flavonoida (38,13 mg/g). Provedeno istraživanje pokazuje da je list motar dobar izvor fenolnih spojeva. Sea fennel is aromatic halophyte which have high content of biologically active compounds and it’s used in nutrition. In this thesis was examine how different solvents effect (water, ethanol (96%), ethanol-water (1:1), acetone (100%), acetone-water (1:1), methanol (100%), methanol-water (1:1), ethanol-acetone-methanol (1:1:1) and ethanol-acetonemethanol- water (1:1:1:1)) on extraction phenolic compounds of sea fennel leaves. Duration of extraction was 1 hour on boiling temperature of each solvent. Extracts were analysed with spectrophotometry (UV/VIS) for total phenols, flavonoids, hydroxycinnamic acid and flavonols content. Results show that solvent has effect on extraction of phenolic compounds, solvents mixed with water have higher capacity to extract phenolics than pure solvents and ethanolwater has the highest capacity of extraction of mass fraction of total polyphenols (77,05 mg/g), hydroxycinnamic acids (53,49 mg/g) and flavonols (38,24 mg/g), and pure solvent, methanol, has the highest capacity of extraction of total flavonoids (38,13 mg/g). These results show that leaves of sea fennel have a high phenolic content.

Ekstrakti kadulje zbog svog visokog udjela polifenolnih spojeva imaju potencijal primjene prirodnih inhibitora rasta oomicetnih patogena. Stoga, istraživan je utjecaj različitih vrsta otapala: voda, etanol (96%), etanol-voda (1:1), aceton (100%), aceton-voda (1:1), metanol (100%), metanol-voda (1:1), etanol-metanol-aceton (1:1:1) i etanol-metanol-aceton-voda (1:1:1:1) na ekstrakciju polifenolnih spojeva samonikle kadulje (Salvia officinalis L). Provedena je ekstrakcija refluksiranjem pri temperaturi ključanja otapala kroz 1 h i maceracija kroz 72 h te su spektrofotometrijskom metodom (UV/VIS) određeni maseni udjeli ukupnih fenola, hidroksicimetnih kiselina, ukupnih flavonoida i flavonola. Neovisno o primijenjenom postupku ekstrakcije, kombinacijom otapala etanol-metanol-aceton-voda ekstrahiran je veći maseni udio ukupnih fenola (134,87 mg/g), hidroksicimetnih kiselina (66,08 mg/g) i flavonola (49,36 mg/g), dok se metanol pokazao najboljim za izolaciju flavonoida. Na temelju dobivenih rezultata može se zaključiti da se bolji učinak ekstrakcije polifenolnih spojeva postiže kombinacijom otapala, te dodatkom vode u otapalo, nego primjenom čistog otapala. Sage extract due to its high proportion of polyphenolic compounds has the potential to be used as a natural inhibitor for the growth of oomycete pathogens. Therefore, the influence of different types of solvents on the extraction of polyphenolic compounds of wild sage (Salvia officinalis L.) was investigated. Solvents used were: water, ethanol (96%), ethanol-water (1:1), acetone (100%), acetone-water (1:1), methanol (100%), methanol-water (1:1), ethanol-methanol-acetone (1:1:1) and ethanol-methanol-acetone-water (1:1:1:1). The extraction was performed by refluxing at the boiling temperature of the solvent for 1 h and by process of maceration for 72 h. By spectrophotometric method (UV/VIS) high levels of total phenols, hydroxycinnamic acid, total flavonoids, and flavonols were determined. Regardless of the extraction procedure used, the highest mass fraction of total phenols (134.87 mg/g), hydroxycinnamic acids (66.08 mg/g) and flavonols (49.36 mg/g) was extracted with the ethanol-methanol-acetone-water solvent combination, while methanol showed best for flavonoids isolation. Based on the obtained results, it can be concluded that the better effect of extraction of polyphenolic compounds is achieved by adding water to the solvent than using a pure solvent.