Exploring Phenolic Compounds Extraction from Saffron (C. sativus) Floral By-Products Using Ultrasound-Assisted Extraction, Deep Eutectic Solvent Extraction, and Subcritical Water Extraction.

Saffron (Crocus sativus) floral by-products are a source of phenolic compounds that can be recovered and used in the nutraceutical, pharmaceutical, or cosmetic industries. This study aimed to evaluate the phenolic compounds' extraction using green extraction techniques (GETs) in saffron floral by-products and to explore the influence of selected extraction techniques on the phytochemical composition of the extracts. Specifically, ultrasound-assisted extraction (UAE), subcritical water extraction (SWE), and deep eutectic solvents extraction (DESE) were used. Phenolic compounds were identified with (HR) LC-ESI-QTOF MS/MS analysis, and the quantitative analysis was performed with HPLC-PDA. Concerning the extraction techniques, UAE showed the highest amount for both anthocyanins and flavonoids with 50:50% v/v ethanol/water as solvent (93.43 ± 4.67 mg/g of dry plant, dp). Among SWE, extraction with 96% ethanol and t = 125 °C gave the best quantitative results. The 16 different solvent mixtures used for the DESE showed the highest amount of flavonoids (110.95 ± 5.55-73.25 ± 3.66 mg/g dp), while anthocyanins were better extracted with choline chloride:butane-1,4-diol (16.0 ± 0.80 mg/g dp). Consequently, GETs can be employed to extract the bioactive compounds from saffron floral by-products, implementing recycling and reduction of waste and fitting into the broader circular economy discussion.

A Novel Approach to Serving Plant-Based Confectionery-The Employment of Spray Drying in the Production of Carboxymethyl Cellulose-Based Delivery Systems Enriched with Teucrium montanum L. Extract.

In this study, spray drying was used as a technological solution for the valorization of Teucrium montanum extract into carboxymethyl cellulose-based delivery systems (CMC), individually or in combination with collagen, guar gum, gum arabic, and kappa-carrageenan. The results showed that the process yield and morphological properties were positively influenced by the introduction of CMC binary blends. The employment of CMC resulted in a high encapsulation efficiency (77-96%) for all phenylethanoid glycosides (PGs) analyzed. Due to the low wettability of the microparticles, a relatively gradual in vitro release of the PGs was achieved. Infusion of the filling with hydrophilic T. montanum extract encapsulated in microparticles with high hydrophobic surface area proved to be a practical route for significant confectionery fortification (5-9 mg PGs per dw serving), ensuring prolonged interaction between the food matrix used and the extract under simulated gastrointestinal conditions. Based on sensory evaluation, the introduction of kudzu starch into the jelly matrix has shown a texture-modifying potential.

Teucrium montanum L.-Unrecognized Source of Phenylethanoid Glycosides: Green Extraction Approach and Elucidation of Phenolic Compounds via NMR and UHPLC-HR MS/MS.

Health-oriented preferences, a demand for innovative food concepts, and technological advances have greatly influenced changes in the food industry and led to remarkable development of the functional food market. Incorporating herbal extracts as a rich source of bioactive compounds (BC) could be an effective solution to meet the high demand of consumers in terms of expanding the high-quality range of functional foods. The aim of this study is the valorization of the bioactive potential of T. montanum L., an understudied Mediterranean plant species, and the in-depth elucidation of a polyphenolic profile with a UHPLC-HR MS/MS and NMR analysis. The total phenolic content (TPC) and antioxidant capacity (AC) were determined on heat-assisted (HAE), microwave-assisted (MAE) and subcritical water (SWE) extracts. In terms of antioxidant capacity, SWE extracts showed the most notable potential (ABTS: 0.402-0.547 mmol eq Trolox g-1 dw, DPPH: 0.336-0.427 mmol eq Trolox g-1 dw). 12 phenolic compounds were identified in the samples of T. montanum from six microlocations in Croatia, including nine phenylethanoid glycosides (PGs) with total yields of 30.36-68.06 mg g-1 dw and 25.88-58.88 mg g-1 dw in HAE and MAE extracts, respectively. Echinacoside, teupolioside, stachysoside A, and poliumoside were the most abundant compounds HAE and MAE extracts, making T. montanum an emerging source of PGs.

Innovative Strategy for Aroma Stabilization Using Green Solvents: Supercritical CO2 Extracts of Satureja montana Dispersed in Deep Eutectic Solvents.

The aim of this work was to establish the potential of natural deep eutectic solvents (NADES) for the stabilization of aroma volatile organic compounds from a natural source. Satureja montana was used as a source of volatile components, as it is rich in terpenes of great commercial and biological importance, such as carvacrol, thymol, and thymoquinone, among others. Supercritical CO2 was used to extract the lipophilic fraction of S. montana, which was further directly dispersed in NADES. The stabilizing capacity of seven different NADES based on betaine and glycerol was analyzed. The stability of the components in NADES was monitored by analyzing the headspace profile during 6 months of storage at room temperature. The changes in the headspace profile over time were analyzed by using different statistical and chemometric tools and the Wilcoxon matched pair test. It was determined that alterations over time occurred such as degradation and oxidation, and they were the most prominent in the control. In addition, the indicator of decreased stability of the control was the formation of the new compounds that could compromise the quality of the product. In the stabilized NADES samples, the changes were significantly less prominent, indicating that the NADES had a stabilizing effect on the volatile compounds. According to Wilcoxon matched pair test, the most efficient stability was achieved by using betaine/ethylene glycol, glycerol/glucose, and betaine/sorbitol/water. Therefore, by applying two green solvents, a sustainable approach for obtaining pure and high-quality S. montana extracts with extended stability at room temperature was established.

Terpenes and Cannabinoids in Supercritical CO2 Extracts of Industrial Hemp Inflorescences: Optimization of Extraction, Antiradical and Antibacterial Activity.

Natural products are increasingly in demand in dermatology and cosmetology. In the present study, highly valuable supercritical CO2 (sCO2) extracts rich in bioactive compounds with antiradical and antibacterial activity were obtained from the inflorescences of industrial hemp. Volatile compounds were analyzed by gas chromatography in tandem with mass spectrometry (GC-MS), while cannabinoids were determined by high performance liquid chromatography (HPLC-DAD). Extraction yields varied from 0.75 to 8.83%, depending on the pressure and temperature applied. The extract obtained at 320 bar and 40 °C with the highest content (305.8 µg mg-1) of cannabidiolic acid (CBDA) showed the best antiradical properties. All tested extract concentrations from 10.42 µg mL-1 to 66.03 µg mL-1 possessed inhibitory activities against E. coli, P. aeruginosa, B. subtilis, and S. aureus. The sCO2 extract with the highest content of cannabidiol (CBD) and rich in α-pinene, ß-pinene, ß-myrcene, and limonene was the most effective. The optimal conditions for sCO2 extraction of cannabinoids and volatile terpenes from industrial hemp were determined. The temperature of 60 °C proved to be optimal for all responses studied, while the pressure showed a different effect depending on the compounds targeted. A low pressure of 131.2 bar was optimal for the extraction of monoterpenes, while extracts rich in sesquiterpenes were obtained at 319.7 bar. A high pressure of 284.78 bar was optimal for the extraction of CBD.

Comprehensive Study of Traditional Plant Ground Ivy (Glechoma hederacea L.) Grown in Croatia in Terms of Nutritional and Bioactive Composition.

In the present study, ground ivy was harvested from different natural habitats in Croatia and subjected to screening analysis for nutritional and bioactive composition. To achieve maximum recovery of phenolic compounds, different extraction techniques were investigated-heat-assisted (HAE), microwave-assisted (MAE) and subcritical water (SWE) extraction. Prepared extracts were analysed by spectrophotometric methods, LC-MS/MS and HPLC-PAD methodologies. Results regarding nutritive analyses, conducted using standard AOAC methods, showed the abundance of samples in terms of insoluble dietary fibre, protein, calcium and potassium, while rutin, chlorogenic, cryptochlorogenic, caffeic and rosmarinic acid were the most dominant phenolic compounds. In addition, LC-MS/MS analysis revealed the presence of apigenin and luteolin in glycosylated form. Maximum recovery of target phenolic compounds was achieved with MAE, while SWE led to the formation of new antioxidants, which is commonly known as neoformation. Moreover, efficient prediction of phenolic composition of prepared extracts was achieved using NIR spectroscopy combined with ANN modelling.

Green Extraction Techniques for Obtaining Bioactive Compounds from Mandarin Peel (Citrus unshiu var. Kuno): Phytochemical Analysis and Process Optimization.

In this study, an efficient utilization and valorization of mandarin peel (Citrus unshiu Marc. var. Kuno) was investigated using innovative and green extraction techniques. The first step of this study included the extraction and analysis of the volatile compounds by performing a supercritical CO2 (SC-CO2) extraction under different operating pressure conditions (100 and 300 bar). The analysis of volatile compounds of the obtained extracts was conducted by gas chromatography-mass spectrometry (GC-MS), and limonene was found to be the dominant volatile component (13.16% at 100 bar; 30.65% at 300 bar). After SC-CO2 treatment, the exhausted citrus peel waste enriched with bioactive compounds was subjected to subcritical water extraction (SWE) in a wide temperature range (130-220 °C) using different solvent-solid ratio (10-30 mL/g) in time periods from 5 to 15 min, in order to obtain bioflavonoids. Identification and quantification of present bioflavonoids was conducted by high-performance liquid chromatography with a with a diode array detector (HPLC), and hesperidin (0.16-15.07 mg/g) was determined as the most abundant flavanon in mandarin peel with other polyphenolic compounds that were possible by-products of thermal degradation. At higher temperatures, the presence of 5-hydroxymethylfurfural (5-HMF) and chlorogenic acid were detected. Antiradical activity and total phenolic content in the extracts were determined using spectrophotometric methods, while the process optimization was performed by response surface methodology (RSM).

Bioprospecting of Less-Polar Constituents from Endemic Brown Macroalga Fucus virsoides J. Agardh from the Adriatic Sea and Targeted Antioxidant Effects In Vitro and In Vivo (Zebrafish Model).

The endemic brown macroalga Fucus virsoides J. Agardh from the Adriatic Sea was in the focus of the present research. The volatiles of fresh (FrFv) and air-dried (DrFv) samples of F. virsoides obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were analyzed by gas chromatography equipped with flame ionization detector and mass spectrometry (GC-FID/MS). The major HS-FrFv compound was pentadecane (61.90-71.55%) followed by pentadec-1-ene (11.00-7.98%). In HS-DrFv, pentadec-1-ene was not present, and few lower aliphatic compounds appeared, as well as benzaldehyde and benzyl alcohol. In HD-FrFv, particularly abundant were alkenes (such as pentadec-1-ene (19.32%), or (E)-pentadec-7-ene (8.35%)). In HD-DrFv, more oxidation products were present (e.g., carbonyl compounds such as tridecanal (18.51%)). The fatty acids profile of freeze-dried sample (FdFv) after conversion to methyl esters was determined by GC-FID, and oleic acid was dominant (42.28%), followed by arachidonic acid (15.00%). High-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (HPLC-ESI-HRMS) was used for the screening of less polar fractions (F3 and F4) of F. virsoides. Mono- and diglycerides of stearic, palmitic, oleic, and arachidonic acids were found. Terpenoids and steroids comprised the compounds C20H30(32)O2 and C29H48O(2). Among carotenoids, fucoxanthin was identified. Chlorophyll derivatives were also found (C55H74(72)N4O(5-7)), dominated by pheophytin a. The antioxidant activity of the fractions was investigated by in vitro assays (oxygen radical absorbance capacity (ORAC), reduction of radical cation (ABTS•+), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay, and ferric reducing antioxidant power (FRAP)) and by in vivo zebrafish model (along with fish embryotoxicity). In vitro experiments proved good radical scavenging abilities of F3 and F4 fractions, which were additionally supported by the protective effect against hydrogen peroxide-induced oxidative stress in zebrafish embryos.

Supercritical Carbon Dioxide Extraction of Allium ursinum: Impact of Temperature and Pressure on the Extracts Chemical Profile.

The aim of this study was to extract Allium ursinum L. for the first time by supercritical carbon dioxide (SC-CO2 ) as green sustainable method. The impact of temperature in the range from 40 to 60 °C and pressure between 150 and 400 bar on the quality of the obtained extracts and efficiency of the extraction was investigated. The highest extraction yield (3.43 %) was achieved by applying the extraction conditions of 400 bar and 60 °C. The analysis of the extracts was performed by gas chromatography and mass spectrometry (GC/MS). The most dominant sulfur-containing constituent of the extracts was allyl methyl trisulfide with the highest abundance at 350 bar and 50 °C. In addition, the presence of other pharmacologically potent sulfur compounds was recorded including S-methyl methanethiosulfinate, diallyl trisulfide, S-methyl methylthiosulfonate, and dimethyl trisulfide. Multivariate data analysis tool was utilized to investigate distributions of the identified compounds among the extracts obtained under various extraction conditions and yields. It was determined that the SC-CO2 extraction can by efficiently used for A. ursinum.

A Two-Year Occurrence of Fusarium T-2 and HT-2 Toxin in Croatian Cereals Relative of the Regional Weather.

To investigate into the T-2 and HT-2 toxin occurrence, 240 samples of unprocessed cereals (maize, wheat, barley, and oats) were sampled from different fields located in three Croatian regions during 2017-2018. In all samples, sum concentrations of T-2/HT-2 toxin were determined using the ELISA method, while the LC-MS/MS was used as a confirmatory method for both mycotoxins in positive samples (>LOD) and the establishment of T-2 over HT-2 toxin ratios. The results showed oats to be the most contaminated cereal, with T-2/HT-2 toxins detected in 70.0% of samples, followed by barley (40.9%), maize (26.8%) and wheat (19.2%), with the mean T-2/HT-2 ratio ranging from 1:2.7 in maize to 1:4.4 in oats. Sum T-2/HT-2 concentrations in two maize samples were higher than the indicative level recommended by the European Commission, necessitating subsequent investigations into the conditions under which these poorly investigated mycotoxins are produced. Statistically significantly (p < 0.05) higher concentrations of T-2/HT-2 toxin were determined in oats throughout study regions as compared to those found in wheat, but not maize and barley, while the concentrations of these mycotoxins were related to the regional weather in Croatia.

Supercritical CO2 extraction of Marrubium vulgare: intensification of marrubiin.

Supercritical carbon dioxide and Soxhlet extraction were employed for delivering Marrubium vulgare extracts. By varying process temperature and pressure, the impact of different densities of carbon dioxide on the extraction yield and chemical profile of the extracts was investigated. The highest extraction yields of M. vulgare were obtained by supercritical carbon dioxide extraction (3.51%) at 300 bar and 60 °C, while the yield obtained with the Soxhlet extraction was 3.23%. The chemical profiles of these two extracts were significantly different and marrubiin, the most dominant component of supercritical extracts was not present in extracts obtained by this conventional method. This labdane diterpene was the most abundant in extracts obtained at 200 bar and temperatures of 40, 50, and 60 °C (62.60, 68.41, and 71.96%, respectively). For the intensification of marrubiin, supercritical fractions were collected in successive time intervals over the course of the extraction (300 bar/60 °C). It was determined that after 1 h of extraction the highest content of marrubiin (75.14%) can be achieved. The similarities between the obtained extracts were estimated and the correlations to the content of identified lipophilic compounds were established using multivariate data analysis tools.

Volatile organic compounds of tobacco leaves versus waste (scrap, dust, and midrib): extraction and optimization.

BACKGROUND: Volatile organic compounds are present at very low concentration but exhibit an important influence on flavor and aroma of tobacco leaves and products. During tobacco processing, at different stages, tobacco wastes occur. Since they are delivered directly from the tobacco plant, they are expected to have a similar aroma profile. RESULTS: The volatile composition of three types of tobacco waste (scrap, dust, and midrib) was characterized for the first time and compared with tobacco leaves' volatile composition. Ultrasound-assisted extraction with hexane followed by gas chromatography-mass spectrometry was successfully applied. Different ultrasound-assisted extraction parameters (temperature, time, and solvent:solid ratio) showed a significant influence on the volatile profiles of the extracts obtained. The most important compounds in tobacco leaves, scrap, and dust with the highest abundance were nicotine (up to 87.5%), 4,8,13-duvatriene-1,3-diol (up to 16.2%), and neophytadiene (up to 9.4%). In midrib, only nicotine was present in all extracts. The most abundant compounds in the extracts were quantified and subjected to optimization using response surface methodology. CONCLUSION: Regression analysis showed that 83-98% of the variation was explained by the models obtained. The experimentally obtained values agreed with those predicted, thus indicating the suitability of the model employed and the success of response surface methodology in optimizing the extraction conditions. © 2020 Society of Chemical Industry.

Trichinella spp. in wild boar (Sus scrofa) populations in Croatia during an eight-year study (2010-2017).

Wild animals represent a constant source of Trichinella spp. infections for domestic animals and humans. To date, four species of Trichinella have been isolated in wild boar populations in Europe: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in addition to several mixed infection types and one hybrid formation between T. britovi and T. spiralis. Meanwhile, insufficiently thermally processed wild boar meat has been reported to be a source of trichinellosis in humans in several European countries. In Croatia, there have been no reported or proven cases of trichinellosis caused by wild boar meat consumption. The aim of this study was to obtain data on the prevalence of Trichinella species present in Croatia and to anticipated the potential risk of infection for humans in specific Croatian regions based on information obtained over an eight-year surveillance period. A veterinary inspection of wild boar carcasses for Trichinella larvae in Croatia has been mandatory since 1989, and the artificial digestion method was introduced as a compulsory test for wild boar samples in 2008. Based on the official data submitted to the Ministry of Agriculture, Directorate of Veterinary Services, in the period 2010-2017, 303 of 183,184 (0.17%) wild boar meat samples tested positive for Trichinella spp. Infected wild boar were found in 18 of 21 counties. Of these positive samples, 85 were submitted by the authorised veterinary inspectors to the National Reference Laboratory for further examination. The intensity of infection in muscle samples was 0.04-152.66 (mean: 23,37) larvae per gram, and Trichinella species were identified as T. spiralis, T. britovi, T. pseudospiralis and T. spiralis + T. britovi. Genetic analysis of T. pseudospiralis isolates demonstrated their belonging to the Palaearctic population.

Chemical Diversity of Codium bursa (Olivi) C. Agardh Headspace Compounds, Volatiles, Fatty Acids and Insight into Its Antifungal Activity.

The focus of present study is on Codium bursa collected from the Adriatic Sea. C. bursa volatiles were identified by gas chromatography and mass spectrometry (GC-FID; GC-MS) after headspace solid-phase microextraction (HS-SPME), hydrodistillation (HD), and supercritical CO2 extraction (SC-CO2). The headspace composition of dried (HS-D) and fresh (HS-F) C. bursa was remarkably different. Dimethyl sulfide, the major HS-F compound was present in HS-D only as a minor constituent and heptadecane percentage was raised in HS-D. The distillate of fresh C. bursa contained heptadecane and docosane among the major compounds. After air-drying, a significantly different composition of the volatile oil was obtained with (E)-phytol as the predominant compound. It was also found in SC-CO2 extract of freeze-dried C. bursa (FD-CB) as the major constituent. Loliolide (3.51%) was only identified in SC-CO2 extract. Fatty acids were determined from FD-CB after derivatisation as methyl esters by GC-FID. The most dominant acids were palmitic (25.4%), oleic (36.5%), linoleic (11.6%), and stearic (9.0%). FD-CB H2O extract exhibited better antifungal effects against Fusarium spp., while dimethyl sulfoxide (DMSO) extract was better for the inhibition of Penicillium expansum, Aspergillus flavus, and Rhizophus spp. The extracts showed relatively good antifungal activity, especially against P. expansum (for DMSO extract MIC50 was at 50 µg/mL).

Screening of Six Medicinal Plant Extracts Obtained by Two Conventional Methods and Supercritical CO2 Extraction Targeted on Coumarin Content, 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity and Total Phenols Content.

Six medicinal plants Helichrysum italicum (Roth) G. Don, Angelica archangelica L., Lavandula officinalis L., Salvia officinalis L., Melilotus officinalis L., and Ruta graveolens L. were used. The aim of the study was to compare their extracts obtained by Soxhlet (hexane) extraction, maceration with ethanol (EtOH), and supercritical CO2 extraction (SC-CO2) targeted on coumarin content (by high performance liquid chromatography with ultraviolet detection, HPLC-UV), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging capacity, and total phenols (TPs) content (by Folin-Ciocalteu assay). The highest extraction yields were obtained by EtOH, followed by hexane and SC-CO2. The highest coumarin content (316.37 mg/100 g) was found in M. officinalis EtOH extracts, but its SC-CO2 extraction yield was very low for further investigation. Coumarin was also found in SC-CO2 extracts of S. officinalis, R. graveolens, A. archangelica, and L. officinalis. EtOH extracts of all plants exhibited the highest DPPH scavenging capacity. SC-CO2 extracts exhibited antiradical capacity similar to hexane extracts, while S. officinalis SC-CO2 extracts were the most potent (95.7%). EtOH extracts contained the most TPs (up to 132.1 mg gallic acid equivalents (GAE)/g from H. italicum) in comparison to hexane or SC-CO2 extracts. TPs content was highly correlated to the DPPH scavenging capacity of the extracts. The results indicate that for comprehensive screening of different medicinal plants, various extraction techniques should be used in order to get a better insight into their components content or antiradical capacity.

Bee community as a source of energy in the production of food, honey-plants in the ecosystem of Croatian Forests' hunting grounds.

In addition to the process of photosynthesis, the bee community is the main source of energy in food production, honey-plants used by game and people in the hunting grounds ecosystem. It is a well-known fact that thousands of plant species depend on the presence of bee communities (pollination, fertilization). In this paper we studied the presence of the bee community in the hunting grounds of Croatian Forests, and their influence on the number of game (wild pigs), as well as the quality of honey, honey plants in the hunting grounds used by people and game. We established the total number of game (wild boars). The honey quality parameters were determined using the Harmonised methods of the European Honey (Bogdanov et al., 1997) and the pollen analysis by were conducted according to Harmonised methods of melissopalynology (Von der Ohe et al., 2004). Research results indicate that the presence of the bee community influences the number of wild boars from 3-18%, and the quality of honey is in line with the European and world standards. The SAS/STAT package was used for the statistical analysis (SAS Institute Inc., 2000). The significance of the differences among the groups was determined by Duncan test.