Nutritional Value, Phytochemical Composition and Biological Activities of Lycium barbarum L. fruits from Serbia.

Cultivation of goji berries (GB), fruits of Lycium barbarum L. (Solanaceae), is expanding worldwide, including in Europe. In this study, a comparative analysis of the nutritional value, chemical composition and in vitro biological activities of GB from different locations in Serbia was performed. Proximate compositions were evaluated according to standard methods. Minerals were assessed by inductively coupled plasma techniques, while fatty acids, sterols, and phenolic profiles were analyzed by gas- and liquid chromatography-based techniques coupled with flame-ionization, mass spectrometry, or diode array detection. The total content of phenolics, flavonoids, carotenoids, and polysaccharides was assessed using spectrophotometric methods. Methanol extracts from GB were examined for their antioxidant, enzyme inhibitory (α-amylase, α-glucosidase, acetylcholinesterase and tyrosinase) and antibacterial activities. Despite significant variations among samples from different locations, the results confirmed that GB are a valuable source of dietary fiber and protein and are characterized by favorable fatty acid profiles. Phytochemical analysis revealed that ß-sitosterol, Δ5-avenasterol, and 24-methyldesmosterol are the predominant sterols and caffeic acid, gallic acid, quercetin and rutin are the main phenols. All GB samples showed both antioxidant and mild antimicrobial activity. A dose-dependent anti-enzymatic activity (IC50 ranging 1.68-6.88 mg/mL) was demonstrated. The results support further promotion of GB cultivation in Serbia and further investigations on their potential applications in various industries.

Chemical Analysis and Investigation of Antimicrobial and Antibiofilm Activities of Prangos trifida (Apiaceae).

Plants of the genus Prangos are intensively investigated as potential new sources of bioactive isolated products. In this work, the chemical composition of volatile constituents (essential oils and headspace volatiles) and dichloromethane extracts, as well as antimicrobial and antibiofilm activities of essential oils and MFDEs (methanol fractions of dichloromethane extracts) of Prangos trifida from Serbia, were investigated. Volatiles of roots, leaves, stems and fruits, and fatty acids and phytosterols in dichloromethane extracts of roots and fruits were analyzed by GC-FID-MS, whereas coumarins in MFDEs by LC-MS and some isolated coumarins by 1H-NMR. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations/minimum fungicidal concentrations (MBCs/MFCs) of essential oils and MFDEs were determined against 13 microorganisms. Antibiofilm activity was assessed against four microorganisms. Additionally, congo red and ergosterol binding assays were conducted to elucidate selected mechanisms of antibiofilm action in the case of Candida albicans. Total of 52 volatile constituents, 16 fatty acids, eight phytosterols and 10 coumarins were identified. Essential oils demonstrated significant activity, surpassing that of commercial food preservatives, against six tested molds from the Aspergillus, Penicillium and Trichoderma genera, as well as against bacteria Staphylococcus aureus and Bacillus cereus. Most of the oils strongly inhibited the formation of biofilms by S. aureus, Listeria monocytogenes and Escherichia coli. MFDEs exhibited noteworthy effects against B. cereus and the tested Aspergillus species, particularly A. niger, and significantly inhibited C. albicans biofilm formation. This inhibition was linked to a marked reduction in exopolysaccharide production, while antifungal mechanisms associated with ergosterol remained unaffected.

Investigation of Anticholinesterase Activity of Chemically Characterised Hieracium s. str. Methanol Extracts and Their Selected Metabolites.

The composition and anticholinesterase activity of the dried MeOH extracts of Hieracium scheppigianum and H. naegelianum underground parts (rhizomes and roots), as well as the anticholinesterase activity of the dried, previously chemically characterised MeOH extracts of the flowering aerial parts of these two and 26 other Hieracium species in the strict sense (s. str.), were investigated. Furthermore, the anticholinesterase activity of 12 selected secondary metabolites of these extracts was evaluated. Using semi-preparative LC-MS, five caffeoylquinic acids and the sesquiterpene lactone crepiside E were isolated from H. scheppigianum underground parts extract. All these compounds were also identified in the underground parts extract of H. naegelianum. Quantitative LC-MS analysis showed that the analysed underground parts extracts were rich in both caffeoylquinic acids (139.77 and 156.62 mg/g of extract, respectively) and crepiside E (126.88 and 116.58 mg/g). In the Ellman method, the tested extracts showed an interesting anti-AChE and/or anti-BChE activity (IC50 =0.56-1.58 mg/mL), which can be explained, at least partially, by the presence of some of their constituents. Among the metabolites tested, the best activity was revealed for the flavonoids apigenin, luteolin and diosmetin, and the sesquiterpene lactone 8-epiixerisamine A (IC50 =68.09-299.37 µM).

Antimicrobial and Cytotoxic Activities of Selected Hieracium L. s. str. (Asteraceae) Extracts and Isolated Sesquiterpene Lactones.

Antimicrobial and cytotoxic activities were tested for dried MeOH extracts of Hieracium calophyllum (CAL), H. coloriscapum (COL), H. pseudoschenkii (PSE), H. valdepilosum (VAL) and H. glabratum (GLA) herbs (flowering aerial parts), their 2 sesquiterpene lactones (SLs) 8-epiixerisamine A and crepiside E, and dried CH2 Cl2 extract of H. scheppigianum (SCH) herb. In microdilution test, extracts showed activity on all tested microorganisms (8 bacteria, 10 fungi). The best effect was exhibited by SCH and CAL on Salmonella Typhimurium (MIC=1.7-2.5 mg/mL MBC=3.4-5.0 mg/mL), and SCH and VAL on Candida albicans (MIC=2.5 mg/mL MFC=5.0 mg/mL). SLs showed notable effect on all tested fungi Aspergillus ochraceus, Penicillium funiculosum, C. albicans and C. krusei (MIC=0.15-0.4 mg/mL MFC=0.3-0.8 mg/mL). In MTT test, extracts inhibited growth of all tested cancer cells (HeLa, LS174 and A549), with the best effect on HeLa (IC50 =148.1 µg/mL for SCH, and 152.3-303.2 µg/mL for MeOH extracts); both SLs were active against HeLa cells (IC50 =46.2 µg/mL for crepiside E and 103.8 µg/mL for 8-epiixerisamine A). Extracts and SLs showed good safety profile on normal MRC-5 cells.

Spasmolytic, Gastroprotective and Antioxidant Activities of Dry Methanol Extract of Ferula heuffelii Underground Parts.

Dry MeOH extract of Ferula heuffelii (Apiaceae) underground parts was tested for spasmolytic, gastroprotective and antioxidant activities. HPLC analysis revealed that chlorogenic acid (CGA; 34.6 mg/g) was its main constituent. Extract in vitro exhibited notable total antioxidant activity (FRAP value=1.0 µmol Fe2+ /mg), and scavenging of DPPH (SC50 =62.5 µg/ml) and • OH radicals (49.5 % at 20 µg/ml in 2-deoxyribose assay). In vitro on isolated rat ileum, extract exhibited significant spasmolytic activity, i. e., it showed 124.6 % of maximal atropine effect on spontaneous contractions (at 100 µg/ml), and reduced spasmogenic effect of KCl (80 mm) to 44.4 % (at 60 µg/ml) and of highest applied concentration of ACh to 26.3 % (at 120 µg/ml). In parallel experiments, spasmolytic effect of CGA was also demonstrated. In acute EtOH-induced gastric ulceration model in rats, extract (100 mg/kg p.o.) showed significant gastroprotective effect (gastric damage score 0.50), similar to ranitidine (20 mg/kg p.o.). Obtained results showed that tested F. heuffelii polar extract represents new herbal preparation with potential use against some gastrointestinal complaints.

Barks of Three Wild Pyrus Taxa: Phenolic Constituents, Antioxidant Activity, and in Vitro and in Silico Investigations of α-Amylase and α-Glucosidase Inhibition.

Dry MeOH extracts of the twig barks of Pyrus communis subsp. pyraster, P. spinosa and their hybrid P.×jordanovii nothosubsp. velenovskyi, collected in wild in Serbia, were analyzed. By LC/MS, the contents of arbutin (99.9-131.0 mg/g), chlorogenic acid (2.2-6.3 mg/g), catechin (1.0-5.3 mg/g) and total dimeric and trimeric procyanidins (42.2-61.3 mg/g), including procyanidin B2 (8.9-17.2 mg/g), were determined. Colorimetrically, high contents of total phenolics (436.2-533.4 mg GAE/g) and tannins (339.4-425.7 mg GAE/g), as well as strong total antioxidant activities (FRAP values 4.5-5.9 mmol Fe2+ /g), and DPPH (SC50 =6.6-7.1 µg/ml) and hydroxyl radical (SC50 =447.1-727.7 µg/ml) scavenging abilities were revealed. In vitro, all extracts exhibited notable inhibition of α-amylase (IC50 =310.8-617.7 µg/ml) and particularly strong inhibition of α-glucosidase (IC50 =2.1-3.7 µg/ml). Molecular docking predicted that among identified compounds procyanidin B2 is the best inhibitor of these carbohydrate-digesting enzymes. Obtained results showed that the barks of investigated Pyrus hybrid and its parent taxa have similar composition and bioactivity.

Investigation of antihyperalgesic and antiedematous activities of three Hieracium species.

Present study investigated triterpene profile, antihyperalgesic and antiedematous activities of Hieracium scheppigianum flowering aerial parts dichloromethane extract (SCH), and antihyperalgesic and antiedematous activities of previously chemically characterised polyphenol-rich H. glabratum and H. calophyllum flowering aerial parts methanol extracts (GLA and CAL, respectively). α- and ß-Amyrin and their acetates, and lupeol acetate were identified and quantified in SCH by GC-FID and GC-MS. In carrageenan-induced localised inflammation model in rats, SCH and GLA (50-200 mg/kg, p.o.) produced significant and dose-dependent antihyperalgesic effect of 26.9%-56.2% (ED50=163.0 ± 26.5 mg/kg) and 25.3%-51.6% (ED50=211.6 ± 70.6 mg/kg), respectively, and CAL (200 mg/kg, p.o.) exhibited effect of 38.1%. Extracts did not significantly reduce paw edema. SCH and GLA, which demonstrated higher (over 50%) antihyperalgesic efficacy, were tested in a rotarod test (200 mg/kg, p.o.) and no alteration of motor coordination was observed. Also, acute administration of SCH and GLA in mice (2000 mg/kg, p.o.) caused neither mortality nor toxicity.

Chemosystematic Significance of Essential Oil Constituents and Furanocoumarins of Underground Parts and Fruits of Nine Heracleum L. Taxa from Southeastern Europe.

The composition of the essential oils and the furanocoumarin profiles of CH2 Cl2 extracts from underground parts and fruits of nine Heracleum taxa (Apiaceae) from Southeastern Europe were statistically analyzed to evaluate their chemosystematic significance. Eight investigated taxa (H. orphanidis and members of H. sphondylium group: H. sphondylium, H. sibiricum, H. montanum, H. ternatum, H. pyrenaicum subsp. pollinianum, H. pyrenaicum subsp. orsinii and H. verticillatum) belong to the type section of the genus. Additionally analyzed taxon, H. austriacum subsp. siifolium, belongs to H. sect. Wendia. Qualitative and quantitative analysis of the essential oils was performed by GC-FID and GC/MS, and of the furanocoumarins by LC/MS. Furanocoumarins were identified using standards and/or based on UV, MS, 1 H- and ROESY NMR spectra. Multivariate statistics (PCA, nMDS, UPGMA) of analyzed metabolites showed that the investigated taxa were grouped according to their taxonomic classification. PCA revealed the significance of some monoterpenes and (Z)-falcarinol of the underground parts oils, octyl esters of the fruit oils, and most of the total of 12 identified furanocoumarins.

Sesquiterpene lactones from the methanol extracts of twenty-eight Hieracium species from the Balkan Peninsula and their chemosystematic significance.

Four sesquiterpene lactones (SLs), including three undescribed proline-SL conjugates, the guaianolides calophyllamine A and 8-epiixerisamine A, and the eudesmanolide calophyllamine B, were isolated from the methanol extract of Hieracium calophyllum R. Uechtr. (Compositae) flowering heads. Another known guaianolide, crepiside E, was detected in Hieracium L. species for the first time. Their structures were elucidated using extensive 1D and 2D NMR spectroscopy in combination with HRMS. The isolated SLs were used as external standards for qualitative and quantitative LC-MS analysis of the dry methanol extracts of the flowering aerial parts of 28 Hieracium species from the Balkan Peninsula. Guaianolides were the dominant SLs in 27 species studied. The chemosystematic significance of detected SLs was evaluated using multivariate statistics (PCA, nMDS and UPGMA). Differentiation between the main groups was well supported. All four compounds significantly and equally contributed to the differences between the species. In addition, the eudesmanolide calophyllamine B could be a significant chemosystematic marker for H. sect. Villosa (Griseb.) Gremli s.l. and Glauciformia (Freyn) Zahn-Italica (Fr.) Av. Touv.

Methanol Extracts of 28 Hieracium Species from the Balkan Peninsula - Comparative LC-MS Analysis, Chemosystematic Evaluation of their Flavonoid and Phenolic Acid Profiles and Antioxidant Potentials.

INTRODUCTION: Hieracium s. str. represents one of the largest and most complex genera of flowering plants. As molecular genetics seems unlikely to disentangle intricate relationships within this reticulate species complex, analysis of flavonoids and phenolic acids, known as good chemosystematic markers, promise to be more reliable. Data about pharmacological activity of Hieracium species are scarce. OBJECTIVE: Evaluation of the chemosystematic significance of flavonoids and phenolic acids of methanol extracts of aerial flowering parts of 28 Hieracium species from the Balkans. Additionally, investigation of antioxidant potentials of the extracts. METHODS: Comparative qualitative and quantitative analysis of flavonoids and phenolic acids was performed by LC-MS. Multivariate statistical data analysis included non-metric multidimensional scaling (nMDS), unweighted pair-group arithmetic averages (UPGMA) and principal component analysis (PCA). Antioxidant activity was evaluated using three colorimetric tests. RESULTS: Dominant phenolics in almost all species were luteolin type flavonoids, followed by phenolic acids. Although the investigated Hieracium species share many compounds, the current classification of the genus was supported by nMDS and UPGMA analyses with a good resolution to the group level. Hieracium naegelianum was clearly separated from the other investigated species. Spatial and ecological distances of the samples were likely to influence unexpected differentiation of some groups within H. sect. Pannosa. The vast majority of dominant compounds significantly contributed to differences between taxa. The antioxidant potential of the extracts was satisfactory and in accordance with their phenolics composition. CONCLUSIONS: Comparative LC-MS analysis demonstrated that flavonoids and phenolic acids are good indicators of chemosystematic relationships within Hieracium, particularly between non-hybrid species and groups from the same location. Copyright © 2017 John Wiley & Sons, Ltd.

Edible wild plant Heracleum pyrenaicum subsp. orsinii as a potential new source of bioactive essential oils.

Many Heracleum L. taxa (Apiaceae) are used as food and spices, and in traditional medicine. In this work, the chemical composition of Heracleum pyrenaicum subsp. orsinii (Guss.) F. Pedrotti and Pignatti root, leaf and fruit essential oils, their antimicrobial activity and cytotoxic effect on malignant and normal cells were investigated for the first time. The composition of the oils was analyzed by GC and GC-MS. Monoterpenes prevailed in the root oil, with ß-pinene (38.6%) being dominant, while in the leaf oil, sesquiterpenes, mostly (E)-nerolidol (20.5%) and (E)-caryophyllene (17.0%), were the most abundant constituents. The fruit oil contained the majority of aliphatic esters, mainly octyl acetate (36.8%) and octyl hexanoate (22.1%). The antimicrobial activity was determined by microdilution method against eight bacteria and eight fungi (standard strains, clinical or food isolates). The best antibacterial activity, better than the activity of ampicillin, was shown by the root oil against Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa. The strongest antifungal activity, stronger than the activity of ketoconazole, was exhibited by the leaf and root oils against Trichoderma viride, and by the root oil against Aspergillus ochraceus. The cytotoxic effect of the oils, determined by MTT test, was prominent against malignant HeLa, LS174 and A549 cells (IC50 = 6.49-14.56 µg/mL). On the other hand, the oils did not show toxicity against normal MRC-5 cells at tested concentrations (IC50 > 200.00 µg/mL). It can be concluded that investigated H. pyrenaicum subsp. orsinii oils represent potential new raw materials for food and pharmaceutical industry.

Laserpitium zernyi Hayek Flower and Herb Extracts: Phenolic Compounds, and Anti-edematous, Antioxidant, and Antimicrobial Activities.

Phenolic compounds and different biological activities of the dry MeOH extracts of the flowers and the herb (aerial parts without flowers) of Laserpitium zernyi Hayek (Apiaceae) were investigated. The total phenolic contents in the extracts were determined spectrophotometrically using Folin-Ciocalteu reagent. In both extracts, apigenin, luteolin, their 7-O-glucosides, and chlorogenic acid were detected by HPLC. Identified phenolics were quantified in both extracts, except luteolin in L. zernyi herb extract. The extracts (p.o.) were tested for anti-edematous activity in a model of carrageenan (i.pl.) induced rat paw edema. Antioxidant activity of the extracts was assessed by FRAP assay and DPPH and • OH radicals scavenging tests. Antimicrobial activity was investigated using broth microdilution test against five Gram-positive and three Gram-negative bacteria, as well as against two strains of Candida albicans. The polyphenol-richer flower extract exerted higher anti-edematous and antioxidant activities. The herb extract exhibited better antimicrobial effect against Micrococcus luteus, Enterococcus faecalis, Bacillus subtilis, and Pseudomonas aeruginosa, while against other tested microorganisms, the activity of both extracts was identical. Demonstrated biological activities of L. zernyi flower and herb extracts represent a good basis for their further investigation as potential new herbal medicinal raw materials.

Essential oils of three cow parsnips - composition and activity against nosocomial and foodborne pathogens and food contaminants.

Although some widespread, native cow parsnips (Heracleum L. spp., Apiaceae) had broad medicinal and culinary applications throughout history, the knowledge about their volatile constituents is insufficient. This work investigates the composition and bioactivities of H. sphondylium L. (HSPH), H. sibiricum L. (HSIB) and H. montanum Schleich. ex Gaudin (HMON) essential oils. The composition was tested by GC and GC-MS. (Z)-ß-Ocimene was the most abundant in HSPH (28.9%) and HMON (20.4%) root oils, while in HSIB root oil, ß-pinene (26.2%), methyl eugenol (22.3%) and elemicin (25.6%) prevailed. Leaf and flower oils were dominated by various sesquiterpenes (germacrene D, ß-sesquiphellandrene, (E)-ß-farnesene and/or (E)-caryophyllene) and/or phenylpropanoids (apiole, methyl eugenol, elemicin and/or (Z)-isoelemicin). Octyl acetate (57.5-67.1%) was the main constituent of all fruit oils. The antimicrobial activity was screened by a microdilution method against eight bacteria and eight fungi. The strongest antimicrobial effect, in several cases better than the activity of antibiotics, was shown by HSPH (MICs = 0.12-3.30 mg mL-1) and HMON (MICs = 0.10-1.30 mg mL-1) flower oils against bacteria, and HSIB fruit oil against fungi (MICs = 0.15-0.40 mg mL-1). The MTT test revealed that the oils were not or weakly cytotoxic against human malignant HeLa, LS174 and/or A549 cells (except HSPH root oil; IC50 = 5.72-24.31 µg mL-1) and that tested oils were not toxic against human normal MRC-5 cells (at 200.00 µg mL-1). Significant activity observed against microorganisms that are the common cause of foodborne diseases, food contamination and/or hospital-acquired infections justifies certain traditional uses of the investigated plants and represents a good basis for further research of these Heracleum oils.

Chemical Composition and Bioactivity of the Essential Oils of Heracleum pyrenaicum subsp. pollinianum and Heracleum orphanidis.

The objective of this research was to analyze the chemical composition, antimicrobial and cytotoxic activity of Heracleun pyrenaicum subsp. pollinianum (Bertol.) F. Pedrotti & Pignatti (HPP) and H. orphanidis Boiss. (HO) essential oils. The composition of the oils was analyzed by GC and GC-MS. ß-Pinene (35.1%) was the most abundant compound in HPP root oil, while (Z)-falcarinol (80.0%) dominated in HO root oil. (E)-Nerolidol (28.5%) was the main constituent in HPP leaf oil. HPP fruit oil, as well as HO leaf and fruit oils mainly contained aliphatic esters, mostly octyl acetate (50.5-84.5%). Antimicrobial screening was performed by microdilution method against eight bacterial and eight fungal strains. The strongest antibacterial activity was shown by both root oils (MICs 0.02-0.60 mg/mL and MBCs 0.04-2.50 mg/mL for HPP, and MICs 0.02-1.25 mg/mL and MBCs 0.04-2.50 mg/mL for HO), while the best antifungal potential was exhibited by HPP fruit oil (MICs 0.30-0.60 mg/mL and MFCs 0.60-1.25 mg/mL) and HO leaf oil (MICs 0.15-0.63 mg/mL and MFCs 0.30-1.25 mg/mL). The tested root and fruit oils exhibited strong cytotoxic effect, which was determined by MTT test against HeLa (IC50 7.53-21.07 µg/mL) and LS174 (IC50 24.16-58.86 µg/mL) cell lines.

Chemical Composition, Antimicrobial and Cytotoxic Activity of Heracleum verticillatum Pancic and H. ternatum Velen. (Apiaceae) Essential Oils.

In this work, the chemical composition, antimicrobial and cytotoxic activity of Heracleum verticillatum Pancic and H. ternatum Velen. root, leaf, and fruit essential oils were investigated. The composition was analyzed by GC and GC/MS. Heracleum verticillatum and H. ternatum root oils were dominated by monoterpenes, mostly ß-pinene (23.5% and 47.3%, respectively). Heracleum verticillatum leaf oil was characterized by monoterpenes, mainly limonene (20.3%), and sesquiterpenes, mostly (E)-caryophyllene (19.1%), while H. ternatum leaf oil by the high percentage of phenylpropanoids, with (Z)-isoelemicin (35.1%) being dominant constituent. Both fruit oils contained the majority of aliphatic esters, mostly octyl acetate (42.3% in H. verticillatum oil and 49.0% in H. ternatum oil). The antimicrobial activity of the oils was determined by microdilution method against eight bacterial and eight fungal strains. The strongest effect was exhibited by H. verticillatum root oil, particularly against Staphylococcus aureus, Salmonella typhimurium (MICs = 0.14 mg/ml, MBCs = 0.28 mg/ml), and Trichoderma viride (MIC = 0.05 mg/ml, MFC = 0.11 mg/ml). Cytotoxic effect was determined by MTT test against malignant HeLa, LS174, and A549 cells (IC50 = 5.9 - 146.0 µg/ml), and against normal MRC-5 cells (IC50 > 120.1 µg/ml). The best effect was exhibited by H. verticillatum root oil on A549 cells (IC50 = 5.9 µg/ml), and H. ternatum root oil against LS174 cells (IC50 = 6.7 µg/ml).