Optimization of Ultrasound-Assisted Extraction of Phenolics from Sideritis raeseri Using Response Surface Methodology.

In this study we define the optimal conditions for ultrasound-assisted extraction of bioactive polyphenols from S. raeseri aerial parts using response surface methodology. The influence of ethanol concentration (10-90%), extraction temperature (20-80 °C), extraction time (10-60 min), and solid-to-solvent ratio (1:10-1:50) on total phenolic content as well as on content of individual flavonoids, and hypolaetin and isoscutellarein derivatives was studied. For the experimental design, a central composite design was chosen. In the obtained extracts, the following ranges of targeted compounds were detected: total phenol from 19.32 to 47.23 mg GAE/g dw, HYP from 1.05 to 11.46 mg/g dw, ISC 1 from 0.68 to 10.68 mg/g dw, and ISC 2 from 0.74 to 15.56 mg/g dw. The optimal extraction conditions were set as: ethanol concentration of 65%, extraction time of 50 min, extraction temperature of 63 °C, and solid-to-solvent ratio of 1:40. Contents of TP, HYP, ISC 1, and ISC 2 in optimal extracts were 47.11 mg GAE/g dw, 11.73 mg/g dw, 9.54 mg/g dw, and 15.40 mg/g dw, respectively. Experimentally set values were in good agreement with those predicted by the response surface methodology model, indicating suitability of the used model, as well as the success of response surface methodology in optimizing the conditions of the extraction.

Antiviral Effects of Pomegranate Peel Extracts on Human Norovirus in Food Models and Simulated Gastrointestinal Fluids.

Human noroviruses (HuNoV) are the dominant cause of viral gastroenteritis in all age groups worldwide. In this study, we investigated the effects of pomegranate peel extract (PPE) on the reduction of HuNoV in different food models, on surfaces of fresh produce (green onion and cherry tomato), in low-fat milk, and simulated gastrointestinal fluids. The antiviral efficacy of PPE against HuNoV was evaluated by quantifying the number of residual virus genomes using a quantitative reverse transcription PCR (qRT-PCR) assay. Pomegranate peel, considered as a waste product of industrial processing, is known for beneficial health effects and broad antimicrobial activity due to the high content of phenolic compounds and tannins. PPE showed significant antiviral properties against HuNoV both in phosphate-buffered saline (PBS) and simulated gastric fluid. The reduction of HuNoV by pomegranate juice was lower than with PPE, which could be attributed to the lower content of antimicrobial compounds. A pretreatment of cherry tomato and green onion surfaces with PPE significantly reduced the amount of HuNoV particles that adhered to those surfaces during subsequent virus suspension treatment. A detrimental effect of PPE on HuNoV structure was confirmed by transmission electron microscopy. Our results indicate that PPE is a natural antiviral agent effective against food-borne noroviruses.

Comparative Study of Subcritical Water and Microwave-Assisted Extraction Techniques Impact on the Phenolic Compounds and 5-Hydroxymethylfurfural Content in Pomegranate Peel.

Two environmentally friendly innovative extraction techniques - subcritical water (SWE) and microwave-assisted extraction (MAE) were applied for the extraction of phenolics from pomegranate peel. The impact of process conditions (SWE: temperature 100-220 °C, extraction time 5-30 min; MAE: solvent water and 50% ethanol, irradiation power 470 and 800 W) on the quality of extracts in terms of the content of total phenolics, total flavonoids, major phenolic constituents (gallic acid, ellagic acid, punicalin, punicalagin), as well as 5-hydroxymethylfurfural(HMF) amount was investigated. For SWE, temperature of 130 °C and 20 min extraction time were found optimal for obtaining high content of bioactive compounds and minimizing the yield of HMF. During MAE, phenolic compounds were effectively extracted by using lower microwave power and 50% ethanol. Comparing two techniques, MAE is more efficient than SWE for the extraction of phenolics from pomegranate peel while obtaining a HMF-free extracts.

Chokeberry polyphenols preservation using spray drying: effect of encapsulation using maltodextrin and skimmed milk on their recovery following in vitro digestion.

Aim: Microencapsulation of chokeberry extracts was performed in order to improve functionality, stability, and bioavailability of extracted polyphenols.Methods: Chokeberry fruits and juice by-product (waste) extracts were spray-dried by using two carriers, maltodextrin and skimmed milk. Morphological and physicochemical characteristics of the obtained powders were analysed. In vitro simulated digestion model was used as an indicator of polyphenolics bioavailability.Results: The moisture content varied between 3.39 and 4.61%, zeta potential had negative values (35-39 mV), maltodetrin powders were smaller (4.27-5.12 µm) compared to skimmed ones (8.50-11.01 µm). All microparticles exhibited high encapsulation efficiency of total polyphenols and anthocyanins (73-97% and 63-96%, respectively). For both extract types, maltodextrin powders released higher phenolics content compared to skimmed milk. During in vitro digestion, maltodextrin exhibited a higher protective effect on both active compounds.Conclusion: Taking into account the obtained results, chokeberry polyphenols stability might be improved using spray drying technique, and maltodextrin showed better properties.

Bioassay-Guided Skin-Beneficial Effects of Fractionated Sideritis raeseri subsp. raeseri Extract.

This study aimed to evaluate the potentials for skin-beneficial effects of the hydroethanolic extract of the aerial parts of ironwort (Sideritis raeseri Boiss. & Heldr. subsp. raeseri) and its fractions (petroleum ether, ethyl acetate, n-butanol and water). For these purposes, chemical analysis and in vitro antioxidant, anti-tyrosinase and antimicrobial assays, as well as determination of sun protection factor (SPF), were performed. The highest total phenolic content and quantity of individual flavonoids, chlorogenic acid and verbascoside were determined in the n-butanol fraction, which is in line with results obtained for antioxidant activity. The greatest antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, the most common causative agents of the skin infections in humans, was exhibited by the ethyl acetate fraction. The strongest anti-tyrosinase activity was shown by the hydroethanolic extract (52.64% of inhibition at 100 µg/mL). Almost all tested samples showed photoprotective activity with SPF higher than 6 obtained at a low concentration of 0.1 mg/mL, which was more than 15 for the butanol fraction. These findings revealed that the hydroethanolic extract of S. raeseri aerial parts could be a valuable ingredient in the formulation of cosmetic and medicinal products intended to overcome various skin disorders.

Gentianella lutescens subsp. carpatica J. Holub.: Shoot Propagation In Vitro and Effect of Sucrose and Elicitors on Xanthones Production.

In vitro shoot culture of the endangered medicinal plant Gentianella lutescens was established from epicotyl explants cultured on MS basal medium with 0.2 mg L-1 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L-1 indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones-mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8-O-glucoside and bellidifolin-8-O-glucoside-in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 µM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2-3-fold compared to the control shoots. Elicitors at 100-300 µM increased the accumulation of mangiferin, demethylbellidifolin-8-O-glucoside, and bellidifolin-8-O-glucoside almost equally, while MeJA at the highest concentration of 500 µM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of G. lutescens and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.

Effect of Type and Concentration of Carrier Material on the Encapsulation of Pomegranate Peel Using Spray Drying Method.

This study aimed to establish a procedure for pomegranate peel (PP) valorization and attainment of stable extracts with preserved bioactive compounds. The technology applied was spray drying with carbohydrate-based (maltodextrin, MD) and protein-based (whey protein, WP) carrier materials in different concentrations (80, 100, and 120%). What was analyzed was the impact of the type and concentration of carrier material on the stability and quality of the final encapsulated powder. The best results were achieved when the PP extract was microencapsulated with the carbohydrate-based carrier (100%), where it had the highest encapsulation efficiency (EE) (88.63%), hygroscopicity (15.17%), and water solubility index (87.04%). The moisture content was in the range of 3.69-4.60% and 4.21-5.84% for MD and WP, respectively, indicating that both are suitable for long-term storage. It was observed that changes in carrier concentration significantly influenced most of the powders' physicochemical properties. Microencapsulation using MD yielded a higher content of punicalin, punicalagin, gallic, and ellagic acid than those with WP. Overall results demonstrated that carbohydrate-based microencapsulation can be utilized efficiently for the protection of powder stability and phytochemical characteristics.

Relaxant effect of the ethanol extract of Helichrysum plicatum (Asteraceae) on isolated rat ileum contractions.

Helichrysum plicatum (Turkish Helichrysum) has been used in folk medicine for the treatment of gastric and hepatic disorders. The aim of the present study was to examine the relaxant activity of an extract of H. plicatum flowers on isolated rat ileum. Segments of ileum of rats were suspended in an organ bath. Cumulative concentrations of H. plicatum ethanol extract induced a relaxant effect on spontaneous rat ileum contractions. H. plicatum extract caused a mean contractile response of 81.68 +/- 6.17% (at a dose of 0.01 mg/mL) and 30.08 +/- 9.07% (at a dose of 1 mg/mL). A similar effect was observed with papaverine (0.01-3 microg/mL). H. plicatum extract (0.01-1 mg/mL) relaxed high K+ (80 mM) precontractions, an effect similar to that caused by papaverine (0.01-3 microg/mL). The plant extract (0.03-0.3 mg/mL) also induced a significant depression of the cumulative concentration response curve for acetylcholine (5-1500 nM) (p < 0.01). Atropine (140 nM) abolished the acetylcholine effect. The extract (0.03-0.3 mg/mL) reduced the histamine (1-300 nM) and BaCl2 (3-900 microM) induced contractions (p < 0.01). Our results showed the relaxant effect of the ethanol extract of Helichrysum plicatum flowers on the isolated rat intestine Extract of H. plicatum can inhibit the spontaneous ileum contractions and contractions induced by acetylcholine, histamine, barium and potassium ions.

Biochemical properties of red currant varieties in relation to storage.

Eleven red currant varieties (Ribes rubrum L.) cultured in Serbia were evaluated for some of their biochemical properties such as total phenolics, anthocyanins, ascorbic acid, invert sugar, soluble solid content, and acidity. The average amount of ascorbic acid varied from 50.5-71.6 mg/100 g FW, while concentration of invert sugars ranged from 6.0%-9.0%. The highest amounts of total phenolics and anthocyanins were detected in variety Redpoll (153.4 mg GAE/100 g FW and 19.3 mg/100 g, respectively). Red currants were processed to juice, and the phenolic and anthocyanin contents changed as a result of processing. Berries and juices were long-term stored at -18 °C and changes in phytochemicals were monitored. In berries, storage caused the decrease of ascorbic acid content up to 49%, and a general reduction of total phenolics was also noticed. In juices, total phenolics content increased after one year of storage. In both berries and juices total anthocyanins increased during storage by up to 85% and 50%, respectively. This study demonstrates that certain varieties, namely Redpoll, Jonkheer and London Market are good source of phytochemicals, retaining the nutritional value during processing and storage.

Gentiana clusii Perr.&Song.: Enhanced production of secondary metabolites by in vitro propagation.

Shoot and root in vitro culture of endemic European species Gentiana clusii was established for the first time. The effects of different concentrations of benzyl adenine (BA), 6-phurphurylaminopurine (KIN), indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) on shoot propagation and rooting of G. clusii were investigated. The optimal in vitro conditions for shoot propagation and long-term maintenance were achieved using woody plant medium (WPM) supplemented with 0.5 mg l-1 KIN, and subsequent application of IBA at 0.5 mg l-1 significantly improved rooting of these shoots. Root culture was established from excised root tips cultured in ½ MS liquid media with increasing concentrations of IBA (0.1-1.0 mg l-1). A high root growth rate and considerable biomass yield were obtained by addition of 1.0 mg l-1 IBA. HPLC analysis revealed that in vitro culture considerably promoted the production of secondary metabolites in G. clusii. The selected protocol for shoot propagation (WPM + 0.5 mg l-1 KIN) increased the content of sweroside, gentiopicrin and norswertianin-1-O-primeveroside (N-1-P) for more than 2-fold compared with the wild plants. IBA promoted N-1-P and norswertianin production in root cultures; their contents were enhanced 6.4- and 18.6-fold, respectively, compared with the wild plants. The extract of these roots displayed the highest antioxidant capacity (IC50 = 66.57 µg ml-1). The established shoot and root propagation protocols facilitate in vitro conservation of G. clusii, and provides a promising tool for the large scale production of valuable secoiridoids and xanthones.

Spontaneous plant regeneration and production of secondary metabolites from hairy root cultures of Centaurium erythraea Rafn.

We have established an efficient protocol for plant regeneration and production of secondary metabolites in hairy root culture of Centaurium erythraea Rafn. Because the hairy roots and regenerated plants produce bitter secoiridoid glucosides and xanthones similar to the plants in nature, the use of in vitro cultures as an alternative source of their production is feasible. This chapter describes a protocol for the induction of adventitious shoots and transgenic plants from hairy root cultures of C. erythraea and their phytochemical analysis.

Antimicrobial activity of Gentiana lutea L. extracts.

Methanolic extracts of flowers and leaves of Gentiana lutea L., together with the isolated compounds mangiferin, isogentisin and gentiopicrin, were used to investigate the antimicrobial activity of the plant. A variety of Gram-positive and Gram-negative bacteria as well as the yeast Candida albicans has been included in this study. Both extracts and isolated compounds showed antimicrobial activity with MIC values ranging from 0.12-0.31 mg/ml. Our study indicated that the synergistic activity of the pure compounds may be responsible for the good antimicrobial effect of the extracts. Quantification of the secondary metabolites was performed using HPLC.

Phenolic content and radical scavenging capacity of berries and related jams from certificated area in Serbia.

Chemical composition and the effects of domestic processing and storage were studied in organically cultivated or wild growing berries, harvested in certificated area in Serbia. The total phenolics content in fresh berries ranged from 380 to 1660 mg GAE/100 g. The phenolic content decreased during the processing to jams, and storage in almost all samples, except in black currant. Among fresh berries, the highest amount of total anthocyanins was found in bilberry (0.61 +/- 0.03 %). Processing and storage caused decrease in anthocyanin content. The most ubiquitous anthocyanin aglycone was found to be cyanidin. Ellagic acid was detected in fresh and frozen black raspberries and related jams, and its content increases during thermal processing to jam. Among fresh berries, bilberry showed the strongest DPPH radical scavenging activity with an IC(50) value of 0.82 mg/ml. However, frozen as well as processed berries in our study still showed comparable high values of radical scavenging activity. After 9 months of storage all investigated jams were microbiologicaly correct. Regardless of the losses in the amount of total phenolics, total anthocyanins and radical scavenging activity, frozen berries and related jams are still excellent sources of nutritional substances with antioxidant potential.

Antiglioma action of xanthones from Gentiana kochiana: Mechanistic and structure-activity requirements.

The present study identifies xanthones gentiakochianin and gentiacaulein as the active principles responsible for the in vitro antiglioma action of ether and methanolic extracts of the plant Gentiana kochiana. Gentiakochianin and gentiacaulein induced cell cycle arrest in G(2)/M and G(0)/G(1) phases, respectively, in both C6 rat glioma and U251 human glioma cell lines. The more efficient antiproliferative action of gentiakochianin was associated with its ability to induce microtubule stabilization in a cell-free assay. Both the xanthones reduced mitochondrial membrane potential and increased the production of reactive oxygen species in glioma cells, but only the effects of gentiakochianin were pronounced enough to cause caspase activation and subsequent apoptotic cell death. The assessment of structure-activity relationship in a series of structurally related xanthones from G. kochiana and Gentianella austriaca revealed dihydroxylation at positions 7, 8 of the xanthonic nucleus as the key structural feature responsible for the ability of gentiakochianin to induce microtubule-associated G(2)/M cell block and apoptotic cell death in glioma cells.

Sandy Everlasting (Helichrysum arenarium (L.) Moench): Botanical, Chemical and Biological Properties.

Sandy everlasting [Helichrysum arenarium (L.) Moench] is herbaceous perennial plant belonging to Asteraceae family and it is native to Europe, Central Asia, and China. It belongs to the section HELICHRYSUM (Asteraceae family, genus Helichrysum) along with H. plicatum DC. Prodr., which very similar phenolic profile and H. italicum (Roth), which is widely used for essential oil extraction. Its flowers have a long tradition in European ethnomedicine as a cholagogue, choleretic, hepatoprotective, and detoxifying herbal drug. The flowers are rich in phenolic compounds including flavonoids, chalcones, phenolic acids, coumarins, and pyrones. Apart from polyphenols, other compounds such as sterols, lignans, and glycosides of aromatic compounds have been also isolated from H. arenarium. The majority of authors confirm that the most important group of compounds responsible for biological activities is flavonoids. Moreover, significant activities of naringenin, one of the main flavonoids of H. arenarium, were reported. On the other hand, there are no clinical data about testing the extracts or preparations based on H. arenarium. Although H. arenarium is well known in phytotherapy for its potential in the treatment of gallbladder disease and are classified as endangered species in a number of European countries, very few data about its cultivation are available in the literature.

Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique.

Traditional maceration method was used for the extraction of polyphenols from chokeberry (Aronia melanocarpa) dried fruit, and the effects of several extraction parameters on the total phenolics and anthocyanins contents were studied. Various solvents, particle size, solid-solvent ratio and extraction time have been investigated as independent variables in two level factorial design. Among examined variables, time was not statistically important factor for the extraction of polyphenols. The optimal extraction conditions were maceration of 0.75mm size berries by 50% ethanol, with solid-solvent ratio of 1:20, and predicted values were 27.7mgGAE/g for total phenolics and 0.27% for total anthocyanins. Under selected conditions, the experimental total phenolics were 27.8mgGAE/g, and total anthocyanins were 0.27%, which is in agreement with the predicted values. In addition, a complementary quantitative analysis of individual phenolic compounds was performed using HPLC method. The study indicated that maceration was effective and simple technique for the extraction of bioactive compounds from chokeberry fruit.

Neuropharmacological evaluation of diethylether extract and xanthones of Gentiana kochiana.

Diethylether extract of aerial parts of Gentiana kochiana mostly consists of two tetraoxygenated xanthones: gentiacaulein (1,7-dihidroxy-3,8-dimethoxyxanthone; 76.1%) and gentiakochianin (1,7,8-trihidroxy-3-methoxyxanthone; 14.2%). The extract and these xanthones were evaluated for the CNS pharmacological activity in rodents. In vitro assays on rat brain preparations revealed insignificant interaction of the compounds with the specific dopamine and serotonin receptors or synaptosomal uptake of serotonin. However, the extract and gentiacaulein strongly inhibited rat microsomal MAO A (IC50=0.22 microg/ml and 0.49 microM, respectively). Their effects on MAO B and a gentiakochianin blocking potential on both MAO enzymes were moderate. Behavioral examinations on mice showed that 10 day s.c. administration of the extract (20 mg/kg) significantly decreased immobility score in a forced swimming test and strongly inhibited ambulation and stereotypy in an open-field test. These effects resembled those induced by 10 mg/kg imipramine. The ex vivo MAO A activity in crude brain mitochondrial fraction of mice treated with 20 mg/kg of the extract was significantly elevated, whilst that outside brain nerve terminals was declined. This study suggests some antidepressant therapeutic potential of G. kochiana, particularly of gentiacaulein, with an ambiguity whether pharmacological mechanism could be related only to the central inhibition of MAO A.

Antioxidant and antiproliferative activity of chokeberry juice phenolics during in vitro simulated digestion in the presence of food matrix.

Chokeberry juice was subjected to in vitro gastric digestion in the presence of food matrix in order to determine the changes in polyphenol content and antioxidant activity. Addition of food matrix immediately decreased the total phenolic content, anthocyanin content, DPPH scavenging activity as well as total reducing power by 36%, 90%, 45% and 44%, respectively. After in vitro digestion, total phenolic content, anthocyanin content and reducing power are slightly elevated, but they are still lower than in initial non-digested juice. The effect of digested juice on Caco-2 cells proliferation was also studied, and the reduction of proliferative rate by approximately 25% was determined. Our results suggested that although a large proportion of chokeberry phenolics undergo transformation during digestion they are still potent as antioxidant and antiproliferative agents.

Biochemical properties of the fresh and frozen black currants and juices.

Thirteen black currant varieties (Ribes nigrum L.) cultured in Serbia were characterized for their pomological properties and chemical composition (total phenolics, total anthocyanins, anthocyanin aglycones, sugars, and vitamin C). The average amount of vitamin C varied from 122.4 to 193.2 mg/100 g fresh weight (FW), while concentration of invert sugars ranged from 6.3% to 11.1%. The highest amounts of total phenolics and anthocyanins were detected in variety Ometa (278.9 mg of gallic acid equivalents per 100 g of FW [mg GAE/100 g FW] and 135.4 mg/100 g, respectively). Quantitative analyses of anthocyanin aglycones in berries were performed using high-performance liquid chromatography, and delphinidin was found to be dominant compound in 11 varieties. Total phenolics and anthocyanins contents decreased during the processing of berry fruits to juices, and the reduction of anthocyanins was more pronounced, 12%-80%. The radical scavenging activity of black currant juices was investigated by 1,1-diphenyl-2-picrylhydrazyl, and the IC(50) value ranged from 1.9 to 4.0 mg/mL. Our results also showed that freezing as a way of preservation and storage could save important phytochemicals and health benefits of berries and berry juices. The amount of total phenolics in berries increased during 1 year of storage by 46.09%-171.76% and in juices by even 107.58%, while the amount of total anthocyanins in berries and juices decrease by 5.63%-52.76% and 13.04%-36.82%, respectively.

Antibacterial activity of Veronica montana L. extract and of protocatechuic acid incorporated in a food system.

This study was designed to evaluate the antibacterial activity of the Veronica montana L. water extract and its main phenolic compound, protocatechuic acid. The antibacterial activity was determined by microdilution assay against six strains of Gram-positive and Gram-negative bacteria. Listeria monocytogenes was the most sensitive of the tested bacterial species. Antibacterial preserving properties of protocatechuic acid were also evaluated after its incorporation in cream cheese, using L. monocytogenes as commonly cheese contaminant. The compound successfully inhibited L. monocytogenes development in cream cheese, at room temperature and in refrigerator (25°C and 4°C, respectively), after 3days of inoculation. Sensory evaluation was carried out in order to validate the mentioned food system. A possible mode of action of the tested compound towards bacterial cells was assessed and appears to be direct lysis of pathogenic cytoplasmic membrane. Prediction of pharmacokinetic properties was also performed using computational analyzes. The obtained results can serve as an important platform for the development of effective natural preservatives.