Biological activity of silver nanoparticles synthesized using viticultural waste.

This research paper presents a novel approach to the green synthesis of silver nanoparticles (AgNPs) using viticultural waste, allowing to obtain NP dispersions with distinct properties and morphologies (monodisperse and polydisperse AgNPs, referred to as mAgNPs and pAgNPs) and to compare their biological activities. Our synthesis method utilized the ethanolic extract of Vitis vinifera pruning residues, resulting in the production of mAgNPs and pAgNPs with average sizes of 12 ± 5 nm and 19 ± 14 nm, respectively. Both these AgNPs preparations demonstrated an exceptional stability in terms of size distribution, which was maintained for one year. Antimicrobial testing revealed that both types of AgNPs inhibited either the growth of planktonic cells or the metabolic activity of biofilm sessile cells in Gram-negative bacteria and yeasts. No comparable activity was found towards Gram-positives. Overall, pAgNPs exhibited a higher antimicrobial efficacy compared to their monodisperse counterparts, suggesting that their size and shape may provide a broader spectrum of interactions with target cells. Both AgNP preparations showed no cytotoxicity towards a human keratinocyte cell line. Furthermore, in vivo tests using a silkworm animal model indicated the biocompatibility of the phytosynthesized AgNPs, as they had no adverse effects on insect larvae viability. These findings emphasize the potential of targeted AgNPs synthesized from viticultural waste as environmentally friendly antimicrobial agents with minimal impact on higher organisms.

Chemical Composition and Antifungal Activity of Myrcia multiflora and Eugenia florida Essential Oils.

The essential oils of three specimens of Myrcia multiflora (A, B and C) and Eugenia florida were extracted by hydrodistillation, and the chemical compositions from the essential oils were identified by gas chromatography and flame ionization detection (CG/MS and CG-FID). The fungicide potential of the EOs against five fungicide yeasts was assessed: Candida albicans INCQS-40175, C. tropicalis ATCC 6258, C. famata ATCC 62894, C. krusei ATCC 13803 and C. auris IEC-01. The essential oil of the specimen Myrcia multiflora (A) was characterized by the major compounds: α-bulnesene (26.79%), pogostol (21.27%) and δ-amorphene (6.76%). The essential oil of the specimen M. multiflora (B) was rich in (E)-nerolidol (44.4%), (E)-γ-bisabolene (10.64%) and (E,E)-α-farnesene (8.19%), while (E)-nerolidol (92.21%) was the majority of the specimen M. multiflora (C). The sesquiterpenes seline-3,11-dien-6-α-ol (12.93%), eremoligenol (11%) and γ-elemene (10.70%) characterized the chemical profile of the EOs of E. florida. The fungal species were sensitive to the essential oil of M. multiflora (B) (9-11 mm), and the lowest inhibitory concentration (0.07%) was observed in the essential oil of M. multiflora (A) against the yeasts of C. famata. Fungicidal action was observed in the essential oils of M. multiflora (A) against C. famata, with an MIC of 0.78 µL/mL and 3.12 µL/mL; C. albicans, with an MFC of 50 µL/mL and M. multiflora (C) against C. albicans; and C. krusei, with a MFC of 50 µL/mL.

Hydroxygenkwanin Increases the Sensitivity of Liver Cancer Cells to Chemotherapy by Inhibiting DNA Damage Response in Mouse Xenograft Models.

Molecules involved in DNA damage response (DDR) are often overexpressed in cancer cells, resulting in poor responses to chemotherapy and radiotherapy. Although treatment efficacy can be improved with the concomitant use of DNA repair inhibitors, the accompanying side effects can compromise the quality of life of patients. Therefore, in this study, we identified a natural compound that could inhibit DDR, using the single-strand annealing yeast-cell analysis system, and explored its mechanisms of action and potential as a chemotherapy adjuvant in hepatocellular carcinoma (HCC) cell lines using comet assay, flow cytometry, Western blotting, immunofluorescence staining, and functional analyses. We developed a mouse model to verify the in vitro findings. We found that hydroxygenkwanin (HGK) inhibited the expression of RAD51 and progression of homologous recombination, thereby suppressing the ability of the HCC cell lines to repair DNA damage and enhancing their sensitivity to doxorubicin. HGK inhibited the phosphorylation of DNA damage checkpoint proteins, leading to apoptosis in the HCC cell lines. In the mouse xenograft model, HGK enhanced the sensitivity of liver cancer cells to doxorubicin without any physiological toxicity. Thus, HGK can inhibit DDR in liver cancer cells and mouse models, making it suitable for use as a chemotherapy adjuvant.

ɛ-polylysine coating with stinging nettle extract for fresh beef preservation.

Combination effects of ɛ-polylysine coating (0.5 and 1%) and stinging nettle extract (3, 6 and 9%) on quality properties and shelf life of beef meat (2 × 2 × 2 cm) was evaluated at 4 °C for 12 days. The results indicated that ɛ-polylysine (ε-PL) coating with stinging nettle extract (SNE) had no significant effects on ash, fat, protein and moisture content among packaged beef samples in polyethylene bags (in atmosphere condition). At the end of storage, beef samples coated with 1% ε-PL and 9% SNE had significantly lower TBARS and TVB-N values compared to those found in control. Furthermore, 1% ε-PL coating with SNE 9% showed the highest inhibitory effects against molds and yeast, total viable counts (TVC) and coliforms during storage. However, sensory evaluation showed that samples coated with 1% ε-PL and 6% SNE had the highest scores for overall acceptability compared to the other groups. Based on the obtained results, ε-PL coating with SNE could be effectively used for extending the beef meat shelf life without negative effects on sensory attributes.

In vitro estimation of superfluid critical extracts of some plants for their antimicrobial potential, phytochemistry, and GC-MS analyses.

BACKGROUND: Along with swift economic evolution and continuous amelioration of lifestyle, people at present are paying more attention to health issues. Synthetic drugs will be compensated with other natural ones that belong to natural origin. Plants have always been considered as sources of several compounds that are used in many fields, especially human and animal health, starting from boosting immunity to the treatment of infectious diseases caused by some pathogenic microbes such as bacteria, fungi as well as viruses. This study aimed to incorporate some types of plants within the antimicrobial portfolio through the examination of different six plants which were Cichorium intybus, Cinnamomum camphora, Commiphora myrrha, Foeniculum vulgare, Nerium oleander, and Spartium junceum. As well, attempting to identify the active constituents of their extracts using GC-MS. MATERIALS AND METHODS: All selected plants were analyzed to determine their phytochemical composition such as phenolics, alkaloids, flavonoids, terpenoids, and so on. The extraction step was done by sophisticated equipment called supercritical fluid extractor SFE through adjustment of specific conditions include temperature, time, flow rate and pressure to change the behavior of CO2. Testing the antimicrobial activity of each plant extract via agar well diffusion method through the formation of clear zones against a wide range of test microorganisms including both Gram-positive and Gram-negative bacteria as well as yeasts. Finally, attempting to primarily identify the constituents of each plant extract using GC-MS. RESULTS AND DISCUSSION: The crude extract of F. vulgare showed the highest potency against C. albicans, E. faecalis and S. typhimurium, it contains some unique compounds such as squalene, eugenol and isoeugenol while, Extract of C. intybus showed a moderate activity especially against C. lipolytica and MRSA and it includes Vitamin A like compound which indicates antioxidant property. CONCLUSION: Conclusively, fennel gave a promising result as a good wide spectrum antimicrobial agent because it contains some compounds act as antimicrobial agents such as eugenol which was used as food preservatives in addition to squalene which acts as an antioxidant and antimycotic agent so, it will be useful especially while it was used in highly purified form excluding all undesirable subcomponents.

Effect of Addition of Pectins from Jujubes (Ziziphus jujuba Mill.) on Vitamin C Production during Heterolactic Fermentation.

Soluble fibers, including pectins from apple and lemon, are commonly used as prebiotic and to prepare functional foods. The present study aimed to investigate the physicochemical and functional properties of pectins extracted from jujubes (Ziziphus jujuba Mill.). Pectins were extracted from jujubes at three stages of harvesting and characterized by FTIR and SEM analyses. Whole milk inoculated with kefir grains was supplemented by 0.25 mg·mL-1 of pectins. The pH value and vitamin C content were evaluated after 24 and 48 h of fermentation. Pectins from jujubes at the first harvesting stage (PJ1K) showed the lowest methoxylation degree. The addition of pectins enhanced the production of vitamin C during heterolactic process. This result was found to depend on jujube harvesting stage as PJ1K stimulated the growth of yeasts in kefir grains yielding to the highest amount of vitamin C (0.83 ± 0.01 µg·mL-1) compared to other samples (0.53-0.60 µg·mL-1) at 24 h. Lactic acid bacteria diminish pH rapidly with respect to control (4.13 ± 0.05), according to the stage of maturation, reducing its initial value by 38.3% in PJ1K. Besides being an excellent prebiotic, pectins from jujubes could be used to enrich kefir with vitamin C.

Selenium-Enriched Yeast Alleviates Oxidative Stress-Induced Intestinal Mucosa Disruption in Weaned Pigs.

To explore the effect of selenium-enriched yeast (SeY) on intestinal barrier functions in weaned pigs upon oxidative stress, a 2 × 2 factorial design was utilized and thirty-two pigs were randomly assigned into four groups. Pigs with or without exposure to oxidative stress (diquat challenge) were fed with a basal diet or a SeY-containing diet. The trial lasted for 21 days, and result showed that SeY supplementation attenuated body-weight reduction and significantly decreased the serum concentrations of diamine oxidase (DAO) and D-lactic acid in pigs upon diquat challenge (P < 0.05). Diquat challenge decreased the villus height and the ratio of villus height to crypt depth (V/C) in the jejunum and ileum (P < 0.05). However, SeY supplementation not only elevated the villus height and the ratio of V/C (P < 0.05) but also improved the distribution and abundance of tight-junction protein ZO-1 in the jejunum epithelium. Interestingly, SeY supplementation acutely decreased the total apoptosis rate of intestinal epithelial cells in pigs upon diquat challenge (P < 0.05). Moreover, SeY elevated the content of antioxidant molecules such as glutathione peroxidase (GSH-Px) and catalase (CAT) but significantly decreased the content of malondialdehyde (MDA) in the intestinal mucosa (P < 0.05). Importantly, SeY elevated the expression levels of critical functional genes such as the nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), sodium/glucose cotransporter 1 (SGLT1), and B-cell lymphoma-2 (BCL-2) in the intestinal mucosa upon diquat challenge (P < 0.05). Moreover, the expression of caspase-3 was downregulated by SeY in the duodenum and jejunum mucosa (P < 0.05). These results indicated that SeY attenuated oxidative stress-induced intestinal mucosa disruption, which was associated with elevated mucosal antioxidative capacity and improved intestinal barrier functions.

Biological Efficacy of Essential Oils and Plant Extracts of Cultivated and Wild Ecotypes of Origanum vulgare L.

Current study describes discrepancy in biological efficacy of methanolic and ethanolic extracts and essential oil procured from cultivated and wild accessions of Origanum vulgare. Simultaneously, quantification of carvacrol, thymol, caryophyllene, ocimene, and terpinen-4-ol contents was determined via GC-MS and GC in both accessions. The results revealed significantly a higher antioxidant potential by methanolic extracts displaying IC50 of 19.9 µg/ml compared to essential oil with IC50 of 10 µg/ml, and ethanolic extracts were found to be less effective even at the concentration of 3 µg/ml. However, essential oil from wild and cultivated accessions of O. vulgare exhibited significantly high antimicrobial activity against all 39 bacteria, 16 fungi, and 2 yeast species tested due to higher concentrations of carvacrol and thymol as revealed by GC analysis. Inhibition of tyrosinase activity in a C6 cell line displayed 81.0%-87.0% depigmentation potential of the methanolic extracts, while ethanolic extracts revealed a maximum of 88.54-99.02% inhibition of reactive oxygen species (ROS) in H2O2-treated cells. Hence, the study determines efficacy of essential oil against microbial pathogenesis, methanolic extracts as potent depigmentation agents, and ethanolic extracts as potent free radical scavenger.

UPLC-ESI-MS/MS based identification and antioxidant, antibacterial, cytotoxic activities of aqueous extracts from storey onion (Allium cepa L. var. proliferum Regel).

Storey onion (Allium cepa L. var. proliferum Regel) is a variety of onion commonly grown in northern China that has not been researched in detail. This study aimed to identify the chemical compositions of storey onion aqueous extracts by UPLC-ESI-MS/MS, as well as characterize the antioxidant, antibacterial and cytotoxic activities, compared with welsh onion and onion. A total of 42 compounds were identified, among which the contents of organosulfur compounds (962.20 ± 34.55 µg/g), polyphenols (100.40 ± 12.55 µg/g) and organic acids (54.04 ± 2.69 µg/g) in storey onion were higher than those in welsh onion and onion. Additionally, the contents of cycloalliin (551.74 ± 8.12 µg/g), ajoene (159.31 ± 5.30 µg/g) and (E)-1-propene-1-sulfenic acid (72.12 ± 2.98 µg/g) in storey onion were the highest. Storey onion had pronounced DPPH• (IC50 = 1.24 ± 0.52 mg/mL) and OH• scavenging activities (IC50 = 14.45 ± 1.29 mg/mL) as well as ferric ion reducing power (absorbance from 0.32 to 2.21). Onion had the highest ABTS•+ scavenging activity (IC50 = 1.64 ± 0.64 mg/mL), while welsh onion had the lowest antioxidant activity. Storey onion had the strongest inhibitory effect on all the tested strains (MIC 31.3-125 mg/mL), and cell viability assays against human liver (HepG2) cancer cell lines also illustrated that aqueous extracts from storey onion significantly inhibited cell proliferation (when incubated for 24 h, IC50 = 33.21 ± 1.12 mg/mL) and induced cell apoptosis. Welsh onion and onion also had weaker antibacterial and anticancer activites, with those of onion being the weakest. The results showed that storey onion with excellent biological activity may benefit to human health and can be developed into functional foods.

Biological Activity of New Cichoric Acid-Metal Complexes in Bacterial Strains, Yeast-Like Fungi, and Human Cell Cultures In Vitro.

Cichoric acid (CA) belongs to the group of polyphenols, which occurs in a variety of plant species and it is characterized by anticancer, antibacterial, and antiviral properties. Selected polyphenols have the ability to combine with metal ions to form chelate complexes that reveal greater biological activity than free compounds. In order to study possible antimicrobial and anticancer effect of CA and its complexes with copper(II)/zinc(II)/nickel(II)/cobalt(II) we decided to conduct cytotoxicity tests to estimate the most effective concentrations of tested compounds. The results of the presented study demonstrated, for the first time, that the treatment with newly synthesized CA-metal complexes has anticancer and antimicrobial effects, which were examined in seven different cell lines: MCF-7, MDA-MB-231, and ZR-75-1 breast cancer cell lines, A375 melanoma cell line, DLD-1 cell line, LN-229 cell line, FN cell line; five bacterial strains: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Proteus vulgaris, Lactobacillus rhamnosus, yeast Sacchcaromyces boulardii, and pathogenic yeast-like fungi Candida albicans. The presented study indicates that CA-metal complexes could be considered as a potential supplementary tool in anticancer therapy, however, because of their possible toxic activity on fibroblasts, they should be used with caution. Some of the tested complexes have also preservative properties and positive influence on normal non-pathogenic microorganisms, which was demonstrated in selected microbial strains, therefore they may serve as food preservatives of natural origin with cytoprotective properties.

Phytochemical composition of wormwood (Artemisia gmelinii) extracts in respect of their antimicrobial activity.

BACKGROUND: Extracts from medicinal plants with phytochemicals with known antimicrobial properties can be an effective adjunct in the complex treatment of infectious diseases. This study aimed to evaluate the antimicrobial activity of wormwood extracts collected in Kazakhstan (Artemisia gmelinii Weber ex Stechm.), along with their phytochemical analysis. METHODS: The ethanolic and chloroform extracts were subjected to HPLC combined with quadrupole time-of-flight mass spectrometry method. For quantitative assessment of antimicrobial activity, minimal inhibitory concentration (MIC) of the tested extracts was determined by micro-dilution broth method for the panel of the reference microorganisms. Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) were also determined. RESULTS: LC/MS analysis showed the presence of 13 compounds in the tested extracts, including flavonoids: apigenin, luteolin, rutin, two O-methylated flavonols (isorhamnetin, rhamnazine), coumarin compounds (umbelliferone, scopoletin and scopolin (scopoletin 7-glucoside), 3-hydroxycoumarin and 4-hydroxycoumarin), chlorogenic acid and two dicaffeoylquinic acid isomers. Quantitative HPLC analysis showed that umbelliferone was dominant in the chloroform extract while chlorogenic acid was identified as a main compound in the ethanolic extract. The antibacterial and antifungal activity of chloroform and ethanolic extracts was comparable. The most sensitive were the Gram-positive bacteria represented by staphylococci, Micrococcus luteus and Bacillus spp. (MIC = 1.25-5 mg/ml) and yeasts represented by Candida spp. (MIC = 2.5-5 mg/ml), irrespective of the assayed extract. CONCLUSIONS: Extracts of wormwood Artemisia gmelinii have shown a wide spectrum of antibacterial and antifungal activity. Luteolin, rutin, isorhamnetin and scopolin were identified in A. gmelinii species for the first time. The determining of the most potential compounds of Artemisia gmelinii can be used to develop effective antibacterial and antifungal agents.

Screening of Chemical Libraries Using a Yeast Model of Retinal Disease.

Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9-cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z' factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.

Natural extracts from grape seed and stem by-products in combination with colloidal silver as alternative preservatives to SO2 for white wines: Effects on chemical composition and sensorial properties.

The search for alternative additives to sulfur dioxide, with antioxidant and antimicrobial activity, in the production of wines is one of the current objectives of the enological industry. In the present study, aqueous extracts obtained from winery byproducts (grape seeds and stems), alone or in combination with colloidal silver complex, have been used in white vinification. The antimicrobial effect of the extracts was similar to that of sulfur dioxide, being more effective on lactic and acetic bacteria in those wines to which colloidal silver was added. The effect on the color, the phenolic compounds and the volatile fraction of the wines was evaluated, as well as their sensory profile. The use of both extracts modified the color of the wines, increasing the chromatic parameters a* and b*, indicating a browning tendency, although no other signs of oxidation were found. Wines with seed extracts contained higher amounts of flavan-3-ols, and a significant increase in some volatile compounds such as fatty acid ethyl esters and benzene compounds, which were identified in the extracts. From a sensorial point of view, the wines with stem extracts were the most similar to those elaborated with SO2, detecting a certain bitterness in wines with seed extracts.

Antimicrobial property and antiproliferative activity of Centaurea babylonica (L.) L. on human carcinomas and cervical cancer cell lines.

INTRODUCTION AND OBJECTIVE: Since antiquity, C. babylonica (L.) L. extracts has been used as a remedy for primary health care in traditional medicine. In this study, a total of seven different crude extracts (acetone, chloroform, hexane, ethylacetate, methanol, ethanol and water) from branches and leaves of C. babylonica (L.) L. were prepared to determine antimicrobial and antiproliferative activity against cancer cell lines. MATERIAL AND METHODS: MIC assay was used for antimicrobial activity against gram positive and gram negative bacteria, and one yeast. MTT assay was applied to screen the antiproliferative activity of seven extracts, and to determine dose- and time-dependent effects of the aceton extract on A549, PC-3, MCF-7, and HeLa cell lines. RESULTS: The aceton extract of C.babylonica (L.) L. showed the best antibacterial activity against Bacillus cereus, P. aeruginosa and C. albicans (MIC: 1.6 mg/mL). GC-MS analyses allowed six compounds to be determined; the main constituents of acetone extract from C. babylonica (L.) L. were diacetone alcohol (53.47 %), 1-dexadecene (10.19 %) and 1-tetradecene (8.67 %). In addition, seven different solvent extracts at 500 µg/mL caused antiproliferative activity between 84% - 88%, compared to control. Dose-dependent effects of the extracts on A549 cells indicated that chloroform, ethyl acetate, and aceton extract were the most effective extracts with the IC50 values of 9, 33, and 36 µg/mL, respectively. CONCLUSIONS: The results clearly demonstrate that C. babylonica (L.) L. exhibited a strong antimicrobial effect and antiproliferative activity against cancer cells in vitro. Further studies are required to isolate and characterize the active pure compounds responsible for the antimicrobial and antiproliferative activities.

Chemical characterization and biological activities of two varieties of xoconostle fruits Opuntia joconostle F.A.C. Weber ex Diguet and Opuntia matudae Scheinvar.

The present work focusses on the chemical characterization and bioactive properties of Opuntia joconostle F.A.C. Weber ex Diguet and Opuntia matudae Scheinvar fruits. This research showed that xoconostle cv. Cuaresmeño (O. joconostle) and xoconostle cv. Rosa (O. matudae) are a good source of PUFAs and tocopherols. Moreover, both fruits revealed the presence of ten phenolic compounds (e.g., ferulic acid hexoside, quercetin-O-di-deoxyhexosyl-hexoside, and kaempferol-O-(di-deoxyhexosyl)-hexoside), as well as other organic acids (oxalic, malic, ascorbic and citric acids), and two betacyanins (betanin and isobetain). The hydroethanolic extracts of both fruits exhibited antioxidant activity, and inhibited the growth of several bacteria strains and of the yeast Candida albicans. As expected, xoconostle cv. Cuaresmeño was the fruit with highest antioxidant potential, since it was also the one that showed the highest content of bioactive compounds, with the exception of betacyanins. Overall, both fruits are revealed to be a good source of nutritive and bioactive compounds.

Bioactivities of Flavonoids from Lopezia racemosa.

Lopezia racemosa Cav. (Onagraceae) has been used in Mexican traditional medicine to alleviate stomachache, biliary colic, urine retention, stomach cancer, and skin, dental, buccal, and urinary infections. The objective of this study was to determine the bioactivities of specific parts of the plant to scientifically confirm its traditional use. Aerial parts and flowers were macerated and subsequently extracted with hexane, chloroform, and methanol. This study was focused on the analysis of polar components, and thus the methanolic fractions were selected for further investigations. These fractions were evaluated for their antimicrobial activity using a panel of bacterial Gram-positive and -negative strains, as well as fungal strains, including filamentous fungi and yeasts. In addition, the cytotoxic activity of the extract was assessed by MTT using the human-derived monocytic THP-1 and the normal human fibroblast cell lines. Various fractions showed antimicrobial activity against various pathogens, although the most relevant were against Pseudomonas aeruginosa and Trichophyton mentagrophytes. No inhibition of yeasts was recorded. Only four fractions showed cytotoxic effects when the human-derived THP-1 and fibroblast cells were assessed. The four flavonoids isolated from the extract were luteolin, luteolin-6-C-hexoside, luteolin-8-C-hexoside, and hyperoside. The biological activities presented in this study validate some traditional uses of the plant.

Mentha piperita L. essential oil inactivates spoilage yeasts in fruit juices through the perturbation of different physiological functions in yeast cells.

This study evaluated the efficacy of the essential oil from Mentha piperita L. (MPEO) to inactivate cells of the potentially spoilage yeasts Candida albicans, Candida tropicalis, Pichia anomala and Saccharomyces cerevisiae in cashew, guava, mango and pineapple juices during 72 h of refrigerated storage. Damage in different physiological functions caused by MPEO in S. cerevisiae in cashew and guava juices were investigated using flow cytometry (FC). The effects of the incorporation of an effective anti-yeast MPEO dose on sensory characteristics of juices were also evaluated. MPEO displayed minimum inhibitory concentration of 1.875 µL/mL against all tested yeasts. A >5 log reduction in counts of C. albicans, P. anomala and S. cerevisiae was observed in cashew and guava juices with 7.5 and 3.75 µL/mL MPEO. Tested MPEO concentrations (1.875, 3.75 and 7.5 µL/mL) were not effective to cause >5 log reduction in counts of target yeasts in mango and pineapple juices during 72 h of exposure. Incorporation of 1.875 µL/mL MPEO in cashew and guava juices strongly compromised membrane permeability, membrane potential, enzymatic activity and efflux pump activity in S. cerevisiae cells. This same MPEO concentration did not affect appearance, odor and viscosity in fruit juices, but negatively affected their taste and aftertaste. These results show the efficacy of MPEO to inactivate potentially spoilage yeasts in fruit juices through disturbance of different physiological functions in yeast cells. However, the combined use of MPEO with other technologies should be necessary to decrease its effective anti-yeast dose in fruit juices and, consequently, the possible negative impacts on specific sensory properties of these products.

A High-Throughput Assay for DNA Replication Inhibitors Based upon Multivariate Analysis of Yeast Growth Kinetics.

Mcm2-7 is the molecular motor of eukaryotic replicative helicase, and the regulation of this complex is a major focus of cellular S-phase regulation. Despite its cellular importance, few small-molecule inhibitors of this complex are known. Based upon our genetic analysis of synthetic growth defects between mcm alleles and a range of other alleles, we have developed a high-throughput screening (HTS) assay using a well-characterized mcm mutant (containing the mcm2DENQ allele) to identify small molecules that replicate such synthetic growth defects. During assay development, we found that aphidicolin (inhibitor of DNA polymerase alpha) and XL413 (inhibitor of the DNA replication-dependent kinase CDC7) preferentially inhibited growth of the mcm2DENQ strain relative to the wild-type parental strain. However, as both strains demonstrated some degree of growth inhibition with these compounds, small and variable assay windows can result. To increase assay sensitivity and reproducibility, we developed a strategy combining the analysis of cell growth kinetics with linear discriminant analysis (LDA). We found that LDA greatly improved assay performance and captured a greater range of synthetic growth inhibition phenotypes, yielding a versatile analysis platform conforming to HTS requirements.

Antifungal activity of well-defined chito-oligosaccharide preparations against medically relevant yeasts.

Due to their antifungal activity, chitosan and its derivatives have potential to be used for treating yeast infections in humans. However, to be considered for use in human medicine, it is necessary to control and know the chemical composition of the compound, which is not always the case for polymeric chitosans. Here, we analyze the antifungal activity of a soluble and well-defined chito-oligosaccharide (CHOS) with an average polymerization degree (DPn) of 32 and fraction of acetylation (FA) of 0.15 (C32) on 52 medically relevant yeast strains. Minimal inhibitory concentrations (MIC) varied widely among yeast species, strains and isolates (from > 5000 to < 9.77 µg mL-1) and inhibition patterns showed a time- and dose-dependencies. The antifungal activity was predominantly fungicidal and was inversely proportional to the pH, being maximal at pH 4.5, the lowest tested pH. Furthermore, antifungal effects of CHOS fractions with varying average molecular weight indicated that those fractions with an intermediate degree of polymerization, i.e. DP 31 and 54, had the strongest inhibitory effects. Confocal imaging showed that C32 adsorbs to the cell surface, with subsequent cell disruption and accumulation of C32 in the cytoplasm. Thus, C32 has potential to be used as a therapy for fungal infections.

Insight into the Influence of Cultivar Type, Cultivation Year, and Site on the Lignans and Related Phenolic Profiles, and the Health-Promoting Antioxidant Potential of Flax (Linum usitatissimum L.) Seeds.

Flaxseeds are a functional food representing, by far, the richest natural grain source of lignans, and accumulate substantial amounts of other health beneficial phenolic compounds (i.e., flavonols, hydroxycinnamic acids). This specific accumulation pattern is related to their numerous beneficial effects on human health. However, to date, little data is available concerning the relative impact of genetic and geographic parameters on the phytochemical yield and composition. Here, the major influence of the cultivar over geographic parameters on the flaxseed phytochemical accumulation yield and composition is evidenced. The importance of genetic parameters on the lignan accumulation was further confirmed by gene expression analysis monitored by RT-qPCR. The corresponding antioxidant activity of these flaxseed extracts was evaluated, both in vitro, using ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and iron chelating assays, as well as in vivo, by monitoring the impact of UV-induced oxidative stress on the lipid membrane peroxidation of yeast cells. Our results, both the in vitro and in vivo studies, confirm that flaxseed extracts are an effective protector against oxidative stress. The results point out that secoisolariciresinol diglucoside, caffeic acid glucoside, and p-coumaric acid glucoside are the main contributors to the antioxidant capacity. Considering the health benefits of these compounds, the present study demonstrates that the flaxseed cultivar type could greatly influence the phytochemical intakes and, therefore, the associated biological activities. We recommend that this crucial parameter be considered in epidemiological studies dealing with flaxseeds.