Hawthorn Crataegus monogyna Jacq. and Crataegus oxyacantha L.; sin. Crataegus laevigata (Poiret) DC., Rosaceae leaves, flowers, and berries are used in traditional medicine in the treatment of ...chronic heart failure, high blood pressure, arrhythmia, and various digestive ailments, as well as geriatric and antiarteriosclerosis remedies. According to European Pharmacopoeia 6.0, hawthorn berries consist of the dried false fruits of these two species or their mixture. The present study was carried out to test free-radical-scavenging, anti-inflammatory, gastroprotective, and antimicrobial activities of hawthorn berries ethanol extract. Phenolic compounds represented 3.54%, expressed as gallic acid equivalents. Determination of total flavonoid aglycones content yielded 0.18%. The percentage of hyperoside, as the main flavonol component, was 0.14%. With respect to procyanidins content, the obtained value was 0.44%. DPPH radical-scavenging capacity of the extract was concentration-dependent, with EC50 value of 52.04 μg/mL (calculation based on the total phenolic compounds content in the extract). Oral administration of investigated extract caused dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. The obtained anti-inflammatory effect was 20.8, 23.0, and 36.3% for the extract doses of 50, 100, and 200 mg/kg, respectively. In comparison to indomethacin, given in a dose producing 50% reduction of rat paw edema, the extract given in the highest tested dose (200 mg/kg) showed 72.4% of its activity. Gastroprotective activity of the extract was investigated using an ethanol-induced acute stress ulcer in rats with ranitidine as a reference drug. Hawthorn extract produced dose-dependent gastroprotective activity (3.8 2.1, 1.9 1.7, and 0.7 0.5 for doses of 50, 100, and 200 mg/kg, respectively), with the efficacy comparable to that of the reference drug. Antimicrobial testing of the extract revealed its moderate bactericidal activity, especially against Gram-positive bacteria Micrococcus flavus, Bacillus subtilis, and Lysteria monocytogenes, with no effect on Candida albicans. All active components identified in the extract might be responsible for activities observed.
L-ascorbic acid represents one of the most potent antioxidant, photoprotective, anti-aging, and anti-pigmentation cosmeceutical agents, with a good safety profile. However, the main challenge is the ...formulation of stable topical formulation products, which would optimize the penetrability of L-ascorbic acid through the skin. The aim of our research was to evaluate the performance of ascorbyl palmitate on the skin, incorporated in creams and emulgels (2%) as carriers, as well as to determine the impact of its incorporation into liposomes on the penetration profile of this ingredient. Tape stripping was used to study the penetration of ascorbyl palmitate into the stratum corneum. In addition, the sensory and textural properties of the formulations were determined. The liposomal formulations exhibited a better penetration profile (p < 0.05) of the active substance compared to the non-liposomal counterpart, leading to a 1.3-fold and 1.2 fold-increase in the total amount of penetrated ascorbyl palmitate in the stratum corneum for the emulgel and cream, respectively. Encapsulation of ascorbyl palmitate into liposomes led to an increase in the adhesiveness and density of the prepared cream and emulgel samples. The best spreadability and absorption during application were detected in liposomal samples. The obtained results confirmed that liposomal encapsulation of ascorbyl palmitate improved dermal penetration for both the cream and emulgel formulations.
A new formulation that gradually released encapsulated Thuja plicata essential oil (TPEO) as an active component from a biopolymer matrix within a given period was obtained. Antimicrobial activity ...was determined in in-vitro tests where pure TPEO successfully inhibited the development of different Phytophthora species. The TPEO essential oil was encapsulated into the biopolymer matrix and an oil-in-water emulsion was formed. FTIR spectra analysis confirmed the formation of electrostatic interaction between these polymers, and hydrogen interactions between active components of TPEO and polymer chains. The stability of the emulsions was confirmed by zeta potential measurements, with a value of about 30 mV, even after 14 days of aging. UV-Vis spectra analysis revealed that >60 % of TPEO remained in the emulsion after 14 days of exposure to ambient conditions, whereas pure TPEO evaporated faster, and around 20 % remained after 6 days. Encapsulated TPEO almost completely inhibited the growth of Phytophthora species during the ten-day day's exposition being statistically significantly improved compared to fungicide treatment. It was demonstrated that the emulsion exhibited a prolonged antimicrobial effect and successfully suppressed the growth of Phytophthora species, and can be considered as a means of protection in forests and crops.A new formulation that gradually released encapsulated Thuja plicata essential oil (TPEO) as an active component from a biopolymer matrix within a given period was obtained. Antimicrobial activity was determined in in-vitro tests where pure TPEO successfully inhibited the development of different Phytophthora species. The TPEO essential oil was encapsulated into the biopolymer matrix and an oil-in-water emulsion was formed. FTIR spectra analysis confirmed the formation of electrostatic interaction between these polymers, and hydrogen interactions between active components of TPEO and polymer chains. The stability of the emulsions was confirmed by zeta potential measurements, with a value of about 30 mV, even after 14 days of aging. UV-Vis spectra analysis revealed that >60 % of TPEO remained in the emulsion after 14 days of exposure to ambient conditions, whereas pure TPEO evaporated faster, and around 20 % remained after 6 days. Encapsulated TPEO almost completely inhibited the growth of Phytophthora species during the ten-day day's exposition being statistically significantly improved compared to fungicide treatment. It was demonstrated that the emulsion exhibited a prolonged antimicrobial effect and successfully suppressed the growth of Phytophthora species, and can be considered as a means of protection in forests and crops.
Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of ...precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.
The aim of this study was to investigate the effect of the use of green solvents, natural deep eutectic solvents (NaDES), in comparison with conventional solvents on the extraction of free unbound ...phenolic compounds and the antioxidant activity of extracts of dried bilberry fruit, bilberry leaves and green tea leaves. After preparation of the extracts via ultrasound-assisted extraction using NaDES and conventional solvents (water and ethanol), spectrophotometric determination of total phenolic and flavonoid content, HPLC analysis of extracted polyphenols and antioxidant determination using FRAP, DPPH and ABTS assays were conducted. The results showed that NaDES have a great potential as agents for the extraction of phenolic compounds with potent antioxidant activity; the highest values of phenolic content and antioxidant activity were detected in the samples obtained by extraction using the NaDES combination betaine + urea. The bilberry leaves exhibited the highest flavonoid content among all extracts and turned out to be more active than bilberry fruits, to which they are often just a by-product during processing. The most active extract of all was the betaine-urea green tea leaves extract. Further research into the most active NaDES extracts should be performed.
High-grade serous ovarian cancer (HGSOC) is the most frequent and aggressive type of epithelial ovarian cancer, with high recurrence rate and chemoresistance being the main issues in its clinical ...management. HGSOC is specifically challenging due to the metastatic dissemination via spheroids in the ascitic fluid. The HGSOC spheroids represent the invasive and chemoresistant cellular fraction, which is impossible to investigate in conventional two-dimensional (2D) monolayer cell cultures lacking critical cell-to-cell and cell-extracellular matrix interactions. Three-dimensional (3D) HGSOC cultures, where cells aggregate and exhibit relevant interactions, offer a promising in vitro model of peritoneal metastasis and multicellular drug resistance. This review summarizes recent studies of HGSOC in 3D culture conditions and highlights the role of multicellular HGSOC spheroids and ascitic environment in HGSOC metastasis and chemoresistance.
Extracts obtained from hops (Humulus lupulus L., Cannabaceae) by supercritical fluid extraction (SFE), SFE followed by isomerization, as well as by conventional technique, were investigated for their ...chemical composition and antibacterial activity against selected foodborne pathogens and microorganisms capable to cause the food spoilage. The antibacterial activity of the extracts was compared with the antibacterial activity of xanthohumol, compound known for its broad pharmacological properties, isolated from the raw material remained after the SFE. Xanthohumol (XH, 96%) proved to posses the most prominent activity against all the tested strains, with the MIC values ranged between 2.5 and 20 μg mL-1. Supercritical hops extract and potassium isomerized supercritical hops extract showed strong antibacterial activity against the tested strains as well. Escherichia coli was not affected by the extracts, meaning that their oral admission would not cause the same problem as antibiotic application in intestinal flora. The chemical composition of the investigated hops extracts was analysed by GC-MS. Contents of α-acids, β-acids, iso-α-acids and xanthohumol in the samples were determined by HPLC.
Sideritis scardica Griseb. (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of gastrointestinal complaints, inflammation, and ...rheumatic disorders. This study aimed to evaluate its gastroprotective and anti-inflammatory activities. Besides, continuously increasing interest in assessing the role of the plant active constituents preventing the risk of cancer was a reason to make a detailed examination of the investigated ethanol, diethyl ether, ethyl acetate, and N-butanol extracts regarding cytotoxicity. Oral administration of the investigated extracts caused a dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. Gastroprotective activity of the extracts was investigated using an ethanol-induced acute stress ulcer in rats. The cytotoxic activity of plant extracts was assessed on PBMC, B16, and HL-60 cells and compared to the cytotoxicity of phenolic compounds identified in extracts. Apoptotic and necrotic cell death were analyzed by double staining with fluoresceinisothiocyanate (FITC)-conjugated annexin V and PI. The developed HPLC method enabled qualitative fingerprint analysis of phenolic compounds in the investigated extracts. Compared to the effect of the positive control, the anti-inflammatory drug indomethacine (4 mg/kg), which produced a 50 % decrease in inflammation, diethyl ether and N-butanol extracts exhibited about the same effect in doses of 200 and 100 mg/kg (53.6 and 48.7 %; 48.4 and 49.9 %, respectively). All investigated extracts produced dose-dependent gastroprotective activity with the efficacy comparable to that of the reference drug ranitidine. The diethyl ether extract showed significant dose-dependent cytotoxicity on B16 cells and HL-60 cells, decreasing cell growth to 51.3 % and 77.5 % of control, respectively, when used at 100 µg/mL. It seems that phenolic compounds (apigenin, luteolin, and their corresponding glycosides) are responsible for the diethyl ether extract cytotoxic effect. It also appears that induction of oxidative stress might be involved in its cytotoxicity, since B16 and HL-60 cells increased their ROS production in response to treatment with diethyl ether extract. Neither of the tested extracts nor any phenolic compounds showed significant cytotoxic effect to human PBMC. These results demonstrated the potent anti-inflammatory and gastroprotective activities, as well as the promising cytotoxicity.
•Recent dCas9-based tools revolutionized the field of (epi)genome editing.•We discuss the newest approaches to gene regulation and epigenetic editing.•Strategies to minimize the off-target effects of ...dCas9-based tools are reviewed.•Characteristics of several Class II Cas9 orthologues are reviewed in detail.
Molecular tools for gene regulation and epigenome editing consist of two main parts: the targeting moiety binding a specific genomic locus and the effector domain performing the editing or regulatory function. The advent of CRISPR-Cas9 technology enabled easy and flexible targeting of almost any locus by co-expression of a small sgRNA molecule, which is complementary to the target sequence and forms a complex with Cas9, directing it to that particular target. Here, we review strategies for recruitment of effector domains, used in gene regulation and epigenome editing, to the dCas9 DNA-targeting protein. To date, the most important CRISPR-Cas9 applications in gene regulation are CRISPR activation or interference, while epigenome editing focuses on targeted changes in DNA methylation and histone modifications. Several strategies for signal amplification by recruitment of multiple effector domains deserve special focus. While some approaches rely on altering the sgRNA molecule and extending it with aptamers for effector domain recruitment, others use modifications to the Cas9 protein by direct fusions with effector domains or by addition of an epitope tag, which also has the ability to bind multiple effector domains. A major barrier to the widespread use of CRISPR-Cas9 technology for therapeutic purposes is its off-target effect. We review efforts to enhance CRISPR-Cas9 specificity by selection of Cas9 orthologs from various bacterial species and their further refinement by introduction of beneficial mutations. The molecular tools available today enable a researcher to choose the best balance of targeting flexibility, activity amplification, delivery method and specificity.
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