Barbara Różalska

Chemical Composition and Biological Activities of Essential Oil from Salvia sclarea Plants Regenerated in vitro

The essential oils obtained by hydrodistillation of dried aerial parts of Salvia sclarea L. plants, regenerated in vitro and reproduced from seeds, were analyzed by GC and GC-MS. The oils from in vitro and in vivo plants were compared in respect to their chemical composition as well as antimicrobial and cytotoxic activities. The chemical profiles of both oils were very similar, although the yield of essential oil from in vitro plants was lower (0.1%, v/w) than the oil yield isolated from in vivo S. sclarea plants (0.2%, v/w). Both oils showed antimicrobial and cytotoxic activity. The oil from in vitro regenerated plants of S. sclarea exhibited stronger cytotoxic action against NALM-6 cell lines in comparison with the essential oil from in vivo plants.

Antiadherent and Antibiofilm Activity of Humulus lupulus L. Derived Products: New Pharmacological Properties

New antimicrobial properties of products derived from Humulus lupulus L. such as antiadherent and antibiofilm activities were evaluated. The growth of gram-positive but not gram-negative bacteria was inhibited to different extents by these compounds. An extract of hop cones containing 51% xanthohumol was slightly less active against S. aureus strains (MIC range 31.2–125.0 μg/mL) than pure xanthohumol (MIC range 15.6–62.5 μg/mL). The spent hop extract, free of xanthohumol, exhibited lower but still relevant activity (MIC range 1-2 mg/mL). There were positive coactions of hop cone, spent hop extracts, and xanthohumol with oxacillin against MSSA and with linezolid against MSSA and MRSA. Plant compounds in the culture medium at sub-MIC concentrations decreased the adhesion of Staphylococci to abiotic surfaces, which in turn caused inhibition of biofilm formation. The rate of mature biofilm eradication by these products was significant. The spent hop extract at MIC reduced biofilm viability by 42.8%, the hop cone extract by 74.8%, and pure xanthohumol by 86.5%. When the hop cone extract or xanthohumol concentration was increased, almost complete biofilm eradication was achieved (97–99%). This study reveals the potent antibiofilm activity of hop-derived compounds for the first time.

Synthesis and biological evaluation of α-methylidene-δ-lactones with 3,4-dihydrocoumarin skeleton

A series of new 3-methylidenechroman-2-ones bearing various aromatic moieties and various substituents at position 4 were synthesized in a three step reaction sequence. Friedel-Crafts alkylation of phenols or naphthols using ethyl 3-methoxy-2-diethoxyphosphorylacrylate in the presence of trifluoromethanesulphonic acid gave 3-diethoxyphosphorylchromen-2-ones. These compounds were employed as Michael acceptors in the reaction with Grignard reagents to give adducts which were finally used as Horner-Wadsworth-Emmons reagents for the olefination of formaldehyde. All obtained 3-methylidenechroman-2-ones were tested against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7 breast cancer and HT-29 colon cancer adenocarcinomas. Several obtained methylidenechromanones displayed high cytotoxic activity with IC(50) values below 1 μM, mainly against leukemia and MCF-7 cell lines. Investigation of structure-activity relationships revealed that the presence of additional, ortho-fused benzene ring and n-butyl or i-propyl group in position 4 enhances the activity. Selected methylidenechromanones were also tested on normal human umbilical vein endothelial cells (HUVEC) and chromanone 14o was found to be eightfold more toxic against MCF-7 than normal cells. Furthermore, antimicrobial assays revealed that chromanone 14n is highly active and bactericidal at concentration equal to MIC or 2MIC against nosocomial and community-associated staphylococci (MRSA) which are resistant to most or all available therapeutic classes of antimicrobial drugs.

New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Candida albicans.

The antifungal activity of the saponin-rich fractions (SFs) from Medicago sativa (aerial parts and roots) and Saponaria officinalis (used as a well-known source of plant saponins) against Candida albicans reference and clinical strains, their yeast-to-hyphal conversion, adhesion, and biofilm formation was investigated. Direct fungicidal/fungistatic properties of the tested phytochemicals used alone, as well as their synergy with azoles (probably resulting from yeast cell wall instability) were demonstrated. Here, to the best of our knowledge, we report for the first time the ability of saponin-rich extracts of M. sativa and S. officinalis to inhibit C. albicans germ tube formation, limit hyphal growth, reduce yeast adherence and biofilm formation, and eradicate mature (24 h) Candida biofilm. Moreover, M. sativa SFs (mainly obtained from aerial parts), in the range of concentrations which were active modulators of Candida virulence factors, exhibited low cytotoxicity against the mouse fibroblast line L929. These properties seem to be very promising in the context of using plant-derived SFs as potential novel antifungal therapeutics supporting classic drugs or as ingredients of disinfectants.

Colony-blot assay with anti-p60 antibodies as a method for quick identification of Listeria in food

The present study evaluated the ability to isolate Listeria from foods, using shortened procedure of sample enrichment followed by immunomagnetic separation or filtration methods, and serological identification of isolated bacteria by colony-blot and Western blot methods with anti-p60 antibodies. By these rapid methods, identification of Listeria was achieved in much shorter time (40-48 h) than with standard cultivation and biochemical identification procedures. The rapid methods used are easy to perform and, what is most important, their specificity is very high and fulfills the expectations. The possibility to select Listeria colonies growing on non-selective media by blotting with anti-p60 antiserum seems to be particularly valuable in examination of food samples containing/not too many Listeria (1-10 CFU/25 g). However, the blot method using anti-PepD mAb specific to unique region of L. monocytogenes p60 is necessary to distinguish L. monocytogenes from other Listeria species.

The Immunomodulatory Activity of Staphylococcus aureus Products Derived from Biofilm and Planktonic Cultures

Biofilms are probably one of the most common structures formed by microorganisms in various environments. The higher resistance of such microbial communities to stress conditions, including antibiotics and host immune response, is recently extensively studied. However, the weak activity of phagocytic cells against microbial biofilm is not yet fully understood and explained. The aim of this study was: (1) a qualitative and quantitative comparison of cell components/products released from Staphylococcus aureus biofilm or planktonic cultures, (2) evaluation of the influence of such cell components/products on murine leukocytes secretory function. For this, mouse peritoneal leukocytes were stimulated with biofilm or planktonic staphylococcal cultures or their acellular filtrates, and then the production of cytokines (TNF-α, IL-6, IL-10, MCP-1 and MIP-1α), hemolytic activity and staphylokinase (SAK) production was determined. It was found that similar staphylococcal components/products possessing the immunomodulatory properties, were present in both, biofilm and planktonic filtrates. Moreover, these compounds were similarly active in the stimulation of TNF-α and MCP-1 release from leukocytes. The hemolytic activity and SAK release by planktonic and biofilm cultures were also comparable. What is interesting, stronger stimulatory activity of biofilm-derived components/products of clinical S. aureus strains in the case of MIP-1α, IL-6 and IL-10 was noticed. On the other hand, taking into consideration the reference strains, MIP-1α production was enhanced by “planktonic filtrates”. Thus, in our study it was proved, first of all, that biofilm is not a structure fully separated from the external environment. Second, the influence of these S. aureus constituents/metabolites on leukocytes seems to be more strain-dependent than culture phenotype-dependent. The lack of one common profile of biofilm and planktonic S. aureus cultures/filtrates biological activity indicates that the disturbances in cytokines’ production could not be the only reason for the so-called “frustrated phagocytosis”, connected with enhanced biofilm resistance.

Biomaterial‐Associated Infection with Candida albicans in Mice

Candida yeasts are frequently isolated from patients with continuous ambulatory peritoneal dialysis peritonitis or other biomaterial‐associated infections. The mouse model of candidal peritonitis was used to study the interaction of Candida cells with end‐point attached heparinized polyethylene (H‐PE) and with polymorphonuclear leukocytes (PMNs) or macrophages (Mφ). Two Candida strains differing in cell surface hydrophobicity and in expression of fibronectin (Fn) binding were used for the study. Cells of both Candida strains adhered at higher numbers to H‐PE surfaces preadsorbed with Fn or with human dialysis fluid (HDF) than to non‐modified H‐PE, supporting a role of Fn in mediating adhesion. C. albicans 4016 cells expressing low hydrophobicity and low binding of soluble Fn demonstrated stronger adhesion to PMNs than the more hydrophobic C. albicans 3248 yeasts, which express high binding of soluble Fn. However, C. albicans 4016 cells were more resistant to phagocytic killing and were hardly eradicated in intraperitoneally infected mice. The animals depleted in PMNs by treatment with CY were neither able to eradicate C. albicans 3248 (rapidly eliminated by normal mice) nor C. albicans 4016 yeasts (with a tendency to persist in the tissues of normal mice).

Synthetic 3-Arylidenefl avanones as Inhibitors of the Initial Stages of Biofilm Formation by Staphylococcus aureus and Enterococcus faecalis

The antimicrobial activity of twenty two synthetic flavonoids is reported. Among them three 3-arylideneflavanones, 2b, 2c, and 2i, were shown to be highly active against Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis reference strains, with MIC (minimal inhibitory concentration) values ranging from 4.68 μg/ml (14.3 μM) to 37.5 μg/ml (119.7 μM). The synergy of oxacillin and vancomycin with 2c, evaluated as fractional inhibitory concentration index (FICI) was shown (against planktonic culture of S. aureus A3 and E. faecium 138/09 clinical strains). The presence of 2c in the culture medium diminished the initial adhesion of bacteria to an abiotic surface. Such an effect resulted in a decrease in biofilm formation during prolonged culture. Unfortunately, 2c failed to eradicate the S. aureus mature biofilm which was already preformed, however, decreased the number of live biofilm cells. The biofilm of E. faecalis was more susceptible to the action of 3-arylideneflavanone 2c than the S. aureus biofilm. The finding that 3-arylideneflavanones are lipophilic, cause bacterial aggregation, and influence the integrity of membranes making them permeable to SYTO 9/propidium iodide dyes may implicate the cytoplasmic membrane as a target site for these compounds activity

In vitro propagation and chemical and biological studies of the essential oil of Salvia przewalskii Maxim.

The procedure of Salvia przewalskii shoot multiplication and the ability of regenerated plants to produce essential oil is reported. The essential oil was obtained by hydrodistillation from leaves and flowering stems of field-grown plants, and their chemical composition was examined by GC, GC-MS and 1H NMR. The differences in yield as well as qualitative and quantitative composition between the oils isolated from in vitro and in vivo plants were observed. S. przewalskii essential oil was tested for its antimicrobial and cytotoxic properties. It was found that cytotoxicity against human leukemia HL-60 cells and antimicrobial activity (especially, against Staphylococcus aureus and S. epidermidis strains) of oils isolated from in vitro plants were higher than those for oils from in vivo S. przewalskii plants.

The cells of monocyte-macrophage lineage in mice with the 2-month polyethylene implantation

End-point-attached heparinized polyethylene (H-PE) was implanted for 2 months into the peritoneum of C57B1/6 mice. The proliferation of bone marrow cells (BMCs) from implanted and non-implanted mice was investigated in M-CSF supplemented medium, in the presence or absence of macrophage-specific monoclonal antibodies (mAbs). The mAb HC 7.67.B, recognizing a surface determinant on immature monocytoid cells, inhibited the proliferation of BMCs from H-PE implanted mice without any influence on the proliferation of BMCs from non-implanted animals. The peritoneal macrophages from H-PE implanted mice demonstrated enhanced production of fibronectin (Fn) in comparison to the macrophages from non-implanted animals. Our results suggest changes in the differentiation of murine monocyte-macrophage lineage in the mice bearing H-PE implants for 2 months.