Iris pseudacorus as an easily accessible source of antibacterial and cytotoxic compounds.

Iris pseudacorus is one of the most widespread iris species and possesses complex secondary metabolites. Our study showed that its rhizomes are abundant with phenolic compounds of which 80 % belong to the tannin group. Methanolic extracts from garden cultured iris rhizomes possessed antibacterial activity against human Gram positive Staphylococcus aureus and Enterococcus faecalis and Gram negative Pseudomonas aeruginosa and Klebsiella pneumoniae pathogens including clinical isolates resistant to commercially available antibiotics. Moreover the extract from rhizome, in concentration 3.125 mg dry weight/mL, containing gallocatechin (1), effectively combats S. aureus biofilm. The same rhizome extract acts against human cancer cell lines, especially against estrogen positive MCF-7 breast cancer cell line (IC50 = 11.75 µg/mL). In vitro culture of excised, anatomical roots of I. pseudacorus excreted three antistaphylococcal compounds into the plant medium, detected by using TLC-overlayer bioautography. By the use of HPLC-DAD-ESIMS system 2 active compounds were identified as 5,7,4'-trihydroxy-6,3'-dimethoxy-isoflavone (7) and unknown dimethoxy-dihydroxy-isoflavone (9). I. pseudacorus as a non-edible plant might be considered to be new, easy accessible, non-wood source of biologically active polyphenolics.

Bersaldegenin-1,3,5-orthoacetate induces caspase-independent cell death, DNA damage and cell cycle arrest in human cervical cancer HeLa cells.

CONTEXT: Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. OBJECTIVE: The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate - a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. MATERIALS AND METHODS: The cytotoxic activity of the compound (at 0.1-20.0 µg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. RESULTS: The compound had strong effect on the cells (IC50 = 0.55 µg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. CONCLUSIONS: Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.

Blue light treatment of Pseudomonas aeruginosa: Strong bactericidal activity, synergism with antibiotics and inactivation of virulence factors.

Pseudomonas aeruginosa is among the most common pathogens responsible for both acute and chronic infections of high incidence and severity. Additionally, P. aeruginosa resistance to conventional antimicrobials has increased rapidly over the past decade. Therefore, it is crucial to explore new therapeutic options, particularly options that specifically target the pathogenic mechanisms of this microbe. The ability of a pathogenic bacterium to cause disease is dependent upon the production of agents termed 'virulence factors', and approaches to mitigate these agents have gained increasing attention as new antibacterial strategies. Although blue light irradiation is a promising alternative approach, only limited and preliminary studies have described its effect on virulence factors. The current study aimed to investigate the effects of lethal and sub-lethal doses of blue light treatment (BLT) on P. aeruginosa virulence factors. We analyzed the inhibitory effects of blue light irradiation on the production/activity of several virulence factors. Lethal BLT inhibited the activity of pyocyanin, staphylolysin, pseudolysin and other proteases, but sub-lethal BLT did not affect the production/expression of proteases, phospholipases, and flagella- or type IV pili-associated motility. Moreover, a eukaryotic cytotoxicity test confirmed the decreased toxicity of blue light-treated extracellular P. aeruginosa fractions. Finally, the increased antimicrobial susceptibility of P. aeruginosa treated with sequential doses of sub-lethal BLT was demonstrated with a checkerboard test. Thus, this work provides evidence-based proof of the susceptibility of drug-resistant P. aeruginosa to BLT-mediated killing, accompanied by virulence factor reduction, and describes the synergy between antibiotics and sub-lethal BLT.

Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.

Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell's susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

Cytotoxic Lignan from the Non-Transformed Root Culture of Phyllanthus amarus.

A new lignan from the non-transformed root in vitro cultures of Phyllanthus amarus was isolated. The structure of the compound was established on the basis of one- and two-dimensional NMR, as well as mass spectrometry data, as 7'-oxocubebin dimethylether (1,4-bis(benzo[d][1,3]dioxol-5-yl)-2,3-bis(methoxymethyl)butan-1-on). The non-transformed root cultures of P. amarus showed to be a selective source of this compound. The lignan revealed strong cytotoxic activity against HeLa cell line with an IC50 value of 3.8 µg/mL.

Chemical composition and biological activity of Rubus idaeus shoots--a traditional herbal remedy of Eastern Europe.

BACKGROUND: The young shoots of Rubus idaeus are traditionally used as a herbal remedy in common cold, fever and flu-like infections yet there is no research concerning this plant material. The aim of the study was to evaluate the chemical composition and biological properties of raspberry shoots from 11 cultivar varieties. METHODS: The methanol extracts were subjected to chromatographic analysis using HPLC-DAD-ESI-MS, and two-dimensional 'comprehensive' LCxLC techniques. The biological activity of the shoot extract from the 'Willamette' cultivar variety was evaluated. Antioxidant activity was tested using DPPH and phosphomolybdenum assay. Antimicrobial activity was estimated towards 15 strains of human pathogenic bacteria using broth microdilution method. Cytotoxic activity was tested using MTT cell viability assay. RESULTS: The dominating compounds identified in the shoots of R. idaeus were ellagic acid (26.1 - 106.8 mg/100 g) and sanguiin H-6 (139.2 - 633.1 mg/100 g). The best separation of compounds present in the analysed polyphenol complex, was achieved by 'comprehensive' LCxLC method using Nucleodur Sphinx RP column in the first dimension and Chromolith Performance column in the second dimension. The shoot extract was found to be a strong antioxidant (EC50 19.4 µg/ml, AAE 427.94 mg/g) and displayed the strongest bactericidal properties towards Corynebacterium diphtheriae. The extract revealed higher cytotoxic activity towards the HL-60 cells (IC50 110 µg/ml) than HeLa (IC50 300 µg/ml). CONCLUSIONS: The shoots of R. idaeus stand out as a valuable source of sanguiin H-6 and ellagic acid and possess a number of biological properties including antioxidative, antimicrobial and cytotoxic.

Isolation and identification of cytotoxic compounds from the rhizomes of Paris quadrifolia L.

BACKGROUND: Paris quadrifolia L. is a medicinal plant which contains steroidal saponins. The present study reports isolation and structural identification of six pennogenyl saponins obtained from P. quadrifolia rhizomes. The four spirostan saponins were obtained from P. quadrifolia for the first time. The cytotoxic effects of the sub-fractions and six compounds isolated from the plant extract were evaluated on tumour cells. MATERIALS AND METHODS: Ethanol extract from the rhizomes of P. quadrifolia were partinioned using column chromatography. The saponins were isolated from the obtained sub-fractions by isocratic RP HPLC and their structures were determined by means of 1D and 2D NMR spectroscopy and MALDI TOF MS. The cytotoxic effects of the sub-fractions and the isolated compounds were tested against human promyelocytic leukaemia cells (HL-60), human cervical adenocarcinoma cells (HeLa) and human breast cancer cells (MCF-7) using the [(3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Six pennogenyl saponins were isolated from P. quadrifolia rhizomes: pennogenin 3-O-ß-D-glucopyranoside (1), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside (2), pennogenin 3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranoside (3), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside (4), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside (5), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside (6). Pennogenyl saponins 5 and 6 exhibited cytotoxic activity against HL-60, HeLa and MCF-7 tumour cells with IC50 values of 1.0 ± 0.04 µg/ml, 1.8 ± 0.072 µg/ml and 2.4 ± 0.096 µg/ml respectively, and 2.0 ± 0.08 µg/ml, 2.5 ± 0.125 µg/ml and 3.2 ± 0.128 µg/ml respectively. CONCLUSION: Compounds 1-4 were isolated from this species for the first time.

Combination of silver nanoparticles and Drosera binata extract as a possible alternative for antibiotic treatment of burn wound infections caused by resistant Staphylococcus aureus.

Staphylococcus aureus is the most common infectious agent involved in the development of skin infections that are associated with antibiotic resistance, such as burn wounds. As drug resistance is a growing problem it is essential to establish novel antimicrobials. Currently, antibiotic resistance in bacteria is successfully controlled by multi-drug therapies. Here we demonstrate that secondary metabolites present in the extract obtained from Drosera binata in vitro cultures are effective antibacterial agents against S. aureus grown in planktonic culture and in biofilm. Moreover, this is the first report demonstrating the synergistic interaction between the D. binata extract and silver nanoparticles (AgNPs), which results in the spectacular enhancement of the observed bactericidal activity, while having no cytotoxic effects on human keratinocytes. Simultaneous use of these two agents in significantly reduced quantities produces the same effect, i.e. by killing 99.9% of bacteria in inoculum or eradicating the staphylococcal biofilm, as higher amounts of the agents used individually. Our data indicates that combining AgNPs with either the D. binata extract or with its pure compound (3-chloroplumbagin) may provide a safe and highly effective alternative to commonly used antibiotics, which are ineffective towards the antibiotic-resistant S. aureus.

Chromatographic analysis of simple phenols in some species from the genus Salix.

INTRODUCTION: Salicis Cortex, made from willow bark is a herbal remedy, which is standardised based on the content of salicin, a compound with analgesic and antiphlogistic properties. However, clinical trials suggest that other compounds also present in Salicis Cortex can contribute to the pharmacological effects. OBJECTIVE: To characterise the composition of phenolic acids in the barks of different species and clones from the genus Salix by use of chromatographic methods--HPTLC and HPLC. METHODOLOGY: The phenolic acid composition was analysed by MGD (multiple gradient development)-HPTLC technique. The separation was performed on HPTLC Diol plates with gradient elution using a mixture of chloroform:hexane:ethyl acetate with increasing concentration of ethyl acetate from 10 to 25%. Derivatisation with thymol reagent was employed for the first time for specific detection of phenolic acids containing methoxyl groups. RESULTS: The presence of all phenolic acids previously reported in the genus Salix was confirmed, namely p-hydroxybenzoic, vanillic, cinnamic, p-coumaric, ferulic and caffeic acids. Furthermore, pyrocatechol as a constituent of willow bark was revealed. The highest concentration of this compound was observed in the S. purpurea bark (2.25 mg/g). CONCLUSION: The presence of a relatively high content of pyrocatechol in Salix species may raise doubts about the safe application of this herbal medicine.