Synergistic antibacterial activity of the combination of the alkaloid sanguinarine with EDTA and the antibiotic streptomycin against multidrug resistant bacteria.

OBJECTIVES: Drug combinations consisting of the DNA intercalating benzophenanthridine alkaloid sanguinarine, the chelator EDTA with the antibiotic streptomycin were tested against several Gram-positive and Gram-negative bacteria, including multi-resistant clinical isolates. METHODS: Microdilution, checkerboard and time kill curve methods were used to investigate the antibacterial activity of the individual drugs and the potential synergistic activity of combinations. KEY FINDINGS: Sanguinarine demonstrated a strong activity against Gram-positive and Gram-negative bacteria (minimum inhibitory concentrations, MIC = 0.5-128 µg/ml), while streptomycin was active against Gram-negative strains (MIC = 2-128 µg/ml). EDTA showed only bacteriostatic activity. Indifference to synergistic activity was seen in the two-drug combinations sanguinarine + EDTA and sanguinarine + streptomycin (fractional inhibitory concentration index = 0.1-1.5), while the three-drug combination of sanguinarine + EDTA + streptomycin showed synergistic activity against almost all the strains (except methicillin-resistant Staphylococcus aureus), as well as a strong reduction in the effective doses (dose reduction index = 2-16 times) of sanguinarine, EDTA and streptomycin. In time kill studies, a substantial synergistic interaction of the three-drug combination was detected against Escherichia coli and Klebsiella pneumoniae. CONCLUSIONS: The combination of drugs, which interfere with different molecular targets, can be an important strategy to combat multidrug resistant bacteria.

Volatile oils from the aerial parts of Eremophila maculata and their antimicrobial activity.

The essential oils isolated from the fresh flowers, fresh leaves, and both fresh and air-dried stems of Eremophila maculata (Scrophulariaceae) were characterized by GC-FID and GC/MS analyses. Sabinene was the major component in most of the oils, followed by limonene, α-pinene, benzaldehyde, (Z)-ß-ocimene, and spathulenol. The leaf and flower essential oils showed antibacterial and antifungal activity against five Gram-positive and four Gram-negative bacterial strains, multi-resistant clinical isolates from patients, i.e., methicillin-resistant Staphylococcus aureus (MRSA), as well as two yeasts. Minimum inhibitory concentrations (MICs) and minimum microbicidal concentrations (MMCs) were between 0.25 and 4 mg/ml.

Anti-infective and cytotoxic properties of Bupleurum marginatum.

BACKGROUND: Bupleurum marginatum Wall. ex DC (Apiaceae) is a perennial herb widely used in traditional Chinese and Kampo medicine for the treatment of various infectious diseases. The biological activities of B. marginatum have not been fully investigated. This study aims to investigate the antitrypanosomal, antimicrobial and antiviral activities of methanol (ME) and dichloromethane (DCM) extracts of B. marginatum aerial parts and the ability of both extracts to inhibit the growth of different cancer cell lines. METHODS: Phytochemical characterization of the extracts was performed by LC-MS profiling. The antitrypanosomal activity was evaluated using the resazurin method. The antimicrobial activity was assessed using agar diffusion and microdilution methods, and the minimum inhibitory concentration (MIC) values were determined. The antiviral activity was determined for 6.25, 12.5, and 50 µg/mL doses using a plaque reduction assay. Cytotoxicity was investigated in eight cancer cell lines (Caco-2, CCL-81, CCRF-CEM, COS-7, HL-60, MIA PaCa-2, MCF-7, and PANC-1) using the MTT assay and the caspase 3/7 activity was determined over the range of 62.5-1000 µg/mL. RESULTS: Phytochemical analyses resulted in the characterization of 15 components, mainly flavonoids and lignans. The DCM extract showed significant antitrypanosomal activity (IC50: 36.21 µg/mL) and moderate activity against Streptococcus pyogenes (MIC value: 0.25 mg/mL). At a dose of 12.5 µg/mL, the DCM extract inhibited 73.6% of the plaque production by hepatitis A virus. CCRF-CEM cells were the most sensitive to both extracts (IC50: 12.5-22.7 µg/mL). The cytotoxicity was mediated by induction of apoptosis (19-fold increase in the cellular caspase 3/7 level after treatment with the DCM extract at 1 mg/mL). CONCLUSIONS: ME and DCM extract of B. marginatum showed anti-infective and antiproliferative effects.

Synergistic interactions in two-drug and three-drug combinations (thymol, EDTA and vancomycin) against multi drug resistant bacteria including E. coli.

Combinations of two or more drugs, which affect different targets, have frequently been used as a new approach against resistant bacteria. In our work we studied the antimicrobial activity (MIC, MBC) of individual drugs (the phenolic monoterpene thymol, EDTA and vancomycin), of two-drug interactions between thymol and EDTA in comparison with three-drug interactions with vancomycin against sensitive and resistant bacteria. Thymol demonstrated moderate bactericidal activity (MBC between 60 and 4000µg/ml) while EDTA only exhibited bacteriostatic activity over a range of 60-4000µg/ml. MICs of vancomycin were between 0.125 and 16µg/ml against Gram-positive and between 32 and 128µg/ml against Gram-negative bacteria. Checkerboard dilution and time-kill curve assays were performed to evaluate the mode of interaction of several combinations against Methicillin-resistant Staphylococcus aureus (MRSA NCTC 10442) and Escherichia coli (ATCC 25922). Checkerboard data indicate indifferent interaction against Gram-positive (FICI=1-1.3) and synergy against Gram-negative bacteria (FICI≈0.4), while time kill analyses suggest synergistic effect in different combinations against both types of bacteria. It is remarkable that the combinations could enhance the sensitivity of E. coli to vancomycin 16-fold to which it is normally insensitive. We have provided proof for the concept, that combinations of known antibiotics with modern phytotherapeutics can expand the spectrum of useful therapeutics.

Synergistic antimicrobial activity of combinations of sanguinarine and EDTA with vancomycin against multidrug resistant bacteria.

A combination of antimicrobial drugs has a potential to overcome multidrug resistant pathogens. In our study we tested the combination of an antimicrobial DNA-intercalating alkaloid (sanguinarine), a chelator (EDTA) with a standard antibiotic (vancomycin), i.e. drugs, which differ in their mode of action. The antibacterial activities of individual substances and of two-drug and three-drug combinations were evaluated for 34 strains of Gram-positive and Gramnegative bacteria (among them 23 clinical isolates) which are not sensitive for vancomycin. MIC and MBC values were determined for each drug individually. Sanguinarine demonstrated a strong activity against all the strains; its activity was comparable to that of antibiotics (MIC = 0.5 - 128 µg/ml). Time kill pharmacokinetics were studied for different concentrations of sanguinarine. A sanguinarine concentration of 16 x MIC was bactericidal against both Gram-positive and Gram-negative strains within 4 to 6 h of incubation. EDTA has only bacteriostatic activity against both Gram-positive and Gram-negative bacteria. As expected, vancomycin is active against Gram-positive bacteria (MIC = 0.125 - 16 µg/ml) but much weaker against Gram-negative bacteria (MIC = 4 - 128 µg/ml). Using the checkerboard design, two- and threedrug combinations were evaluated. Additive and synergistic effects were recorded for all sanguinarine + EDTA and sanguinarine + EDTA + vancomycin combinations against Gram-negative bacteria. Time kill assays indicated that only the combination of 1 x MIC sanguinarine + 1 x MIC EDTA + 1 x MIC vancomycin resulted in a synergistic interaction against MRSA. In the combination assays Gram-negative bacteria became sensitive for vancomycin. More experiments are needed to demonstrate that such a combination strategy also works under in vivo conditions and is clinically relevant.

Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities.

The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 µg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC50 = 13-15 µg/ml).

Antimicrobial activity of a traditionally used complex essential oil distillate (Olbas(®) Tropfen) in comparison to its individual essential oil ingredients.

Plant extracts and essential oils have been widely studied and used as antimicrobial agents in the last decades. In our study we investigated the antimicrobial activities of Olbas(®) Tropfen (in the following named Olbas), a traditionally used complex essential oil distillate, in comparison to its individual essential oil ingredients. Olbas (10 g) consists of three major components such as peppermint oil (5.3 g), eucalyptus oil (2.1 g), and cajuput oil (2.1 g) and of two minor constituents like juniper berry oil (0.3 g) and wintergreen oil (0.2 g). The composition of Olbas and the five individual essential oils were characterized by GLC-MS. According to GLC-MS analysis 1,8-cineol is the main component of the complex essential oil distillate followed by menthol and menthone. The minimum inhibitory and minimum microbicidal concentrations of Olbas and each of the single essential oils were evaluated in 17 species/strains of bacteria and fungi. Time-kill assay was performed to compare the microbicidal activity of Olbas and peppermint oil during several time intervals. Olbas displayed a high antimicrobial activity against all test strains used in this study, among them antibiotic resistant MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococcus). Its antimicrobial activity was comparable to that of peppermint oil which was the most potent one of all individual essential oils tested. In the time kill assay Olbas as well as peppermint oil demonstrated similar microbicidal activities. Based on its wide antimicrobial properties Olbas can be a useful agent for the treatment of uncomplicated infections of skin and respiratory tract.

Flavonoids in Scutellaria immaculata and S. ramosissima (Lamiaceae) and their biological activity.

OBJECTIVES: The aim of this study was to investigate the flavonoid composition of Scutellaria immaculata and S. ramosissima (Lamiaceae) and the in-vitro biological activity of their extracts and flavonoids. METHODS: The flavonoid composition of S. immaculata (Si) and S. ramosissima (Sr) were analysed using LC-MS. Antimicrobial activity was studied in vitro against a range of bacteria and fungi using diffusion and microdilution methods. Anti-trypanosomal and cell proliferation inhibitory activity of the extracts and flavonoids was assessed using MTT. The antioxidant activity of the flavonoids and extracts were evaluated using DPPH* test. KEY FINDINGS: LC-MS investigation of Si and Sr plants allowed the identification, for the first time, of an additional 9 and 16 flavonoids, respectively. The methanol, chloroform and water extracts from these plants and six flavonoids (scutellarin, chrysin, apigenin, apigenin-7-O-glucoside, cynaroside and pinocembrine) exhibited significant inhibition of cell growth against HeLa, HepG-2 and MCF-7 cells. The chloroform extract of Sr showed potent cytotoxic effects with IC50 (drug concentration which resulted in a 50% reduction in cell viability) values of 9.25 ± 1.07 µg/ml, 12.83 ± 1.49 µg/ml and 17.29 ± 1.27 µg/ml, respectively. The highest anti-trypanosomal effect against T. b. brucei was shown by the chloroform extract of Sr with an IC50 (drug concentration which resulted in a 50% inhibition of the biological activity) of 61 µg/ml. The pure flavonoids showed an IC50 range between 3 and 29 µm, with cynaroside as the most active compound with an IC50 value of 3.961 ± 0.133 µm. The chloroform extract of Sr has potent antimicrobial activity against Streptococcus pyogenes (minimum inhibitory concentration, MIC = 0.03 mg/ml). Pinocembrine exhibited a strong activity against the all bacteria except Escherichia coli and yeasts. Water extracts of Sr and Si exhibited potent antioxidant activity with IC50 values of 5.62 ± 0.51 µg/ml and 3.48 ± 0.02 µg/ml, respectively. Scutellarin exerted stronger antioxidant activity than other flavonoids. CONCLUSIONS: This is the first study reporting an in-vitro biological investigation for Si and Sr. Especially the chloroform extract of Sr showed potent anticancer and antimicrobial activity. Cynaroside had a highly selective and strong cytotoxicity against T. b. brucei while showing only mild effects against cancer cells.

Carlina oxide--a natural polyacetylene from Carlina acaulis (Asteraceae) with potent antitrypanosomal and antimicrobial properties.

Carlina acaulis (Asteraceae) has a long history of medicinal use in Europe due to its antimicrobial properties. The strong activity of Carlina oxide, themain compound of the essential oil of C. acaulis against two MRSA strains, Streptococcus pyogenes, Pseudomonas aeruginosa, Candida albicans, and C. glabrata was confirmed. A strong and selective activity against Trypanosoma brucei brucei with an IC50 of 1.0 µg/mL and a SI of 446 compared to human HeLa cells was recorded. The selective toxicity of Carlina oxide makes it a promising lead compound for the development of drugs to treat African trypanosomiasis and multiresistant gram-positive bacteria.