Synergistic interaction of PMAP-36 and PRW4 with aminoglycoside antibiotics and their antibacterial mechanism

The antimicrobial peptide PMAP-36 is a highly cationic and amphipathic α-helical peptide. PRW4 is a truncated analog that replaces paired lysine residues with tryptophan along the N-terminal and deletes the C-terminal hydrophobic tail of PMAP-36. Studies on the two peptides have already been performed. However, whether there is a synergistic effect with antibiotics has not been investigated, and the study of the antibacterial mechanism of the peptides is inadequate. In this study, antibiotic-peptide combinations were tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, and the confocal laser scanning microscopy (LSCM) and DNA gel retardation were measured. The results indicated synergy between the peptides and gentamicin when tested against E. coli [fractional lethal concentration (FLC) < 0.5]; partial synergy was observed between the peptides and gentamicin against S. aureus (0.5 < FLC < 1); and streptomycin showed no reaction with the peptides against E. coli and S. aureus (1 < FLC < 4). LSCM and DNA binding suggest that PMAP-36 was able to translocate across the bacterial membranes and interact with intracellular DNA, but PRW4 presented no DNA-binding ability. These results indicate that the combination of PMAP-36 and PRW4 with aminoglycosides may provide useful information for clinical application, and the antibacterial mechanism of peptides likely does not solely involve cytoplasmic-membrane permeabilization.     

Effect of salt stress on the antimicrobial activity of <Emphasis Type="Italic">Ruta chalepensis</Emphasis> essential oils

In the current investigation, the biological activities of essential oils obtained from organs of Ruta chalepensis plants grown under salt stress (0, 50 and 100 mM NaCl) were analyzed. Their chemical composition was often investigated by GC/FID and GC–MS and the antimicrobial activities towards eight bacteria (Salmonella All, Salmonella K, Escherichia coli 45AG, Escherichia coli 45AI, Staphylococcus aureus 9402, Staphylococcus aureus 02B145, Listeria 477 and Pseudomonas aeruginosa ATCC 10145) and five fungi strains (Aspergillus, Saccharomycee crvisiale, Streptomyces griseus, Fusarium solani and Penicillium thomii) were studied. Results revealed that salt increased essential oil production in leaves at 50 and 100 mM NaCl. A total of 20 compounds were identified in leaves, undecan-2-one, nonan-2-one and geijerene being the dominant ones. In stems, 21 compounds were found; they were dominated by decan-2-one, geijerene, nonan-2-one and undecan-2-one. In contrast, roots exhibited a large variation with 25 volatile compounds and octyl acetate, methyl decanoate, phytyl acetate were the major ones. Salt stress induced significant antibacterial activity changes, mainly in leaves and stems. In leaves, the minimum inhibitory and bactericidal concentration decreased at 100 mM NaCl against Listeria 477, the two strains of E. coli (45AG and 45AI) and P. aeruginosa but it increased versus other bacteria. In stems, salt increased oil antibacterial activity against all strains except P. aeruginosa ATCC 10145. Root oil showed the least antibacterial activity under saline conditions versus Listeria 477 and P. aeruginosa ATCC 10145. As regards antifungal activity, NaCl reduced the antifungal activity of essential oils against the majority of fungi strains.     

Antimicrobial activity and carbohydrate specificity of new mycelial lectins from Fusarium sp.

In the present study, ten Fusarium sp. were screened for the presence of lectins by hemagglutination assay using human and animal erythrocytes. Amongst them nine species, namely F. acuminatum, F. chlamydosporium, F. coeruleum, F. compactum, F. concolor, F. crookwellense, F. culmorum, F. decemcellulare and F. dimerum were found to possess lectin activity. Neuraminidase treatment to rabbit erythrocytes considerably augmented hemagglutination titre, but no such effect was observed with protease-treated erythrocytes. Lectins were tested for inhibition of hemagglutination activity against a panel of carbohydrates. Majority of the lectins were inhibited by L-fucose, D-galactose, bovine submaxillary mucin and dextran. γ-Globulin was inhibitory against lectins from F. acuminatum, F. chlamydosporium, F. compactum and F. culmorum at a concentration of >250 μg/mL, whereas bovine submaxillary mucin and porcine stomach mucin were observed to be strongest inhibitors of lectin from F. compactum with minimum inhibitory concentration of 7.18 μg/mL and 15.6 μg/mL, respectively. Most of the lectins displayed antimicrobial activity against Bacillus cereus, Escherichia coli, Staphylococcus aureus and Aspergillus niger. Lectins from F. chlamydosporium, F. culmorum and F. crookwellense have also exhibited antimicrobial activity against Candida albicans. These findings illustrate the significance of Fusarium sp. lectins in clinical applications.     

Antibacterial activities of nemonoxacin against clinical isolates of Staphylococcus aureus: an in vitro comparison with three fluoroquinolones

In comparison with ciprofloxacin, levofloxacin and moxifloxacin, antimicrobial activity of nemonoxacin against ciprofloxacin-susceptible/-resistant methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) was determined with the availability to select resistant mutants evaluated. Minimum inhibitory concentrations and mutant prevention concentrations of quinolones were determined by agar dilution method, that concentrated bacterial cells were spread onto Mueller–Hinton agar plates containing antibacterials at different concentrations. Selection index (SI) was calculated. Minimum inhibitory concentration and mutant prevention concentration of nemonoxacin were 0.063 and 0.25 μg/mL for ciprofloxacin-susceptible MSSA and those were 0.5 and 4.0 μg/mL for ciprofloxacin-resistant MSSA, lower than observations of three fluoroquinolones distinctly. SI of nemonoxacin and moxifloxacin were similar, with narrower mutant selective window than levofloxacin and ciprofloxacin. Minimum inhibitory concentration and mutant prevention concentration of nemonoxacin were 0.25 and 2.0 μg/mL for ciprofloxacin-susceptible MRSA, which were 0.5 and 16.0 μg/mL for ciprofloxacin-resistant MRSA. Values were lower than those determined from fluoroquinolones. Nemonoxacin presents good antimicrobial activity against clinical isolates of S. aureus, especially for ciprofloxacin-resistant strains. But stepwise mutant accumulation of ciprofloxacin-resistant MRSA can be hardly inhibited by nemonoxacin with pharmacokinetic parameters considered.     

Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus

Background

The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins.

Methodology/Principal Findings

A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA).

Conclusions/Significance

The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory pathogens

Thymus longicaulis C. Presl is a small aromatic perennial herb used as a traditional remedy for cold, flu and cough. Composition of the essential oil of T. longicaulis from Croatia and its in vitro antimicrobial activity against the most common respiratory pathogens were evaluated. The yield of essential oil obtained by hydrodistillation from aerial plant parts was 1.2%. According to the GC-MS analysis, a total of forty one compounds (99%) were identified. Thymol (46.3%), ??-terpinene (16.2%), thymyl methyl ether (11.4%), and p-cymene (9.4%) were the main components. Antimicrobial activity of the essential oil against six clinically isolated bacterial and yeast strains was determined using standard disc agar diffusion method and microdilution broth assay. The essential oil exhibited antimicrobial activity towards all tested respiratory pathogens. The most sensitive strains were Haemophilus influenzae and Streptococcus pneumoniae(MIC=0.78 mg/mL), while Staphylococcus aureus was the most resistant (MIC>25.00 mg/mL). Our results indicate that T. longicaulis essential oil could be effective against clinically relevant respiratory pathogens which have the ability to develop resistance to antimicrobial drugs.     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

Biological activity of Cymbopogon schoenanthus essential oil

IntroductionA number of plant species, including Cymbopogon schoenanthus, are traditionally used for the treatment of various diseases. C. schoenanthus is currently, traded in the Saudi markets, and thought to have medicinal value. This study aimed at investigating the biological activities of C. schoenanthus against both Gram-positive and Gram-negative bacteria and to identify its chemical ingredients.Materials and methodsThe inhibitory effects of water extracts of C. schoenanthus essential oils were evaluated against ten isolates of both Gram-positive and Gram-negative bacteria using the agar well diffusion and dilution methods. The minimum inhibitory concentration (MIC) was assayed using the Broth microdilution test on five of the ten isolates. The death rates were determined by the time kill assay, done according to the Clinical Laboratory Standards Institute (CLSI) guidelines. The chemical composition of the essential oils of the plant was performed using GC/MS.ResultsThe C. schoenanthus essential oil was effective against Escherichia coli, Staphylococcus aureus, methicillin-sensitive (MSSA) S. aureus (MRSA) and Klebsiella pneumoniae. The essential oil was not effective against Staphylococcus saprophyticus at the highest concentration applied of >150 μg/ml. The MIC values were as follows: 9.37 μg/ml for E. coli 4.69 μg/ml for S. aureus (MRSA), 2.34 mg/ml for MSSA and 2.34 μg/ml for K. pneumoniae. The time-kill assay indicated that there was a sharp time dependent decline in K. pneumoniae counts in the presence of the oil. This is in contrast to a gradual decline in the case of S. aureus under the same conditions. The eight major components of the essential oil were: piperitone (14.6%), cyclohexanemethanol (11.6%), β-elemene (11.6%), α-eudesmol (11.5%), elemol (10.8%), β-eudesmol (8.5%), 2-naphthalenemethanol (7.1%) and γ-eudesmol (4.2%).ConclusionThe results of the present study provide a scientific validation for the traditional use of C. schoenanthus as an antibacterial agent. Future work is needed to investigate and explore its application in the environmental and medical fields. In addition, to evaluating the efficacy of the individual ingredients separately to better understand the underlying mechanism.     

玫瑰精油的化学成分及其抗菌活性

通过水蒸汽同步蒸馏法提取玫瑰精油,采用GC-MS方法分析了玫瑰精油的化学组成,共鉴定出其中14个化学成分并测定其相对含量,占总含量的95.25%。香茅醇为玫瑰精油的主要成分,相对含量为90.37%。体外抑菌实验表明,玫瑰精油除对黑曲霉没有抗菌活性外,对其它7种供试菌均具有不同程度的抑制作用,其中对表皮葡萄球菌、金黄色葡萄球菌和大肠杆菌的最小抑菌浓度(MIC)为0.063%(v/v),对枯草芽孢杆菌、变形杆菌和白色念珠菌的最小抑菌浓度（MIC）为0.125%(v/v),而对绿脓杆菌(Pseudomonas aeruginosa)的抗菌活性相对较弱,MIC为0.5%(v/v)。抑菌直径结果也表明了玫瑰精油除对黑曲霉、绿脓杆菌的抗菌活性较弱外,对其它6种菌株的抑菌直径都大于8.5 mm。考察了玫瑰精油对3种敏感菌株包括金黄色葡萄球菌(革兰氏阳性菌)、大肠杆菌(革兰氏阴性菌)和白色念珠菌(真菌)的杀菌动态过程,为玫瑰精油的应用提供了理论依据。     

Anticancer Activities of Antimicrobial BmKn2 Peptides Against Oral and Colon Cancer Cells

There is considerable current interest in developing antimicrobial and anticancer agents with a new mode of action. The antimicrobial peptides are regarded as a potential solution for treating cancer cells. The antimicrobial effect of 6 synthetic peptides against 7 bacterial species was evaluated. The result showed that IsCT, BmKn2 and BMAP-28 exhibited broad range of action against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, methicillin resistant S. aureus DMST 20651, Staphylococcus epidermidis ATCC 12228, Acinetobacter baumanii ATCC 19066, Escherichia coli ATCC 25922 and Salmonella typhi DMST 562 at minimal inhibitory concentrations (MIC) of 2.97–24.28 μM. Neither AMP induced significant hemolysis, or showed cytotoxic on dental pulp stem cells and smooth muscle cells at their MICs. In addition, BmKn2 inhibited growth of human oral squamous carcinoma HSC4 cells and human colon cancer SW620 cells with IC₅₀ of 17.26 and 40 µM, respectively. Taken together, BmKn2 peptide from scorpion venom may offer a novel therapeutic strategy for development of cationic antimicrobial and anticancer peptides as potential new therapeutic agents.     

Assessment of synergistic antibacterial activity of combined biosurfactants revealed by bacterial cell envelop damage

Besides potential surface activity and some beneficial physical properties, biosurfactants express antibacterial activity. Bacterial cell membrane disrupting ability of rhamnolipid produced by Pseudomonas aeruginosa C2 and a lipopeptide type biosurfactant, BS15 produced by Bacillus stratosphericus A15 was examined against Staphylococcus aureus ATCC 25923 and Escherichia coli K8813. Broth dilution technique was followed to examine minimum inhibitory concentration (MIC) of both the biosurfactants. The combined effect of rhamnolipid and BS15 against S. aureus and E. coli showed synergistic activity by expressing fractional inhibitory concentration (FIC) index of 0.43 and 0.5. Survival curve of both the bacteria showed bactericidal activity after treating with biosurfactants at their MIC obtained from FIC index study as it killed > 90% of initial population. The lesser value of MIC than minimum bactericidal concentration (MBC) of the biosurfactants also supported their bactericidal activity against both the bacteria. Membrane permeability against both the bacteria was supported by amplifying protein release, increasing of cell surface hydrophobicity, withholding capacity of crystal violet dye and leakage of intracellular materials. Finally cell membrane disruption was confirmed by scanning electron microscopy (SEM). All these experiments expressed synergism and effective bactericidal activity of the combination of rhamnolipid and BS15 by enhancing the bacterial cell membrane permeability. Such effect of the combination of rhamnolipid and BS15 could make them promising alternatives to traditional antibiotic in near future.     

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μm) than against E. coli (64 μm) and an intermediate cytotoxicity against hRBC (30 μm). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.     

Influence of bovine lactoferrin on the growth of selected probiotic bacteria under aerobic conditions

Bovine lactoferrin (bLf) is a natural glycoprotein, and it shows broad-spectrum antimicrobial activity. However, reports on the influences of bLf on probiotic bacteria have been mixed. We examined the effects of apo-bLf (between 0.25 and 128 mg/mL) on both aerobic and anaerobic cultures of probiotics. We found that bLf had similar effects on the growth of probiotics under aerobic or anaerobic conditions, and that it actively and significantly (at concentrations of >0.25 mg/mL) retarded the growth rate of Bifidobacterium bifidum (ATCC 29521), B. longum (ATCC 15707), B. lactis (BCRC 17394), B. infantis (ATCC 15697), Lactobacillus reuteri (ATCC 23272), L. rhamnosus (ATCC 53103), and L. coryniformis (ATCC 25602) in a dose-dependent manner. Otherwise, minimal inhibitory concentrations (MICs) were 128 or >128 mg/mL against B. bifidum, B. longum, B. lactis, L. reuteri, and L. rhamnosus (ATCC 53103). With regard to MICs, bLf showed at least four-fold lower inhibitory effect on probiotics than on pathogens. Intriguingly, bLf (>0.25 mg/mL) significantly enhanced the growth of Rhamnosus (ATCC 7469) and L. acidophilus (BCRC 14065) by approximately 40–200 %, during their late periods of growth. Supernatants produced from aerobic but not anaerobic cultures of L. acidophilus reduced the growth of Escherichia coli by about 20 %. Thus, bLf displayed a dose-dependent inhibitory effect on the growth of most probiotic strains under either aerobic or anaerobic conditions. An antibacterial supernatant prepared from the aerobic cultures may have significant practical use.     

Molecular docking,PASS analysis,bioactivity score prediction,synthesis, characterization and biological activity evaluation of a functionalized 2-butanone thiosemicarbazone ligand and its complexes

2-Butanone thiosemicarbazone ligand was prepared by condensation reaction between thiosemicarbazide and butanone. The ligand was characterized by ¹H NMR, ¹³C NMR, FT-IR, mass spectrometry and UV spectroscopic studies. Docking studies were performed to study inhibitory action against topoisomerase II (Topo II) and ribonucleoside diphosphate reductase (RR) enzymes. Inhibition constants (K _i ) of the ligand were 437.87 and 327.4 μM for the two enzymes, respectively. The ligand was tested for its potential anticancer activity against two cancer cell lines MDA-MB-231 and A549 using MTT assay and was found to exhibit good activity at higher doses with an IC₅₀ = 80 μM against human breast cancer cell line MDA-MB-231. On the other hand, no significant activity was obtained against the lung carcinoma cell line A549. Antibacterial activity of the ligand was tested against Staphylococcus aureus and E. coli using the disc diffusion method. Ligand did not exhibit any significant antibacterial activity. Four complexes of Co(III), Fe(II), Cu(II), and Zn(II) were prepared with the ligand and characterized by various spectroscopic studies. Low molar conductance values were obtained for all complexes displaying non-electrolyte nature except in Co(III) complex. As expected, complexation with metal ions significantly increased the cytotoxicity of the ligand against the tested cell lines viz. IC₅₀ values of <20 μM for Co, Fe, and Zn complexes and approx. 80 μM against MDA cells versus IC₅₀ value of <20 μM for Co and Cu complexes and that of 30 and 50 μM for Fe and Zn complexes, respectively, against A549 cells. The Cu complex was found to be active against E. coli and S. aureus with MIC values in the range of 6–10 mg/mL. Other than Cu, only Co complex was found to possess antibacterial activity with MIC values of 5–10 mg/mL when tested against S. aureus. Bioactivity score and Prediction of Activity Spectra for Substances (PASS) analysis also depicted the drug-like nature of ligand and complexes.     

Compositional studies and Biological activities of Perovskia abrotanoides Kar. oils

Background

Current study has been designed to evaluate the chemical composition of essential and fixed oils from stem and leaves of Perovskia abrotanoides and antioxidant and antimicrobial activities of these oils.

Results

GC-MS analysis of essential oil identified 19 compounds with (E)-9-dodecenal being the major component in stem and hexadecanoic acid in leaves. In contrast, GC-MS analysis of fixed oil showed 40 constituents with α-amyrin the major component in stem and α-copaene in leaves. The antioxidant activity showed the highest value of 76.7% in essential oil from leaves in comparison with fixed oil from stem (45.9%) through inhibition of peroxidation in linoleic acid system. The antimicrobial assay tested on different microorganisms (e.g. E. coli, S. aureus, B. cereus, Nitrospira, S. epidermis, A. niger, A. flavus and C. albicans) showed the higher inhibition zone at essential oil from leaves (15.2 mm on B. cereus) as compared to fixed oil from stem (8.34 mm on S. aureus) and leaves (11.2 mm on S. aureus).

Conclusions

The present study revealed the fact that essential oil analyzed from Perovskia abrotanoides stem and leaves could be a promising source of natural products with potential antioxidant and antimicrobial activities, as compared to fixed oil.     

Carvacrol Codrugs: A New Approach in the Antimicrobial Plan

Objective

The increasing prevalence of antibiotic-resistant bacterial infections led to identify alternative strategies for a novel therapeutic approach. In this study, we synthesized ten carvacrol codrugs – obtained linking the carvacrol hydroxyl group to the carboxyl moiety of sulphur-containing amino acids via an ester bond – to develop novel compounds with improved antimicrobial and antibiofilm activities and reduced toxicity respect to carvacrol alone.

Method

All carvacrol codrugs were screened against a representative panel of Gram positive (S. aureus and S. epidermidis), Gram negative (E. coli and P. aeruginosa) bacterial strains and C. albicans, using broth microdilution assays.

Findings

Results showed that carvacrol codrug 4 possesses the most notable enhancement in the anti-bacterial activity displaying MIC and MBC values equal to 2.5 mg/mL for all bacterial strains, except for P. aeruginosa ATCC 9027 (MIC and MBC values equal to 5 mg/mL and 10 mg/mL, respectively). All carvacrol codrugs 1-10 revealed good antifungal activity against C. albicans ATCC 10231. The cytotoxicity assay showed that the novel carvacrol codrugs did not produce human blood hemolysis at their MIC values except for codrugs 8 and 9. In particular, deepened experiments performed on carvacrol codrug 4 showed an interesting antimicrobial effect on the mature biofilm produced by E. coli ATCC 8739, respect to the carvacrol alone. The antimicrobial effects of carvacrol codrug 4 were also analyzed by TEM evidencing morphological modifications in S. aureus, E. coli, and C. albicans.

Conclusion

The current study presents an insight into the use of codrug strategy for developing carvacrol derivatives with antibacterial and antibiofilm potentials, and reduced cytotoxicity.     

<Emphasis Type="Italic">Acorus calamus</Emphasis> Linn.: phytoconstituents and bactericidal property

Acorus calamus Linn. of the family Araceae (Acoraceae), commonly known as Sweet Flag and Vacha. The rhizome of this plant has medicinal properties against bugs, moths, lice and emetic stomach in dyspepsia. Chemical composition of the hydro-distilled essential oil obtained from the rhizomes of A. calamus was analyzed by gas chromatography equipped with flame ionization detector and gas chromatography coupled with mass spectrometry. The essential oil of A. calamus and its major compound β-asarone were tested against five Gram-positive, eight Gram-negative bacteria, and three fungi by the tube-dilution method at a concentration rang of 5.0–0.009 mg/mL. Forty constituents were identified which comprised 98.3 % of the total oil. The major compound β-asarone (80.6 %) was identified and confirm by NMR (¹H– & ¹³C–) in rhizome oil of A. calamus. The organism Micrococcus luteus was found to be more susceptible to the oil with minimum bactericidal concentration (MBC) value of 0.032 ± 0.004 mg/mL, followed by Aspergillus fumigatus, Aspergillus niger and Micrococcus flavus with MBC values of 0.104 ± 0.016, 0.117 ± 0.017 and 0.143 ± 0.013 mg/mL, respectively. The compound β-asarone was susceptible to the microorganism A. niger with MBC value 0.416 ± 0.065 mg/mL. The present study revealed that tetraploid variety of A. calamus is growing in this region with substantial amount of β-asarone. The oil showed bactericidal property against tested bacteria and fungi. The β-asarone exhibited poorer bactericidal activity against test microorganisms.     

Screening of Indonesian peat soil bacteria producing antimicrobial compounds

The development and world-wide spread of multidrug-resistant (MDR) bacteria have a high concern in the medicine, especially the extended-spectrum of beta-lactamase (ESBL) producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). There are currently very limited effective antibiotics to treat infections caused by MDR bacteria. Peat-soil is a unique environment in which bacteria have to compete each other to survive, for instance, by producing antimicrobial substances. This study aimed to isolate bacteria from peat soils from South Kalimantan Indonesia, which capable of inhibiting the growth of Gram-positive and Gram-negative bacteria. Isolates from peat soil were grown and identified phenotypically. The cell-free supernatant was obtained from broth culture by centrifugation and was tested by agar well-diffusion technique against non ESBL-producing E. coli ATCC 25922, ESBL-producing E. coli ATCC 35218, methicillin susceptible Staphylococcus aureus (MSSA) ATCC 29,213 and MRSA ATCC 43300. Putative antimicrobial compounds were separated using SDS-PAGE electrophoresis and purified using electroelution method. Antimicrobial properties of the purified compounds were confirmed by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). In total 28 isolated colonies were recovered; three (25PS, 26PS, and 27PS) isolates produced proteins with strong antimicrobial activities against both reference strains. The substance of proteins from three isolates exerted strong antimicrobial activity against ESBL-producing E. coli ATCC 35,218 (MIC = 2,80 µg/mL (25PS), 3,76 µg/mL (26PS), and 2,41 µg/mL (27PS), and MRSA ATCC 43,300 (MIC = 4,20 µg/mL (25PS), 5,65 µg/mL (26PS), and 3,62 µg/mL (27PS), and also had the ability bactericidal properties against the reference strains. There were isolates from Indonesian peat which were potentials sources of new antimicrobials.     

Stability of the hybrid plasmid pIM138 and its curing by some eliminating agents

Hybrid plasmid pIM138 was constructed by insertion of a chromosomal fragment with the threonme operon fromEscherichia coli into the pBR322 vector. Molar mass of pIM138 was 2.8 Mg/mol. Heteroduplexes between pBR322 vector and pIM138 hybrid DNA molecules were prepared. The hybrid plasmid shows a high stability against the curing effect of rifampicin and clorobiocm inE. coli SK1590thr host.     

Chemical Composition and In Vitro Cytotoxicity of Essential Oils from Leaves and Flowers of Callistemon citrinus from Western Himalayas

Background

Plant-based traditional system of medicine continues to play an important role in healthcare. In order to find new potent source of bioactive molecules, we studied the cytotoxic activity of the essential oils from the flowers and leaves of Callistemon citrinus. This is the first report on anticancer potential of essential oils of C. citrinus.

Methods

Cytotoxicity of essential oil was evaluated using sulfo-rhodamine B (SRB) assay against human lung carcinoma (A549), rat glioma (C-6), human colon cancer (Colo-205) and human cervical cancer (SiHa) cells. Apoptosis induction was evaluated by caspase-3/7 activity which was further confirmed by western blotting. Percentage cell apoptosis was determined by Annexin V based dead cell assay followed by DNA content as cell cycle analysis against A549 and C-6 cells. While 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to check the toxicity against normal human peripheral blood mononuclear cells (PBMCs), the immunomodulatory activity on mouse splenocytes was evaluated using SRB assay.

Results

The GC and GC-MS analysis of these essential oils revealed high content of α-pinene (32.3%), limonene (13.1%) and α-terpineol (14.6%) in leaf sample, whereas the flower oil was dominated by 1,8-cineole (36.6%) followed by α-pinene (29.7%). The leaf oil contained higher amount of monoterpene hydrocarbons (52.1%) and sesquiterpenoids (14%) as compared to flower oil (44.6% and 1.2%, respectively). However, the flower oil was predominant in oxygenated monoterpenes (43.5%). Although both leaf and flower oils showed highest cytotoxicity on A549 cells (61.4%±5.0 and 66.7%±2.2, respectively), only 100 μg/mL flower oil was significantly active against C-6 cells (69.1%±3.1). Interestingly, no toxicity was recorded on normal cells.

Conclusion

Higher concentration of 1,8-cineole and/or synergistic effect of the overall composition were probably responsible for the efficacy of flower and leaf oils against the tested cells. These oils may form potential source of natural anti-cancer compounds and play important role in human health.