A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin

A new potent antibacterial protein, for which we propose the name royalisin, was found in royal jelly of the honeybee Apis mellifera L. and purified to homogeneity for the first time by acid extraction, gel filtration, and reverse-phase high pressure liquid chromatography. The primary structure of royalisin was determined to consist of 51 residues, with three intramolecular disulfide linkages, having a calculated molecular mass of 5523 Da. Royalisin is an amphipathic protein, with the C-terminal half of the molecule being rich in charged amino acids; and it showed extensive sequence homology to two other antibacterial proteins, sapecin from embryonic Sarcophaga peregrina cells and phormicins from Phormia terranovae larvae. Royalisin was found to have potent antibacterial activity against Gram-positive bacteria at low concentrations, but not against Gram-negative bacteria. Royalisin may be involved in a defense system active against bacterial invasion of the honeybee.     

Antibacterial activity of major royal jelly proteins of the honeybee (Apis mellifera) royal jelly

Major royal jelly proteins (MRJPs) are the protein components in royal jelly (RJ). MRJPs 1–7 are detected in the honeybee Apis mellifera RJ. Although A. mellifera MRJP (AmMRJP) 2 exhibited antibacterial activity, the other MRJPs with antimicrobial activities in A. mellifera RJ remains largely unknown. Here, we compared the antibacterial activity of recombinant AmMRJPs 1–7 expressed in baculovirus-infected insect cells. Antibacterial assays of recombinant AmMRJPs 1–7 against the gram-negative bacterium Escherichia coli revealed that AmMRJPs 2–5 and 7 exhibited antibacterial activity, whereas AmMRJPs 1 and 6 displayed almost no antibacterial activity. Consistent with the antibacterial activity of AmMRJPs, AmMRJPs 2–5 and 7 are bound to bacterial cell walls. These results indicated that AmMRJPs 2–5 and 7 contribute directly to the antibacterial property of RJ, suggesting that MRJPs play a role in the antimicrobial property of RJ.     

De-Novo Design of Antimicrobial Peptides for Plant Protection

This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of “healthy” food, these peptides might serve as templates for novel antibacterial and antifungal agents.     

Rational Design of Berberine-Based FtsZ Inhibitors with Broad-Spectrum Antibacterial Activity

Inhibition of the functional activity of Filamenting temperature-sensitive mutant Z (FtsZ) protein, an essential and highly conserved bacterial cytokinesis protein, is a promising approach for the development of a new class of antibacterial agents. Berberine, a benzylisoquinoline alkaloid widely used in traditional Chinese and native American medicines for its antimicrobial properties, has been recently reported to inhibit FtsZ. Using a combination of in silico structure-based design and in vitro biological assays, 9-phenoxyalkyl berberine derivatives were identified as potent FtsZ inhibitors. Compared to the parent compound berberine, the derivatives showed a significant enhancement of antibacterial activity against clinically relevant bacteria, and an improved potency against the GTPase activity and polymerization of FtsZ. The most potent compound 2 strongly inhibited the proliferation of Gram-positive bacteria, including methicillin-resistant S. aureus and vancomycin-resistant E. faecium, with MIC values between 2 and 4 µg/mL, and was active against the Gram-negative E. coli and K. pneumoniae, with MIC values of 32 and 64 µg/mL respectively. The compound perturbed the formation of cytokinetic Z-ring in E. coli. Also, the compound interfered with in vitro polymerization of S. aureus FtsZ. Taken together, the chemical modification of berberine with 9-phenoxyalkyl substituent groups greatly improved the antibacterial activity via targeting FtsZ.     

Bacteriocin-Producing Strain of Lactococcus lactis subsp. diacitilactis S50

Lactococcus lactis subsp. diacitilactis S50 produces a bacteriocin, designated bacteriocin S50, which has a narrow antibacterial spectrum. It was active only against Lactococcus species, including a nisin producer exhibiting a bactericidal effect. The activity of bacteriocin S50 was sensitive to proteases. It retained antimicrobial activity after being heated to 100°C for up to 60 min and in the pH range 2 to 11.     

Synergistic Actions of Nisin, Sublethal Ultrahigh Pressure, and Reduced Temperature on Bacteria and Yeast

Nisin in combination with ultrahigh-pressure treatment (UHP) showed strong synergistic effects against Lactobacillus plantarum and Escherichia coli at reduced temperatures (<15°C). The strongest inactivation effects were observed when nisin was present during pressure treatment and in the recovery medium. Elimination (>6-log reductions) of L. plantarum was achieved at 10°C with synergistic combinations of 0.5 μg of nisin per ml at 150 MPa and 0.1 μg of nisin per ml at 200 MPa for 10 min. Additive effects of nisin and UHP accounted for only 1.2- and 3.7-log reductions, respectively. Elimination was also achieved for E. coli at 10°C with nisin present at 2 μg/ml, and 10 min of pressure at 200 MPa, whereas the additive effect accounted for only 2.6-log reductions. Slight effects were observed even against the yeast Saccharomyces cerevisiae with nisin present at 5 μg/ml and with 200 MPa of pressure. Combining nisin, UHP, and lowered temperature may allow considerable reduction in time and/or pressure of UHP treatments. Kill can be complete without the frequently encountered survival tails in UHP processing. The slightly enhanced synergistic kill with UHP at reduced temperatures was also observed for other antimicrobials, the synthetic peptides MB21 and histatin 5. The postulated mode of action was that the reduced temperature and the binding of peptides to the membrane increased the efficacy of UHP treatment. The increases in fatty acid saturation or diphosphatidylglycerol content and the lysylphosphatidyl content of the cytoplasm membrane of L. plantarum were correlated with increased susceptibility to UHP and nisin, respectively.     

Lantibiotic Immunity: Inhibition of Nisin Mediated Pore Formation by NisI

Nisin, a 3.4 kDa antimicrobial peptide produced by some Lactococcus lactis strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, Lactococcus lactis is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC₅₀ of 73±10 nM, an 8–10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the Lactococcus lactis cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.     

Facilitative production of an antimicrobial peptide royalisin and its antibody via an artificial oil-body system

Royalisin found in the royal jelly of Apis mellifera is an antimicrobial peptide (AMP). It has a molecular weight of 5.5 kDa, which contains six cysteine residues. In this study, royalisin was overexpressed in Escherichia coli AD494 (DE3) as two oleosin-fusion proteins for preparation of its antibodies and functional purification. The recombinant royalisin, fused with oleosin central hydrophobic domain in both N- and C-termini, was reconstituted with triacylglycerol and phospholipids to form artificial oil bodies (AOBs). The AOBs were then purified to raise the antibodies. These antibodies could recognize both the native and recombinant royalisins, but not oleosin. Another oleosin-intein S-fusion protein was purified by AOBs system, and royalisin was subsequently released from the AOBs through self-splicing of the intein. The recombinant royalisin exhibited high antibacterial activity, which suggested that it was refolded to its functional structure. These results demonstrated that AOBs system is an efficient method to functionally express and purify small AMPs. In addition, it also provides a facile platform for the production of antibodies against small peptides.     

Screening and Characterisation of Antimicrobial Properties of Semisynthetic Betulin Derivatives

Betulin (lup-20(29)-ene-3β, 28-diol) is a naturally occurring triterpene, which is found in substantial amounts from the outer bark of birch trees. A library of 51 structurally diverse semisynthetic betulin derivatives was screened against five bacterial strains, Enterobacter aerogenes, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus and a fungal strain Candida albicans, using broth microdilution assays. Primary antimicrobial screening at 50 µM concentration led to the identification of five compounds showing antimicrobial properties (inhibition of growth by >70% against one or more microbial strains). According to the dose-response results, 28-O-(N-acetylanthraniloyl)betulin (compound 5) was the most active, showing MIC₉₀ of 6.25 µM against two Gram-positive bacteria, E. faecalis and S. aureus. However, the activity of this compound was affected by albumin binding, which was demonstrated by the loss of activity in a host-pathogen co-culture assay as well as in the antibacterial assay in the presence of increased concentration of albumin. Furthermore, the effects on mammalian cells were evaluated by cytotoxicity assessment on hepatocyte cell culture after 24 h exposure to the compounds. Betulinic aldehyde (18), betulin-28-oxime (31) and hetero cycloadduct with acetoxy groups at carbon atoms 3 and 28 and ethyl substituent at the triazolo ring (43) displayed cytotoxicity towards hepatocytes, with IC₅₀ values of 47, 25 and 16 µM, respectively. The IC₅₀ value for 28-O-(N-acetylanthraniloyl)betulin (5) was 56 µM. The current study presents an insight into using betulin scaffold for developing derivatives with antibacterial potential, and furthermore the necessity of in-depth analysis of found actives through selectivity profiling and follow-up studies including in silico ADMET predictions.     

Antibacterial Activities of Nisin Z Encapsulated in Liposomes or Produced In Situ by Mixed Culture during Cheddar Cheese Ripening

This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7°C for 6 months. In some trials, L. innocua was added to cheese milk at 10⁵ to 10⁶ CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 ± 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 ± 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.     

Repurposing Salicylanilide Anthelmintic Drugs to Combat Drug Resistant Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacterium that has become the leading cause of hospital acquired infections in the US. Repurposing Food and Drug Administration (FDA) approved drugs for antimicrobial therapy involves lower risks and costs compared to de novo development of novel antimicrobial agents. In this study, we examined the antimicrobial properties of two commercially available anthelmintic drugs. The FDA approved drug niclosamide and the veterinary drug oxyclozanide displayed strong in vivo and in vitro activity against methicillin resistant S. aureus (minimum inhibitory concentration (MIC): 0.125 and 0.5 μg/ml respectively; minimum effective concentration: ≤ 0.78 μg/ml for both drugs). The two drugs were also effective against another Gram-positive bacteria Enterococcus faecium (MIC 0.25 and 2 μg/ml respectively), but not against the Gram-negative species Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes. The in vitro antimicrobial activity of niclosamide and oxyclozanide were determined against methicillin, vancomycin, linezolid or daptomycin resistant S. aureus clinical isolates, with MICs at 0.0625-0.5 and 0.125-2 μg/ml for niclosamide and oxyclozanide respectively. A time-kill study demonstrated that niclosamide is bacteriostatic, whereas oxyclozanide is bactericidal. Interestingly, oxyclozanide permeabilized the bacterial membrane but neither of the anthelmintic drugs exhibited demonstrable toxicity to sheep erythrocytes. Oxyclozanide was non-toxic to HepG2 human liver carcinoma cells within the range of its in vitro MICs but niclosamide displayed toxicity even at low concentrations. These data show that the salicylanilide anthelmintic drugs niclosamide and oxyclozanide are suitable candidates for mechanism of action studies and further clinical evaluation for treatment of staphylococcal infections.     

Antioxidant capacity of major royal jelly proteins of honeybee (Apis mellifera) royal jelly

Major royal jelly proteins (MRJPs) of honeybee royal jelly (RJ) exhibit antimicrobial and antioxidant activities. Although MRJPs of Apis mellifera RJ (AmMRJPs) responsible for antibacterial activity have been identified, AmMRJPs with antioxidant effects remain to be elucidated. Here we identified and compared the antioxidant activities of purified recombinant AmMRJPs 1–7, which are expressed in baculovirus-infected insect cells. Antioxidant assays of recombinant AmMRJPs 1–7 against H₂O₂ revealed that AmMRJPs reduce caspase-3 activity and oxidative stress-induced cell apoptosis and lead to increased cell viability. Consistent with these results, AmMRJPs 1–7 exhibit 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and protect against oxidative DNA damage. These results indicate that AmMRJPs play a role as antioxidants in A. mellifera RJ.     

Antimicrobial Activity and Probable Mechanisms of Action of Medicinal Plants of Kenya: Withania somnifera,Warbugia ugandensis,Prunus africana and Plectrunthus barbatus

Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus are used traditionally in Kenya for treatment of microbial infections and cancer. Information on their use is available, but scientific data on their bioactivity, safety and mechanisms of action is still scanty. A study was conducted on the effect of organic extracts of these plants on both bacterial and fungal strains, and their mechanisms of action. Extracts were evaluated through the disc diffusion assay. Bacteria and yeast test strains were cultured on Mueller-Hinton agar and on Sabouraud dextrose agar for the filamentous fungi. A 0.5 McFarland standard suspension was prepared. Sterile paper discs 6 mm in diameter impregnated with 10 µl of the test extract (100 mg/ml) were aseptically placed onto the surface of the inoculated media. Chloramphenicol (30 µg) and fluconazole (25 µg) were used as standards. Discs impregnated with dissolution medium were used as controls. Activity of the extracts was expressed according to zone of inhibition diameter. MIC was determined at 0.78–100 mg/ml. Safety studies were carried using Cell Counting Kit 8 cell proliferation assay protocol. To evaluate extracts mechanisms of action, IEC-6 cells and RT-PCR technique was employed in vitro to evaluate Interleukin 7 cytokine. Investigated plants extracts have both bactericidal and fungicidal activity. W. ugandensis is cytotoxic at IC₅₀<50 µg/ml with MIC values of less than 0.78 mg/ml. Prunus africana shuts down expression of IL 7 mRNA at 50 µg/ml. W. somnifera has the best antimicrobial (1.5625 mg/ml), immunopotentiation (2 times IL 7 mRNA expression) and safety level (IC₅₀>200 µg/ml). Fractions from W. ugandensis and W. somnifera too demonstrated antimicrobial activity. Mechanisms of action can largely be attributed to cytotoxicity, Gene silencing and immunopotentiation. Use of medicinal plants in traditional medicine has been justified and possible mechanisms of action demonstrated. Studies to isolate and characterize the bioactive constituents continue.     

Antimicrobial and Antimycobacterial Activities of Methyl Caffeate Isolated from Solanum torvum Swartz. Fruit

Abstract

Solanum torvum Swartz. (Solanaceae) fruit is traditionally used for the treatment of bacterial and fungal infections. The methanolic extract was subjected to activity guided fractionation by column chromatography over silica gel. The structure of the compound was elucidated using physical and spectroscopic data. The antimicrobial activity was screened using five Gram-positive bacteria, six Gram-negative bacteria, seven clinical isolates and four fungi. Antimycobacterial activity was screened against two Mycobacterium strains. The zone of inhibition by methyl caffeate ranged from 0 to 22 mm. The lowest minimum inhibitory concentration (MIC) values of methyl caffeate were: 50 μg/ml against P. vulgaris, 25 μg/ml against K. pneumoniae (ESBL-3971), 8 μg/ml against M. tuberculosis (H³⁷Rv) and 8 μg/ml against M. tuberculosis (Rif^R). Methyl caffeate showed moderate antimicrobial and prominent antimycobacterial activities. Methyl caffeate can be evaluated further for drug development.     

Anti-tubercular agents. Part 8: Synthesis,antibacterial and antitubercular activity of 5-nitrofuran based 1,2,3-triazoles

A series of 5-nitrofuran–triazole conjugates were synthesized and evaluated for their antimicrobial activity against both Gram-positive and Gram-negative bacterial strains. All the compounds exhibited promising inhibition towards Gram-positive pathogenic strains, while mild inhibitory effects were observed towards Gram-negative bacterial strains. Some of the compounds 8a, 8b, 8e, 8f, 8h are most active among the series exhibiting MIC value of 1.17 μg/ml against different bacterial strains. The bactericidal activity is found to be in accordance with the bacterial growth inhibition data. Compound 8e was found to be equipotent to the standard drug Ciprofloxacin displaying MBC value of 1.17 μg/ml against the bacterial strain Bacillus subtilis. The compounds have also demonstrated promising antibacterial activity against the resistant strain MRSA and were found to be effective inhibitors of biofilm formation. The compound 8b exhibited excellent anti-biofilm activity with IC₅₀ value as low as 0.8 μg/ml. These conjugates were also screened for antitubercular activity against Mycobacterium tuberculosis H₃₇Rv strain. Compound 8e showed promising antitubercular activity with MIC value of 0.25 μg/ml. Most of these compounds are less toxic to normal mammalian cells than the widely used antibacterial drug Ciprofloxacin.     

Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus

Background

Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin.

Results

The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 μg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 μg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI) <0.5. The killing curve assay had confirmed that the viability of ARSA was dramatically reduced from 5x10⁵ cfu/ml to 10³ cfu/ml within 6 h after exposure to SSE (2 mg/ml) plus ampicillin (0.15 μg/ml) combination. Electron microscopic study clearly revealed that these ARSA cells treated with this combination caused marked morphological damage, peptidoglycan and cytoplasmic membrane damage, and average cell areas significant smaller than control. Obviously, Immunofluorescence staining and confocal microscopic images confirmed that the peptidoglycan of these cells were undoubtedly disrupted by this combination. Furthermore, the CM permeability of ARSA was also increased by this combination. Enzyme assay demonstrated that SSE had an inhibitory activity against β-lactamase in concentrations manner.

Conclusions

So, these findings provide evidence that SSE has the high potential to reverse bacterial resistance to originate traditional drug susceptibility of it and may relate to three modes of actions of SSE: (1) inhibits peptidoglycan synthesis, resulting in morphological damage, (2) inhibits β-lactamases activity, and (3) increases CM permeability. It is widely recognized that many types of drugs are derived from alkaloids. So, this SSE offers the prominent potential to develop a novel adjunct phytopharmaceutical to ampicillin for the treatment of ARSA. Further active ingredients study, toxicity of it, and the synergistic effect on blood and tissue should be performed and confirmed in an animal test or in humans.     

Antimicrobial Activity of Novel Synthetic Peptides Derived from Indolicidin and Ranalexin against Streptococcus pneumoniae

Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in order to defeat multidrug-resistant bacteria such as Streptococcus pneumoniae. In this study, thirteen antimicrobial peptides were designed based on two natural peptides indolicidin and ranalexin. Our results revealed that four hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8, and RN7-IN6 possess potent antibacterial activity against 30 pneumococcal clinical isolates (MIC 7.81-15.62µg/ml). These four hybrid peptides also showed broad spectrum antibacterial activity (7.81µg/ml) against S. aureus, methicillin resistant S. aureus (MRSA), and E. coli. Furthermore, the time killing assay results showed that the hybrid peptides were able to eliminate S. pneumoniae within less than one hour which is faster than the standard drugs erythromycin and ceftriaxone. The cytotoxic effects of peptides were tested against human erythrocytes, WRL-68 normal liver cell line, and NL-20 normal lung cell line. The results revealed that none of the thirteen peptides have cytotoxic or hemolytic effects at their MIC values. The in silico molecular docking study was carried out to investigate the binding properties of peptides with three pneumococcal virulent targets by Autodock Vina. RN7IN6 showed a strong affinity to target proteins; autolysin, pneumolysin, and pneumococcal surface protein A (PspA) based on rigid docking studies. Our results suggest that the hybrid peptides could be suitable candidates for antibacterial drug development.     

Structure-Activity Relationship of Benzophenanthridine Alkaloids from Zanthoxylum rhoifolium Having Antimicrobial Activity

Zanthoxylum rhoifolium (Rutaceae) is a plant alkaloid that grows in South America and has been used in Brazilian traditional medicine for the treatment of different health problems. The present study was designed to evaluate the antimicrobial activity of the steam bark crude methanol extract, fractions, and pure alkaloids of Z. rhoifolium. Its stem bark extracts exhibited a broad spectrum of antimicrobial activity, ranging from 12.5 to 100 µg/mL using bioautography method, and from 125 to 500 µg/mL in the microdilution bioassay. From the dichloromethane basic fraction, three furoquinoline alkaloids (1–3), and nine benzophenanthridine alkaloids (4–12) were isolated and the antimicrobial activity of the benzophenanthridine alkaloids is discussed in terms of structure-activity relationships. The alkaloid with the widest spectrum of activity was chelerythrine (10), followed by avicine (12) and dihydrochelerythrine (4). The minimal inhibitory concentrations of chelerythrine, of 1.50 µg/mL for all bacteria tested, and between 3.12 and 6.25 µg/mL for the yeast tested, show this compound to be a more powerful antimicrobial agent when compared with the other active alkaloids isolated from Z. rhoifolium. To verify the potential importance of the methylenedioxy group (ring A) of these alkaloids, chelerythrine was selected to represent the remainder of the benzophenanthridine alkaloids isolated in this work and was subjected to a demethylation reaction giving derivative 14. Compared to chelerythrine, the derivative (14) was less active against the tested bacteria and fungi. Kinetic measurements of the bacteriolytic activities of chelerythrine against the bacteria Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative) were determined by optical density based on real time assay, suggesting that its mechanism of action is not bacteriolytic. The present study did not detect hemolytic effects of chelerythrine on erythrocytes and found a protective effect considering the decrease in TBARS and AOPP (advanced oxidized protein products) levels when compared to the control group.     

In vivo antipyretic,antiemetic, in vitro membrane stabilization,antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

Background

The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh.

Results

In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 μg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC₅₀ value 48.978 μg/ml) activity, whereas ethyl acetate fraction showed mild (LC₅₀ value 216.77 μg/ml) cytotoxic activity.

Conclusions

Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.     

Improved Biosurfactant Production by Enterobacter cloacae B14, Stability Studies,and its Antimicrobial Activity

This work aimed to optimize carbon and nitrogen sources for the growth of Enterobacter cloacae B14 and its biosurfactant (BS) production via One-Variable-At-a-Time (OVAT) method. The BS stability under a range of pH and temperatures was assessed. Antimicrobial activity against Gram-positive and Gram-negative pathogens was determined by the agar well diffusion method. The results showed that the optimum carbon and nitrogen sources for BS production were maltose and yeast extract, respectively, with a maximum BS yield of (39.8 ± 5.2) mg BS/g biomass. The highest emulsification activity (E24) was 79%, which is significantly higher than in the previous studies. We found that B14 BS can withstand a wide range of pH values from 2 to10. It could also function under a range of temperatures from 30–37°C. Thin Layer Chromatography (TLC) and Fourier Transform Infrared Spectrometry (FTIR) analysis confirmed that B14 BS is a glycolipid-like compound, which is rarely found in Enterobacter spp. Cell-free broth showed inhibition against various pathogens, preferable to Gram-positive ones. It had better antimicrobial activity against Bacillus subtilis than a commonly-used antibiotic, tetracycline. Furthermore, B14 broth could inhibit the growth of a tetracycline-resistant Serratia marcescens. Our results showed promising B14 BS applications not only for bioremediation but also for the production of antimicrobial products.Key words: biosurfactant, cultivation media, Enterobacter cloacae, antimicrobial activity, stability