In situ TEM and SEM studies on the antimicrobial activity and prevention of Candida albicans biofilm by Cassia spectabilis extract

The inhibitory effect of Cassia spectabilis methanol leaf extract was evaluated against biofilm forming Candida albicans, which was sensitive to 6.25 mg/ml concentration of the extract. Transmission (TEM) and scanning electron microscope (SEM) observations were used to study the anticandidal activity and prevention of biofilm formation by the C. spectabilis extract. SEM analysis further revealed reduction in C. albicans biofilm in response to the extract. The main abnormalities noted via TEM study was the alterations in morphology and complete collapse of the yeast cells after 36 h of exposure to the extract. The significant antifungal activity shown by this methanol extract of C. spectabilis suggests its potential against infections caused by C. albicans.     

In situ SEM,TEM and AFM studies of the antimicrobial activity of lemon grass oil in liquid and vapour phase against Candida albicans

Inhibition of Candida albicans growth was shown by lemon grass oil (LGO) and lemon grass oil vapour (LGO vapour) at 288 μg/ml and 32.7 μg/ml concentration, respectively. The assessment of cell damage by LGO and LGO vapour was done through scanning electron microscope (SEM), transmission electron microscope (TEM) and atomic force microscope (AFM) observations. SEM analysis showed complete rupture of C. albicans cells treated with LGO vapour while in those treated with LGO in broth, only shrinkage was observed. TEM study showed the alterations in morphology upon treatment with LGO while complete degradation of the Candida cells was observed in case of LGO vapour. Further three dimensional morphological changes and roughness of the cells have also been evaluated with AFM after the treatment with LGO & LGO vapour. Roughness (root mean square value) was significantly higher in control C. albicans cells (211.97 nm) than LGO (143 nm) and LGO vapour (5.981 nm) treated cells. The results for the first time demonstrate relatively higher efficacy of LGO vapours for inhibition and cellular damage of C. albicans cells as compared to the LGO in liquid phase. This suggests the potential application of LGO vapour phase against infections caused by C. albicans.     

Physalis angulata induces death of promastigotes and amastigotes of Leishmania (Leishmania) amazonensis via the generation of reactive oxygen species

Leishmaniasis are a neglected group of emerging diseases that have been found in 98 countries and are caused by protozoa of the genus Leishmania. The therapy for leishmaniasis causes several side effects and leads to drug-resistant strains. Natural products from plants have exhibited activities against Leishmania in various experimental models. Physalis angulata is a widely used plant in popular medicine, and in the literature it has well-documented leishmanicidal activity. However, its mechanism of action is still unknown. Thus, this study aims to evaluate the mechanism driving the leishmanicidal activity of an aqueous extract of P. angulata root (AEPa). AEPa was effective against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. This effect was mediated by an increase of reactive oxygen species (ROS), but not of nitric oxide (NO). The increased production of ROS induces cell death by phenotypes seems by apoptosis cell death in Leishmania, but not autophagy or necrosis. In addition, morphological analysis of macrophages showed that AEPa induced a high number of cytoplasmic projections, increased the volume of cytoplasm and number of vacuoles, caused cytoskeleton alterations and resulted in high spreading ability. AEPa also promoted superoxide anion (O₂⁻) production in both uninfected macrophages and those infected with Leishmania. Therefore, these results revealed that AEPa causes cell death by phenotypes seems by apoptosis cell death in L. amazonensis and modulates macrophage activation through morphofunctional alterations and O₂⁻ generation to induce Leishmania death.     

Preparation and characterization of antibacterial Au/C core-shell composite

An environment-friendly oxidation-reduction method was used to prepare Au/C core-shell composite using carbon as core and gold as shell. The chemical structures and morphologies of Au/C core-shell composite and carbon sphere were characterized by X-ray diffraction, transmission electron microscope, energy dispersion X-ray spectrometry (EDS) and X-ray photoelectron spectroscopy (XPS). The antibacterial properties of the Au/C core-shell composite against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) were examined by the disk diffusion assay and minimal inhibition concentration (MIC) methods. In addition, antibacterial ability of Au/C core-shell composite was observed by atomic force microscope. Results demonstrated that gold homogeneously supported on the surface of carbon spheres without aggregation and showed efficient antibacterial abilities.     

Colorimetric Sensing of Toxic Metal and Antibacterial Studies by Using Bioextract Synthesized Silver Nanoparticles

Here, we report the simple and cost effective colorimetric technique for the determination of toxic metals (Hg²⁺) in aqueous sample by using bioextract silver nanoparticles (AgNPs). The indigenous AgNPs were synthesised by green and ecologically friendly style using extract of fig (Ficus carica) leaf. The synthesized AgNPs were confirmed by UV–vis spectroscopy, FT-IR spectroscopy, and scanning electron microscopy methods. The synthesis of AgNPs was observed by its colour changing from light yellow to dark brownish. The existence of furanocoumarins bioactive materials in the fig leaf extract, which act as bio-reducing and capping agent, help in the formation of stabilized silver nanoparticles. In addition, the bacterial activity of the synthesized silver nanoparticles was tested against gram-negative (Klebsiella oxytocam, Pseudomonas aeruginosam, Shigella flexneri and Proteus mirabilis), gram-positive (Staphylococcus aureus and Micrococcus luteus) and one Candida (Candida albicans) human pathogen and the results showed moderate activity.     

Antifungal effects of 3D scaffold type gelatin/Ag nanoparticles biocomposite prepared by solution plasma processing

The 3D scaffold type biocomposites of gelatin/silver nanoparticles were prepared through the silver nanoparticles (Ag NPs) formation in gelatin solution using solution plasma process (SPP) and their antifungal activity was evaluated. The mixture of 3% gelatin solution and silver precursor (AgNO₃; 1–10 mM) was subject to discharge at high voltage (1600 V) under the controlled conditions to form the suspension of Ag NPs in the gelatin matrix. The freeze-drying process of lyophilization was employed to fabricate the 3D scaffold type biocomposite of gelatin/Ag NPs from the suspension. The water-insoluble property was improved by cross-linking using UV-irradiation (λ = 254 nm for 15 min). The physical and chemical characteristics of the biocomposite were investigated using UV–vis spectroscopy, EDS, FE-SEM, and TEM. The results indicated that the 3D scaffold biocomposite of gelatin/Ag NPs had spherical shape with approximately 11–12 nm of diameter. The antifungal activity analysis suggested that the biocomposite with Ag NPs could inhibit the growth of Candida albicans as well as that of hyphae and spores of Aspergillus parasiticus significantly. MIC of the biocomposite for C. albicans and A. parasiticus was determined as 80 μg/ml and 240 μg/ml of Ag NPs, respectively. The growth inhibition of 92.8% was observed in the biocomposite with 10 mM Ag against C. albicans.     

Sonochemical fabrication of edible fragrant antimicrobial nano coating on textiles and polypropylene cups

We report on a simple and effective ultrasound-assisted deposition of vanillin nanoparticles (∼50 nm in size), raspberry ketone (RK) nanoparticles (∼40 nm in size) and camphor nanoparticles (width ∼30 nm, length ∼40 nm in size) on textiles and on polypropylene surfaces. The excellent antibacterial and antifungal activity of the fragrant coatings on cotton bandages, and polypropylene surface against Escherichia coli (E. coli), Salmonella paratyphi A (S. paratyphi A) and the yeast Candida albicans (C. albicans) cultures was demonstrated. It is worth pointing out that these fragrant materials are edible, making them very useful for packaging. The mechanism of the edible fragrant coating formation and adhesion to the textile was discussed, and finally an up-scaling of the sonochemical process for textile coating was carried out.     

Synthesis,characterization, and antimicrobial evaluation of a small library of ferrocene-containing acetoacetates and phenyl analogs: the discovery of a potent anticandidal agent

A library of 16 2-substituted methyl acetoacetates containing ferrocenyl or phenyl units was designed to disclose differences in the antimicrobial activity of ferrocene-containing compounds and their phenyl analogs. Two methyl acetoacetates, whose structures do not contain an aromatic nucleus, were also included in order to probe the inherent activity of the scaffold itself. The acetoacetates were synthesized (low-to-good yields) and fully characterized by spectral (MS, IR, UV–Vis, 1D and 2D NMR) and electrochemical (cyclic voltammetry) techniques. Single-crystal X-ray analysis has been performed for methyl 2-acetyl-2-(ferrocenylmethyl)-5-methylhex-4-enoate. All compounds have demonstrated in vitro antimicrobial activity against six bacterial (three Gram-positive and three Gram-negative) and two fungal strains with minimal inhibitory concentration values of 0.0050–20.6  \(\upmu \hbox {mol mL}^{-1}\) . The most active compound was 2-acetyl-2-(ferrocenylmethyl)-4-methylpent-4-enoate whose activity was comparable to that of nystatin against the yeast Candida albicans. Agglomerative hierarchical clustering statistical analysis of the antimicrobial assay data demonstrated that ferrocene-containing compounds have statistically different and greater antimicrobial activity when compared to their phenyl analogs.     

Sulfur-containing components of supercritical garlic extracts and their synthetic analogs as potential biocides

We have shown that a specific component of supercritical (SC) garlic extract and some synthetic disulfide analogs are active as antimicrobials. In the course of this study, six compounds (diallyl disulfide, 1-propenylallyl disulfide, methylallyl disulfide, diallyl trisulfide, 2-vinyl-4H-1,3-dithiin, and 3-vinyl-4H-1,2-dithiin) were isolated from SC garlic extract and identified. Also, two novel sulfoxide-containing garlic analogs were synthesized. All isolated and synthetic substances were tested as potential antimicrobials against gram-positive bacteria Bacillus cereus, Mycobacterium smegmatis, and Micrococcus luteus and gram-negative bacteria Pseudomonas aurantiaca. Diallyl disulfide and diallyl trisulfide exhibited the best activity against both gram-positive and gram-negative bacteria.     

Persistence of pierisin-1 activities in the adult cabbage white butterfly,Pieris rapae,during storage after killing

Crude extracts from larvae, pupae and adults of cabbage white butterflies, Pieris rapae and Pieris brassicae, and green-veined butterfly, Pieris napi, have an ability to induce apoptosis in the human cancer cell lines. As apoptosis inducing protein, pierisin-1 and -2 have been isolated from pupae of P. rapae and P. brassicae, respectively, and shown to exhibit DNA ADP-ribosylating activity. Although the highest activity was detected in the late phase of larvae and early phase of pupae, certain activity was found in adult butterflies. In order to investigate distribution of substances having pierisin-like activities in butterflies, many species need to be analyzed. However, fresh samples of larvae and pupae are hard to obtain, especially if samples are of scarce species or from overseas. The usage of adult butterflies is practical to examine the distribution of pierisin-like activity in many species. In this study, we examined the cytotoxicity of crude extracts from adults of P. rapae against HeLa cells and DNA ADP-ribosylation ability during storage for 1, 2 and 8 weeks at room temperature after killing adult butterflies after eclosion. Body weights decreased to 18% for 8 weeks through dehydration. Cytotoxicity of samples from butterfly kept for 1, 2 and 8 weeks decreased to 47, 39 and 22%, respectively, of the control value. DNA ADP-ribosylating activity of the samples also decreased to 30, 27 and 23%. Similar reduction was observed on western blot analysis with anti-pierisin-1 antibody. Fortunately, these results suggest that cytotoxic and DNA ADP-ribosylating activity persists to some extent in the body after killing, at least for 8 weeks. Thus, butterfly adult samples kept for two months at room temperature can still be useful for examination of the presence of substance having pierisin-like activity.     

Cell-based screening strategy in the search for bioactive microbial secondary metabolites Utilization of microbial and animal cells with special functions

Here we present results obtained in our group from cell-based screenings of microbial secondary metabolites using intact microbial and mammalian cells having specific functions. We present summaries of the following five categories of compounds and the strategies that were used to identify them. (1) Antibacterial agents targeting cell wall peptidoglycan synthesis as well as active agents against organisms other than bacteria were identified by a combination of Bacillus subtilis and Mycoplasma as test organisms. (2) Antimetabolites, such as herbicidal agents targeting glutamine synthase, were identified using B. subtilis grown on minimal medium with or without the addition of glutamine and folate inhibitors by combination of B. subtilis and Enterococcus faecium, which have distinct folate metabolic pathways. (3) Among inhibitors of lipid metabolism, acyl-CoA synthetase inhibitors were identified using two mutants of C. lipolytica with different deletion sites in the fatty acid metabolic pathways, HMG-CoA synthase inhibitors were identified using Vero cells cultured in the presence and absence of mevalonate, and anti-atherosclerosis agents were identified using macrophages. (4) IL-6 inhibitors were detected by a combination of IL- 6-dependent and independent murine hybridoma MH60 cells, and (5) Small molecules with TNF-like activities were identified using a mouse neuroblastoma cell line. Some advantages of such screening method and the significance of the identified compounds are discussed.     

Growth and investigation of antifungal properties of ZnO nanorod arrays on the glass

In this study, we have investigated the antifungal activity of ZnO nanorods prepared by the chemical solution method against Candida albicans. In the study, Zinc oxide nanorods have been deposited on glass substrates using the chemical solution method. The as-grown samples are characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). X-ray diffraction (XRD) showed zinc oxide nanorods grown in (0 0 2) orientation. The antifungal results indicated that ZnO nanorod arrays exhibit stable properties after two months and play an important role in the growth inhibitory of Candida albicans.     

TOF-SIMS imaging of chlorhexidine-digluconate transport in frozen hydrated biofilms of the fungus Candida albicans

The diffusion of the anti-microbial chlorhexidine digluconate (CHG) has been studied in C. albicans biofilms by time-of-flight secondary-ion mass spectrometry (TOF-SIMS). C. albicans has been shown to become resistant to common anti-microbial agents, including CHG, when growing as a biofilm. Mass transport resistance within biofilms has commonly been suggested as a resistance mechanism, but measurement of transport for most anti-microbial agents in biofilms has proven extremely difficult because of the heterogeneity of the biofilms and the difficulty in detecting these agents within an intact biofilm. In this study, TOF-SIMS has been used to study the transport of CHG and glucose in a frozen hydrated biofilm. The TOF-SIMS images reveal a progression of CHG from the top of the biofilm to its base with time. Images suggest that there are channels within the biofilm and show preferential binding of CHG to cellular components of the biofilm. Additionally, both living and dead cells can be identified in the TOF-SIMS images by the sequestration of K⁺ and the presence of cell markers. This study demonstrates that TOF-SIMS has the unique potential to simultaneously observe the presence of an antimicrobial agent, concentration of nutrients, and the viability of the cell population.     

Green synthesis of garlic oil nanoemulsion using ultrasonication technique and its mechanism of antifungal action against Penicillium italicum

Penicillium italicum (P. italicum) can cause significant economic loss of fruits and vegetables. Although garlic oil (GO) is an effective antimicrobial agent, the unstability and hydrophobicity limit its use as an environmentally friendly alternative to the conventional antibiotics against P. italicum. In this study, we focused on the fabrication and characterization of a functional GO nanoemulsion (NE) using ultrasonic technique and revealed the antifungal mechanism of the GO NE on P. italicum based on morphological, structural and molecular analyses. The optimal hydrophilic lipophilic balance (HLB) value determined for GO was 14 through the combination of Tween 80 and Span 80. Then the Box-Benhnken Design (BBD) was applied to produce the GO NE and the effects of different fabrication parameters on the particle size were evaluated. The optimal GO NE was selected with the GO concentration of 5.5%, the Smix concentration of 10%, the ultrasonic time of 5 min and the power of 50%. This GO NE had the smallest particle size of 52.27 nm, the best antifungal effect and the most stability. Furthermore, the antifungal mechanism of the GO NE on P. italicum was evaluated by extracellular conductivity, micro-Raman spectra, fluorescence imaging and scanning electron microscopy (SEM) imaging. The results presented that the GO NE retained the antifungal active ingredients. The fungal cell structure and morphology were malformed after treated with the GO NE and the lipids, nucleic acids and protein of P. italicum were destructed. Finally, the optimal GO NE was applied in vivo and P. italicum in citrus was successfully inhibited. It indicated that the optimal GO NE had the better antifungal activity against P. italicum than the pure GO. Besides, the minimum inhibitory concentration (MIC) of GO after preparing into the NE was changed from 3.7% to 0.01265% with about 300 times improvement of bioavailability. Therefore, the synthetic GO NE which promoted the bioavailability of GO was recommended as a promising alternative to inhibit P. italicum in vegetables and fruits.     

Synthesis, crystal structure and biological activity of novel anthranilic diamide insecticide containing alkyl ether group

In searching for environmentally benign insecticides with high activity, low toxicity and low residue, two series of novel anthranilic diamide containing methyl ether and isopropyl ether group were designed and synthesized. All of the compounds were characterized by ¹H NMR spectroscopy, ¹³C NMR spectroscopy and elemental analysis. The single crystal structure of 19j was determined by X-ray diffraction. The insecticidal activities of the new compounds were evaluated. The results showed that some compounds exhibited excellent insecticidal activities against Lepidoptera pests. Among this series, compound, 18l showed 100?% larvicidal activity against Mythimna separate Walker, Plutella xylostella Linnaeus and Laphygma exigua Hubner at the test concentration, which was equal to the available chlorantraniliprole.     

Antioxidative and antiproliferative activities of novel pyrido[1,2-<Emphasis Type="Italic">a</Emphasis>]benzimidazoles

A series of pyrido[1,2-a]benzimidazoles has been designed, and novel examples are synthesized and evaluated for their potential antiproliferative activity against four human tumour cell lines—cervical (HeLa), colorectal (SW620), breast (MCF-7) and hepatocellular carcinoma (HepG2). In addition, their antioxidative potency has been evaluated by in vitro spectrophotometric assays. Preliminary structure–activity relationships among the synthesized compounds are discussed. Evaluation of their antioxidative capacity has shown that two compounds (25 and 26) possess promising reducing characteristics and free radical scavenging activity. Selective antiproliferative effect in the single-digit micromolar range was observed for compound 25 on MCF-7 \((\hbox {IC}_{50} = 6\,{\upmu }\hbox {M})\) and HeLa \((\hbox {IC}_{50} = 8\,{\upmu }\hbox {M})\) cell lines, comparable to the standards 5-fluorouracil and cisplatin. The combination of the radical scavenging activity and antiproliferative activity of compound 25 positions this compound as a potential lead candidate for further optimization.     

Optimization of ultrasound induced emulsification on the formulation of palm-olein based nanoemulsions for the incorporation of antioxidant β-d-glucan polysaccharides

Polysaccharides of β-d-glucan configuration have well-known antioxidant activity against reactive free radicals generated from the oxidation of metabolic processes. In this study, β-d-glucan-polysaccharides extracted from Ganoderma lucidum were incorporated in palm olein based nanoemulsions which act as carrier systems to enhance the delivery and bioactivity of these polysaccharides and could be potentially useful for skin care applications. Initially response surface statistical design (Central Composite Design – CCD) was subjected to optimize the formulation variables of oil-in-water (O/W) nanoemulsions induced by ultrasound. The optimal formulation variables as predicted by CCD resulted in considerably improving the physical characteristics of ultrasonically formulated nanoemulsions by minimizing their droplet size, polydispersity index and viscosity. Moreover, the β-d-glucan-loaded nanoemulsions exhibited good stability over 90 days under different storage conditions (4 °C and 25 °C). The studies using palm olein based β-d-glucan-loaded nanoemulsion generated using ultrasound confirm higher antioxidant activity as compared to free β-d-glucan.     

Effect of sonication on different quality parameters of Pinus massoniana pollen

A study was initiated with the objective of evaluating the effects of sonication treatment on important quality parameters of extract of Pinus massoniana pollen. Sonication of extract was done (frequency 20 kHz and various amplitude levels) for 10, 30, 50 min, respectively. As results, total polysaccharide, phenolics and flavonoids significantly increased (P < 0.05). And sonicated P. massoniana pollen displays strong immuno-stimulating activity by increasing proliferations of splenic lymphocytes and subsets of CD4+ T cells (CD3+CD4+), CD8 T cells (CD3+CD8+), and increased Ig secretion. Sonicated P. massoniana pollen also showed anti-tumor function by inhibition of tumor cell proliferation, inhibition of ROS production, up-regulation of GSH/GSSG ration, up-regulating the gene expression of P53, Bax and down-regulating the gene expression of Bcl-2. Findings of the present study suggested the sonication treatment of P. massoniana pollen could improve the quality and bioactivity of P. massoniana pollen, indicating that sonication is effective in processing of pollen and could be a potential process in tumor prevention and treatment.     

Preparation and characterization of a novel green tea essential oil nanoemulsion and its antifungal mechanism of action against Magnaporthae oryzae

Blast is one of the most devastating fungal diseases of rice caused by Magnaporthe oryzae. Plant essential oil (EO) can function as antifungal agents and are regarded as a safe and acceptable method for plant disease control. However, EOs are unstable and hydrophobic, which limits its use. In the present study, we aimed for the preparation and characterization of a nanoemulsion (NE) from green tea essential oil (GTO) by ultrasonication method and determined the antifungal activity of NE on M. oryzae. The particle size and zeta potential of the NE were 86.98 nm and −15.1 mV, respectively. The chemical composition and functional groups of GTO and NE were studied by using GC–MS analysis, portable Raman spectroscopy, and FTIR coupled with chemometric analysis. GC–MS analysis showed the major components in GTO and NE were n-Hexyl cinnamaldehyde and L-α-Terpineol. Both GTO and NE showed good antioxidant activity and total phenol content. Moreover, the NE showed good antifungal activity against M. oryzae which was further confirmed by scanning electron microscopy (SEM) examination. Also, confocal Raman micro-spectroscopy (CRM) revealed the antifungal mechanism of GTO and NE on M. oryzae which proves the cell damage. To the best of our knowledge, this is the first study on the antifungal activity of GTO and NE against M. oryzae and also the use of CRM for the evaluation of the chemical changes in single fungal hyphae in a holistic approach. This study suggests that the prepared NE could be a potential candidate for use as a substitute for synthetic fungicides.     

Ultrasound irradiation: A robust approach for direct functionalization of graphene oxide with thermal and antimicrobial aspects

Sonochemical waves as mechanochemical energy was employed to exfoliate graphite oxide and functionalized graphene oxide (GrO), through a reaction of solvent and accountable for top-down and bottom-up approach respectively. The in situ formation of ester intermediate was inferred and a polymeric surface of GrO was further functionalized with 6-Aminoindazole (6-AIND) through sonochemical nucleophilic substitution reaction. As compared to conventional method the effect of ultrasound was verified for the direct functionalization of GrO. The conventional hazardous acylation step for functionalization of GrO was deleted in ultrasound assisted formation of f-(6-AIND) GrO nanocomposite, prepared by stereoselective exploitation of carboxyl groups at edges of GrO. The characterization has ascertained a covalent attachment of 6-AIND onto GrO surface with ATR-FTIR, XPS, SSNMR, TGA, DSC, XRD, AFM, RAMAN, EDX, SEM, BET and elemental analyzer. A weight loss in TGA depicts enhanced thermal stability of f-(6-AIND) GrO and a thermally sensitive behavior. The f-(6-AIND) GrO was studied for in vitro antimicrobial activity to ensure health and environmental safety. Antibacterial activity was identified against human pathogenic gram-positive (Staphylococcus aureus; ATCC 25923) and gram-negative bacteria (Escherichia coli; ATCC 25922). The antifungal activity was observed against Candida albicans (ATCC 10231).