Development of alkaline-stable nanoformulation of nisin: special insights through cytotoxic and antibacterial studies.

Nisin, a thermostable, approved food preservative, has limited therapeutic applications because of its high pH and proteolytic enzyme instability. The unavailability of a rapid, simple method of detection also restricts the research of nisin. The objective of this study was to adapt the simple, rapid protein estimation method of detection for nisin formulation and to formulate and evaluate site-specific nanoformulation for therapeutic applications, viz. colon cancer, and anti-bacterial action. Three nanoformulations of nisin with chitosan, gellan gum, and dextran (ECN, EGN, and EDN) were prepared and characterized in vitro. Among three, EGN was selected as a good formulation based on its size surface charge, morphology, drug loading, and release characteristics. FT-IR and DSC revealed the interaction pattern and stability nature. The stability of nisin in an alkaline environment was confirmed by CD. Its therapeutic applications were proved by efficiency against colon cancer cells evaluated by MTT assay and AO/EB staining using Caco-2 cell lines. The in situ sol-gel mechanism imparted by gellan gum was proved the sole reason for the stability and activity of nisin in EGN at lower GIT. This was confirmed (using rheometer) by shear-thickening characteristics of formulation EGN in simulated colon fluid. The antibacterial activity against Staphylococcus aureus by disk diffusion method was also performed to confirm the retention of antimicrobial activity of nisin in EGN. Hence, gellan gum-nisin colloidal nanoparticles are found good candidates for drug delivery at lower GIT and stabilizing alkaline food materials.

Phytol loaded PLGA nanoparticles ameliorate scopolamine-induced cognitive dysfunction by attenuating cholinesterase activity, oxidative stress and apoptosis in Wistar rat.

OBJECTIVE: Alzheimer's disease (AD) is an acquired neurological disorder of cognitive and behavioral impairments, with a long and progressive route. Currently, efforts are being made to develop potent drugs that target multiple pathological mechanisms that drive the successful treatment of AD in human beings. The development of nano-drug delivery systems has recently emerged as an effective strategy to treat AD. METHODS: In the present study, the protective effect of Phytol and Phytol loaded Poly Lactic-co-Glycolic Acid nanoparticles (Phytol-PLGANPs) were evaluated in Wistar rat scopolamine model of AD. RESULTS AND DISCUSSION: The consumption of Phytol and Phytol-PLGANPs significantly ameliorated the cognitive deficits caused by scopolamine on spatial and short term memory. Phytol and Phytol-PLGANPs significantly enhanced the cholinergic effect by inhibiting both acetylcholinesterase and butyrylcholinesterase (AChE & BuChE), ß-secretase 1 (BACE1) activity, attenuating macromolecular damage, reducing reactive oxygen species (ROS) and reactive nitrogen species (RNS) level by activating antioxidative defense system (Superoxide dismutase and catalase) and restoring glutathione metabolizing enzyme systems (Glutathione S-transferase) and also regulating the apoptotic mediated cell death. Moreover, in vivo toxicity study suggests that Phytol and Phytol-PLGANPs did not cause any adverse pathological alteration in rats treated with a higher concentration of Phytol-PLGANPs (200 mg/kg). Pharmacokinetic study revealed that Phytol-PLGANPs enhanced the biodistribution and sustained the release profile of phytol in the brain and plasma. CONCLUSION: Overall, the outcome of the study suggests that Phytol and Phytol-PLGANPs act as a potent candidate with better anti-amnesic effects and multi-faceted neuroprotective potential against scopolamine-induced memory dysfunction in Wistar rats.

In vitro study on aspects of molecular mechanisms underlying invasive aspergillosis caused by gliotoxin and fumagillin, alone and in combination.

Gliotoxin (GT) and fumagillin (FUM) are mycotoxins most abundantly produced by Aspergillus fumigatus during the early stages of infection to cause invasive aspergillosis (IA). Therefore, we hypothesized that GT and FUM could be the possible source of virulence factors, which we put to test adopting in vitro monoculture and the novel integrated multiple organ co-culture (IdMOC) of A549 and L132 cell. We found that (i) GT is more cytotoxic to lung epithelial cells than FUM, and (ii) GT and FUM act synergistically to inflict pathology to the lung epithelial cell. Reactive oxygen species (ROS) is the master regulator of the cytotoxicity of GT, FUM and GT + FUM. ROS may be produced as a sequel to mitochondrial damage and, thus, mitochondria are both the source of ROS and the target to ROS. GT-, FUM- and GT + FUM-induced DNA damage is mediated either by ROS-dependent mechanism or directly by the fungal toxins. In addition, GT, FUM and GT + FUM may induce protein accumulation. Further, it is speculated that GT and FUM inflict epithelial damage by neutrophil-mediated inflammation. With respect to multiple organ cytotoxicity, GT was found to be cytotoxic at IC50 concentration in the following order: renal epithelial cells < type II epithelial cells < hepatocytes < normal lung epithelial cells. Taken together, GT and FUM alone and in combination contribute to exacerbate the damage of lung epithelial cells and, thus, are involved in the progression of IA.

ß-Sitosterol targets Trx/Trx1 reductase to induce apoptosis in A549 cells via ROS mediated mitochondrial dysregulation and p53 activation.

ß-Sitosterol (BS), a major bioactive constituent present in plants and vegetables has shown potent anticancer effect against many human cancer cells, but the underlying mechanism remain elusive on NSCLC cancers. We found that BS significantly inhibited the growth of A549 cells without harming normal human lung and PBMC cells. Further, BS treatment triggered apoptosis via ROS mediated mitochondrial dysregulation as evidenced by caspase-3 & 9 activation, Annexin-V/PI positive cells, PARP inactivation, loss of MMP, Bcl-2-Bax ratio alteration and cytochrome c release. Moreover, generation of ROS species and subsequent DNA stand break were found upon BS treatment which was reversed by addition of ROS scavenger (NAC). Indeed BS treatment increased p53 expression and its phosphorylation at Ser15, while silencing the p53 expression by pifithrin-α, BS induced apoptosis was reduced in A549 cells. Furthermore, BS induced apoptosis was also observed in NCI-H460 cells (p53 wild) but not in the NCI-H23 cells (p53 mutant). Down-regulation of Trx/Trx1 reductase contributed to the BS induced ROS accumulation and mitochondrial mediated apoptotic cell death in A549 and NCI-H460 cells. Taken together, our findings provide evidence for the novel anti-cancer mechanism of BS which could be developed as a promising chemotherapeutic drug against NSCLC cancers.

A novel biphenolic ligand for selective Mg2+ and Zn2+ ions sensing followed by colorimetric, spectroscopic and cell imaging methods.

The (E)-2-((2-hydrohy-5-methylphenylimino) methyl) phenol ligand was synthesized. The receptor was characterized by IR, 1H and 13C NMR and CHN analysis. The ligand exhibits colorimetric and fluorometric sensing of Zn2+ and Mg2+ ions in semi-aqueous medium (DMSO-H2O). The receptor was tested with series of transition metal ions (Cr2+, Fe2+, Ni2+, Co2+, Cu2+, Zn2+) and heavy metal ions (Sn2+, Pd2+, Ce2+, Hg2+, Cd2+) and the essential human body elements like Ca2+, Mg2+, Na+ and K+ ions. The naked eye colorimetric sensing was absorbed only for Zn2+ and Mg2+. Both ions (ZnCl2 and MgCl2 in H2O), when added to the colorless solutions of the receptor of about 1 equivalence in incremental additions turn the solution into bright turmeric yellow. All other ions remain inactive, in colorimetric sensing. Further the Zn2+ and Mg2+ ions were probed by absorption and emission spectroscopy through incremental addition of respective metal ions. The in-situ deprotonation of the ligand on both Mg2+ and Zn2+ ions binding was confirmed by 1H NMR titration studies. The imino nitrogen of the receptor is not coordinated to the metal ions. The Job's plot studies reveal the 1:2 binding ratio of metal ions to the receptor. The high fold fluorescence output on metal ions binding was positively used to sense the Zn2+ and Mg2+ ions, separately and together in HeLa cancer cells through cell imaging.

Anti-amyloidogenic and anti-apoptotic effect of α-bisabolol against Aß induced neurotoxicity in PC12 cells.

Alzheimer's disease (AD) is a life-threatening neurodegenerative disorder leading to dementia, with a progressive decline in memory and other thinking skills of elderly populace. Of the multiple etiological factors of AD, the accumulation of senile plaques (SPs) particularly as Aß oligomers correlates with the relentlessness cognitive impairment in AD patients and play a vital role in AD pathology. Since natural essential oil constituents have successfully served as a source of drugs for AD treatment, the present study aims at the in vitro and in silico investigation of anti-amyloidogenic potential and anti-apoptotic property of the α-bisabolol against Aß25-35 induced neurotoxicity in PC12 cells. Treatment with α-bisabolol (5 µg/ml) after 24 h incubation with Aß25-35 reduced the aggregation propensity of Aß (p < 0.05), as observed by the reduced fluorescence intensity of thioflavin T (ThT). Confocal laser scanning microscopy (CLSM) analysis, Transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopic analysis and molecular dynamics simulation study also substantiated the Aß fibril formation hampering ability of α-bisabolol even after 9 days of incubations. The results of antiaggregation and disaggregation assay showed an increase in fluorescence intensity in Aß treated group, whereas the co-treatment of α-bisabolol (5 µg/ml) with Aß25-35 showed an extensive decrease in the fluorescence intensity, which suggests that α-bisabolol prevents the oligomers formation as well as disaggregates the matured fibrils. FACS analysis of the cells revealed the competency of α-bisabolol in rescuing the PC12 cells from Aß induced neurotoxicity and chromosomal damage and clonogenic assay proved its ability to retain the colony survival of cells. Overall, the anti-amyloidogenic and anti-apoptotic effect of α-bisabolol proves that it could be used as an excellent therapeutic drug to combat AD.

Phytol-loaded PLGA nanoparticle as a modulator of Alzheimer's toxic Aß peptide aggregation and fibrillation associated with impaired neuronal cell function.

Alzheimer's disease (AD) is an unfavourable neurological condition of the brain leading to the loss of behavioural and cognitive skills of the aging population. At present, drugs representing cholinesterase inhibitors provide lateral side effects to AD patients. Hence, there is a need for improved fabrication of drugs without side effects, for which nanoencapsulated bioactive compounds that can cross the blood-brain barrier offer new hope as novel alternative treatment strategy for AD. This study involved synthesis of phytol loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles by solvent evaporation method. Physico-chemical characterization of phytol-PLGA NPs through the field emission scanning electron microscope, dynamic laser scattering (DLS) measurement revealed that the particles were nanosize range with smooth surface and spherical morphology. Furthermore, the biocompatibility of drug/polymer ratio was investigated by power X-ray diffraction (PXRD) and Fourier-transform infrared spectroscopic (FT-IR) analysis. The in vitro drug release study showed that the phytol was released in a sustained manner. Moreover, phytol-PLGA NPs were able to disrupt amyloid aggregates, exhibit anti-cholinesterase and anti-oxidative property and are non-cytotoxic in Neuro2a cells.

Exploring the Anti-quorum Sensing and Antibiofilm Efficacy of Phytol against Serratia marcescens Associated Acute Pyelonephritis Infection in Wistar Rats.

Quorum Sensing (QS) mechanism, a bacterial density-dependent gene expression system, governs the Serratia marcescens pathogenesis through the production of virulence factors and biofilm formation. The present study demonstrates the anti-quorum sensing (anti-QS), antibiofilm potential and in vivo protective effect of phytol, a diterpene alcohol broadly utilized as food additive and in therapeutics fields. In vitro treatment of phytol (5 and 10 µg/ml) showed decreasing level of biofilm formation, lipase and hemolysin production in S. marcescens compared to their respective controls. More, microscopic analyses confirmed the antibiofilm potential of phytol. The biofilm related phenomenons such as swarming motility and exopolysccharide productions were also inhibited by phytol. Furthermore, the real-time analysis elucidated the molecular mechanism of phytol which showed downregulation of fimA, fimC, flhC, flhD, bsmB, pigP, and shlA gene expressions. On the other hand, the in vivo rescue effect of phytol was assessed against S. marcescens associated acute pyelonephritis in Wistar rat. Compared to the infected and vehicle controls, the phytol treated groups (100 and 200 mg/kg) showed decreased level of bacterial counts in kidney, bladder tissues and urine samples on the 5th post infection day. As well, the phytol treatment showed reduced level of virulence enzymes such as lipase and protease productions compared to the infected and vehicle controls. Further, the infected and vehicle controls showed increasing level of inflammatory markers such as malondialdehyde (MDA), nitric oxide (NO) and myeloperoxidase (MPO) productions. In contrast, the phytol treatment showed decreasing level of inflammatory markers. In histopathology, the uninfected animal showed normal kidney and bladder structure, wherein, the infected animals showed extensive infiltration of neutrophils in kidney and bladder tissues. In contrast, the phytol treatment showed normal kidney and bladder tissues. Additionally, the toxic effect of phytol (200 mg/kg) was assessed by single dose toxicity analysis. No changes were observed in hematological, biochemical profiles and histopathological analysis of vital organs in phytol treated animals compared to the untreated controls. Hence, this study suggested the potential use of phytol for its anti-QS, antibiofilm and anti-inflammatory properties against S. marcescens infections and their associated inflammation reactions.

Beta sitosterol and Daucosterol (phytosterols identified in Grewia tiliaefolia) perturbs cell cycle and induces apoptotic cell death in A549 cells.

Lung cancer is the leading cause of cancer related deaths both in developed and developing countries. Since majority of the existing therapeutic methods harms both normal and malignant cells, a viable alternative is to switch into safe and beneficial traditional medicinal plants. Hence the present study was framed to identify selective anti-lung cancer agents from the medicinal plant Grewia tiliaefolia (GT). Cell viability experiments showed that benzene extract of GT (BGT) leaf effectively inhibited the growth of A549 cells, while being non-toxic to normal human lung L132 and PBMC cells. Ames and comet assays demonstrated that BGT is of non-mutagenic and non-genotoxic nature in untransformed cells. The hematological and histopathological profiles of the in vivo acute and sub-acute toxicity studies demonstrated that BGT is safe and tolerable. Importantly, western blot analysis and Annexin V-FITC staining confirmed that BGT promotes mitochondrial dependent apoptotic cell death in A549 cells by arresting cell cycle at G2/M phase. Bio-assay guided fractionation revealed the presence of phytosteols (ß-sitosterol and daucosterol) which significantly inhibited the growth of A549 cells both alone and in combination. This study warrants that these phytosterols in alone or in combination can be considered as safe and potential drug candidates for lung cancer treatment.

Exploitation of novel gum Prunus cerasoides as mucoadhesive beads for a controlled-release drug delivery.

The present study deals with the formulation of pH-sensitive mucoadhesive beads using natural gum isolated from Prunus cerasoides (PC) in combination with sodium alginate (SA) for the controlled release of diclofenac sodium (DS). PC and SA composite (PC-SA), DS loaded SA (DS-SA) and DS loaded PC-SA (DS-PC-SA) beads were prepared by ionotropic gelation method. The absence of interaction between DS and PC-SA was shown by FTIR, DSC and TGA analyses. The optimized DS-PC-SA formulation exhibited mucoadhesive property and the controlled release of DS was achieved 68% in 12h. The in vitro release kinetics follows zero order with anomalous diffusion mechanism. Therefore, the formulated mucoadhesive beads with the novel gum are preferable for the controlled release of DS by prolonging the residence time of the drug in the gastrointestinal tract, overcoming the problems associated with the immediate release dosage forms of DS.

A versatile effect of chitosan-silver nanocomposite for surface plasmonic photocatalytic and antibacterial activity.

Chitosan-silver (CS-Ag) nanocomposite was green synthesised without the aid of any external chemical-reducing agents. The synthesised nanocomposite was characterised by UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermo-gravimetric analysis (TGA), differential scanning calorimetry (DSC), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), high-resolution transmission electron microscopy (HR-TEM) with selected area electron diffraction (SAED), X-ray photoelectron spectroscopy (XPS), and zeta potential analyser. The particle size of the synthesised CS-Ag nanocomposite was around 20 nm and was found to be thermally stable in comparison with pure chitosan. The prepared nanocomposite acts as a photocatalyst for dye decolourisation, with a maximum of 81% of methyl orange (MO) decolourisation that occurred under visible light irradiation. The kinetics was found to follow pseudo-first-order according to Langmuir-Hinshelwood (L-H) model. The nanocomposite also proved to be an excellent antimicrobial agent against both Gram-positive and Gram-negative bacteria, possessing a broad spectrum of antimicrobial activity. The zone of inhibition ranged between 16.000 ± 1.000 and 19.333 ± 1.155 (mm), proving its high susceptibility than chitosan itself. The minimum inhibitory concentration (MIC) values were from 8 to 64 µg/mL, whereas the minimum bactericidal concentration (MBC) values ranged from 16 to 128 µg/mL, with the highest antibacterial activity shown against Gram-positive Staphlococcus aureus. This report illustrates the eco-friendly approach for the reduction of silver using chitosan as a reducing agent, and its potential to dye decay and microbial contaminants.

Formulation and optimization of Zidovudine niosomes.

Zidovudine (AZT) is commonly used to treat patients with AIDS, but it is limited by toxicity and high dosing needs. Alternative formulations have been proposed to overcome these drawbacks. The objective of this study was to evaluate process-related variables like hydration and sonication time, rotation speed of evaporation flask, and the effects of charge-inducing agent and centrifugation on zidovudine entrapment and release from niosomes. Formulation of zidovudine niosomes was optimized by altering the proportions of Tween, Span and cholesterol. The effect of process-related variables like hydration time, sonication time, charge-inducing agent, centrifugation and rotational speed of evaporation flask on zidovudine entrapment and release from niosomes was evaluated. The effect of changes in osmotic shock and viscosity were also evaluated. Non-sonicated niosomes were in the size range of 2-3.5 µm and sonicated niosomes formulated with Tween 80 and dicetylphosphate (DCP) had a mean diameter of 801 nm. Zidovudine niosomes formulated with Tween 80 entrapped high amounts of drug and the addition of DCP enhanced drug release for a longer time (88.72% over 12 h). The mechanism of release from Tween 80 formulation was the Fickian type and obeyed first-order release kinetics. Niosomes can be formulated by proper adjustment of process parameters to enhance zidovudine entrapment and sustainability of release. These improvements in zidovudine formulation may be useful in developing a more effective AIDS therapy.

Tissue distribution, pharmacokinetics and stability studies of zidovudine delivered by niosomes and proniosomes.

Niosomes containing zidovudine (ZDV), an anti-HIV drug for intravenous administration were formulated by a thin-film hydration technique. Proniosomes were prepared in the form of a slurry using beta-cyclodextrin as carrier. The effect of the surfactants Tween and Span and the negative charge-inducing agent dicetylphosphate (DCP) on tissue distribution of niosomes and proniosomes was studied. The distribution of ZDV in lungs, kidney, heart, liver and spleen of mice after intravenous bolus injection was higher in Tween 80 niosomes without DCP than either niosomes with DCP or Tween 80 proniosomes. The amount of ZDV in plasma was low in Tween 80 niosomes without DCP. The results of a pharmacokinetic study in rabbits confirmed that Tween 80 formulations with DCP were cleared from the circulation within five hours. An increased half-life of 202 minutes and mean residence time of 212.1 minutes was observed in Tween 80 formulation. A stability study showed that after 90 days of storage, the drug leakage from Tween 80 formulations stored at room temperature was significant (p < 0.001) compared to niosomes stored at 4 degrees C. Encapsulation ZDV in proniosomes reduced drug leakage from vesicles stored at room temperature. These results demonstrate that niosomes are a promising vehicle for targeted delivery of ZDV to macrophages in spleen and liver.