Antitubercular study on stem bark of Albizia procera (ROXB.) BENTH using Microplate Alamar Blue assay (MABA)

Albizia procera commonly known as white siris is a large deciduous tree of the family Mimosideae, widely distributed throughout India. Traditionally the plant is used in convulsions, pain, delirium, cancer and septicemia. The decoction of bark is given for rheumatism, hemorrhage and is considered useful in treating pregnancy problems, for stomach ache and sinus.The preliminary phytochemical screening of ethanol extract of Albizia procera shows the presence of active constituents like flavonoids, phenolic compounds and tannins. Earlier studies show the flavonoids and phenolic compounds play major role in antitubercular activity. Based on this, we made an attempt to evaluate the antitubercular potential of this plant. The ethanolic extract and its fractions (Hexane and Ethyl acetate) were investigated for antitubercular activity using Alamar Blue dye method (MABA), in which Mycobacterium tuberculosis was sensitive upto 3.12μg/ml.The extract and fractions show better anti tubercular activity than streptomycin and potentially equal to pyrazinamide and ciprofloxacin. The observed activity may be due to the presence of flavonoids and phenolic compound which is confirmed by HPTLC. This investigation on stem bark of Albizia procera has the potential to be developed further into a natural Anti-TB drug.

Ozone ultrafine bubble water inhibits the early formation of Candida albicans biofilms

This study aimed to investigate the effect of ozone ultrafine bubble water (OUFBW) on the formation and growth of Candida albicans (C. albicans) biofilms and surface properties of denture base resins. OUFBWs were prepared under concentrations of 6 (OUFBW6), 9 (OUFBW9), and 11 ppm (OUFBW11). Phosphate buffered saline and ozone-free electrolyte aqueous solutions (OFEAS) were used as controls. Acrylic resin discs were made according to manufacturer instructions, and C. albicans was initially cultured on the discs for 1.5 h. A colony forming unit (CFU) assay was performed by soaking the discs in OUFBW for 5 min after forming a 24-h C. albicans biofilm. The discs after initial attachment for 1.5 h were immersed in OUFBW and then cultured for 0, 3, and 5 h. CFUs were subsequently evaluated at each time point. Moreover, a viability assay, scanning electron microscopy (SEM), Alamar Blue assay, and quantitative real-time polymerase chain reaction (qRT-PCR) test were performed. To investigate the long-term effects of OUFBW on acrylic resin surface properties, Vickers hardness (VH) and surface roughness (Ra) were measured. We found that OUFBW9 and OUFBW11 significantly degraded the formed 24-h biofilm. The time point CFU assay showed that C. albicans biofilm formation was significantly inhibited due to OUFBW11 exposure. Interestingly, fluorescence microscopy revealed that almost living cells were observed in all groups. In SEM images, the OUFBW group had lesser number of fungi and the amount of non-three-dimensional biofilm than the control group. In the Alamar Blue assay, OUFBW11 was found to suppress Candida metabolic function. The qRT-PCR test showed that OUFBW down-regulated ALS1 and ALS3 expression regarding cell-cell, cell-material adhesion, and biofilm formation. Additionally, VH and Ra were not significantly different between the two groups. Overall, our data suggest that OUFBW suppressed C. albicans growth and biofilm formation on polymethyl methacrylate without impairing surface properties.

Comparative analysis of methods for assessing the safety of Dunaliella salina Teodoresco and Chlorococcum dissectum Korshikov (Chlorophyta) microalgae cultures after exposure to stress factors

The possibility of using various methods for determining the viability of cultures of microalgae Dunaliella salina and Chlorococcum dissectum before and after freezing-warming was investigated and analyzed. It has been established that the selection of an effective method should be carried out individually for each culture. For an integral assessment of the proliferative and metabolic activity of cells of both species of the studied microalgae, Alamar Blue-test and the ability to grow on liquid nutrient media can be used. The use of the Koch plate method, MTT-test and TTC staining is possible only for the microalga C. dissectum. Vital staining with trypan blue was found to be incorrect.

The photoprotective properties of α-tocopherol phosphate against long-wave UVA1 (385 nm) radiation in keratinocytes in vitro

UVA1 radiation (340–400 nm), especially longwave UVA1 (> 370 nm), is often ignored when assessing sun protection due to its low sunburning potential, but it generates reactive oxygen species (ROS) and is poorly attenuated by sunscreens. This study aimed to investigate if α-tocopherol phosphate, (α-TP) a promising new antioxidant, could protect against long-wave UVA1 induced cell death and scavenge UVA1 induced ROS in a skin cell model. HaCaT keratinocyte cell viability (24 h) was assessed with Alamar Blue and Neutral Red assays. The metabolism of α-TP into α-T, assessed using mass spectrometry, and the compound's radical scavenging efficacy, assessed by the dichlorodihydrofluorescein (H2DCFDA) ROS detection assay, was monitored in HaCaTs. The mechanism of α-TP ROS scavenging was determined using non-cell based DPPH and ORAC assays. In HaCaT keratinocytes, irradiated with 226 J/cm2 UVA1 in low-serum (2%, starved) cell culture medium, pretreatment with 80 µM α-TP significantly enhanced cell survival (88%, Alamar Blue) compared to control, whereas α-T pre-treatment had no effect survival (70%, Alamar Blue). Pre-treatment of cells with 100 μM α-TP or 100 μM α-T before 57 J/cm2 UVA1 also significantly reduced ROS generation over 2 h (24.1% and 23.9% respectively) compared to the control and resulted in α-TP bioconversion into α-T. As α-TP displayed weak antioxidant activity in the cell-free assays thus its photoprotection was assigned to its bioconversion to α-T by cellular phosphatases. Through this mechanism α-TP prevented long-wave UVA1 induced cell death and scavenged UVA1 induced ROS in skin cells when added to the starved cell culture medium before UVA1 exposure by bioconversion into α-T.

The Synthesis of Carbohydrates for the Treatment of Disease

<p>In this thesis I investigated two aspects of glycobiology. In the first, I investigated the potential of α-GalCer analogues to be used in cancer immunotherapy. Two 4-deoxy α-GalCer analogues, with either a sphinganine or a sphingosine base, were synthesised using a convergent strategy. The α-GalCer sphinganine derivative was synthesised in 14 steps from D-arabinose, and in an overall 13% yield. The α-GalCer sphingosine analogue was synthesised in 13 steps also in 13% yield. Biological analysis revealed that both 4-deoxy analogues possessed comparable activity to α-GalCer in mice, however demonstrated significantly reduced hNKT cell activity. The reduced activity was attributed to species-specific differences in iNKT cell glycolipid recognition rather than reduced CD1d presentation. From these results we suggest that glycolipids developed for potent CD1d-iNKT cell activity in humans should contain a ceramide base with the 4-hydroxyl present. The second part of this thesis focused on protecting group free methodology for the synthesis of sugar mimetics that have proven potential as glycosidase inhibitors. In this work I developed an efficient, high yielding and diastereoselective strategy for the synthesis of a number of five and six membered azasugars. This strategy utilises two novel reaction methodologies. The first enabled the stereoselective formation of cyclic carbamates from olefinic amines, the transition states controlling the stereoselectivity during this reaction are discussed. The second reaction facilitated the synthesis of primary amines without the need for protecting groups, the scope of this reductive amination methodology is also investigated. The five membered azasugars 1,4-dideoxy-1,4-imino-Dxylitol, 1,4-dideoxy-1,4-imino-L-lyxitol, 1,4-dideoxy-1,4-imino-L-xylitol and 1,2,4-trideoxy-1,4-imino-L-xylitol were prepared in 5 steps, in good overall yields (57%, 55%, 54% and 48% respectively), and without the need for protecting groups. The six membered azasugar DGJ was prepared over six steps in 33% yield using similar methodology. The synthesised compounds were also tested for anti-tubercular activity using a BCG alamar blue assay.</p>

The Synthesis of Carbohydrates for the Treatment of Disease

<p>In this thesis I investigated two aspects of glycobiology. In the first, I investigated the potential of α-GalCer analogues to be used in cancer immunotherapy. Two 4-deoxy α-GalCer analogues, with either a sphinganine or a sphingosine base, were synthesised using a convergent strategy. The α-GalCer sphinganine derivative was synthesised in 14 steps from D-arabinose, and in an overall 13% yield. The α-GalCer sphingosine analogue was synthesised in 13 steps also in 13% yield. Biological analysis revealed that both 4-deoxy analogues possessed comparable activity to α-GalCer in mice, however demonstrated significantly reduced hNKT cell activity. The reduced activity was attributed to species-specific differences in iNKT cell glycolipid recognition rather than reduced CD1d presentation. From these results we suggest that glycolipids developed for potent CD1d-iNKT cell activity in humans should contain a ceramide base with the 4-hydroxyl present. The second part of this thesis focused on protecting group free methodology for the synthesis of sugar mimetics that have proven potential as glycosidase inhibitors. In this work I developed an efficient, high yielding and diastereoselective strategy for the synthesis of a number of five and six membered azasugars. This strategy utilises two novel reaction methodologies. The first enabled the stereoselective formation of cyclic carbamates from olefinic amines, the transition states controlling the stereoselectivity during this reaction are discussed. The second reaction facilitated the synthesis of primary amines without the need for protecting groups, the scope of this reductive amination methodology is also investigated. The five membered azasugars 1,4-dideoxy-1,4-imino-Dxylitol, 1,4-dideoxy-1,4-imino-L-lyxitol, 1,4-dideoxy-1,4-imino-L-xylitol and 1,2,4-trideoxy-1,4-imino-L-xylitol were prepared in 5 steps, in good overall yields (57%, 55%, 54% and 48% respectively), and without the need for protecting groups. The six membered azasugar DGJ was prepared over six steps in 33% yield using similar methodology. The synthesised compounds were also tested for anti-tubercular activity using a BCG alamar blue assay.</p>

The effect of cartilage decellularized extracellular matrix-chitosan compound on treating knee osteoarthritis in rats

Knee osteoarthritis (KOA) refers to a common disease in orthopaedics, whereas effective treatments have been rarely developed. As indicated from existing studies, chondrocyte death, extracellular matrix degradation and subchondral bone injury are recognized as the pathological basis of KOA. The present study aimed to determine the therapeutic effect of decellularized extracellular matrix-chitosan (dECM-CS) compound on KOA. In this study, rat knee cartilage was decellularized, and a satisfactory decellularized extracellular matrix was developed. As suggested from the in vitro experiments, the rat chondrocytes co-cultured with allogeneic dECM grew effectively. According to the results of the alamar blue detection, dECM did not adversely affect the viability of rat chondrocytes, and dECM could up-regulate the genes related to the cartilage synthesis and metabolism. As reported from the animal experiments, dECM-CS compound could protect cartilage, alleviate knee joint pain in rats, significantly delay the progress of KOA in rats, and achieve high drug safety. In brief, dECM-CS compound shows a good therapeutic effect on KOA.

Semisynthetic Derivatives of Selected Amaryllidaceae Alkaloids as a New Class of Antimycobacterial Agents

The search for novel antimycobacterial drugs is a matter of urgency, since tuberculosis is still one of the top ten causes of death from a single infectious agent, killing more than 1.4 million people worldwide each year. Nine Amaryllidaceae alkaloids (AAs) of various structural types have been screened for their antimycobacterial activity. Unfortunately, all were considered inactive, and thus a pilot series of aromatic esters of galanthamine, 3-O-methylpancracine, vittatine and maritidine were synthesized to increase biological activity. The semisynthetic derivatives of AAs were screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra and two other mycobacterial strains (M. aurum, M. smegmatis) using a modified Microplate Alamar Blue Assay. The most active compounds were also studied for their in vitro hepatotoxicity on the hepatocellular carcinoma cell line HepG2. In general, the derivatization of the original AAs was associated with a significant increase in antimycobacterial activity. Several pilot derivatives were identified as compounds with micromolar MICs against M. tuberculosis H37Ra. Two derivatives of galanthamine, 1i and 1r, were selected for further structure optimalization to increase the selectivity index.

Antimicrobial and cytotoxic activity of fungal mycelial extracts from aquatic environments in the Amazon

Fungi are a prolific source of biologically active metabolites, including a wide range of clinically important drugs. Therefore, this study aims to evaluate the antimicrobial and cytotoxic activity of secondary metabolites extracted from fungal mycelia isolated from freshwater samples in the state of Amazonas. Mycelial extracts from 12 fungal were used, extracted with MeOH/AcOEt (1:1) according to the criteria established by Souza et al. (2004). For antimicrobial activity, the extracts were tested against the pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus feacalis, Candida albicans and C. tropicalis. To identify the minimum inhibitory dosage (MID) the microdilution method was used. To perform the cytotoxicity assay, the VERO strain (ATCC® CCL-81TM) was used. The assays were determined by the Alamar Blue method according to Ahmed et al. (1994). The tested extracts did not show antibacterial activity. Five extracts (41.7%), obtained from the fungi Aspergillus - 1283, Chrysoporther - 1169, Diaporther – 1203, Fusarium – 1085, and Trichoderma, showed antifungal activity against C. albicans. Diaporther extract (8.3%) - 1203 was active against C. tropicalis. In the cytotoxicity assay, 58.3% of the evaluated extracts showed no significant toxic effect. Five extracts, Cladosporium - 1135, Chrysoporther - 1169, Cytospora - 1098, Fusarium - 1085, and Talaromyces - 1244, showed cytotoxic potential, exhibiting viability lower than 70%. The results obtained suggest that mycelial extracts of fungi isolated from water samples from the Amazon region have potential against yeasts of medical interest. Only two of the active extracts were revealed potentially cytotoxic.