Canarium patentinervium Miq. belongs to the family of Burseraceae best known for producing resins of economic, medicinal, and cultural values such as frankincense, myrrh, and copal. This family consists of 18 genera and 700 species of trees. In the Asia-Pacific region, about 20 species of Burseraceae are used to treat haemorrhoids, heal wounds and to treat skin infections. This plant has been used to heal wounds amongst the indigenous people of Malaysia. Furthermore no pharmacological and phytochemical studies have been reported on this species. This study was undertaken to screen the phytochemical and pharmacological aspects of this plant. Qualitative phytochemical properties of the crude extract was determined for the presence of tannins, flavonoids, alkaloids, saponins or sterols. Phytochemical analysis of Canarium patentinervium Miq. revealed presence of tannins and flavonoids in the ethanol extract of leaves and barks. Bioassay guided fractionation led to isolation of eight secondary metabolites by chromatography which were identified by NMR techniques. Two coumarins (scopoletin and scoparone), five phenols (cynaroside, hyperin, (+)-catechin, lioxin and syringic acid) and a norsesquiterpene with cyclohexenone ring (vomifoliol). The latter three compounds were isolated for the first time from the genus Canarium. The plant and the isolated compounds were then subjected to six biological assays comprising of antibacterial, antioxidant, anticancer, anti-inflammatory, anti-acetylcholinesterase and anti-parasitic activities. Infectious diseases remain the leading cause of death worldwide and bacteria have become more resistant to conventional antibiotic and the search for novel antimicrobial agents from medicinal plants has become crucial. Antibacterial test was done using disc diffusion method, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and death kinetic assay with ampicillin as the positive control. The ethanol extract of leaves and the hexane extract of bark displayed remarkable antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria. All isolated compounds tested against S.aureus ATCC 11632 showed bacterial growth inhibition. Scopoletin, scoparone, hyperin, cynaroside and syringic acid had bactericidal effect <100 µg/ml. Only scopoletin had bactericidal effect and complete kill at MBC 50.00 µg/ml. Bacterial infections have been known to generate extensive formation of free radicals which is becoming increasingly recognized in the pathogenesis of the many human diseases. The role of free radicals and active oxygen is becoming increasingly recognized in the pathogenesis of the many human diseases, including cancer, neurodegenerative diseases, ageing, and atherosclerosis. Five various antioxidant assays with different mode of action [2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), β-carotene bleaching assay and superoxide dismutase (SOD) assay] were used to test the antioxidant scavenging abilities of this plant. Vitamin C (L-ascorbic acid), quercetin and trolox were used as positive controls. The ethanol extract of leaves and barks displayed superior antioxidant capacities. The EC50 values of the samples were consistently low in SET methods (ABTS, DPPH and FRAP) superior to standard as opposed to HAT method (β-carotene bleaching assay). Hyperin and (+)-catechin exhibited the most consistent free radical scavenging capability across the five antioxidant assay. Hyperin and (+)-catechin have significantly lower IC50 (0.75±0.03 µg/ml and 0.94±0.27 µg/ml respectively) compared to SOD enzyme (IC50 1.59±04 µg/ml). Scopoletin exhibited potent antioxidant activity compared to scoparone with significantly lower EC50 values in ABTS (IC50 1.08±0.03 µg/ml) compared to ascorbic acid (EC50 1.54±0.03 µg/ml) and lower values in FRAP assay (FRAP value 49.00±0.64 µg/ml) than quercetin (FRAP value 86.00±0.24 µg/ml) and ascorbic acid (FRAP value 347.00±0.23 µg/ml). Vomifoliol had potent β-carotene bleaching activity with IC50 6.85±0.37 µg/ml. Infections and free radical generation are recognized in the pathogenesis of cancer. The ethanol and chloroform extract of barks showed significant anticancer activities with GI50 values of 34.40µg/ml and 23.44µg/ml. The most susceptible cell lines were found to be the breast cancer cell line, MDA 468. Scopoletin displayed potent anticancer effect against breast cancer cell line MDA 468 (GI50 0.09±0.25 µg/ml) and colorectal cancer cell line HT-29 (GI50 0.17±0.05 µg/ml), the latter being more significant that positive control doxorubicin (GI50 0.66±0.60 µg/ml). In recent years, roles have been identified for several inflammatory cells and for a large number of inflammatory mediators in important pathologies not previously linked to inflammation, such as Alzheimer’s disease and cardiovascular disorders including atherosclerosis, as well as cancer. Recently, reports have appeared regarding so-called “dual inhibitors,” agents that inhibit not only cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), but also 5-lipoxygenase (5-LOX). Chloroform extract of the barks had the lowest 5-LOX inhibition (IC50=29.53±0.0 3μg/ml) when compared to NDGA (IC50=29.19±0.02 μg/ml). Ethanol extract of leaves had superior COX-1 inhibition (IC50 = 0.60±0.01µg/ml) compared to COX-2 inhibition (IC50 = 1.07±0.01 µg/ml), whereas the barks had superior COX-2 inhibition (IC50 = 9.39±0.03 µg/ml) as opposed to COX-1 (IC50 = 11.41±0.03 µg/ml). All isolated compounds exhibited significantly lower 5-LOX inhibition than NDGA. Scopoletin and scoparone were potent inhibitors of 5-LOX recording lowest IC50 values (IC50 0.34±0.01 µg/ml and 0.20±0.01 µg/ml respectively). However (+)-catechin had a more comprehensive anti-inflammatory activity with dual inhibition of 5-LOX (IC50 16.10±0.03 µg/ml) and COX (COX-1; IC50 12.08±0.02 µg/ml, COX-2; IC50 83.89±0.03 µg/ml). Syringic acid exhibited potent 5-LOX inhibition (IC50 1.38±0.03 µg/ml) and moderate COX-1 inhibition (IC50 34.89±0.02 µg/ml). Furthermore, oxidative and inflammatory processes are among the pathological features associated with the central nervous system in Alzheimer’s disease. There is evidence that acetyl cholinesterase (AChE) inhibitors have an anti-inflammatory role through action against free radicals and amyloid toxicity, as well as through decreasing release of cytokines from activated microglia in the brain and blood. Chloroform extract of the barks displayed the best activity (IC50 = 88.59±0.14 μg/ml) as opposed to positive control, galanthamine (IC50 = 0.74±0.06 μg/ml). The ethanol extract of barks and leaves follow through with IC50 = 186.00±0.15 μg/ml and IC50 = 201.24±0.15 μg/ml respectively. Only scopoletin, scoparone, vomifoliol and syringic acid showed AChE inhibition at IC50 <100 µg/ml. Syringic acid exhibited good AChE inhibition (IC50 29.53±0.19 µg/ml), lowest of all compounds tested. Choline is the precursor of phosphatidylcholine (PC), a main component of Leishmania promastigote membranes. Therefore, inhibition of choline formation may de¬crease Leishmania survival. This hypothesis can be tested by using inhibitors of the acetylcholinesterase enzyme (AChE), which catalyzes the hydrolysis of acetylcholine to choline and acetic acid, as leishmanicidal compounds. The hexane extract of leaves showed moderate antileshmanial activity with IC50 values of 257.40±0.30 μg/ml. Only ethanol extracts showed activity against Giardia intestinalis and Entamoeba histolytica at concentration of 500 μg/ml. Scopoletin was tested against all three parasite amd it was more potent against Leishmania donovani (IC50 163.30±0.32 µg/ml) and MIC of >200 µg/ml for both Giardia intestinalis and Entamoeba histolytica. Six kinds of major biological effects were evident in the crude and compounds namely, antioxidant, antibacterial, anti-inflammatory, anti-AChE, anti-parasitic, and anticancer all of which were reported for the first time from this plant. Given the aforementioned evidence it is tempting to speculate that Canarium patentinervium Miq. represents an exciting scaffold from which to develop leads for treatment of inflammatory and oxidative stress related diseases.

Malaysia is ranked the 12th richest country for its biological diversity of plant species by the Convention on Biological Diversity and this project aims to contribute to existing knowledge of Malaysian rainforest plants, Acalypha wilkesiana, Duabanga grandiflora, Archidendron ellipticum and Pseuduvaria macrophylla, for the treatment of cancer. A. wilkesiana (Euphorbiaceae) whole plant EtOH and EtOAc extracts inhibited growth of breast cancer MDA-MB-468 cells (GI50: 22.7 and 15.9 μg/ml) and revealed preference over non-transformed MRC5 fibroblasts (GI50: 46.6 and 53.3 μg/ml, respectively). EtOH and HEX extracts were able to impair cell survival and colony-forming abilities in MDA-MB-468 cells after 24 h. Detection of increased MDA-MB-468 sub-G1 cell populations after 48 h treatment to EtOH and HEX extracts, suggest that cells may be undergoing apoptosis. A. ellipticum (Leguminosae) crude polar bark and leaf extracts inhibited MDA-MB-468 cell growth (GI50 of bark EtOH, EtOAc extracts: 1.7 and 40.4 μg/ml, respectively and leaf EtOH and EtOAc extracts: 9.3 and 9.3 μg/ml, respectively). However, MDA-MB-468 cell growth was unaffected by HEX extracts (> 200 μg/ml). Separation of crude extracts revealed sub-fractions of greater activity, in particular a 50-fold enhanced potency in sub-fractions of HEX extract thus overcoming masking or antagonistic activity in the crude mixture. Following 24 h, bark and leaf extracts impaired MDA-MB-468 cells’ proliferative and colony-forming abilities at 1X and 2X GI50 values suggesting significant damage was induced leading to observed cellular senescence and inhibition of cell proliferation. After 48 h exposure to EtOH and HEX extracts, MDA-MB-468 cells accumulated cellular damage, possibly affecting microtubule functions resulted in activation of apoptosis as shown by increased of sub-G1 and G2/M MDA-MB-468 cell populations and presence of phosphatidyl serine on the outer membrane of cells. Modest levels of flavonoid and phenolic compounds were found in bark and leaf extracts, which correlated to moderate level of free radical scavenging activity observed. D. grandiflora (Lythraceae) bark and leaf extracts revealed growth inhibitory effects against colorectal cancer HCT116 cells (GI50 of bark Water, EtOH, EtOAc and HEX extracts: 42.4, 37.5, 21.69 and 28.9 μg/ml, respectively; leaf Water, EtOH, EtOAc and HEX extracts GI50: 38.0, 40.9, 24.7 and > 200 μg/ml, respectively). Separation of crude bark extracts resulted in fractions of greater activity, whereas separation of leaf extracts revealed reduced activity suggesting synergistic activity in the mixture. Following 24 h, bark and leaf extracts impaired HCT116 cells’ proliferative and colony-forming abilities at 1X and 2X GI50 values indicating significant damage incurred leading to observed cellular senescence and cell proliferation inhibition. After 48 h exposure to D. grandiflora extracts, increased sub-G1 HCT116 cell population and a G1/0 cell cycle block accompanied by decreased S and G2/M cell populations were measured. Detection of phosphatidyl serine on cells’ outer membrane and activated apoptotic caspase 3 protein confirmed D. grandiflora extracts induced apoptosis. Highest levels of flavonoid and phenolic compounds were found in polar bark and leaf D. grandiflora extracts, which may correlate to the highest free radical scavenging activity measured. P. macrophylla (Annonaceae) extracts collectively displayed greatest HCT116 cell growth inhibition (EtOH, EtOAc and HEX extracts GI50: 5.2, 1.6 and 5.4 μg/ml) and separation of EtOH and EtOAc extracts revealed even greater activity in sub-fractions. After 24 h, polar extracts at 2X GI50 completely impaired HCT116 cells’ proliferative and colony-forming abilities suggesting extract-induced significant damage led to inability of cells to proliferate. Analysis of cell cycle distribution revealed increased sub-G1 HCT116 cell population and high levels of early apoptotic HCT116 cells following 48 h exposure to P. macrophylla extracts coupled with detection of caspase 3 activation confirming execution of apoptosis. Modest levels of flavonoid and phenolic compounds were detected in EtOH, EtOAc and HEX extracts, which correlated to modest free radical scavenging activity. The findings in this project should justify further separation and in vitro investigations.

A tropical shrub from Euphorbiaceae (Juss.) family, namely Acalypha wilkesiana var. macafeana hort. was investigated for its antioxidant and antibacterial properties. The antioxidant properties were assayed by Ferric Reducing Antioxidant Power (FRAP) assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and β-carotene bleaching assay. Assessment of its antibacterial properties was conducted with pour plate disc diffusion assay and dilution methods. The plant was collected, dried, grinded and soaked continuously in four different solvent starting from non-polar to polar solvent: hexane, ethyl acetate, ethanol and water. The crude extracts were concentrated under pressure and kept in -20 °C prior to investigation. The ethanol extract of A. wilkesiana var. macafeana hort. exhibited good antioxidant and antibacterial activities with results more potent than the standards used. To further locate the bioactive constituents of the plant, we fractionated the ethanol extract leading to five fractions, namely F1, F2, F3, F4 and F5. Both antioxidant and antibacterial assays were conducted upon all the five fractions. Results showed profound activity from F5 of both antioxidant and antibacterial properties which warrants further investigation. To shed light on the active constituents of F5, identification was done with high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR). Separation was obtained with reversed phase HPLC which showed one major compound and 6 minor compounds. The major compound was collected with a fraction collector and identified as geraniin via interpretation and comparison of its NMR shifts, while the other 3 compounds were identified by fragmentations of LC-MS. The compounds identified are β-glucogallin, potentillin and sanguiin H-6. All identified compounds are ellagitannins, except for β-glucogallin which is a gallotannin. The in vitro cell-based assay was performed to HepG2 cells to assess the ability of antioxidants like ellagitannins to protect cells against oxidative insults, and F5 was observed to be able to protect cells against cell death induced by t-BHP insults in a dose-dependent manner. F5 was also found non-toxic in the concentration needed to protect the cells, which is 100 µg/mL. We then explored the synergistic property of the tannin fraction, F5 with commercial antibiotics and observed F5 and ampicillin inhibit the growth of Staphylococcus aureus synergistically. Field Emission Scanning Electron Microscopy (FESEM) analysis was able to demonstrate that the bactericidal mechanism of F5 involves cell wall lysis as the result illustrates indentation of the cell surface and some showed total collapse of the cells. To explore its ability to be formulated and used as a topical agent for treating bacterial infections, a preliminary formulation was made incorporating F5, and formulated with 3 different bases. The formulation made with the paraffin base was observed able to exert the antibacterial property of F5 against Staphylococcus aureus in the in-vitro assay. In vivo animal study on guinea pigs with an incised cut infected with Staphylococcus aureus and treated with the formulation showed that the closure and healing of the wound was faster than Burnol®. Our results indicate possible use of ellagitannins from A. wilkesiana var. macafeana hort. with ampicillin to treat Staphylococcus aureus infections as it is bactericidal via a mechanism involving cell lysis. It also illustrates the possibility to be used as a topical wound healing agent with respect to its antibacterial and antioxidant properties. Ellagitannins from A. wilkesiana var. macafeana hort. can be viewed as a possible bactericidal agent that can contribute to the development of topical antibacterial drug or cosmetics in tropical countries.

ON01910.Na (Rigosertib, Estybon®), a styryl benzylsulfone, is a Phase III stage anti-cancer agent. This non-ATP competitive kinase inhibitor has multi-targeted activity, promoting mitotic arrest and apoptosis. Extensive Phase I/II studies with ON01910.Na, conducted in patients with solid tumours and haematological cancers demonstrate excellent efficacy. However, issues remain affecting its development. These include incomplete understanding of anti-tumour mechanisms, low oral bioavailability and unpredictable pharmacokinetics. In an attempt to improve drug-likeness and ADME properties of ON01910.Na analogues, a novel series of (E)-styrylsulfonyl methylpyridine derivatives was designed and synthesised. The SAR of this novel series is discussed. The lead compounds TL-68, TL-77, and AH-123 are highly potent mitotic inhibitors. Their selective cytotoxicity to cancer cells was identified in the screening cascade. Impressively, TL-77 possesses excellent pharmaceutical properties, with improved oral bioavailability when compared to ON01910.Na. The detailed cellular mechanisms of TL-77 were further investigated in comparison with ON01910.Na. TL-77 exhibits potent anti-proliferative activity against a wide range of human tumour cell lines, and demonstrated > 2 fold greater potency in cancer cell lines over normal cells. Cell cycle analyses reveal that TL-77 evokes profound G2/M cell cycle arrest at ≥ 6 h in cancer cells, followed by the onset of apoptosis. In cell-free conditions, TL-77 as well as ON01910.Na potently inhibits tubulin polymerization. Mitotically arrested cells display multipolar spindles and misalignment of chromosomes, indicating TL-77 interfere mitotic spindle assembly in cancer cells. These effects are accompanied by induction of DNA damage, inhibition of Cdc25c (Ser198) phosphorylation [indicative polo-like kinase 1 (Plk1) inhibition], and downstream inhibition of cyclin B1. However, kinase assays failed to confirm the inhibition of Plk1. Non-significant effects on PI3K/AKT signal transduction are observed after TL-77 treatment. Analysis of apoptotic signalling pathways reveals that TL-77 down-regulates expression of B-cell lymphoma 2 (Bcl-2) family proteins [Bid (BH3 interacting-domain death agonist), Bcl-xl (B-cell lymphoma-extra large) and Mcl-1 (induced myeloid leukaemia cell differentiation protein)] and stimulates caspase activation. These effects are comparable to those elicited by ON01910.Na. Unlike ON01910.Na, however, TL-77 causes preferential toxicity in cancer cells when compared to normal cells and mediates rapid mitotic inhibitory effects. In summary, selective in vitro anti-tumour activity and multi-faceted mechanisms of action of a novel molecule TL-77 have been identified, presenting a strong rationale for further development of (E)-styrylsulfonyl methylpyridine derivatives as therapeutic agents for cancer.

In the field of Photovoltaic technologies the organic solar cells are particularly attractive because of their ease of processing, mechanical flexibility and potential low cost production techniques. So far, the reported efficiencies are not high enough to allow to be competitive in the market, however with the introduction of new photoactive materials, device architectures and light management structures, the power conversion efficiencies, at laboratory scale, has rapidly reached the 12%, showing a great potential and a bright future for organic solar cells. Nevertheless, in view of commercial products, two main problems are still unresolved: the relatively low performance of the modules and their lifetime. In sight of this, the present Ph.D thesis has a double goal: 1) a better understanding of the relationship between devices performance and photoactive materials structures 2) a deep investigation on device degradation processes, with particular attention on the effects induced by temperature and incident light. As a result, promising approaches to further optimize the polymer’s optoelectrial properties, and thus the corresponding device performance, are proposed. About the lifetime, first the thermal degradation mechanisms involved on the active layer was investigated and it has been demonstrated the role of the other layers and interfaces in the solar cell thermal stability. In this contest an innovative fast capacitance based thermal test has been developed in order to obtain information regarding the limit operating temperature above which the device becomes thermally unstable. In the end, a preliminary study on the photostability issue was carried out demostrating that the photodegradation of organic solar cells not depends just on the photostability of the donor polymer, but is connected also with the composition of the active layer solution and on the interaction with the adjacent layers. Solutions to limit or prevent the devices degradation processes are proposed.

The research reported in this manuscript concerns the structural characterization of graphene membranes and single-walled carbon nanotubes (SWCNTs). The experimental investigation was performed using a wide range of transmission electron microscopy (TEM) techniques, from conventional imaging and diffraction, to advanced interferometric methods, like electron holography and Geometric Phase Analysis (GPA), using a low-voltage optical set-up, to reduce radiation damage in the samples. Electron holography was used to successfully measure the mean electrostatic potential of an isolated SWCNT and that of a mono-atomically thin graphene crystal. The high accuracy achieved in the phase determination, made it possible to measure, for the first time, the valence-charge redistribution induced by the lattice curvature in an individual SWCNT. A novel methodology for the 3D reconstruction of the waviness of a 2D crystal membrane has been developed. Unlike other available TEM reconstruction techniques, like tomography, this new one requires processing of just a single HREM micrograph. The modulations of the inter-planar distances in the HREM image are measured using Geometric Phase Analysis, and used to recover the waviness of the crystal. The method was applied to the case of a folded FGC, and a height variation of 0.8 nm of the surface was successfully determined with nanometric lateral resolution. The adhesion of SWCNTs to the surface of graphene was studied, mixing shortened SWCNTs of different chiralities and FGC membranes. The spontaneous atomic match of the two lattices was directly imaged using HREM, and we found that graphene membranes act as tangential nano-sieves, preferentially grafting achiral tubes to their surface.

No description available.

We present a study of the metal sites of different proteins through X-ray Absorption Fine Structure (XAFS) spectroscopy. First of all, the capabilities of XAFS analysis have been improved by ab initio simulation of the near-edge region of the spectra, and an original analysis method has been proposed. The method subsequently served ad a tool to treat diverse biophysical problems, like the inhibition of proton-translocating proteins by metal ions and the matrix effect exerted on photosynthetic proteins (the bacterial Reaction Center, RC) by strongly dehydrate sugar matrices. A time-resolved study of Fe site of RC with μs resolution has been as well attempted. Finally, a further step aimed to improve the reliability of XAFS analysis has been performed by calculating the dynamical parameters of the metal binding cluster by means of DFT methods, and the theoretical result obtained for MbCO has been successfully compared with experimental data.

No description available.

Nowadays alternative energies are an extremely important topic and the possibility of using hydrogen as an energy carrier must be explored. Many problems infer the technological application of this abundant and powerful resource, one of them the possibility of storage. In the framework of suitable materials for hydrogen storage, magnesium has been the center of this study because it is cheap and the amount of stored hydrogen that it achieves (7.6 wt%) is extremely appealing. Nanostructure helps to overcome the slow hydrogen diffusion and the functionalization of surfaces with transition metals or oxides favors the hydrogen molecule dissociation/recombination. The aim of this research is the investigation of the metal-hydride transformation in magnesium nanoparticles synthesized by inert-gas condensation, exploiting the fact that they are a simple model system. The so produced nanostructured powder has been analyzed in response to nanoparticles surface functionalization by transition metal clusters, specifically palladium, nickel and titanium, chosen on the basis of their completely different Mg-related phase diagrams. The role of the intermetallic phases formed upon heating and hydrogenation treatments will be presented to provide a comprehensive picture of hydrogen sorption in this class of nanostructured storage materials.