Class of 2017 Abstract / Objectives: Evaluate thymoquinone (TQ) and thymol (THY) inhibitory activities against α-glucosidase enzyme by using an in vitro assay and determine the IC50 (concentration of TQ/THY to inhibit 50% of maximum enzyme activity). Methods: Various concentrations of thymoquinone and thymol were incubated, separately, with one concentration of the substrate - p-nitrophenol-α-D-glucopyranoside (PNPG) (<<Km) in presence of α-glucosidase enzyme. A positive and a negative control consisting of acarbose, and buffer, respectively, were included in the incubation as well. The incubation time was set at 30 min in a 37 °C controlled water bath. The enzyme activity was determined by detecting and quantitating the levels of p-nitrophenol using a spectrophotometer set at 405 nm. The percent inhibition exhibited by any studied drug was calculated as shown in equation 1. % inhibition = Absorbance Substrate Alone – Absorbance of Substrate + Inhibitor Absorbance Substrate Alone Results: Results of the in vitro incubation of thymoquinone, thymol and acarbose revealed “statistically” significant inhibition of -glucosidase (p<0.05). At 400 g/ml, thymoquinone inhibited the enzyme activity by ~52 % whereas the enzyme inhibition by thymol and acarbose were calculated to be ~84% and 57%, respectively. IC50 were tentatively determined although the maxima inhibitions of the inhibitors were not reached fully. IC50s were calculated as 234 μg/ml, 304 μg/ml and 157 μg/ml for each of thymoquinone, thymol and acarbose, respectively. Conclusions: Thymoquinone and thymol do exhibit antagonistic pharmacological activity against α-glucosidase.

BACKGROUND: Thymoquinone (TQ) is one of the most active ingredients of Nigella sativa seed. TQ has a variety of pharmacologic properties including possess antinociceptive, antibacterial, anti-inflammatory, anti-oxidative activities and osteogenic effects on bone cells. Because of these properties, TQ might play an important role in odontogenic effects on human dental pulp cells and be used as a pulp capping material. PURPOSE: This study evaluates the effect of TQ on the attachment efficiency, proliferation, and odontogenic differentiation of Human Dental Pulp Cells (HDPC's). The effect of TQ on odontogenic differentiation was also determined by evaluating alkaline phosphatase (ALP) activity and Dentin Sialoprotein (DSP) expression. METHODS: Human dental pulp cells were cultured in triplicate using growth media with various TQ concentrations: 5 μM, 10μM, 15μM, 30μM, and 0μM as a control group at 7 and 21 days. Crystal violet staining was used to determine cell attachment efficiency and cell proliferation. The proliferation rates were normalized to cell numbers of each group at 16 hours. Cell differentiation was assessed by evaluating ALP activity and DSP expression. The data were normalized on per million cells basis. Univariate analyses including ANOVA and Student’s t-test were conducted. RESULTS: Higher cell attachment efficiency was shown in all TQ groups at 16 hours (P<. 0001) except for the 5 μM group. A significantly higher cell proliferation rate was shown with low TQ concentration 5 μM at 7 days (P<. 0001) and at 21days (P<. 0.05). However, the cell proliferation rates decreased significantly with higher TQ concentrations at both time intervals (P< 0.0001). Similarly, prolifration rates decreased at 21 days TQ =10 μM (P< 0.0001) and TQ =15 μM (P=0.0006). Significantly higher levels of alkaline phosphatase activity were observed in all TQ groups at 7 days (P <0.0001) and at 21 days (P <0.0001). Dentin sialoprotein expression was significantly down regulated in all TQ groups at 7 days compared to the control (P <0.05), however, at day 21 only TQ =15 μM exhibited significant down regulation of DSP compared to the control (P <0.0001). CONCLUSION: All tested TQ concentrations significantly enhanced cell attachment efficiency at 16 hours except TQ =5 μM. Cell proliferation rates were increased significantly by low TQ concentration 5 μM, and decreased significantly with higher TQ concentration 30 μM at 7 and 21 days. TQ exhibits odontogenic potential by inducing a significant increase in ALP activity at 7 and 21 days. TQ did not have any effect on DSP expression at 21 days except TQ =15 μM which significantly decreased DSP expression.

Cancer is a group of diseases that are the second leading cause of human mortality in the United States. Discovering new therapies is vital to conquer cancer. Thymoquinone (TQ) is found in the plant Nigella sativa. TQ was found to be cytotoxic to the human ovarian cancer cell lines PA-1, CAOV-3 and SKOV-3, which have varying p53 status. PA-1 cells were the most sensitive, indicating that TQ was effective against cells having wild-type (WT) p53. Western blots indicated an increase in p53 in cell lines having WT p53. TQ when given concurrently with cisplatin resulted in antagonism for PA-1, A172 and H460 cell lines. Sequential exposure to TQ followed by cisplatin resulted in synergy or additive effects in these cell lines. Sequential exposure to cisplatin followed by TQ resulted in additive or moderate antagonism in these cell lines. Concurrent exposure to TQ and paclitaxel showed synergy in PA-1 and H460 cells. Sequential exposure to TQ followed by paclitaxel resulted in synergism or antagonism in A172, PA-1, and H460 cells. Paclitaxel followed by TQ resulted in antagonism or synergism in these cells. These results demonstrate that TQ has a potential as an antineoplastic agent and may affect p53 levels.

Une meilleure compréhension des mécanismes moléculaires impliqués dans la malignité des lymphome B diffus à grande cellules (LBDGC) et dans leur résistance aux traitements devraient permettre de développer de nouvelles opportunités thérapeutiques innovantes et d’envisager dans un futur proche de nouveaux agents de thérapie ciblée. Jusqu’à ce jour, le traitement standard se solde encore par un échec chez un nombre significatif de patients. Les rechutes post traitement et les cas réfractaires restent le défi majeur pour améliorer le taux de survie global. Plusieurs méchanismes ont été décrits comme responsables de ces échecs, dont, pour une grande part, le dysfonctionnement de la machinerie apoptotique. Dès lors, des stratégies thérapeutiques activant des voies non-apoptotiques plus sélectivement dans les cellules malignes sont absolument nécessaires. La Thymoquinone (TQ), un principe actif isolé de la plante médicinale Nigell a Sativa, a démontré des propiétés anticancéreuses in vitro ainsi que dans des modèles animaux, en agissant via plusieurs mécanismes incluant principalement des effets antiprolifératifs et pro-apoptotiques. Malgré les nombreuses investigations, les mécanismes de destruction cellulaire sous-jacents à l’effet anti-cancéreux de la TQ restent ambigus et peu élucidés, notament sur les voies de mort cellulaire non-apoptotiques. L’objectif de notre travail vise l’évaluation de la sélectivité de la TQ vis-à-vis des lignées cellulaires de lymphomes B diffus à grandes cellules (LBDGC), et l’élucidation des méchanismes critiques responsables de la destruction cellulaire. Dans une première partie, nous avons démontré la capacité de la TQ à produire un effet cytotoxique plus prononcé dans des lignées cellulaires lymphomateuses (LBDGC) par rapport aux cellules normales provenant de donneurs sains. Le profil de sélectivité de la TQ s’est avéré intéressant par rapport aux agents de chimiothérapie conventionnelle dans le LBDGC. A l’échelle moléculaire, on a mis en évidence l’implication de voies de mort cellulaire non-apoptotiques, en supplément des voies apoptotiques, dans l’effet cytotoxique de la TQ dans la plupart des lignées étudiées. Nos investigations ont d’abord révélé un effet pro-apoptotique vraisemblablement sous-jacent à l’effet génotoxique de la TQ. L’importance de cette modalité de mort cellulaire dans l’effet cytotoxique de la TQ est toutefois limitée aux lignées les moins sensibles. Nos observations ont révélé la capacité de la TQ à activer la voix mitochondriale des caspases suite à la libération du cytochrome c. La mort cellulaire produite par la TQ n’est toutefois pas freinée par l’inhibition des caspases, et nos investigations ont également exclu l’implication des voies apoptotiques caspases-indépendantes. Paradoxalement, ces investigations ont montré le rôle critique des voies de mort cellulaire non-apoptotiques, en particulier dans les lignées sensibles à l’effet de la TQ. En étudiant l'origine de cette voie alternative de mort cellulaire, on a constaté que la TQ provoque le dysfonctionnement réticulaire qui se manifeste par l’activation des voies de transduction UPR et se produit de façon plus significative dans les lignées sensibles. En effet, dans cette dernière catégorie nos investigations ont démontré que la TQ produit un effet important par l’augmentation du calcium cytosolique principalement via la dépletion de celui-ci à partir des compartiments réticulaires. En accord avec ces observations, nous avons demontré que l’augmentation du calcium cytosolique joue un rôle critique, plus particulièrement dans l’effet non-apoptotique de la TQ opérant principalement dans les lignées fort sensibles aux effets de la TQ. Afin de mieux caractériser les mécanismes de susceptibilité et de résistance des lignées LBDGC, la deuxième partie de ce travail a été consacrée à l’évaluation de l'implication de l’histone déacétylase SIRT1 ( silent information regulator 1) dans la sensibilité des lignées aux effets de la TQ. SIRT1 intervient notament dans la résistance au stress oxydatif et à l’apoptose. SIRT1 serait également protectrice contre les dommages à l'ADN. Rappelons que l’expression de SIRT1 est considérée comme un facteur de mauvais pronostic pour les patients atteints de LBDGC. En focalisant nos investigations sur les lignées résistantes aux traitementx standard et moins sensibles à la TQ, nous avons démontré l'effet protecteur de SIRT1 vis-à-vis de l'effet pro-apoptotique et génotoxique de la TQ. En résumé, la capacité de la TQ à déclencher des modalités de mort cellulaire non apoptotiques pourrait constituer une stratégie prometteuse pour surmonter les mécanismes anti-apoptotiques responsables en partie de l’échec du traitement standard. Tenant compte du rôle critique de l’effet génotoxique dans le traitement des LBDGC, notre travail suggère que l'inhibition de SIRT1 pourrait être une stratégie préventive pour surmonter la résistance native ou acquise. D’autre part, nos résultats contribuent à une compréhension plus précise des mécanismes critiques responsables de l’effet cytotoxique de la TQ. Nos travaux pourraient d’autre part permettre l’étude de combinaisons innovantes de médicaments incluant la TQ in vivo. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

In the past two decades, few reports have confirmed the various antibacterial activities of total extract from the seeds of Nigella sativa (Black cumin). In attempts to identify the active ingredients in this extract, the seeds were extracted with hexane. The volatile oil obtained from the crude extract was shown to contain at least three distinct compounds, thymoquinone, p-cymene, and α-pinene, as confirmed by GC/MS and NMR spectroscopy. While p-cymene and α-pinene showed no antibacterial activities, thymoquinone, exhibited remarkable inhibition of the growth of various strains of bacteria. For instance, the IC50 and the Minimal Inhibitory Concentration (MIC) for thymoquinone were found to be 1.31 μg/mL and 3.6 μg/mL respectively, when tested against the gram-positive bacteria, S. aureus. It is also found that thymoquinone significantly inhibites the protein and RNA synthesis in S. aureus.

Cette thèse traite du rôle de la protéine FAK (Focal Adhésion Kinase) dans les mécanismes d'invasion cellulaire. / This thesis is about the role of the protein FAK (Focal Adhesion Kinase) in the cellular mechanisms of invasion.

Axonal damage, demyelination and inflammation of the central nervous system are the major pathological features of multiple sclerosis (MS). MS is thought to be due to an abnormal T cell mediated immune response. Oxidative stress plays an important role in the advancement of MS. The reduced glutathione (GSH) has very important role in the management of oxidative stress. In our experiment we used Experimental autoimmune encephalomyelitis (EAE) animal model that mimic human MS and tested the effect of Thymoquinone (TQ) a constituent of oil of Nigella Sativa also known as black seed. Thirty female mice strain C57BL/6J between 6 to 12 weeks of age were placed into 3 groups of 10 and MOG was used subcutaneously (s.c) to induce EAE. Group A, the control group. Group B, received MOG (s.c) and TQ intraperiotoneally (i.p) from day 1 till day 50. Group C, received MOG (s.c) and TQ (i.p) was given on the appearance of first sign and symptoms of Chronic relapsing EAE (CR-EAE). All Mice were examined daily for behavioral deficits and all euthanized and sacrificed on day 50. In this study we found mice belonging to group C (EAE with TQ treatment after the appearance of chronic symptoms) were observed to have the highest mean clinical scores in both the acute and chronic phases of EAE with symptom reduction following the TQ injections. Group B (which received daily TQ injections) had decreased symptoms compared to Group A and C. Glutathione level dropped significantly in the control group (p < 0.05) and increased (p > 0.05) in groups B and C mice who received TQ injections. We also noted that EAE clinical signs correlated well with the extent of perivascular lymphocyte infiltrate compared with normal histology following TQ injections. Our results indicate that TQ, due to its anti-oxidant effects is almost 80% preventive and 50% curative in CR-EAE. These results could assist further studies on the mechanism of the action of TQ in CR-EAE and on the possibility of treating the chronic- relapsing phase of human multiple sclerosis. It seems within the realm of possibility that TQ may be as, if not more, therapeutically efficacious as interferon â and glatiramer acetate.

Axonal damage, demyelination and inflammation of the central nervous system are the major pathological features of multiple sclerosis (MS). MS is thought to be due to an abnormal T cell mediated immune response. Oxidative stress plays an important role in the advancement of MS. The reduced glutathione (GSH) has very important role in the management of oxidative stress. In our experiment we used Experimental autoimmune encephalomyelitis (EAE) animal model that mimic human MS and tested the effect of Thymoquinone (TQ) a constituent of oil of Nigella Sativa also known as black seed. Thirty female mice strain C57BL/6J between 6 to 12 weeks of age were placed into 3 groups of 10 and MOG was used subcutaneously (s.c) to induce EAE. Group A, the control group. Group B, received MOG (s.c) and TQ intraperiotoneally (i.p) from day 1 till day 50. Group C, received MOG (s.c) and TQ (i.p) was given on the appearance of first sign and symptoms of Chronic relapsing EAE (CR-EAE). All Mice were examined daily for behavioral deficits and all euthanized and sacrificed on day 50. In this study we found mice belonging to group C (EAE with TQ treatment after the appearance of chronic symptoms) were observed to have the highest mean clinical scores in both the acute and chronic phases of EAE with symptom reduction following the TQ injections. Group B (which received daily TQ injections) had decreased symptoms compared to Group A and C. Glutathione level dropped significantly in the control group (p < 0.05) and increased (p > 0.05) in groups B and C mice who received TQ injections. We also noted that EAE clinical signs correlated well with the extent of perivascular lymphocyte infiltrate compared with normal histology following TQ injections. Our results indicate that TQ, due to its anti-oxidant effects is almost 80% preventive and 50% curative in CR-EAE. These results could assist further studies on the mechanism of the action of TQ in CR-EAE and on the possibility of treating the chronic- relapsing phase of human multiple sclerosis. It seems within the realm of possibility that TQ may be as, if not more, therapeutically efficacious as interferon â and glatiramer acetate.

Type 2 Diabetes Mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia, which develops as a consequence of peripheral insulin resistance and defective insulin secretion from pancreatic β-cells. A high calorie diet coupled with physical inactivity are known risk factors for the development of T2DM; however, these alone fail to account for the rapid rise of the disease. Recent attention has turned to the role of environmental pollutants in the development of metabolic diseases. PBDEs (polybrominated diphenyl ethers) are environmental pollutants that have been linked to the development of type 2 diabetes (T2D), however, the precise mechanisms are not clear. In particular, their direct effect on insulin secretion is unknown. In this study, we show that two PBDE congeners, BDE-47 and BDE-85, potentiate glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 cells. This effect of BDE-47 and BDE-85 on GSIS was dependent on thyroid receptor (TR). Both BDE-47 and BDE-85 (10 μM) activated Akt during an acute exposure. The activation of Akt by BDE-47 and BDE-85 plays a role in their potentiation of GSIS, as pharmacological inhibition of PI3K, an upstream activator of Akt, significantly lowers GSIS compared to compounds alone. This study suggests that BDE-47 and BDE-85 directly act on pancreatic β-cells to stimulate GSIS, and that this effect is mediated by the thyroid receptor (TR) and Akt activation. This can cause the β-cells to oversecrete insulin, potentially leading to hyperinsulinemia, insulin resistance, and high blood glucose. In contrast to the potential diabetogenic effects of POPs, there are several naturally-derived compounds which accomplish just the opposite, exerting sensitizing effect on the peripheral tissues and sparing effect on β-cells. TQ, a natural occurring quinone and the main bioactive component of plant Nigella sativa, undergoes intracellular redox cycling and re-oxidizes NADH to NAD+. TQ administration (20 mg/kg/bw/day) to the Diet-Induced Obesity (DIO) mice reduced their diabetic phenotype by decreasing fasting blood glucose and fasting insulin levels, and improved glucose tolerance and insulin sensitivity as evaluated by oral glucose and insulin tolerance tests (OGTT and ITT). Furthermore, TQ decreased serum cholesterol levels and liver triglycerides, increased protein expression of phosphorylated Akt, decreased serum levels of inflammatory markers resistin and MCP-1, and decreased the NADH/NAD+ ratio. These changes were paralleled by an increase in phosphorylated SIRT-1 and AMPKα in liver and phosphorylated SIRT-1 in skeletal muscle. TQ also increased insulin sensitivity in insulin-resistant HepG2 cells via a SIRT-1-dependent mechanism These findings are consistent with the TQ-dependent re-oxidation of NADH to NAD+, which stimulates glucose and fatty acid oxidation and activation of SIRT-1-dependent pathways. Taken together, these results demonstrate that TQ ameliorates the diabetic phenotype in the DIO mouse model of type 2 diabetes.

Thymoquinone (TQ), a volatile oil component of black seed oil (derived from Nigella sativa), has been shown to have various biological effects including disease treatment and prevention. TQ is believed to share similar properties to the benzoquinones already in use as therapeutic drugs. Based on previous reports on the anti-inflammatory properties of black seed oil and TQ, it was originally hypothesized that TQ would inhibit lipopolysaccharide (LPS)-induced cellular sialidase activity in an anti-inflammatory manner. Sialidase activity was tested on live mouse bone marrow derived primary macrophage cells, BMC-2 macrophage cells, human embryonic kidney epithelial (HEK293) cells and human fibroblast cells using an assay that measures the cleavage of the sialidase specific fluorescent substrate 2’-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (4-MUNANA). The cleavage of 4-MUNANA causes the release of free 4-methylumbelliferone, which fluoresces at 450nm (blue) after excitation at 365nm. Unexpectedly, TQ induced sialidase activation in all three cell lines and wild type primary macrophage cells. TQ was unable to induce sialidase activity in primary macrophage cells isolated from Neu4 knockout mice suggesting that the TQ activates Neu4 sialidase enzyme. TQ-induced sialidase activity in these live cells was found to occur through intermediate GPCR-associated guanine nucleotide Gαi subunit and matrix metalloproteinase 9 (MMP9) by using specific inhibitors. In addition, TQ was found to induce sialidase activity in Toll-like receptor-deficient HEK293 cells. These latter data suggested that TQ may be activating GPCR Gαi and MMP9 signaling associated with Neu4 sialidase independent of TLRs. It is proposed that TQ-induced sialidase activity may activate Toll-like receptors in macrophage cells and the subsequent production of pro-inflammatory cytokines in the absence of LPS. Immunocytochemical staining of BMC-2 cells shows that TQ induced NFκB activation. NFκB activation was confirmed with electrophoretic mobility shift assay (EMSA) and western immunoblotting techniques. Cytokine arrays were used to test the pro-inflammatory cytokine response induced in mice by 5 hour treatment of TQ, compared to LPS. Mice treated with TQ exhibited an increase in IL-1β, IL-6 and TNF-α production, similar to LPS treatment. Taken together, the findings in these studies suggest that TQ is a novel ligand for Neu4 sialidase activation which consequently induces pro-inflammatory cytokine responses. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-04-21 17:38:10.413