Doses Of Thymoquinone Research Articles

English

The present study was carried out to evaluate the role of thymoquinone (TQ) in animal models of schizophrenia. TQ (20 mg/kg, intraperitoneally) was administered daily for 28 days in mice. Different models of schizophrenia such as haloperidol-induced catalepsy, apomorphine-induced climbing behaviour and elevated plus maze test were used. After the last dose of TQ on the 28th day, behavioural tests were performed followed by biochemical estimations. Pre-treatment of TQ alone and in combination with haloperidol observed cataleptic behaviour. In apomorphine induced climbing behaviour model, administration of TQ reduced maximum time of single climb and climbing index (p < 0.001). Scopolamine-induced prolongation of transfer latency (TL) was reduced by TQ (p < 0.001). There was no change in the percentage alternation in TQ pre-treated group of animals in elevated plus maze test. However, a significant increase in possible alternation was observed (p < 0.001), suggesting its anti-amnesic effect. The anti-amnesic effect of TQ was further confirmed with a decrease in acetyl cholinesterase (AChE) enzymatic activity in mice brain. A decrease in thiobarbituric acid reactive substance (TBARS) levels and increase in glutathione (GSH) and catalase levels were observed in all models used, thus, confirming its antioxidant properties. TQ administration also showed reduction in dopamine levels indicating the involvement of dopamine receptors in all three models; hence, demonstrating its antipsychotic like activities. The present study observed antipsychotic like actions in different animal models of schizophrenia and also improved memory. Our results are preliminary, further research is warranted to establish its role as a new candidate in schizophrenia. Key words: Schizophrenia, thymoquinone, dopamine, antioxidant markers.

The New Miracle of Habbatus Sauda: Its Major Component Thymoquinone can be Used in the Management of Autoimmune Diseases

Prophet Muhammad (SWS) once stated that habbatus sauda (black seed) can heal every disease except death. To our knowledge the effect of this seed on autoimmunity process has never been examined.This study was carried out in UiTM Malaysia to investigate the potential immunomodulatory effect of administration of the major component of Habbatus Sauda; (Thymoquinone) in the autoimmune disease process of type 1 diabetes mellitus as a common example of autoimmune diseases. We have evaluated with the help of ELISA kits the levels of anti- islet cell autoantibodies (ICA), Pan T lymphocytes marker, Pan B lymphocytes marker, and Pan innate cells marker in male Sprague-Dawley rats with Streptozocin-induced the autoimmune disease process of type 1 diabetes mellitus. The four groups (6 rats each) under study received or not different doses of Thymoquinone. The results have been compared to the ones obtained from healthy and non treated diabetic rats. The administration of Thymoquinone especially in high doses to the type1 diabetic rats leads to a significant decreases in the levels of all immunological parameters and stop the disease process (p<0.001). The data may provide new strategies about Habbatus Sauda to be recommended in the clinical management of autoimmune diseases as one of documented halal prescription.

Thymoquinone Protects Against EGCG/LPS Induced Hepatotoxicity in Mice

Green tea (GT) is a widely used beverage and folk medicine. Epigallocatechin-3-gallate (EGCG), the main polyphenol of GT has been demonstrated to impact a number of biological targets. Our previous work has shown that the combination of LPS and EGCG in male mice causes hepatotoxicity, perhaps related to a prooxidant effect. Thymoquinone (TQ) is a prominent constituent of “black seed”, Nigella sativa, and is known for a number of biological activities, including potent antioxidant and anti-inflammatory activities. It has been extensively used in the Middle East as a protectant against hepatic and renal injuries. In the current study we treated mice with oral doses of TQ before and concurrently with EGCG/LPS. Thymoquinone ameliorated the rise in the plasma concentrations of ALT, ALP, TB, Amylase and the decrease of A/G ratio to normal after exposure to EGCG/LPS. It also blunted the weight loss. No histopathological lesions or mortality was observed. It can be concluded from this study that TQ showed a hepatoprotective effect in a murine model of EGCG/LPS induced hepatotoxicity. This hepatoprotective effect may be related to the antioxidant properties of TQ, but this conclusion needs further evaluation.

Thymoquinone treatment against acetaminophen-induced hepatotoxicity in rats

AimIn this study, we aimed to examine the efficacy of thymoquinone (TQ) treatment in acetaminophen-induced liver toxicity in rats. MethodsForty Wistar Albino rats were used for the study (four groups, with 10 rats for each group). Animals in the control group were not given any medication. In the thymoquinone (TQ) group, animals were given three times 5 mg/kg oral thymoquinone for every six hours, which equals to a total dose of 15 mg/kg. In the acetaminophen (APAP) group, animals were given APAP at a single dose of 500 mg/kg orally. In the APAP + TQ group, animals were given 500 mg/kg APAP orally followed by three doses of TQ at a 15 mg/kg total dose in an 18-h time interval. All animals were sacrificed at the 24th hour. Alanine amino transferase (ALT), aspartat amino transferase (AST), superoxide dismutase (SOD), oxidized glutathione (GSSG), glutathione peroxides (GSH-Px), and malondialdehyde (MDA) activities were measured in rat blood. Histopathological examination was also performed. ResultsSerum ALT, AST levels, GSSG, and SOD activity as well as the serum and tissue MDA levels were found to be higher in the APAP group than in the control group (p ≤ 0.001). Likewise, serum GSH-Px activity was found to be lower in the APAP group (p ≤ 0.001). In contrast, in the APAP + TQ group, serum ALT, AST levels, GSSG, SOD activity and the serum and tissue MDA levels were found to be lower compared to that of the APAP group. This difference was statistically significant (p ≤ 0.001). In the APAP + TQ group, the GSH-Px activity was found to be significantly higher compared to the APAP group (p < 0.05). In contrast to this finding, the GSH-Px activity in the APAP + TQ group was found to be lower than that of the control group (p ≤ 0.001). Histopathological analysis revealed significant liver necrosis and toxicity with a high dose of APAP where TQ treatment was related with significantly lower liver injury scores. ConclusionTQ treatment may have an important therapeuthic effect via the upregulation of antioxidant systems in the APAP-induced liver hepatotoxicity in rats.

Neuroprotective Effect of Thymoquinone on Repeated Immobilization Stress-Induced Oxidative Stress in Rats

The present study was designed to investigate the effect of Thymoquinone (TQ) in restraint stress- induced biochemical alterations in Wistar albino rats. Restraint stress was applied for 21 days (4 h/day) and 60 min before every stress exposure the rats were treated with two doses of TQ (5 and 10 mg/kg). In serum, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Interleukin-1β (IL-1β) Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α) were estimated. Thiobarbituric Acid Reactive Substances (TBARS), reduced Glutathione (GSH), nitrite and nitrate levels and Super Oxide Dismutase (SOD), Catalase (CAT), Glutathione-S-Transferase (GST), Glutathione-Reductase (GR) and Glutathione Peroxidase (GSH-Px) activities were estimated in brain. Interleukins and enzymatic activities in serum significantly elevated by the restraint stress and were ameliorated by both the doses of TQ treatment. In brain TBARS, nitrite and nitrate levels significantly elevated due to restraint stress, the TQ treatment brings back to normal levels. Restraint stress significantly decreased in oxidative enzyme activities as the SOD, CAT, GST, GSH-Px and GR. TQ treatments significantly enhanced the activities compared to untreated stressed rats. Present results revealed that, TQ reduced the restraint stress-induced oxidative process in terms of above mentioned biochemical parameters. Thus, in view of its antioxidative nature, TQ may be developed as an effective therapeutic agent for stress-induced oxidation and CNS depression.

Phosphorylated IκBα predicts poor prognosis in activated B-cell lymphoma and its inhibition with thymoquinone induces apoptosis via ROS release.

Activated B-cell lymphoma (ABC), one of the three subtypes of Diffuse Large B-cell Lymphoma (DLBCL) has the worst survival rate after upfront chemotherapy and is characterized by constitutively activated NFκB. We therefore studied the role of NFκB In a cohort of clinical DLBCL samples and ABC cell lines. In our clinical tissue microarray cohort of DLBCL samples, p-IκBα was detected in 38.3% of ABC DLBCL and was an independent prognostic marker for poor survival. In vitro, we found that Thymoquinone (TQ), a natural compound isolated from Nigella sativa caused release of ROS in ABC cells. TQ-mediated release of ROS in turn inhibited NFκB activity by dephosphorylating IκBα and decreased translocation of p65 subunit of NFκB in the nuclear compartment in ABC cell lines. This led to inhibition of cell viability and induction of mitochondrial dependent apoptosis in ABC-DLBCL cell lines. Additionally, TQ treatment also caused up-regulation of death receptor 5 (DR5), however, up-regulation of DR5 did not play a role in TQ-induced apoptosis. Finally, combination of sub-optimal doses of TQ and TRAIL induced efficient apoptosis in ABC-DLBCL cell lines. These data show that p-IκBα can be used as a prognostic marker and target for therapy in this aggressive sub-type of DLBCL and TQ may play an important role in the management of DLBCL in the future.

Cellular responses with thymoquinone treatment in human breast cancer cell line MCF-7

Background:Nigella sativa or black seed extract has been reported to show various medicinal benefits. Thymoquinone which is an active compound of its seed has been reported to contain anti-cancer properties.Objective:The study addressed the anti-cancer efficiency of long-term in vitro treatment with thymoquinone towards human breast cancer cell lines MCF-7.Materials and Methods:Cell proliferation was determined with CellTiter 96 Aqueous. Non-Radioactive Cell Proliferation Assay Kit. It was followed with trypan blue exclusion test to determine the percentage of viable cells. The study incorporated cell cycle assay to distinguish cell distribution at various cell cycle phases using Cycletest Plus DNA Reagent Kit. The apoptosis detection kit was used to determine the percentage of apoptotic and necrotic cells using flow cytometry.Results:The 50% inhibitory concentration (IC50) value determined using the proliferation assay was 25 μM thymoquinone. Late apoptotic cell percentage increased rapidly when treatment duration was increased to 24 h with 25 and 100 μM thymoquinone. Further analysis using cell cycle assay showed thymoquinone inhibition of breast cancer cell proliferation at minimal dose 25 μM and led to S phase arrest significantly at 72 h treatment (P = 0.009). It was also noted elevation sub-G1 peak following treatment with 25 μM thymoquinone for 12 h. Increase in thymoquinone to 50 μM caused G2 phase arrest at each time-point studied.Conclusion:In general thymoquinone showed sustained inhibition of breast cancer cell proliferation with long-term treatment. Specificity of phase arrest was determined by thymoquinone dose.

Anti-inflammatory and antioxidant activity of thymoquinone in a rat model of acute bacterial prostatitis

Prostatitis plays a major role in morbidity and mortality related to prostate diseases. The aim of this study was to detect whether thymoquinone (TQ) could ameliorate oxidative damage and the proliferative response induced by Escherichia coli (E. coli) in rats. A total of 42 adult male Wistar rats were used. The rats were randomly divided into seven groups (three treatment groups, three infected groups and one control group). Control group received saline and was killed 24 h after saline administration. Infected rats were killed after 24, 48 and 72 h following direct injection of E. coli into the prostate. Treatment groups were administered with 10 mg/kg dose of TQ intraperitoneally following E. coli injection and after 24 and 48 h following E. coli injection. The rats were killed at 24, 48 and 72 h after the first drug administration. Each group was compared with each other and with the control group. In addition, infected groups were compared with treatment groups. Our findings show that the treatment with TQ has a protective effect against bacterial prostatitis-induced tissue injury. Increase in malondialdehyde levels and histological damage caused by E. coli were improved markedly with TQ treatment. TQ treatment particularly increased the activity of glutathione peroxidase and decreased the activities of catalase and superoxide dismutase. These observations might be attributed, at least in part, to the antioxidant effect of TQ and suggest that it could be a clinically valuable agent in the prevention of acute prostatitis caused by E. coli.

Abstract 170: Thymoquinone-mediated suppression of NF- αB activity causes inhibition of cell viability and induces apoptosis in activated B-cell sub-type of diffuse large B-cell lymphoma

Abstract Diffuse Large B-cell Lymphoma (DLBCL) is the most common lymphoid malignancy and constitutes approximately 40% of all cases of non-Hodgkin's lymphoma (NHL). Despite improvement in treatment protocols, a large number of DLBCL cases remain refractory to treatment. DLBCL can be further classified as three distinct subtypes: germinal center B cell-like (GCB)-, activated B cell-like (ABC)-, and primary mediastinal B-cell lymphoma (PMBL). ABC DLBCL has a worse survival after upfront chemotherapy and is characterized by constitutive activation of the anti-apoptotic nuclear factor-kappa B (NF-κB) pathway. Yet the implication of NF-κB inhibition in ABC DLBCL need to be further elucidated. We have previously shown that Thymoquinone (TQ), a natural compound isolated from Nigella sativa, induces growth inhibition and induces apoptosis in several NHL cell lines. However its role in inhibition of growth and apoptosis has not been fully elucidated in ABC sub-type of DLBCL cell lines. We found that TQ treatment inhibits cell viability and induces apoptosis in a dose dependent manner in various ABC cell lines. Further more, we found that TQ treatment causes inactivation of IκBα and consequently inhibits NF-κB activity by decreasing the expression of p65 subunit of NF-κB in the nuclear compartment of ABC cell lines. In-activation of NF-κB survival pathway leads to induction of apoptosis via the mitochondrial apoptotic pathway initiated by conformational changes in the Bax protein leading to changes in the mitochondrial membrane potential and release of cytochrome c in the cytosole. Once cytochrome c is released, it leads to activation and cleavage of caspases-9 and -3 and cleavage of PARP. This leads to caspase dependent apoptosis in ABC cell lines. In addition, we also found that TQ treatment causes up-regulation of death receptor 5 (DR5), an important and essential receptor for tumor necrosis factor related apoptosis inducing ligand (TRAIL). However, up-regulation of DR5 does not play a role in TQ-induced apoptosis in ABC cell lines. Interestingly, combination of sub-toxic doses of TQ and TRAIL induces efficient significant apoptosis in ABC cell lines. These data show that TQ can be used alone or in combination with TRAIL to induce apoptosis in these aggressive sub-types of DLBCL and may play an important role in the management of DLBCL in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 170. doi:1538-7445.AM2012-170

Modulation of Hepatic Drug Metabolizing Enzymes by Dietary Doses of Thymoquinone in Female New Zealand White Rabbits

Herbal medicines can affect drug metabolizing enzymes. Therefore the effect of thymoquinone (TQ), the active ingredient of black seeds, was examined on rabbit liver drug metabolizing enzymes. Two groups of New Zealand female rabbits received TQ at 10 and 20 mg/kg/day orally and a control group of six animals each were killed after 8 weeks. Blood and livers were harvested and the activity of cytochrome P450 (CYP) and phase II enzymes in the microsomal and cytosolic preparations were measured by HPLC and ELISA methods. The liver enzymes ALT/AST and albumin were similar in the three groups. CYP1A2, CYP3A4, but not CYP2E1, were significantly diminished by TQ treatment. Of the phase II enzymes, glutathione-S-transferase (GST) and glutathione peroxidase (GPx) were significantly induced by the high TQ dose, while the total glutathione levels were unaffected. Glutathione reductase (GR), on the other hand, was significantly induced in the two experimental groups. Thymoquinone has differential effects on CYP and phase II enzymes. Inhibition of some CYP enzyme activities may lead to a hazardous herb-drug interaction. Induction of GR activity may explain the salutatory effect of the black seeds in inhibiting the generation of bioactive metabolites known to promote carcinogenesis and oxidative cell damage.

Acute effects of thymoquinone on the pregnant rat and embryo-fetal development

The effect of a single intraperitoneal (i.p.) injection of thymoquinone (TQ) on the pregnant rat and embryo-fetal development was investigated. Pregnant female Wistar rats received 15, 35, and 50 mg/kg body weight of TQ i.p. on day 11 or 14 of gestation, and on day 18 of gestation they were sacrificed and laparotomized. Results showed that TQ induces maternal and embryonic toxicities in a dose- and time-dependent manner. With a dose of 50 mg/kg, treated rats experienced a significant decrease in maternal body weight and complete fetal resorption when the dose was given on day 11 of gestation. On the other hand, 46.2% of implants were resorbed and the viable fetuses showed no TQ-related malformations when the dose was given on day 14 of gestation. At a lower TQ dose of 35 mg/kg, maternal and embryonic toxicities were observed only when it was given on day 11 of gestation. The dose of 15 mg/kg was considered to be a dose with no observed adverse effect level for maternal and embryo-fetal toxicities when it was given day 11 or 14 of gestation. Based on the results of this study, TQ, at doses of 50 and 35 mg/kg, has a potentially disruptive effect on embryonic development during the second trimester of rat pregnancy.

Thymoquinone suppresses growth and induces apoptosis via generation of reactive oxygen species in primary effusion lymphoma

We provide evidence that thymoquinone (TQ), a natural compound isolated from Nigella sativa, induces growth inhibition and apoptosis in several primary effusion lymphoma (PEL) cell lines. Our data demonstrate that TQ treatment results in down-regulation of constitutive activation of AKT via generation of reactive oxygen species (ROS) and it causes conformational changes in Bax protein, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. This leads to activation of caspase-9, caspase-3, and polyadenosine 5′-diphosphate ribose polymerase cleavage, leading to caspase-dependent apoptosis. Pretreatment of PEL cells with N-acetylcysteine, a scavenger of ROS, prevented TQ-mediated effects. In addition, subtoxic doses of TQ sensitized PEL cells to TRAIL via up-regulation of DR5. Altogether, these findings demonstrate that TQ is a potent inducer of apoptosis in PEL cells via release of ROS. They also raise the possibility that incorporation of TQ in treatment regimens for primary effusion lymphomas may provide a novel approach to sensitizing malignant cells and provide a molecular basis for such future translational efforts.

The protective effect of thymoquinone against sepsis syndrome morbidity and mortality in mice

Sepsis and septic shock are life threatening complications and most common cause of death in intensive care units. Thymoquinone, a constituent of Nigella sativa (black seed), holds exceptional promise as an anti-cancer and anti-inflammatory agent. No evidence has been published, however, whether this compound has a protective effect from sepsis-related morbidity, mortality and associated organ dysfunction. To examine this, two sets of mice (n=12 per group), with parallel control groups, were acutely treated with thymoquinone intraperitoneal injections of 1.0 and 2.0mg/kg body weight, and were subsequently challenged with endotoxin Gram-negative bacteria (LPS O111:B4). In another set of experiments, thymoquinone was administered at doses of 0.75 and 1.0mg/kg/day for three consecutive days prior to sepsis induction with live Escherichia coli. Survival of various groups was computed, and renal, hepatic and sepsis markers were quantified. Thymoquinone reduced mortality by 80–90% and improved both renal and hepatic biomarker profiles. The concentrations of IL-1α with 0.75mg/kg thymoquinone dose was 310.8±70.93 and 428.3±71.32pg/ml in the 1mg/kg group as opposed to controls (1187.0±278.64pg/ ml; P<0.05). Likewise, IL-10 levels decreased significantly with 0.75mg/kg thymoquinone treatment compared to controls (2885.0±553.98 vs. 5505.2±333.96pg/ml; P<0.01). Mice treated with thymoquinone also exhibited relatively lower levels of TNF-α and IL-2 (P values=0.1817 and 0.0851, respectively). This study gives strength to the potential clinical relevance of thymoquinone in sepsis-related morbidity and mortality reduction and suggests that human studies should be performed.

Studies on molecular mechanisms of growth inhibitory effects of thymoquinone against prostate cancer cells: role of reactive oxygen species

Thymoquinone (TQ), an active ingredient of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against a variety of experimental tumors. However, the precise mechanism of action of TQ is not known. We investigated the mechanism of action of TQ in androgen receptor (AR)-independent (C4-2B) and AR naïve (PC-3) prostate cancer cells, as models of aggressive prostate cancers. Exposure (24-48 h) to TQ (25-150 micromol/L) inhibited the growth of both C4-2B and PC-3 cells, with IC(50) values of approximately 50 and 80 micromol/L, respectively. Within one hour, TQ increased reactive oxygen species (ROS) levels (3-fold) and decreased glutathione (GSH) levels (60%) in both cell types. Pretreatment with N-acetylcysteine (NAC) inhibited both TQ-induced ROS generation and growth inhibition. TQ did not increase the activity of caspases and the caspase inhibitor, z-VAD-FMK did not decrease TQ-induced apoptosis. Furthermore, although TQ treatment resulted in the activation of Jun kinase (JNK), pretreatment with the JNK inhibitor, SP600125, did not protect cells from TQ. However, TQ significantly up-regulated the expressions of growth arrest and DNA damage inducible gene (GADD45alpha) and apoptosis-inducing factor-1 and down-regulated the expressions of several Bc12-related proteins, such as BAG-1, Bcl2, Bcl2A1, Bcl2L1 and BID. In C4-2B cells, TQ dose dependently inhibited both total and nuclear AR levels (4-5 fold) and AR-directed transcriptional activity (10-12 fold). Interestingly, this suppressive effect on AR was not prevented by NAC, which clearly suggested that TQ-induced cytotoxicity is not due to changes in AR regulation. These data suggest that TQ-induced cell death is primarily due to increased ROS generation and decreased GSH levels, and is independent of AR activity.

Thymoquinone is active in vitro against P. larvae and exhibits low toxicity against adult honey bees

American foulbrood (AFB) is a serious worldwide spreading disease of honeybees caused by the spore-forming, Gram-positive bacterium Paenibacillus larvae. Antimicrobial natural products may provide a safe and acceptable alternative in prevention and treatment of AFB. The inhibiting action of thymoquinone (TQ) on P. larvae was previously studied in vitro [1] (MIC 8–16µg/mL). Laboratory and field trials were conducted to evaluate the acute oral toxicity and transfer of TQ to royal jelly or to honey. We determined in vivo acute oral toxicity on adult honey bees by technique ICBR (1993) [2], expressed as LD50. The defined amounts of TQ were dissolved in a feeding solution (50% v/v sucrose in distilled water). The second experiment transfer to royal jelly was conducted from September to October. Non-toxic dose of TQ was fed on honey bee colony. Amount of TQ was given with the aid of GC-MS methods.

Attenuation of the development of hypercholesterolemic atherosclerosis by thymoquinone.

Thymoquinone (TQ), derived from Nigella sativa seed, is an antioxidant. The present study investigated whether TQ attenuates the development of atherosclerosis, and/or reduces the serum lipid levels and oxidative stress in rabbits. New Zealand white female rabbits were assigned to four groups of six animals each: group I, control; group II, 1% cholesterol diet; group III, 1% cholesterol plus TQ (10 mg/kg/day; through a nasogastric tube) diet; and group IV, 1% cholesterol plus TQ (20 mg/kg/day; through a nasogastric tube) diet. Blood samples were collected at baseline and after four and eight weeks on the experimental diets for measurement of serum lipids, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), TC/HDL-C ratio and oxidative stress biomarkers (malondialdehyde [MDA] and protein carbonyls). At the end of the eight weeks, the aorta was removed for the assessment of atherosclerotic changes, MDA and protein carbonyls. Group II animals developed atherosclerosis (45%±11% of the intimal surface of aorta was covered with atherosclerotic plaques), which was associated with an increase in the serum TC, TG, LDL-C, HDL-C, TC/HDL-C, MDA and protein carbonyls. In group III, TQ decreased serum TC, LDL-C, MDA and protein carbonyls by 26%, 29%, 85% and 62%, respectively, and aortic MDA by 73%, which was associated with a 40% reduction of the development of aortic atherosclerosis. The higher dose of TQ in group IV had effects similar to the lower dose (group III), except that this dose further decreased serum TG. It is concluded that TQ attenuates hypercholesterolemic atherosclerosis and this effect is associated with a decrease in serum lipids and oxidative stress.

Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models

We have shown that thymoquinone (TQ) is a potent antitumor agent in human colorectal cancer cells. In this study, we evaluated TQ's therapeutic potential in two different mice colon cancer models [1,2-dimethyl hydrazine (DMH) and xenografts]. We also examined TQ effects on the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor invasion in vitro. Mice were treated with saline, TQ, DMH, or combinations once per week for 30 weeks and the multiplicity, size and distribution of aberrant crypt foci (ACF) and tumors were determined at weeks 10, 20 and 30. TQ injected intraperitoneally (i.p.) significantly reduced the numbers and sizes of ACF at week 10; ACF numbers were reduced by 86%. Tumor multiplicity was reduced at week 20 from 17.8 in the DMH group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long-term; tumors did not re-grow even when TQ injection was discontinued for 10 weeks. In a xenograft model of HCT116 colon cancer cells, TQ significantly (P < 0.05) delayed the growth of the tumor cells. Using a matrigel artificial basement membrane invasion assay, we demonstrated that sub-cyto-toxic doses of TQ (40μM) decreased C26 cell invasion by 50% and suppressed growth in three-dimensional spheroids. Apoptotic signs seen morphologically were increased significantly in TQ-treated spheroids. TUNEL staining of xenografts and immunostaining for caspase 3 cleavage in DMH tumors confirmed increased apoptosis in mouse tumors in response to TQ. These data should encourage further in vivo testing and support the potential use of TQ as a therapeutic agent in human colorectal cancer.

Beneficial Interaction of Thymoquinone and Sodium Valproate in Experimental Models of Epilepsy: Reduction in Hepatotoxicity of Valproate

The antiepileptic potential of thymoquinone (TQ) was studied in mice by using pentylenetetrazole (PTZ) and maximal electroshock seizure (MES)-induced convulsion models. In this investigation, the combined treatment of TQ and sodium valproate (SVP) was also studied. The aim of this part was to minimize SVPinduced hepatotoxic implications, by reducing its antiepileptic dose. TQ in both PTZ- and MES- models increased SVP potency. The experiments dealing with the effects of TQ on the ED50 of SVP revealed that TQ reduced the ED50 of SVP in both the models. However, this potentiation of SVP antiepileptic response was relatively more significant in PTZ model at both 50 and 100 mg/kg doses of TQ. Although very well tolerated and effective, SVP is well known for its hapatotoxic implications. In the experiment dealing with subacute treatment, SVP (1300-1500mg/kg/day in drinking water) for 21 days, produced hepatotoxicity in mice, characterized by elevated serum ALT and AST, reduced levels of non protein sulfhydryls and increase in lipid peroxidation in hepatic cells. Hepato-protection was successfully achieved when TQ (5-5.5mg/kg/day) was combined with SVP in drinking water for the same duration. The protective activity of TQ was evident by averting any serum ALT rise seen in SVP treatment. Though there is no prevention to the rise in lipid peroxidation in hepatic tissue but the same time a significant recovery in glutathione was evident by TQ joint treatment. However, a combination of TQ and low dose of SVP may be useful strategy to minimize adverse reactions of SVP. From the results of the present study that disclosed a protective role of TQ against SVP toxic damage to the liver, it could be mentioned safely that TQ behaves as an antioxidant and protected liver against its harmful effects. It also protected against the liver enzyme induction seen in the case of SVP alone.

Gastroprotective activity of Nigella sativa oil and its constituent, thymoquinone, against gastric mucosal injury induced by ischaemia/reperfusion in rats

Ischaemia/reperfusion (I/R) induced gastric lesion, is known to be linked with free radical (FR) formation. Therefore, this model was used to assess the antioxidant effects of Nigella sativa oil (N.O) and thymoquinone (TQ) on gastric mucosal redox state and gastric lesions, 1 and 24 h after reperfusion. Male Wistar rats were subjected to I/R and were injected with either N.O (2.5 and 5 ml/kg, p.o) or TQ (5, 20, 50 and 100 mg/kg, p.o). The results showed that I/R elevated the levels of lipid peroxide (LPX) and lactate dehydrogenase (LDH), while decreased those of reduced glutathione (GSH) and superoxide dismutase (SOD). These biochemical changes were accompanied by an increase in the formation of gastric lesions, which was reduced by either treatment. N.O tended to normalize the level of LDH, GSH and SOD. However, its effect to restore LPX was only seen 24 h after reperfusion. Moreover, the aforementioned parameters were nearly reinstated by TQ. On the other hand, high doses of TQ (50 and 100 mg/kg) severely reduced the GSH content, 1 h after reperfusion. These results indicate that both N.O and TQ possess gastroprotective effect against gastric lesions which may be related to the conservation of the gastric mucosal redox state.

Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action.

The present investigation focused, firstly, on the effects of oral administration of thymoquinone (TQ) on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase activity in hepatic, cardiac and kidney tissues of normal mice. Superoxide dismutase (SOD; E.C:1.15.1.1), catalase (CAT; E.C:1.11.1.6), glutathione peroxidase (GSH-Px; E.C:1.11.1.9), glutathione-S-transferase (GST; E.C:2.5.1.18), and DT-diaphorase (E.C:1.6.99.2) enzyme activities in each tissue type were determined. Treatment of mice with the different doses of TQ (25, 50 and 100 mg kg(-1) day(-1) orally) for 5 successive days, produced significant reductions in hepatic SOD, CAT and GSH-Px activities. In addition cardiac SOD activity was markedly inhibited with the higher doses of TQ, (namely 50 and 100 mg kg(-1)). Moreover, TQ (100 mg kg(-1)) significantly reduced hepatic and cardiac lipid peroxidation as compared with the respective control group. Conversely, TQ (50,100 mg kg(-1)) and TQ (100 mg kg(-1)) enhanced cardiac and renal DT-diaphorase activity respectively. However, the selected doses of TQ neither produced any change in GST activity nor influenced reduced glutathione content in all tissues studied. TQ was tested, secondly, as a substrate for hepatic, cardiac and renal DT-diaphorase of normal mice in the presence of NADPH. Kinetic parameters for the reduction of TQ to dihydrothymoquinone (DHTQ) indicated that DT-diaphorase of different tissues can efficiently reduce TQ to DHTQ. K(m) and V(max) values revealed that hepatic DT-diaphorase exhibited the higher values, while the lower values were associated with renal DT-diaphorase. TQ and DHTQ were tested, thirdly, as specific scavengers for superoxide anion (generated biochemically) or as general scavengers for free radicals (generated photochemically). The results revealed that TQ and DHTQ acted not only as superoxide anion scavengers but also as general free radical scavengers. The IC(50) for TQ and DHTQ in biochemical and photochemical assays were in the nanomolar and micromolar range respectively. Our data may explain at least partly the reported beneficial in vivo protective effects of TQ through the combined antioxidant properties of TQ and its metabolite DHTQ.