Unveiling the therapeutic potential of thymol from Nigella sativa L. seed: selective anticancer action against human breast cancer cells (MCF-7) through down-regulation of Cyclin D1 and proliferative cell nuclear antigen (PCNA) expressions.

BACKGROUND: Breast adenocarcinoma cells (MCF-7) are characterized by the overexpression of apoptotic marker genes and proliferative cell nuclear antigen (PCNA), which promote cancer cell proliferation. Thymol, derived from Nigella sativa (NS), has been investigated for its potential anti-proliferative and anticancer properties, especially its ability to suppress Cyclin D1 and PCNA expression, which are crucial in the proliferation of cancer cells. METHODS: The cytotoxicity of thymol on MCF-7 cells was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release methods. Thymol was tested at increasing concentrations (0-1000 µM) to evaluate its impact on MCF-7 cell growth. Additionally, Cyclin D1 and PCNA gene expression in thymol-treated and vehicle control groups of MCF-7 were quantified using real-time Polymerase Chain Reaction (RT-qPCR). Protein-ligand interactions were also investigated using the CB-Dock2 server. RESULTS: Thymol significantly inhibited MCF-7 cell growth, with a 50% inhibition observed at 200 µM. The gene expression of Cyclin D1 and PCNA was down-regulated in the thymol-treated group relative to the vehicle control. The experimental results were verified through protein-ligand interaction investigations. CONCLUSIONS: Thymol, extracted from NS, demonstrated specific cytotoxic effects on MCF-7 cells by suppressing the expression of Cyclin D1 and PCNA, suggesting its potential as an effective drug for MCF-7. However, additional in vivo research is required to ascertain its efficacy and safety in medical applications.

The effect of Nigella sativa (black seed) on biomarkers of inflammation and oxidative stress: an updated systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: This study was conducted to assess the effect of Nigella sativa (N. sativa) supplementation on inflammatory and oxidative markers among the adult population. METHODS: We carried out a comprehensive, systematic search of Scopus, Embase, Cochrane Library, Web of Science, PubMed, and Google Scholar till December 2022. A random-effects model was used to estimate the overall effect size. RESULTS: In total, twenty trials consisting of 1086 participants were included in the meta-analysis. Findings from 20 RCTs included in the meta-analysis suggest that N. sativa supplementation could significantly reduce serum C-reactive protein (CRP) (SMD = - 2.28; 95% CI - 3.20, - 1.37, p < 0.001), tumour necrosis factor α (TNFα) (SMD = - 1.21; 95% CI - 2.15, - 0.26; p = 0.013), and malondialdehyde (MDA) (SMD = - 2.15; 95% CI - 3.37, - 0.93, p < 0.001) levels, and significantly improves total antioxidant capacity (TAC) (SMD = 2.28; 95% CI 1.29, 3.27, p < 0.001), glutathione peroxidase (GPx) (SMD = 1.23, 95% CI 0.25, 2.22; p = 0.014) and superoxide dismutase (SOD) (SMD = 2.05; 95% CI 1.22, 2.88, p < 0.001) levels. However, no significant reduction was found in interleukin 6 (IL-6) levels (SMD = - 1.13; 95% CI - 2.72, 0.46, p = 0.162). CONCLUSION: N. sativa supplementation had beneficial effects on CRP, TNF-α, MDA, SOD, GPx, and TAC. Thus, Nigella sativa can be recommended as an adjuvant anti-oxidant agent and anti-inflammatory.

In Vitro Assessment of Anti-Adipogenic and Anti-Inflammatory Properties of Black Cumin (Nigella sativa L.) Seeds Extract on 3T3-L1 Adipocytes and Raw264.7 Macrophages.

Background and Objectives: This study evaluated the in vitro anti-adipogenic and anti-inflammatory properties of black cumin (Nigella sativa L.) seed extract (BCS extract) as a potential candidate for developing herbal formulations targeting metabolic disorders. Materials and Methods: We evaluated the BCS extract by assessing its 2,2-diphenyl-1-picrohydrazyl (DPPH) radical scavenging activity, levels of prostaglandin E2 (PGE2) and nitric oxide (NO), and mRNA expression levels of key pro-inflammatory mediators. We also quantified the phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) signaling molecules. To assess anti-adipogenic effects, we used differentiated 3T3-L1 cells and BCS extract in doses from 10 to 100 µg/mL. We also determined mRNA levels of key adipogenic genes, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/BEPα), adipocyte protein 2 (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), and sterol-regulated element-binding protein 1c (SREBP-1c) using real-time quantitative polymerase chain reaction (qPCR). Results: This study showed a concentration-dependent DPPH radical scavenging activity and no toxicity at concentrations up to 30 µg/mL in Raw264.7 cells. BCS extract showed an IC50 of 328.77 ± 20.52 µg/mL. Notably, pre-treatment with BCS extract (30 µg/mL) significantly enhanced cell viability in lipopolysaccharide (LPS)-treated Raw264.7 cells. BCS extract treatment effectively inhibited LPS-induced production of PGE2 and NO, as well as the expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), interleukin (IL)-1ß and IL-6, possibly by limiting the phosphorylation of p38, p65, inhibitory κBα (I-κBα), and c-Jun N-terminal kinase (JNK). It also significantly attenuated lipid accumulation and key adipogenic genes in 3T3-L1 cells. Conclusions: This study highlights the in vitro anti-adipogenic and anti-inflammatory potential of BCS extract, underscoring its potential as a promising candidate for managing metabolic disorders.

PI3K-AKT Pathway Modulation by Thymoquinone Limits Tumor Growth and Glycolytic Metabolism in Colorectal Cancer.

Colorectal cancer (CRC) is the third leading cause of death in men and the fourth in women worldwide and is characterized by deranged cellular energetics. Thymoquinone, an active component from Nigella sativa, has been extensively studied against cancer, however, its role in affecting deregulated cancer metabolism is largely unknown. Further, the phosphoinositide 3-kinase (PI3K) pathway is one of the most activated pathways in cancer and its activation is central to most deregulated metabolic pathways for supporting the anabolic needs of growing cancer cells. Herein, we provide evidence that thymoquinone inhibits glycolytic metabolism (Warburg effect) in colorectal cancer cell lines. Further, we show that such an abrogation of deranged cell metabolism was due, at least in part, to the inhibition of the rate-limiting glycolytic enzyme, Hexokinase 2 (HK2), via modulating the PI3/AKT axis. While overexpression of HK2 showed that it is essential for fueling glycolytic metabolism as well as sustaining tumorigenicity, its pharmacologic and/or genetic inhibition led to a reduction in the observed effects. The results decipher HK2 mediated inhibitory effects of thymoquinone in modulating its glycolytic metabolism and antitumor effects. In conclusion, we provide evidence of metabolic perturbation by thymoquinone in CRC cells, highlighting its potential to be used/repurposed as an antimetabolite drug, though the latter needs further validation utilizing other suitable cell and/or preclinical animal models.

Effect of Black Cumin Cake Addition on the Chemical Composition, Glycemic Index, Antioxidant Activity, and Cooking Quality of Durum Wheat Pasta.

Pasta is a good carrier for plant enrichment substances due to its popularity among consumers. The purpose of the study was to investigate the functional potential and optimize the recipe of pasta made from durum semolina with the addition of black cumin cake at the level of 5, 10, 15, 20, and 25%. The use of black cumin cake resulted in a statistically significant (p ≤ 0.05) increase in the content of protein, fat, ash, and fiber, including both the insoluble and soluble fractions. A reduction in the digestible carbohydrate content, in vitro starch hydrolysis index (HI), was observed. Pasta with a reduced glycemic index (GI) compared to the semolina control was obtained. The content of polyphenols, including flavonoids, in the cake-enriched pasta increased significantly (p ≤ 0.05), which resulted in higher antioxidant activity against DPPH. The increase in the iron content was over 2.5 times higher in the sample with the 25% addition of black cumin cake than in the control sample. The functional addition significantly (p ≤ 0.05) increased the loss of dry matter and influenced the cooking time of pasta.

The Potential Role of Nigella sativa Seed Oil as Epigenetic Therapy of Cancer.

Nigella sativa oil, commonly known as black seed oil (BSO), is a well-known Mediterranean food, and its consumption is associated with beneficial effects on human health. A large number of BSO's therapeutic properties is attributed to its pharmacologically active compound, thymoquinone (TQ), which inhibits cell proliferation and induces apoptosis by targeting several epigenetic players, including the ubiquitin-like, containing plant homeodomain (PHD) and an interesting new gene, RING finger domains 1 (UHRF1), and its partners, DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1). This study was designed to compare the effects of locally sourced BSO with those of pure TQ on the expression of the epigenetic complex UHRF1/DNMT1/HDAC1 and the related events in several cancer cells. The gas chromatographs obtained from GC-MS analyses of extracted BSO showed that TQ was the major volatile compound. BSO significantly inhibited the proliferation of MCF-7, HeLa and Jurkat cells in a dose-dependent manner, and it induced apoptosis in these cell lines. BSO-induced inhibitory effects were associated with a significant decrease in mRNA expression of UHRF1, DNMT1 and HDAC1. Molecular docking and MD simulation showed that TQ had good binding affinity to UHRF1 and HDAC1. Of note, TQ formed a stable metal coordinate bond with zinc tom, found in the active site of the HDAC1 protein. These findings suggest that the use of TQ-rich BSO represents a promising strategy for epigenetic therapy for both solid and blood tumors through direct targeting of the trimeric epigenetic complex UHRF1/DNMT1/ HDAC1.

GSH Protects the Escherichia coli Cells from High Concentrations of Thymoquinone.

The aim of the present study was to evaluate the potential protective effect of glutathione (GSH) on Escherichia coli cells grown in a high concentration of thymoquinone (TQ). This quinone, as the main active compound of Nigella sativa seed oil, exhibits a wide range of biological activities. At low concentrations, it acts as an antioxidant, and at high concentrations, an antimicrobial agent. Therefore, any interactions between thymoquinone and glutathione are crucial for cellular defense against oxidative stress. In this study, we found that GSH can conjugate with thymoquinone and its derivatives in vitro, and only fivefold excess of GSH was sufficient to completely deplete TQ and its derivatives. We also carried out studies on cultures of GSH-deficient Escherichia coli strains grown on a minimal medium in the presence of different concentrations of TQ. The strains harboring mutations in gene ΔgshA and ΔgshB were about two- and fourfold more sensitive (256 and 128 µg/mL, respectively) than the wild type. It was also revealed that TQ concentration has an influence on reactive oxygen species (ROS) production in E. coli strains-at the same thymoquinone concentration, the level of ROS was higher in GSH-deficient E. coli strains than in wild type.

In Silico Studies on Zinc Oxide Based Nanostructured Oil Carriers with Seed Extracts of Nigella sativa and Pimpinella anisum as Potential Inhibitors of 3CL Protease of SARS-CoV-2.

Coming into the second year of the pandemic, the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants continue to be a serious health hazard globally. A surge in the omicron wave, despite the discovery of the vaccines, has shifted the attention of research towards the discovery and use of bioactive compounds, being potential inhibitors of the viral structural proteins. The present study aimed at the green synthesis of zinc oxide (ZnO) nanoparticles with seed extracts of Nigella sativa and Pimpinella anisum-loaded nanostructured oil carriers (NLC)-using a mixture of olive and black seed essential oils. The synthesized ZnO NLC were extensively characterized. In addition, the constituent compounds in ZnO NLC were investigated as a potential inhibitor for the SARS-CoV-2 main protease (3CLpro or Mpro) where 27 bioactive constituents, along with ZnO in the nanostructure, were subjected to molecular docking studies. The resultant high-score compounds were further validated by molecular dynamics simulation. The study optimized the compounds dithymoquinone, δ-hederin, oleuropein, and zinc oxide with high docking energy scores (ranging from -7.9 to -9.9 kcal/mol). The RMSD and RMSF data that ensued also mirrored these results for the stability of proteins and ligands. RMSD and RMSF data showed no conformational change in the protein during the MD simulation. Histograms of every simulation trajectory explained the ligand properties and ligand-protein contacts. Nevertheless, further experimental investigations and validation of the selected candidates are imperative to take forward the applicability of the nanostructure as a potent inhibitor of COVID-19 (Coronavirus Disease 2019) for clinical trials.

The ameliorative effects of Nigella sativa, thymoquinone, and bentonite against aflatoxicosis in broilers via AFAR and Nrf2 signalling pathways, and down-regulation of caspase-3.

1. Aflatoxins (AFs) are metabolites which especially have toxic effects on proteins, and are detoxified by the aflatoxin-B1 aldehyde reductase (AFAR) pathway. In this pathway, the aldo-keto reductase family 7, member A2 (AKR7A2) enzyme, which is controlled by nucleic-related erythroid factor 2 (Nrf2), plays an active role. However, data on the efficacy of this critical pathway in broilers is limited.2. The aim of the following study was to investigate the changes in the expression levels of AKR7A2, Nrf2, and caspase-3, and the effects of Nigella sativa seeds (NS), thymoquinone (TMQ), and bentonite (BNT) in broilers exposed to AFs.3. One-hundred broilers were divided into ten groups (control (CNT); AF; NS; TMQ; BNT; AF+TMQ; AF+NS; AF+BNT; AF+BNT+NS; AF+BNT+TMQ) and fed for 28 d. AF, TMQ, NS and BNT were added to diets at levels of 2 mg/kg, 300 mg/kg, 50 g/kg and 10 g/kg respectively.4. The addition of AF to the diet decreased AKR7A2 and Nrf2 levels dramatically, but increased caspase-3 (P < 0.01). TMQ, NS and BNT additions to the diet eliminated all negative effects caused by AF (P < 0.01); and AKR7A2 and Nrf2 were further raised in TMQ and NS groups when compared to the control group. TMQ and NS showed a positive effect on detoxification parameters when given together with BNT.5. Supplementation with NS and TMQ enhanced AF detoxification via the AFAR pathway, by increasing AKR7A2 and Nrf2 levels, in addition to reducing hepatocyte apoptosis.

Protective Effects of Thymoquinone, an Active Compound of Nigella sativa, on Rats with Benzo(a)pyrene-Induced Lung Injury through Regulation of Oxidative Stress and Inflammation.

Benzopyrene [B(a)P] is a well-recognized environmental carcinogen, which promotes oxidative stress, inflammation, and other metabolic complications. In the current study, the therapeutic effects of thymoquinone (TQ) against B(a)P-induced lung injury in experimental rats were examined. B(a)P used at 50 mg/kg b.w. induced lung injury that was investigated via the evaluation of lipid profile, inflammatory markers, nitric oxide (NO), and malondialdehyde (MDA) levels. B(a)P also led to a decrease in superoxide dismutase (SOD) (34.3 vs. 58.5 U/mg protein), glutathione peroxidase (GPx) (42.4 vs. 72.8 U/mg protein), catalase (CAT) (21.2 vs. 30.5 U/mg protein), and total antioxidant capacity compared to normal animals. Treatment with TQ, used at 50 mg/kg b.w., led to a significant reduction in triglycerides (TG) (196.2 vs. 233.7 mg/dL), total cholesterol (TC) (107.2 vs. 129.3 mg/dL), and inflammatory markers and increased the antioxidant enzyme level in comparison with the group that was administered B(a)P only (p < 0.05). B(a)P administration led to the thickening of lung epithelium, increased inflammatory cell infiltration, damaged lung tissue architecture, and led to accumulation of collagen fibres as studied through haematoxylin and eosin (H&E), Sirius red, and Masson's trichrome staining. Moreover, the recognition of apoptotic nuclei and expression pattern of NF-κB were evaluated through the TUNEL assay and immunohistochemistry, respectively. The histopathological changes were found to be considerably low in the TQ-treated animal group. The TUNEL-positive cells increased significantly in the B(a)P-induced group, whereas the TQ-treated group showed a decreased apoptosis rate. Significantly high cytoplasmic expression of NF-κB in the B(a)P-induced group was seen, and this expression was prominently reduced in the TQ-treated group. Our results suggest that TQ can be used in the protection against benzopyrene-caused lung injury.

Comparative Protective Effect of Nigella sativa Oil and Vitis vinifera Seed Oil in an Experimental Model of Isoproterenol-Induced Acute Myocardial Ischemia in Rats.

The study's aim was to characterize the composition of Nigella sativa seed (NSO) and grape seed (GSO) oils, and to evaluate their cardioprotective and anti-inflammatory effect on isoproterenol (ISO)-induced ischemia in rats. Materials and Methods: NSO and GSO supplements were physicochemically characterized. Liquid chromatography-mass spectrometry (HPLC-MS), Fourier-transform infrared spectroscopy (FTIR), and gas chromatography-mass spectrometry (GC-MS) analyses were used to determine the phytochemical composition in the oils. Total polyphenol content (TPC) and in vitro antioxidant activity were also determined. Pretreatment with 4 mL/kg/day NSO or GSO was administered to rats for 14 days. The experimental ischemia was induced by a single administration of ISO 45 mg/kg after 14 days. An electrocardiogram (ECG) was performed initially and 24 h after ISO. Biological evaluation was done at the end of experiment. Results: The HPLC-MS, GC-MS, and FTIR analyses showed that both NSO and GSO are important sources of bioactive compounds, especially catechin and phenolic acids in GSO, while NSO was enriched in flavonoids and thymol derivatives. Pretreatment with GSO and NSO significantly reduced ventricular conduction, prevented the cardiotoxic effect of ISO in ventricular myocardium, and reduced the level of proinflammatory cytokines and CK-Mb. Conclusion: Both NSO and GSO were shown to have an anti-inflammatory and cardioprotective effect in ISO-induced ischemia.

Black Edible Films from Protein-Containing Defatted Cake of Nigella sativa Seeds.

Black biodegradable/edible protein-based films were prepared from defatted cake waste obtained from Nigella sativa (black cumin) seeds as by-product of oil extraction process. The effects of pH, glycerol concentrations, and transglutaminase-catalyzed protein cross-linking activity on the stability of film-forming solutions were studied to determine the best experimental conditions to produce handleable films. Proteins contained in the analyzed defatted cake were shown to be able to act as transglutaminase acyl donor and acceptor substrates being polymerized when incubated in vitro in the presence of the enzyme. Film-forming solutions containing 20% glycerol and casted at pH 8.0 after treatment with the enzyme gave rise to morphologically more homogeneous films possessing mechanical and barrier properties, as well as antimicrobial activity, compatible with their possible applications as food packaging materials and mulching sheets. These findings confirm the validity of the strategy to consider the seed oil processed cakes as protein-based renewable sources to produce not only fertilizers, animal feed, or culinary food but also further valuable products such as bioplastics.

Identification of Novel Proteins from Black Cumin Seed Meals Based on 2D Gel Electrophoresis and MALDI-TOF/TOF-MS Analysis.

The amount of cold press oil manufacture is globally rising, which in turn leads to the accumulation of deoiled plant seeds at significant quantities and consequent manufacture of plant protein products. In this study, we made an attempt to analyze the protein profile of black cumin seed protein concentrates prepared by the alkali extraction-acid precipitation technique (AE-IP). The analytical strategy relied on gel-based proteome mapping which included two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). 14 different protein bands were identified, and in gel-trypsinolysis was carried out for the corresponding gel spots. Using the MASCOT database, current findings on 10 proteins were compared with the existing data. The highest similarity was 46 which was obtained between the highest pI black cumin protein observed here and the cyclin dependent kinase inhibitor of Arabidopsis thaliana. The molecular mass of the intact protein was determined by linear MALDI-TOF/TOF-MS as 23,711.2186 Da. The peptide constructs of this protein have been further studied in order to identify potential biological activity. Matching sequences generated bioactive peptides in silico such as IR, AL, and SL dipeptides during sequential enzymatic digestion with pepsin and trypsin. Since the majority of bioactivity investigations on black cumin seeds have been related to black cumin oil and its oil soluble components, the structure and bioactivities of black cumin proteins deserve further research.

Nigella Sativa Oil Protects the Rat Ovary from Oxidative Injury Due to Ischemia-Reperfusion.

BACKGROUND The aim of this study was to evaluate the effects of Nigella sativa (N. sativa) oil (NSO) on ovarian oxidative damage following ischemia-reperfusion injury, using a rat model of ovarian torsion. MATERIAL AND METHODS Forty-eight female albino Wistar rats were divided into six groups: (Group 1) laparotomy only; (Group 2) intraperitoneal NSO (2 ml/kg), 1 hour following laparotomy; (Group 3) 3 hours of ovarian ischemia; (Group 4) 3 hours of ovarian ischemia followed by 3 hours of reperfusion; (Group 5) 3 hours of ovarian ischemia and 2 ml/kg of NSO 1 hour before laparotomy; (Group 6) 3 hours of reperfusion after 3 hours of ovarian ischemia and 2 ml/mg of NSO 1 hour before laparotomy. RESULTS The antioxidant status, ceruloplasmin level, native thiol, total thiol, and disulfide levels of the control group (Group 1) were significantly increased compared with the ovarian ischemia-reperfusion group treated with NSO (Group 6) (p=0.003, p=0.002, p=0.006, p=0.001 and p=0.003, respectively); these levels in the ovarian ischemia group (Group 3) and ischemia-reperfusion group (Group 4) were statistically similar to those of the ovarian ischemia + NSO group (Group 5) and ovarian ischemia-reperfusion + NSO group (Group 6). CONCLUSIONS In this preliminary rat study, administration of NSO shortly after the onset of ovarian ischemia-reperfusion injury, did not significantly reduce levels of markers of oxidative injury. Further studies are required to evaluate the ovarian changes at the tissue level, and to determine the optimum dose of NSO.

Effect of black cumin seeds on growth performance, nutrient utilization, immunity, gut health and nitrogen excretion in broiler chickens.

BACKGROUND: Use of antibiotic growth promoters (AB) as feed additives in broiler chickens poses risks due to cross-resistance amongst pathogens and residues in tissues. The aim of this study was to evaluate the effect of dietary supplementation of black cumin seeds (BCS) as a natural growth promoter in chickens on nutrient utilization, intestinal microbiota and morphology, immunity, antioxidant status, protein deposition in muscles and nitrogen excretion. RESULTS: Broiler chickens were fed BCS at 0, 5, 10 and 20 g kg-1 diet. Body weight gain tended to increase (P = 0.10) and daily feed intake increased quadratically with increasing concentrations of BCS in the diets. Supplementation of BCS resulted in a tendency to decrease feed conversion efficiency on days 28-42. Metabolizability of nutrients increased linearly with increasing doses of BCS. Protein deposition in thigh and breast muscles was increased and nitrogen excretion was reduced by BCS and AB compared with the control (CON). Intestinal morphology in the duodenum, jejunum and ileum was not generally affected by BCS. Counts of total bacteria, Escherichia coli, Lactobacillus spp. and Clostridium spp. were not affected by BCS and AB compared with CON, but Salmonella spp. decreased linearly (P = 0.05) with increasing doses of BCS. Antibody titers against Newcastle disease virus on day 35 increased quadratically (P < 0.001) with increasing doses of BCS. Concentrations of glucose and triglyceride in blood were not affected by BCS. Concentrations of cholesterol decreased linearly while the concentration of total protein increased linearly with increasing doses of BCS. CONCLUSION: The use of dietary BCS may improve growth performance, immunity and nutrient utilization in broiler chickens. © 2017 Society of Chemical Industry.

Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate.

The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients' sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4(+) and cytotoxic CD8(+)- T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV infection. Also, the immunostimulatory effects induced by the two plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV infection. The neutralizing capacity of the induced antibodies is a subject of future investigations. Furthermore, the predicted B- and T-cell epitopes may be useful for tailoring future diagnostics and candidate vaccines against various HCV genotypes.

Effect of different levels of black cumin (Nigella sativa L.) on performance, intestinal Escherichia coli colonization and jejunal morphology in laying hens.

This study was conducted to investigate the effects of different levels of black cumin seeds (Nigella sativa L.) on performance, intestinal Escherichia coli count and morphology of jejunal epithelial cells in laying hens. A total of 100 Leghorn laying hens (Hy-Line W-36) of 49 weeks old were randomly distributed among five cage replicates of five birds each. Experimental diets consisted of different levels (0%, 1%, 2% and 3% of diet) of dietary black cumin inclusion. The experimental period lasted for a total of 10 weeks, and egg quality indexes and laying hens' performance were measured as two 35-day trial periods. At the final day, two hens per replicate were slaughtered to investigate the influence of dietary treatments on intestinal E. coli colonization and morphology of jejunal cells. Although dietary black cumin in all supplementation levels decreased (p < 0.05) the enumeration of ileal E. coli, the morphological and histological alterations in small intestine such as enhancement of villus height to crypt depth ratio, increased goblet cell numbers and proliferation of lamina propria lymphatic follicles were observed after dietary supplementation with at least 2% black cumin. Dietary treatments decreased (p < 0.05) the concentration of serum cholesterol and triglycerides and increased (p < 0.05) serum HDL concentration and relative weight of pancreas; however, the egg yolk cholesterol was not influenced by dietary treatments. In addition, dietary supplementation with black cumin improved (p < 0.05) eggshell quality and Haugh unit. The best feed conversion ratio was obtained when diets were supplemented with 2% black cumin. This improvement was due to the increase (p < 0.05) in egg mass and contemporaneous decrease (p < 0.01) in feed consumption. The present results indicated that regardless of supplementation level, dietary inclusion of black cumin decreased E. coli enumeration in ileal digesta and improved serum lipid profile and eggshell quality, whereas the best intestinal health indices and haying hens' performance were obtained by at least 2% black cumin seeds.

Hepatoprotective impact of Nigella sativa silver nanocomposite against genotoxicity, oxidative stress, and inflammation induced by thioacetamide.

Protection from liver damage and the repercussion of that harm is thought to be crucial for reducing the number of deaths each year. This work was developed to evaluate the possible role of silver nanocomposite prepared using Nigella sativa (N. sativa) aqueous extract against the hepatic damage brought on by thioacetamide (TAA), with particular attention to how they affect the NF-κß, TNF-α, IL-1ß, and COX-2 signaling pathways. There were seven groups of male Wistar rats used as follows: control, saline, N. sativa aqueous extract (NSAE; 200 mg/kg/d), N. sativa silver nanocomposite (NS-AgNC; 0.25 mg/kg/d), TAA (100 mg/kg; thrice weekly), NSAE + TTA, and NS-AgNC + TAA, respectively. The experiment continued for six weeks. The results showed that NS-AgNPs significantly enhanced liver functions (p<0.05) (albumin, ALP, LDH, AST, total protein, ALT, and globulin) and oxidant/antioxidant biomarkers (p<0.05) (H2O2, MDA, PCC, NO, SOD, CAT, GPx, GR, GST and, GSH), contrasted with TAA group. Moreover, a significant (p<0.05) downregulation of the gene expressions (COX-2, TNF-α, IL-1ß, and NF-κß) was also achieved by using silver nanocomposite therapy. These findings have been supported by histological analysis. Collectively, NS-AgNC exhibits more prominent and well-recognized protective impacts than NSAE in modulating the anti-inflammatory, genotoxicity and oxidative stress effects against TAA-induced liver injuries.

Study on the potential toxicity of a thymoquinone-rich fraction nanoemulsion in Sprague Dawley rats.

Toxicological studies constitute an essential part of the effort in developing an herbal medicine into a drug product. A newly developed thymoquinone-rich fraction nanoemulsion (TQRFNE) has been prepared using a high pressure homogenizer. The purpose of this study was to investigate the potential acute toxicity of this nanoemulsion in Sprague Dawley rats. The acute toxicity studies were conducted as per the OECD guidelines 425, allowing for the use of test dose limit of 20 mL TQRFNE (containing 44.5 mg TQ)/kg. TQRFNE and distilled water (DW) as a control were administered orally to both sexes of rats on Day 0 and observed for 14 days. All the animals appeared normal, and healthy throughout the study. There was no observed mortality or any signs of toxicity during the experimental period. The effects of the TQRFNE and DW groups on general behavior, body weight, food and water consumption, relative organ weight, hematology, histopathology, and clinical biochemistry were measured. All the parameters measured were unaffected as compared to the control (DW) group. The administration of 20 mL TQRFNE /kg was not toxic after an acute exposure.

Effects of resistance training and nigella sativa on type 2 diabetes: implications for metabolic markers, low-grade inflammation and liver enzyme production.

CONTEXT: Proper nutrition and exercise are effective strategies to improve overall metabolic health in diabetic patients. OBJECTIVE: This study evaluated the effects of Nigella sativa (NS) supplementation during resistance training (RT) on some biochemical variables in type 2 diabetes patients. METHODS: Forty patients were assigned to groups: RT + NS (RN), NS, RT + placebo (RP), and control (CO). RT was performed and NS was consumed for 8 weeks. Blood samples were collected at rest immediately before and after the 8 week intervention. RESULTS: RT or NS by themselves reduced HOMA-IR, insulin, glucose, TG, TC, LDL, ESR, CRP, AST, ALT and ALP, and increased HDL and HOMA-S. The combination of RT and NS, rather than each intervention alone, had significant effects on reduction of HOMA-IR, insulin, ESR and CRP as well as increases in HDL, HOMA-ß/S. CONCLUSION: RT combined with NS is sometimes a better strategy compared to single interventions for improving diabetes related biomarkers in type 2 diabetic patients.