Antioxidant Rich Extracts of Terminalia ferdinandiana Inhibit the Growth of Foodborne Bacteria.

Terminalia ferdinandiana (Kakadu plum) is a native Australian plant containing phytochemicals with antioxidant capacity. In the search for alternatives to synthetic preservatives, antioxidants from plants and herbs are increasingly being investigated for the preservation of food. In this study, extracts were prepared from Terminalia ferdinandiana fruit, leaves, seedcoats, and bark using different solvents. Hydrolysable and condensed tannin contents in the extracts were determined, as well as antioxidant capacity, by measuring the total phenolic content (TPC) and free radical scavenging activity using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. Total phenolic content was higher in the fruits and barks with methanol extracts, containing the highest TPC, hydrolysable tannins, and DPPH-free radical scavenging capacity (12.2 ± 2.8 g/100 g dry weight (DW), 55 ± 2 mg/100 g DW, and 93% respectively). Saponins and condensed tannins were highest in bark extracts (7.0 ± 0.2 and 6.5 ± 0.7 g/100 g DW). The antimicrobial activity of extracts from fruit and leaves showed larger zones of inhibition, compared to seedcoats and barks, against the foodborne bacteria Listeria monocytogenes, Bacillus cereus, Methicillin resistant Staphylococcus aureus, and clinical isolates of Pseudomonas aeruginosa. The minimum inhibitory concentration and minimum bactericidal concentration in response to the different extracts ranged from 1.0 to 3.0 mg/mL. Scanning electron microscopy images of the treated bacteria showed morphological changes, leading to cell death. These results suggest that antioxidant rich extracts of Terminalia ferdinandiana fruits and leaves have potential applications as natural antimicrobials in food preservation.

Indospicine cytotoxicity and transport in human cell lines.

Indospicine, a non-proteinogenic analogue of arginine, occurs only in Indigofera plant species and accumulates in the tissues of animals grazing on Indigofera. Canine deaths have resulted from the consumption of indospicine-contaminated meat but only limited information is available regarding indospicine toxicity in humans. In this study three human cell lines, Caco-2 (colorectal adenocarcinoma), HT29-MTX-E12 (colorectal adenocarcinoma) and HepG2 (hepatocellular carcinoma), were used to investigate the cytotoxicity of indospicine and its metabolite 2-aminopimelic acid in comparison to arginine. Indospicine and 2-aminopimelic acid were more cytotoxic than arginine, displaying the highest toxicity in HepG2 liver cells. Intestinal transport in vitro also revealed a 2-fold higher transport rate of indospicine compared to arginine. The sensitivity of HepG2 cells to indospicine is consistent with observed canine hepatotoxicity, and considering the higher in vitro transport of indospicine across an intestinal barrier, it is possible that similar ill effects could be seen in humans consuming contaminated meat.

The tert-butylhydroquinone-mediated activation of the human thioredoxin gene reveals a novel promoter structure.

Thioredoxin is a redox-active protein that plays multiple roles in regulating cell growth, cell signalling and apoptosis. Here, we have demonstrated that a complex mechanism involving multiple regulatory elements is involved in the tBHQ [tert-butylhydroquinone or 2,5-di-(t-butyl)-1,4-hydroquinone]-mediated activation of the thioredoxin gene. Luciferase assays, utilizing various wild-type and mutated thioredoxin promoter fragments, revealed roles for the ORE (oxidative stress responsive element), ARE (antioxidant responsive element), three Sp1 (specificity protein 1)-binding sites and the TATA box in the activation of the thioredoxin gene by tBHQ. The ORE required the presence of the ARE to elicit its response, whereas the independent removal of three Sp1-binding sites and the TATA box also decreased activation of the thioredoxin gene, with mutation of the TATA box having the greatest effect. Real-time RT (reverse transcriptase)-PCR analysis also revealed varying roles for two TSSs (transcription start sites) in the activation of the thioredoxin gene by tBHQ. Transcription was initiated from both TSSs; however, different response rates and fold inductions were observed. Together, these results suggest that the thioredoxin gene is controlled by a novel arrangement of two overlapping core promoter regions, one containing a TATA box and the other TATA-less. Altering the intracellular levels of thioredoxin in a breast cancer cell line also influenced the induction of thioredoxin transcription in response to tBHQ. Stable transfections with a redox-inactive thioredoxin mutant produced 3.6 times higher induction levels of thioredoxin transcription compared with control cells, indicating an intrinsic form of control of promoter activity by the thioredoxin system itself.

Thioredoxin-mediated redox control of the transcription factor Sp1 and regulation of the thioredoxin gene promoter.

In recent years, redox control has emerged as a fundamental mechanism of gene regulation through transcriptional control. Thioredoxin (Trx) is a dithiol-reducing enzyme known to be involved in the redox regulation of a number of transcription factors, and in this study, we have investigated the redox-dependent regulation of the DNA binding activity of Sp1 by thioredoxin. Electrophoretic mobility shift assays were used to show that both recombinant Sp1 produced in Escherichia coli and endogenous Sp1 expressed by MDA-MB-231 breast cancer cells is subject to redox regulation. We found that thioredoxin alone or in conjunction with the full thioredoxin system (comprising thioredoxin, thioredoxin reductase [TR], and alpha-nicotinamide adenine dinucleotide phosphate [NADPH]) can increase Sp1 DNA binding activity in vitro to an oligonucleotide containing the Sp1 consensus sequence. Furthermore, we have provided evidence that recombinant Sp1 can bind to Sp1 consensus sequences within a 330-base pair (bp) thioredoxin promoter fragment and that this interaction can also be enhanced by the presence of thioredoxin. Luciferase reporter assays using this same minimal thioredoxin promoter region demonstrated that both Sp1 and Sp3 can bind to the promoter and act to enhance transcription. When the three identified Sp1 consensus sequences within the reporter construct were deleted, there was a loss of basal promoter activity, showing that these closely positioned sites are important for regulation of thioredoxin gene expression.