Phytochemical and Safety Evaluations of Finger Lime, Mountain Pepper, and Tamarind in Zebrafish Embryos.
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SUBMITTER: Caceres-Velez PR
PROVIDER: S-EPMC9311898 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Australian finger lime (Citrus australasica L.) has become increasingly popular due to its potent antioxidant capacity and health-promoting benefits. This study aimed to determine the chemical composition, antibacterial characteristics, and mechanism of finger lime extract. The finger lime extracts were obtained from the fruit of the Australian finger lime by the ethanol extraction method. The antibacterial activity of the extract was examined by detecting the minimum inhibitory concentration (MIC) for two Gram-positive and four Gram-negative bacterial strains in vitro, as well as by assessing variations in the number of bacteria for Candidatus Liberibacter asiaticus (CLas) in vivo. GC-MS analysis was used to identify the antibacterial compounds of the extract. The antibacterial mechanisms were investigated by assessing cell permeability and membrane integrity, and the bacterial morphology was examined using scanning electron microscopy. The extract demonstrated significant antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and Gram-negative bacterial species, such as Escherichia coli, Agrobacterium tumefaciens, Xanthomonas campestris, Xanthomonas citri, and CLas. Among the six strains evaluated in vitro, B. subtilis showed the highest susceptibility to the antimicrobial effects of finger lime extract. The minimum inhibitory concentration (MIC) of the extract against the tested microorganisms varied between 500 and 1000 μg/mL. In addition, the extract was proven effective in suppressing CLas in vivo, as indicated by the lower CLas titers in the treated leaves compared to the control. A total of 360 compounds, including carbohydrates (31.159%), organic acid (30.909%), alcohols (13.380%), polyphenols (5.660%), esters (3.796%), and alkaloids (0.612%), were identified in the extract. We predicted that the primary bioactive compounds responsible for the antibacterial effects of the extract were quinic acid and other polyphenols, as well as alkaloids. The morphology of the tested microbes was altered and damaged, leading to lysis of the cell wall, cell content leakage, and cell death. Based on the results, ethanol extracts from finger lime may be a fitting substitute for synthetic bactericides in food and plant protection.
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Project description:Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a comprehensive methodology was developed wherein test compounds (i.e., parathion, malathion and chloramphenicol) were first exposed in vitro to rat liver microsomes (RLM) for 1 h at 37 °C. After adding methanol, the mixture was ultrasonicated, placed for 2 h at -20 °C, centrifuged and the supernatant evaporated. The pellet was resuspended in water for the quantification of the metabolic conversion and the detection of the presence of metabolites using ultra high performance liquid chromatography-Ultraviolet-Mass (UHPLC-UV-MS). Next, three days post fertilization (dpf) zebrafish eleuthero embryos were exposed to the metabolic mix diluted in Danieau's medium for 48 h at 28 °C, followed by a stereomicroscopic examination of the adverse effects induced, if any. The novelty of our method relies in the possibility to quantify the rate of the in vitro metabolism of the parent compound and to co-incubate three dpf larvae and the diluted metabolic mix for 48 h without inducing major toxic effects. The results for parathion show an improved predictivity of the toxic potential of the compound.
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Project description:BackgroundZebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed.ResultsAs a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins.ConclusionThe provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.
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Project description:A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.
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Project description:Diflubenzuron is an insecticide that serves as a chitin inhibitor to restrict the growth of many harmful larvae, including mosquito larvae, cotton bollworm and flies. The residue of diflubenzuron is often detected in aquaculture, but its potential toxicity to aquatic organisms is still obscure. In this study, zebrafish embryos (from 6 h to 96 h post-fertilization, hpf) were exposed to different concentrations of diflubenzuron (0, 0.5, 1.5, 2.5, 3.5 and 4.5 mg/L), and the morphologic changes, mortality rate, hatchability rate and average heart rate were calculated. Diflubenzuron exposure increased the distance between the venous sinus and bulbar artery (SV-BA), inhibited proliferation of myocardial cells and damaged vascular development. In addition, diflubenzuron exposure also induced contents of reactive oxygen species (ROS) and malondialdehyde (MDA) and inhibited the activity of antioxidants, including SOD (superoxide dismutase) and CAT (catalase). Moreover, acridine orange (AO) staining showed that diflubenzuron exposure increased the apoptotic cells in the heart. Q-PCR also indicated that diflubenzuron exposure promoted the expression of apoptosis-related genes (bax, bcl2, p53, caspase3 and caspase9). However, the expression of some heart-related genes were inhibited. The oxidative stress-induced apoptosis damaged the cardiac development of zebrafish embryos. Therefore, diflubenzuron exposure induced severe cardiotoxicity in zebrafish embryos. The results contribute to a more comprehensive understanding of the safety use of diflubenzuron.
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