Bioactivity guided isolation and identification of phenolic compounds from Citrus aurantium L. with anti-colorectal cancer cells activity by UHPLC-Q-TOF/MS.

Natural plants are rich sources of various bioactive compounds. Consequently, the efficiently isolation of these bioactive components has always attracted considerable attention. Our work aims to demonstrate a framework for bioactivity guided isolation of potential effective compounds from the complex food materials. We demonstrated its application for isolation of phenolic compounds with anti-proliferative activity against colorectal cancer cells (CRCs) from Citrus aurantium L. Firstly, phenolic rich fraction was successfully identified as the main effective components that could simultaneously suppress the growth of CRCs and inhibit Wnt signaling. In order to obtain the bioactive phenolic constituents, a detailed study was performed by optimizing the purification conditions. Two phenolic rich fractions (40% and 60% ethanol elution fractions) were then obtained by AB-8 macroporous resins under optimized condition. Finally, the main components (65 compounds) were tentatively identified from the 40% ethanol eluant by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) analysis. Notably, there were five of the phytochemicals (Feruloylagmatine, Haploside C, Sagittatin A, Linderagalactone C and Koparin-2'-methyl ether) which were hitherto unidentified in Citrus aurantium L. fruit. In conclusion, this study showed that under the principle of bioactivity guided strategy, phenolic constituents with potential anti-CRCs activity were isolated from Citrus aurantium L.

Bioactivity-Oriented Purification of Polyphenols from Cinnamomum cassia Presl. with Anti-Proliferation Effects on Colorectal Cancer Cells.

Cinnamomum cassia Presl. (CCP) is a popular natural spice possessing various pharmacological properties. We obtained polyphenol-rich fraction (CCP-P) from CCP by bioactivity-oriented purification method and evaluated its Wnt signaling inhibition activity. Firstly, the phenolic components were identified as the main bioactive compounds with anti-colorectal cancer activity. Then, we compared the anti-colorectal cancer activity of CCP extract obtained from different solvent by cell morphology alteration and EdU assay. Ethanol extract showed higher antiproliferative activity compared to water extract on HCT116 cells, with proliferating cells reducing to 41.12 and 21.83% at 156.00 µg GAE/mL, respectively. Next, separation and enrichment of polyphenols from ethanol extract was performed on AB-8 macroporous resins under optimal conditions. Further evaluation of the CCP-P bioactivity revealed that it exerted more potent antiproliferative activity on RKO and HCT116 cells, showing higher selectivity for Wnt-dependent colorectal cancer cells (CRCs). Ten major polyphenols were identified in the CCP-P by UPLC-ESI-MS/MS. In summary, this study presents evidence that CCP-derived polyphenols are promising potential candidates as functional food ingredients against CRC.

The response of Escherichia coli to the alkylating agents chloroacetaldehyde and styrene oxide.

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.

A Quantitative Toxicogenomics Assay for High-throughput and Mechanistic Genotoxicity Assessment and Screening of Environmental Pollutants.

The ecological and health concern of mutagenicity and carcinogenicity potentially associated with an overwhelmingly large and ever-increasing number of chemicals demands for cost-effective and feasible method for genotoxicity screening and risk assessment. This study proposed a genotoxicity assay using GFP-tagged yeast reporter strains, covering 38 selected protein biomarkers indicative of all the seven known DNA damage repair pathways. The assay was applied to assess four model genotoxic chemicals, eight environmental pollutants and four negative controls across six concentrations. Quantitative molecular genotoxicity end points were derived based on dose response modeling of a newly developed integrated molecular effect quantifier, Protein Effect Level Index (PELI). The molecular genotoxicity end points were consistent with multiple conventional in vitro genotoxicity assays, as well as with in vivo carcinogenicity assay results. Further more, the proposed genotoxicity end point PELI values quantitatively correlated with both comet assay in human cell and carcinogenicity potency assay in mice, providing promising evidence for linking the molecular disturbance measurements to adverse outcomes at a biological relevant level. In addition, the high-resolution DNA damaging repair pathway alternated protein expression profiles allowed for chemical clustering and classification. This toxicogenomics-based assay presents a promising alternative for fast, efficient and mechanistic genotoxicity screening and assessment of drugs, foods, and environmental contaminants.

Toxicity mechanisms identification via gene set enrichment analysis of time-series toxicogenomics data: impact of time and concentration.

The advance in high-throughput "toxicogenomics" technologies, which allows for concurrent monitoring of cellular responses globally upon exposure to chemical toxicants, presents promises for next-generation toxicity assessment. It is recognized that cellular responses to toxicants have a highly dynamic nature, and exhibit both temporal complexity and dose-response shifts. Most current gene enrichment or pathway analysis lack the recognition of the inherent correlation within time series data, and may potentially miss important pathways or yield biased and inconsistent results that ignore dynamic patterns and time-sensitivity. In this study, we investigated the application of two score metrics for GSEA (gene set enrichment analysis) to rank the genes that consider the temporal gene expression profile. One applies a novel time series CPCA (common principal components analysis) to generate scores for genes based on their contributions to the common temporal variation among treatments for a given chemical at different concentrations. Another one employs an integrated altered gene expression quantifier-TELI (transcriptional effect level index) that integrates altered gene expression magnitude over the exposure time. By comparing the GSEA results using two different ranking metrics for examining the dynamic responses of reporter cells treated with various dose levels of three model toxicants, mitomycin C, hydrogen peroxide, and lead nitrate, the analysis identified and revealed different toxicity mechanisms of these chemicals that exhibit chemical-specific, as well as time-aware and dose-sensitive nature. The ability, advantages, and disadvantages of varying ranking metrics were discussed. These findings support the notion that toxicity bioassays should account for the cells' complex dynamic responses, thereby implying that both data acquisition and data analysis should look beyond simple traditional end point responses.