Effect of production system and inhibitory potential of aroma volatiles on polyphenol oxidase and peroxidase activity in tomatoes.

BACKGROUND: Polyphenol oxidase (PPO) and peroxidase (POD) are key enzymes associated with shelf life and defense responses. Thus, the activity of PPO and POD enzymes is usually assessed to check the quality of food samples and to understand the physiological responses of plants to different stresses. However, the outcomes of PPO and POD activity assessment studies are highly dependent on assay conditions. Hence, in this study, we initially optimized PPO and POD extraction and high-throughput 96-well plates-based enzymatic activity assessment methods to evaluate the inhibitory potential of tomato volatile compounds. Later, we explored the effects of net-house and open-field growing on the PPO and POD activity in tomato fruits of eight cultivars. RESULTS: We found 150 mM of catechol and pH 7.0 were the optimal conditions for the maximum activity for the PPO assay. Conversely, 24 mM guaiacol with 12 mM H2 O2 and pH 6.0 was the best condition for the POD assay. Thermal inactivation studies confirmed that tomato POD is more resistant to heat than PPO. We found that the production systems had a considerable genotype-specific impact on tomato PPO and POD activity. Moreover, amongst the volatiles that were studied, ß-damascenone and d-limonene showed 50% PPO inhibition at 40 and 80 mM, respectively. CONCLUSION: The results of this study can be used to improve the shelf-life of fresh tomato fruit and its products. The findings also underscore the significance of PPO and POD enzymes as physiological trait markers in the tomato crop and fruit quality improvement programs. © 2020 Society of Chemical Industry.

Improved Sample Preparation and Optimized Solvent Extraction for Quantitation of Carotenoids.

Accurate, rapid quantitation of key antioxidants such as carotenoids is important for assessment of food quality. Carotenoids are lipid-soluble pigments that are susceptible to oxidation due to their highly conjugated carbon-carbon double bonds. Therefore, the present work focuses on improving sample preparation to facilitate rapid analysis of carotenoids. The method involves optimized carotenoid extraction followed by direct HPLC analysis without further concentration and redissolution. For extraction, we tested the effect of blending time (1, 3 and 5 min) and 12 different solvent combinations for carotenoid extraction from cantaloupe (Cucumis melo var. cantalupensis) and oranges (Citrus sinensis), two popular fruits that are high in carotenoids. The identification of carotenoids was performed by LC-APCI-QTOF-HR-MS in positive-ionization mode. In melon, 1 min blending time gave significantly higher ß-carotene content with CHCl3: Ace (1:1) solvent. The optimized method was validated with tomato, watermelon, oranges, grapefruit, melon varieties and commercial products such as fruit juices. Among the different melon varieties, Western Shipper had significantly higher ß-carotene (25.1 ± 0.4 µg/g) contents. In oranges, ß-carotene and (all-E)-lycopene contents were 4.4 ± 0.1and 3.8 ± 0.1 µg/g, respectively. The optimized method has fewer unit operations and is reproducible for the quantitation of carotenoids and their isomers. This is the first report on the identification of ζ-carotene isomers, and lycopene isomers from cantaloupe varieties and lycopene from oranges. Graphical Abstract.

Hormonal and metabolites responses in Fusarium wilt-susceptible and -resistant watermelon plants during plant-pathogen interactions.

BACKGROUND: Fusarium oxysporum f. sp. niveum (FON) causes Fusarium wilt in watermelon. Several disease-resistant watermelon varieties have been developed to combat Fusarium wilt. However, the key metabolites that mount defense responses in these watermelon varieties are unknown. Herein, we analyzed hormones, melatonin, phenolic acids, and amino acid profiles in the leaf tissue of FON zero (0)-resistant (PI-296341, Calhoun Grey, and Charleston Grey) and -susceptible (Sugar Baby) watermelon varieties before and after infection. RESULTS: We found that jasmonic acid-isoleucine (JA-Ile) and methyl jasmonate (MeJA) were selectively accumulated in one or more studied resistant varieties upon infection. However, indole-3-acetic acid (IAA) was only observed in the FON 0 inoculated plants of all varieties on the 16th day of post-inoculation. The melatonin content of PI-296341 decreased upon infection. Conversely, melatonin was only detected in the FON 0 inoculated plants of Sugar Baby and Charleston Grey varieties. On the 16th day of post-inoculation, the lysine content in resistant varieties was significantly reduced, whereas it was found to be elevated in the susceptible variety. CONCLUSIONS: Taken together, Me-JA, JA-Ile, melatonin, and lysine may have crucial roles in developing defense responses against the FON 0 pathogen, and IAA can be a biomarker of FON 0 infection in watermelon plants.

CIRCADIAN CLOCK-ASSOCIATED1 Controls Resistance to Aphids by Altering Indole Glucosinolate Production.

CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), a well-known central circadian clock regulator, coordinates plant responses to environmental challenges. Its daily rhythmic expression in Arabidopsis (Arabidopsis thaliana) confers host resistance to the caterpillar Trichoplusia ni However, it is unclear whether CCA1 plays a role in defense against phloem sap-feeding aphids. In this study, we showed that green peach aphid (Myzus persicae) displayed an intrinsic circadian feeding rhythm. Under constant light, wild-type Columbia-0 (Col-0) Arabidopsis plants coentrained with aphids in the same light/dark cycles exhibited greater antixenotic activity than plants preentrained in the opposite cycle from the aphids. Consistently, circadian mutants cca1-1, cca1-11, lhy-21, ztl-1, ztl-4, and lux-2 suffered more severe damage than Col-0 plants when infested by aphids, suggesting that the Arabidopsis circadian clock plays a defensive role. However, the arrhythmic CCA1 overexpression line (CCA1-OX) displayed strong antixenotic and antibiotic activities despite its loss of circadian regulation. Aphids feeding on CCA1-OX plants exhibited lower reproduction and smaller body size and weight than those on Col-0. Apparently, CCA1 regulates both clock-dependent and -independent defense responses. Systematic investigation based on bioinformatics analyses indicated that resistance to aphids in CCA1-OX plants was due primarily to heightened basal indole glucosinolate levels. Interestingly, aphid feeding induced alternatively spliced intron-retaining CCA1a/b transcripts, which are normally expressed at low levels, whereas expression of the major fully spliced CCA1 transcript remained largely unchanged. We hypothesize that posttranscriptional modulation of CCA1 expression upon aphid infestation maximizes the potential of circadian-mediated defense and stress tolerance while ensuring normal plant development.

In vitro and in silico elucidation of antidiabetic and anti-inflammatory activities of bioactive compounds from Momordica charantia L.

Bitter melon (Momordica charantia) has been used to manage diabetes and related conditions in various parts of the world. In the present study, ten compounds were isolated from acetone and methanol extracts of bitter melon. The chemical structures of compounds were unambiguously elucidated by 1D, 2D NMR, and high-resolution mass spectra. Identified compounds 1-7 exhibited significant inhibition of α-amylase and moderate inhibition of α-glucosidase activities. Momordicoside G and gentisic acid 5-O-ß-d-xyloside showed the highest inhibition of α-amylase (70.5%), and α-glucosidase (56.4%), respectively. Furthermore, molecular docking studies of isolated compounds 1-7 were able to bind to the active sites of both enzymes. Additionally, the isolated compounds 1-7 significantly attenuated lipopolysaccharide (LPS)-induced inflammation, downregulating the expression of pro-inflammatory markers NF-κB, INOS, IL-6, IL-1ß, TNF-α, and Cox-2 in murine macrophage RAW 264.7 cells. One phenolic derivative, gentisic acid 5-O-ß-d-xyloside, was isolated and identified for the first time from bitter melon, and significantly suppressed the expression of Cox-2 and IL-6 compared to the LPS-treated group. α-Amylase and α-glucosidase are targets of anti-diabetes drugs, our findings suggest that compounds purified from bitter melon may have potential to use as functional food ingredients for the prevention of type 2 diabetes and related inflammatory conditions.

Cucurbitane-type compounds from Momordica charantia: Isolation, in vitro antidiabetic, anti-inflammatory activities and in silico modeling approaches.

Momordica charantia L., commonly known as bitter melon, belongs to the Cucurbitaceae family. Various in vitro and in vivo studies have indicated that extracts of bitter melons have anti-diabetic properties. However, very little is known about the specific purified compounds responsible for these antidiabetic properties. In the present study, 3ß,7ß,25-trihydroxycucurbita-5,23(E)-dien-19-al, charantal, charantoside XI, and 25ξ-isopropenylchole-5, 6-ene-3-O-d-glucopyranoside were isolated from bitter melon fruit. The structures of the purified compounds were elucidated by HR-ESIMS, 1D, and 2D NMR experiments. All compounds exhibited significant inhibition of α-amylase and α-glucosidase comparable to acarbose. Molecular docking studies demonstrated that purified compounds were able to bind to the active sites of proteins. Additionally, the purified compounds showed significant anti-inflammatory activity, downregulating the expression of NF-κB, iNOS, IL-6, IL-1ß, TNF-α, and Cox-2 in lipopolysaccharide-activated macrophage RAW 264.7 cells. Our findings suggest that the purified compounds have potential anti-diabetic and anti-inflammatory activities and therefore hold promise for the development of plant-based management for diabetic and inflammatory conditions.

Evaluation of bitter melon (Momordica charantia) cultivars grown in Texas and levels of various phytonutrients.

BACKGROUND: In the USA, Momordica charantia is relatively unknown and is usually found in specialty markets. In the present study, cultivation of five bitter melon cultivars grown under field conditions in College Station (TX, USA), was evaluated. Additionally, ascorbic acid, amino acids and phenolic compounds were quantified from various cultivars grown in different years. RESULTS: The yield of the first year of evaluation was comparable to other bitter melon growing regions, ranging from 9371.5 kg ha-1 for the Japanese Spindle cultivar to 20 839.1 kg ha-1 for the Hong Kong Green cultivar. Multivariate analysis suggests a strong correlation between yield and growth degree days, water use efficiency and organic matter, as well as an inverse correlation with the amount or precipitation during the growing season. The highest levels of total ascorbic acid were shown in the Japanese Spindle cultivar (162.97 mg 100 g-1 fresh fruit), whereas the lowest levels were expressed in the Hong Kong Green cultivar (42.69 mg 100 g-1 fresh fruit). The highest levels of total phenolics were consistently found the Indian White cultivar, in the range 10.6-12.5 mg g-1 catechin equivalents. Seven phenolics and organic acids were identified and quantified by liquid chromatography-mass spectrometry and high-performance liquid chromatography, respectively. Additionally, the highest levels of total amino acids were found in the Large Top cultivar. CONCLUSION: The current 3-year field study demonstrates that it is feasible to grow bitter melon commercially in Texas with proper climatic and agronomic conditions. Bitter melon is a rich source for ascorbic acid, amino acids and phenolic compounds, which makes it a valuable food source with respect to improving human health. © 2018 Society of Chemical Industry.

Production system influences volatile biomarkers in tomato.

INTRODUCTION: In recent years, growers have used various production types, including high-tunnel systems, to increase the yield of tomatoes (Lycopersicon esculentum). However, the effect of high-tunnel cultivation, in comparison to conventional open-field production, on aroma and flavor volatiles is not fully understood. OBJECTIVES: To optimize the extraction and quantification conditions for the analysis of tomato volatiles using headspace solid phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS), and study the effect of production systems on volatile profiles using metabolomics approach. METHODS: The HS-SPME conditions were optimized for extraction and GC-MS was used to quantify the volatiles from four tomato varieties grown in open-field and high-tunnel systems. Univariate and multivariate analyses were performed to identify the influence of production system on tomato volatiles. RESULTS AND CONCLUSIONS: The extraction of 2 g tomato samples at 60 °C for 45 min using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber gave the maximum amounts of volatiles. This optimized method was used to identify and quantify 41 volatiles from four tomato varieties. The levels of ß-damascenone were higher in the high-tunnel tomatoes and geranylacetone was higher in open-field tomatoes. These two volatile compounds could be considered as biomarkers for tomatoes grown in high-tunnel and open-field production systems. This study is the first report comparing volatiles in tomatoes grown in high-tunnel and open-field conditions, and our results confirmed that there is a critical need to adopt biomarker-specific production systems to improve the nutritional and organoleptic properties of tomatoes.

Effect of harvest time on the levels of phytochemicals, free radical-scavenging activity, α-amylase inhibition and bile acid-binding capacity of spinach (Spinacia oleracea).

BACKGROUND: Spinach is a green leafy vegetable that is rich in health-promoting compounds. The present study analyzed the levels of phytochemicals and health-promoting properties of spinach harvested at 20, 30, 40, 50 and 60 days after planting. RESULTS: The time of harvest had a significant effect on nitrate levels, which increased from 1909 ± 70.6 µg g-1 (20 days) to 3668 ± 101.3 µg g-1 (40 days) and then decreased to 974 ± 164 µg g-1 (60 days). Lutein and chlorophylls a and b were found to be maximum at 60 days, whereas ß-carotene was higher at 50 days. Liquid chromatography/high-resolution quadrupole time-of-flight tandem mass spectrometry (LC/HR-QTOF-MS) was used to identify 12 flavonoids, and their tentative fragmentation pathways have been proposed. Spinach harvested at 30 and 60 days exhibited significantly higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical-scavenging activities and inhibition of amylase. The levels of total phenolics ranged from 885 ± 35.1 to 1162 ± 112.4 µg g-1 in the samples. In vitro bile acid-binding capacity showed that glycochenodeoxycholate and glycodeoxycholate were bound to maximum levels in all spinach samples. CONCLUSION: The harvest time has a major effect on the levels of phytochemicals and health-beneficial properties, which indicates that consumption of both baby and mature spinach will provide maximum health benefits. © 2017 Society of Chemical Industry.

Rapid ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry and selected reaction monitoring strategy for the identification and quantification of minor spinacetin derivatives in spinach.

RATIONALE: Spinach is green leafy vegetable which is a rich source of flavonoids, phenolic acids, carotenoids, and vitamins A, C and E. It contains unique flavonoids which have significant anticarcinogenic, antiinflammatory and free radical scavenging activities. The present study reports the systematic identification and quantification of novel flavonoids by ultra-high-performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (UHPLC/HR-QTOFMS). METHODS: An ultrasonication technique was used for the extraction of flavonoids from spinach. A rapid and reliable analytical method was established for the identification of flavonoids from methanolic extract. Flavonoids were characterized by their ultraviolet (UV) spectra, high-resolution accurate masses and MS/MS fragmentation pathways obtained using electrospray ionization (ESI). Furthermore, precursor ions from the intact molecule, and the resulting product ions, were monitored by selected reaction monitoring (SRM) with different collision energies in positive and negative ion mode. RESULTS: For the first time, five minor spinacetin derivatives were identified under optimized SRM and broadband collision-induced dissociation (+bbCID) conditions. Fragmentation pathways were proposed for spectra obtained in ESI positive ion mode. The use of HR-QTOFMS and SRM allowed us to differentiate between molecules with the same nominal mass. The identified spinacetin derivatives were found to be acylated with ferulic and coumaric acids. CONCLUSIONS: UHPLC interfaced with HR-QTOFMS in combination with SRM provides a rapid, reliable and highly sensitive method for the identification of flavonoids, and potentially other bioactive compounds, in a complex matrix.

Homeostatic responses of colonic LGR5+ stem cells following acute in vivo exposure to a genotoxic carcinogen.

Perturbations in DNA damage, DNA repair, apoptosis and cell proliferation in the base of the crypt where stem cells reside are associated with colorectal cancer (CRC) initiation and progression. Although the transformation of leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)(+) cells is an extremely efficient route towards initiating small intestinal adenomas, the role of Lgr5(+) cells in CRC pathogenesis has not been well investigated. Therefore, we further characterized the properties of colonic Lgr5(+) cells compared to differentiated cells in Lgr5-EGFP-IRES-creER(T2) knock-in mice at the initiation stage of carcinogen azoxymethane (AOM)-induced tumorigenesis using a quantitative immunofluorescence microscopy approach. At 12 and 24h post-AOM treatment, colonic Lgr5(+) stem cells (GFP(high)) were preferentially damaged by carcinogen, exhibiting a 4.7-fold induction of apoptosis compared to differentiated (GFP(neg)) cells. Furthermore, with respect to DNA repair, O(6)-methylguanine DNA methyltransferase (MGMT) expression was preferentially induced (by 18.5-fold) in GFP(high) cells at 24h post-AOM treatment compared to GFP(neg) differentiated cells. This corresponded with a 4.3-fold increase in cell proliferation in GFP(high) cells. These data suggest that Lgr5(+) stem cells uniquely respond to alkylation-induced DNA damage by upregulating DNA damage repair, apoptosis and cell proliferation compared to differentiated cells in order to maintain genomic integrity. These findings highlight the mechanisms by which colonic Lgr5(+) stem cells respond to cancer-causing environmental factors.

A comparison of juice extraction methods in the pungency measurement of onion bulbs.

BACKGROUND: Onion pungency is estimated by measuring the pyruvic acid content in juice extracted from fresh tissues. We compared pyruvic acid content and its variation in the juices extracted by the pressing, maceration, blending with no water, or blending with water (blend/water) methods. RESULTS: There were considerable differences in the pyruvic acid content and coefficient of variation (CV) among these methods, and there was an interaction between the onion cultivars and the juice extraction methods. The pressing method showed over 30% CV in the quartered or composite samples. The blend/water method showed the greatest pyruvic acid content in the shortday-type ('TG1015Y' and 'Texas Early White') onions, while the pressing method showed the greatest pyruvic acid content in the longday-type onions. The blend/water method, which gave ratios between 1:1 and 1:4 (w/w), showed the same pyruvic acid content. The blending (no water) method had the highest correlation, followed by the maceration method. The lowest correlations were found with the pressing method and the blend/water method. CONCLUSIONS: Complete homogenisation of tissues with 1:1 or greater ratios of water was necessary for the maximum consistency and full development of the pyruvic acid reaction for onion pungency measurement.

Inhibition of Escherichia coli O157:H7 motility and biofilm by ß-sitosterol glucoside.

BACKGROUND: Escherichia coli O157:H7 (EHEC) is a food borne pathogen, which causes diarrhea and hemolytic uremic syndrome (HUS). There is an urgent need of novel antimicrobials for treatment of EHEC as conventional antibiotics enhance shiga toxin production and potentiate morbidity and mortality. METHODS: Six bioactive compounds were isolated, identified from citrus and evaluated for the effect on EHEC biofilm and motility. To determine the possible mode of action, a series of genes known to affect biofilm and motility were overexpressed and the effect on biofilm/motility was assessed. Furthermore, the relative expression of genes involved in motility and biofilm formation was measured by qRT-PCR in presence and absence of phytochemicals, to examine the repression caused by test compounds. RESULTS: The ß-sitosterol glucoside (SG) was identified as the most potent inhibitor of EHEC biofilm formation and motility without affecting the cell viability. Furthermore, SG appears to inhibit the biofilm and motility through rssAB and hns mediated repression of flagellar master operon flhDC. CONCLUSION: SG may serve as novel lead compound for further development of anti-virulence drugs. GENERAL SIGNIFICANCE: Plant sterols constitute significant part of diet and impart various health benefits. Here we present the first evidence that SG, a plant sterol has significant effect on EHEC motility, a critical virulence factor, and may have potential application as antivirulence strategy.

Continuous-Flow High-Pressure Homogenization of Blueberry Juice Enhances Anthocyanin and Ascorbic Acid Stability during Cold Storage.

Blueberries (Vaccinium section Cyanococcus) have a wealth of bioactive compounds, including anthocyanins and other antioxidants, that offer significant health benefits. Preserving these compounds and maintaining the sensory and nutritional qualities of blueberry products such as juice during cold market storage is critical to meet consumer expectations for nutritious, safe, and minimally processed food. In this study, we compared the effects of two preservation processing techniques, high-temperature short-time (HTST) and continuous flow high-pressure homogenization (CFHPH), on blueberry juice quality during storage at 4 °C. Our findings revealed that inlet temperature (Tin) of CFHPH processing at 4 °C favored anthocyanin retention, whereas Tin at 22 °C favored ascorbic acid retention. After 45 days of storage, CFHPH (300 MPa, 1.5 L/min, 4 °C) juice retained up to 54% more anthocyanins compared to control at 0 day. In contrast, HTST treatment (95 °C, 15 s) initially increased anthocyanin concentrations but led to their subsequent degradation over time, while also significantly degrading ascorbic acid. Furthermore, CFHPH (300 MPa, 4 °C) juice had significantly lower polyphenol oxidase activity (>80% less than control), contributing to the overall quality of the juice. This innovative processing technique has the potential to improve commercial blueberry juice, and help meet the rising demand for healthy and appealing food choices.

Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States.

The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of ≥ 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers' health.

Daily consumption of grapefruit for 6 weeks reduces urine F2-isoprostanes in overweight adults with high baseline values but has no effect on plasma high-sensitivity C-reactive protein or soluble vascular cellular adhesion molecule 1.

Individuals with obesity and metabolic syndrome (MetS) are at increased risk of cardiovascular disease, in part due to heightened inflammatory/oxidative processes. Results from epidemiologic and experimental studies suggest that citrus, and grapefruit in particular, may have a role in promoting vascular health, although clinical trial data are lacking. Here, we evaluated the anti-inflammatory/antioxidant effects of habitual grapefruit consumption in 69 overweight/obese men and women and in a subsample of participants with MetS (n = 29). Participants were randomly assigned to either a grapefruit group in which they consumed a low bioactive diet plus 1.5 grapefruit/d for 6 wk (n = 37, n = 14 with MetS) or to a control condition in which a low bioactive diet devoid of citrus was consumed (n = 32, n = 15 with MetS). Plasma soluble vascular adhesion molecule-1 (sVCAM-1), plasma high-sensitivity C-reactive protein (hsCRP), and urinary F2-isoprostanes were evaluated before and after the intervention phase. F2-isoprostane concentrations were not different in the grapefruit versus control arm after the intervention (12.4 ± 6.4 vs. 15.9 ± 9.0 ng/mg creatinine, P = 0.16), whereas plasma hsCRP concentrations tended to be lower in the grapefruit versus control arm postintervention (2.1 ± 1.5 vs. 2.8 ± 2.0 mg/L, P = 0.09). In adults with MetS, grapefruit consumption tended to result in lower postintervention F2-isoprostane concentrations compared with the control condition (12.0 ± 4.5 vs. 18.3 ± 10.9 ng/mg creatinine, P = 0.06). Furthermore, those with high baseline F2-isoprostane concentrations experienced significant reductions in this biomarker in response to grapefruit consumption (P = 0.021). Change in sVCAM-1 concentrations did not vary by treatment arm nor were there differences between arms postintervention. These results suggest that intake of grapefruit twice daily for 6 wk does not significantly reduce inflammation and oxidative stress, although there is a suggestion of favorable modulation of oxidative stress in overweight and obese adults with MetS or those with high baseline urine F2-isoprostane concentrations.

5-Geranyloxy-7-methoxycoumarin inhibits colon cancer (SW480) cells growth by inducing apoptosis.

For the first time, three coumarins were isolated from the hexane extract of limes (Citrus aurantifolia) and purified by flash chromatography. The structures were identified by NMR (1D, 2D) and mass spectral analyses as 5-geranyloxy-7-methoxycoumarin, limettin, and isopimpinellin. These compounds inhibited human colon cancer (SW-480) cell proliferation, with 5-geranyloxy-7-methoxycoumarin showing the highest inhibition activity (67 %) at 25 µM. Suppression of SW480 cell proliferation by 5-geranyloxy-7-methoxycoumarin was associated with induction of apoptosis, as evidenced by annexin V staining and DNA fragmentation. In addition, 5-geranyloxy-7-methoxycoumarin arrested cells at the G0/G1 phase, and induction of apoptosis was demonstrated through the activation of tumour suppressor gene p53, caspase8/3, regulation of Bcl2, and inhibition of p38 MAPK phosphorylation. These findings suggest that 5-geranyloxy-7-methoxycoumarin has potential as a cancer preventive agent.

Obacunone represses Salmonella pathogenicity islands 1 and 2 in an envZ-dependent fashion.

Obacunone belongs to a class of unique triterpenoids called limonoids, present in Citrus species. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling in Vibrio harveyi and Escherichia coli O157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogen Salmonella enterica serovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone represses Salmonella pathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated through hilA. Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to repress hilA in an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect on S. Typhimurium and may serve as a lead compound for development of antivirulence strategies for S. Typhimurium.

Isolimonic acid interferes with Escherichia coli O157:H7 biofilm and TTSS in QseBC and QseA dependent fashion.

BACKGROUND: E. coli O157:H7 (EHEC) is an important human pathogen. The antibiotic treatment of EHEC reportedly results in release of Shiga toxin and is therefore discouraged. Consequently, alternative preventive or therapeutic strategies for EHEC are required. The objective of the current study was to investigate the effect of citrus limonoids on cell-cell signaling, biofilm formation and type III secretion system in EHEC. RESULTS: Isolimonic acid and ichangin were the most potent inhibitors of EHEC biofilm (IC25=19.7 and 28.3 µM, respectively) and adhesion to Caco-2 cells. The qPCR analysis revealed that isolimonic acid and ichangin repressed LEE encoded genes by ≈3 to 12 fold. In addition, flhDC was repressed by the two limonoids (≈3 to 7 fold). Further studies suggested that isolimonic acid interferes with AI-3/epinephrine activated cell-cell signaling pathway. Loss of biofilm inhibitory activity of isolimonic acid in ΔqseBC mutant, which could be restored upon complementation, suggested a dependence on functional QseBC. Additionally, overexpression of qseBC in wild type EHEC abated the inhibitory effect of isolimonic acid. Furthermore, the isolimonic acid failed to differentially regulate ler in ΔqseA mutant, while plasmid borne expression of qseA in ΔqseA background restored the repressive effect of isolimonic acid. CONCLUSIONS: Altogether, results of study seem to suggest that isolimonic acid and ichangin are potent inhibitors of EHEC biofilm and TTSS. Furthermore, isolimonic acid appears to interfere with AI-3/epinephrine pathway in QseBC and QseA dependent fashion.

Inhibition of prostate cancer (LNCaP) cell proliferation by volatile components from Nagami kumquats.

Fresh Nagami kumquats (Fortunella margarita) were subjected to hydrodistillation using a Clevenger-type apparatus to obtain volatile oil. The chemical composition of the volatile oil was analyzed by GC-MS using Rtx-5 Sil MS and DB Wax columns. A total of 25 volatile compounds were identified by mass spectra, retention index, and comparison with known standards. The major identified compounds are d-limonene (41.64 %), ß-myrecene (16.54 %), linalyl propionate (9.55 %), and germacrene-D (5.93 %) from the Rtx-5 Sil MS column; d-limonene and ß-myrecene were also separated as major compounds on the DB wax column. The oil is rich in hydrocarbons (77.41 %) consisting of 60.05 % monoterpenes and 17.36 % sesquiterpenes. Interestingly, oxygenated hydrocarbons (17.6 %) were also found in kumquat volatile oil. Certain volatile compounds were also confirmed by positive chemical ionization and NMR spectra. Further, the volatile oil demonstrated good DPPH radical scavenging activity and antioxidant capacity. Kumquat volatile oil at 200 ppm concentration exhibited 55 %, 61 %, and 63.4 % inhibition of human prostate cancer (LNCaP) cell proliferation at 24, 48, and 72 h, respectively, by cell count assays. Significant increases in expression of bax/bcl2 and p53 proteins confirmed that volatile oil induces apoptosis. In addition, inhibition of inflammatory markers such as NF-κB and Cox-2 was observed. The cleavage of caspase-8 in the LNCaP cells treated with volatile oil demonstrated that apoptosis occurred through an extrinsic pathway. This is the first report of the identification and possible mechanisms of in vitro antiproliferative effects of kumquat volatile components on human prostate cancer (LNCaP) cells.