Bioactive compounds, antioxidant capacity and anti-inflammatory activity of native fruits from Brazil.

The purpose of this study was to extract, identify, and quantify the phenolic compounds in grumixama (Eugenia brasilienses Lam.) and guabiju (Myrcianthes pungens), native fruits from southern region of Brazil, and to explore their antioxidant and anti-inflammatory properties. The phenolic compounds were extracted with acidified water and acidified methanol and evaluated for their bioactive constituents, antioxidant capacity, and anti-inflammatory properties. Spectrophotometric quantification shows tannins to be the most prevalent at 2.3 to 5.8 g/100g fresh fruit with acidified methanol containing higher concentrations of different phenolics than acidified water. HPLC analysis indicates that gallic acid, catechin, vanillic acid, and ellagic acid are the most prevalent phenolics in the two fruits extracts. Scavenging of DPPH and NO radicals showed inhibition by as much as 95% and 80%, respectively, at 2.5 gallic acid equivalent (GAE)/mL of the extract. At 50 µg GAE/mL, the release of pro-inflammatory molecules NO and IL-6 was significantly reduced with acidified methanol extract having higher inhibitory activity. Our results revealed that these native fruits, grown in the south of Brazil, are rich sources of phenolic compounds and have great antioxidant and anti-inflammatory activity.

Physicochemical, functional and bioactive properties of hempseed (Cannabis sativa L.) meal, a co-product of hempseed oil and protein production, as affected by drying process.

Hempseed meal after protein isolation (HM-PI) is a co-product obtained from hempseed. The objectives were to characterize and determine the effect of drying on HM-PI. HM-PI was produced using three drying methods: freeze (FD), vacuum oven (VOD), and oven drying (OD). HM-PI contained over 70% protein and had similar or higher level of essential amino acids than recommended values for human adults. Osborne fractionation indicated that glutelin was the most dominant fraction in HM-PI. FD HMPI has a significant lower surface hydrophobicity and higher in vitro protein digestibility than OD and VOD HM-PI. FD HM-PI demonstrated better functional properties than OD and VOD HM-PI. Pepsin-pancreatin digestion of VOD, FD and OD resulted in comparable and considerable antioxidant and anti-inflammatory properties. This is the first report on the characterization of HM-PI, a co-product of hempseed processing. HM-PI could serve as a novel food protein ingredient resulting in increase utilization of hempseed.

A Comparative Study on Phenolic Content, Antioxidant Activity and Anti-Inflammatory Capacity of Aqueous and Ethanolic Extracts of Sorghum in Lipopolysaccharide-Induced RAW 264.7 Macrophages.

Sorghum is an important cereal with diverse phenolic compounds that have potential health promoting benefits. The current study comparatively characterized the phenolic contents of two novel black-seeded sorghum lines (SC84 and PI570481) using different extraction systems (water, ethanol and their acidified counterparts) and evaluated their antioxidant and anti-inflammatory activities. Phenolic compositions were determined by spectrophotometric assays and HPLC analysis. Antioxidant activities were assessed by radical scavenging effects on nitric oxide (NO) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, and the oxygen radical absorbance capacity (ORAC). Anti-inflammatory capacity was estimated by measuring levels of pro-inflammatory markers produced by lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Results showed that effects of solvent types and HCl on extraction efficiency differed among phenolic compounds and sorghum samples. Tannins were the most dominant polyphenols in the studied extracts (11.11-136.11 mg epicatechin equivalent/g sorghum). Sorghum extracts exerted more potent scavenging activity on DPPH than NO radicals. In LPS-activated RAW264.7 cells, sorghum extracts dose-dependently inhibited the production of NO, interleukin-6 (IL-6), and intracellular reactive oxygen species (ROS), with ethanolic extracts showing greater anti-inflammatory activity. Positive correlations were noted between tannin content and DPPH radical scavenging activity, and anti-inflammatory capacity. These results suggest the potential role of tannin-rich sorghum extracts against inflammation and associated diseases.

Bovine Milk Exosomes Affect Proliferation and Protect Macrophages against Cisplatin-Induced Cytotoxicity.

BACKGROUND: Exosomes are extracellular vesicles involved in intercellular communication. The objectives were to characterize bovine milk exosomes (BME) and determine its effect on RAW 264.7 macrophages. METHODS: BME were isolated using differential centrifugation and characterized by particle size and the presence of exosomal markers Alix, TSG101, and CD81. The effect of in vitro digestion and different pH on the stability of BME was investigated. The biological activity of BME in RAW 264.7 macrophages was conducted by assessing proliferation and cell cycle. Moreover, the protective effect of exosomes on cisplatin-induced cytotoxicity was evaluated. RESULTS: BME have an average particle size of 106.8 ± 3.4 nm and expressed Alix, TSG101, and CD81. TSG101 was detected after digestion and exposure to different pH values. Cell-cycle analysis showed that BME reduced the percentage of apoptotic cells while arresting the cells in G2/M phase accompanied by differential expression of proliferation markers p53, p21, cyclin D1, and ß-catenin. Exosomes protected macrophages against cisplatin-induced cytotoxicity. CONCLUSION: Our results showed for the first time the effect of BME on the proliferation of RAW 264.7 macrophages and its protective effect against chemotherapeutic drug-induced cytotoxicity. Potential effect of BME on immune system must be studied.

Effect of heating on the digestibility of isolated hempseed (Cannabis sativa L.) protein and bioactivity of its pepsin-pancreatin digests.

The objective was to investigate the effects of heat pretreatment and simulated gastrointestinal digestion on potential antioxidant, anticancer and anti-inflammatory activities of hempseed (Cannabis sativa L.) proteins. Unheated isolated hempseed protein (IHP) and its heated counterparts (100 °C, 15 min and 30 min, termed as HP15D and HP30D) were hydrolyzed sequentially with pepsin and pancreatin and analyzed for digestibility and bioactivity (antioxidant, anti-proliferative and anti-inflammatory properties). Heat pretreatment led to an increase of low molecular weight proteins and degree of hydrolysis, and decrease of concentration of soluble protein, which means heat pretreated can significantly improve the digestibility of IHP. Pepsin-pancreatin digests released from heat pretreated IHP possessed less antioxidant, antiproliferative and anti-inflammatory properties than digests from unheated IHP. In conclusion, heat pre-treatment improved the digestibility of IHP but the resulting digests from heated IHP had lower bioactivity.

Sorghum Phenolics Inhibits Inflammasomes in Lipopolysaccharide (LPS)-Primed and Adenosine Triphosphate (ATP)-Activated Macrophages.

Sorghum contains phenolic compounds with reported biological activities. The objective was to evaluate the ability of sorghum phenolic extract to inhibit inflammasomes in THP-1 human macrophages. THP-1 human macrophages was pre-treated with sorghum phenolics and the inflammasome was activated by lipopolysaccharide and adenosine triphosphate treatment. Treatment of macrophages with 50 µg sorghum extract/mL reduced IL-1ß and IL-18 secretion by 59.7 and 32.0%, respectively, associated with caspase-1 activity reduction. Moreover, the production of intracellular reactive oxygen species was reduced. Our data showed the potential role of sorghum phenolics in diseases associated with aberrant inflammasomes activation.

BG-4, a novel bioactive peptide from momordica charantia, inhibits lipopolysaccharide-induced inflammation in THP-1 human macrophages.

BACKGROUND: Bitter melon (Momordica charantia) is a commonly used food crop for management of a variety of diseases most notably for control of diabetes, a disease associated with aberrant inflammation. PURPOSE: To evaluate the anti-inflammatory property of BG-4, a novel bioactive peptide isolated from the seed of bitter melon. METHODS: Differentiated THP-1 human macrophages were pre-treated with BG-4 and stimulated with lipopolysaccharide. Pro-inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. The mechanism of action involving activation of NF-κB and phosphorylation of ERK and STAT3 was measured by western blot and immunofluorescence. The production of intracellular reactive oxygen species was evaluated by fluorescence microscopy and fluorescence spectrophotometry. RESULTS: BG-4 dose dependently reduce the production of pro-inflammatory cytokines IL-6 and TNF-α. The ability of BG-4 to reduce production of cytokines are associated with reduced phosphorylation of ERK and STAT3 accompanied by reduced nuclear translocation of p65 NF-κB subunit. The mechanism of action is reduction of LPS-induced production of intracellular reactive oxygen species. CONCLUSION: Our results demonstrated the ability of BG-4, a novel peptide from the seed of bitter melon, to exert anti-inflammatory action. This could explain the traditional use of bitter melon against diseases associated with aberrant and uncontrolled inflammation.

Impact of ultrasonication on the physicochemical properties of sorghum kafirin and in vitro pepsin-pancreatin digestibility of sorghum gluten-like flour.

Sorghum is a nutritionally dense grain but its nutritional quality is limited by its poor digestibility. The objective was to determine the effects of ultrasonication on the physicochemical properties of kafirin, major protein in sorghum, and its stability against pepsin-pancreatin hydrolysis (PPH). Ultrasonication for 10min at 40% amplitude increased the solubility of purified kafirin from 6.5µg/mL to 173.3µg/mL. Ultrasonication altered the secondary structure of kafirin as evaluated by circular dichroism and Fourier-transform infrared red spectroscopy. In pepsin-pancreatin hydrolysates, 14.7% increase in molecules with molecular weight between 0.075 and 0.5kDa was detected in size-exclusion chromatogram after ultrasonication at 40% amplitude, 10min. The degree of hydrolysis was also increased after ultrasonication by 17.7%, 127.6%, 346.6% as measured by o-phthaldialdehyde derivatization, trichloroacetic acid precipitation and hydrochloric acid hydrolysis, respectively with improved antioxidant property. Our results showed the potential of ultrasonication to improve digestibility and biological properties of sorghum flour.

Kafirin from Sorghum bicolor inhibition of inflammation in THP-1 human macrophages is associated with reduction of intracellular reactive oxygen species.

Aberrant inflammation as a result of activation of the transmembrane protein Toll-like receptor 4 belonging to pattern recognition receptor and subsequent phosphorylation of signaling proteins facilitated by reactive oxygen species has been linked to a myriad of diseases. Sorghum is a drought-resistant cereal with health promoting properties associated with its biologically active substances such as kafirin. Kafirin is an alcohol soluble protein and accounts for as much as 70% of the total proteins in sorghum. The objective was to determine the effect of kafirin on lipopolysaccharide (LPS)-induced inflammation in THP-1 human macrophages. THP-1 human monocytic leukemia cells were differentiated into macrophages by phorbol-12-myristate 13-acetate followed by treatment of LPS with or without 50 µg/mL or 100 µg/mL concentrations of kafirin. Kafirin at 100 µg/mL reduced the production of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α by 28.3%, 74.0%, and 81.4%, respectively. Kafirin reduced production of intracellular reactive oxygen species is associated with reduced phosphorylation of extracellular regulated kinase1/2 and c-JUN N-terminal kinase and nuclear translocation of p65 and c-JUN transcription factors. Our results showed for the first time the anti-inflammatory property of kafirin purified from sorghum in LPS-induced THP-1 human macrophages.

Pepsin-pancreatin hydrolysis reduced the ability of lunasin-enriched material to inhibit activation of the inflammasomes in THP-1 human macrophages.

Inflammation caused by the NLRP3 inflammasome has been linked to many diseases. Lunasin is a bioactive peptide from soybeans with reported anti-inflammatory properties. The objective of this work was to determine the effect of pepsin-pancreatin hydrolysis (PPH) on the ability of lunasin-enriched preparation (LEP) to inhibit inflammasome activation in differentiated THP-1 human macrophages. THP-1 macrophages were treated with different concentrations of LEP (0.0625 to 0.25 mg mL-1), primed with 1 µg mL-1 lipopolysaccharide for 6 h and activated by 5 mM adenosine triphosphate for 1 h. LEP reduced secretion of IL-1ß and IL-18. In addition, LEP treatment inhibited the production of intracellular reactive oxygen species (ROS) in THP-1 human macrophages without affecting the expressions of NLRP3 and ASC proteins involved in inflammasomes. PPH reduced the ability of LEP to inhibit production of intracellular ROS. Our results showed that LEP inhibited activation of inflammasomes by reducing intracellular ROS in vitro which was reduced by PPH.

Epithelial-to-Mesenchymal Transition in Paclitaxel-Resistant Ovarian Cancer Cells Is Downregulated by Luteolin.

Ovarian cancer (OVCA) is the deadliest of all gynecological cancers which is attributed to late presentation, persistence, and development of chemoresistance. The objectives were to evaluate the association between OVCA paclitaxel-resistance and epithelial-to-mesenchymal transition (EMT) and to determine the capability of luteolin to chemosensitize OVCA cells. X10 and X22 cells were 11.8-25.3-fold and 7.8-8.6-fold resistant to paclitaxel than 1AP cells. X10 and X22 cells exhibited a mesenchymal phenotype, while 1AP has an epithelial characteristics. Furthermore, the expression of the epithelial marker E-cadherin was downregulated, while mesenchymal markers Vimentin and N-cadherin were upregulated in X10 and X22 cells when compared to 1AP cells. Transcription factors Snail, Slug, and Twist1 were upregulated in X10 cells, while Twist1 was highly expressed in X22 cells. Luteolin treatment caused cytotoxicity being most potent to X10 OVCA cells. Treatment of non-cytotoxic dose of luteolin at 15.625 µM chemosensitized X10 and X22 OVCA cells to paclitaxel as evidenced by reduced ED50 values from 11.8 to 0.2 µM and 8.6 to 3.6 µM for X10 and X22 cells, respectively. Moreover, luteolin treatment led to a more epithelial phenotype of X10 and X22 cells and modification of EMT markers indicating reversal of EMT. The mechanism involved is through reduction of phosphorylation of FAK and ERK leading to reduced nuclear translocation of p65. Our results highlight the significance of EMT in OVCA resistance to paclitaxel and warrant the investigation of luteolin as a potential therapeutic agent in chemoresistant OVCA. J. Cell. Physiol. 232: 391-401, 2017. © 2016 Wiley Periodicals, Inc.

Phytochemical concentrations and biological activities of Sorghum bicolor alcoholic extracts.

Sorghum is an important cereal with reported health benefits. The objectives of this study were to measure the biological activities of alcoholic extracts of ten sorghum varieties and to determine the association between the color of the extracts and their biological activities. Variation on concentrations of bioactives among sorghum varieties was observed with ethanolic extracts giving higher concentrations than methanolic extracts. The color of the extracts significantly correlated with the concentrations of bioactives and with nitric oxide scavenging activity. Freeze-dried ethanol extract is more potent than freeze-dried methanol extract and caused cytotoxicity to A27801AP and PTX-10 OVCA with ED50 values of 0.69 and 1.29 mg mL(-1), respectively. Pre-treatment of OVCA with ethanol extract led to chemosensitization to paclitaxel and the proliferation and colony formation of OVCA cells were reduced by 14.7 to 44.6% and 36.4 to 40.1%, respectively. Sorghum is a potential source of colorants with health promoting properties. This is the first report on the capability of sorghum alcoholic extracts to cause cytotoxicity and chemosensitize ovarian cancer cells in vitro.

Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis.

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor (BBI) and Kunitz trypsin inhibitor (KTI) concentrations on the stability of lunasin against pepsin-pancreatin hydrolysis (PPH). In addition, the effect of KTI mutation on lunasin stability against PPH was determined. LEP were produced by calcium and pH precipitation methods of 30% aqueous ethanol extract from defatted soybean flour. LEP, lunasin-enriched commercially available products and KTI control and mutant flours underwent PPH and samples were taken after pepsin and pepsin-pancreatin hydrolysis. The concentrations of BBI, KTI, and lunasin all decreased after hydrolysis, but they had varying results. BBI concentration ranged from 167.5 to 655.8µg/g pre-hydrolysis and 171.5 to 250.1µg/g after hydrolysis. KTI concentrations ranged from 0.3 to 122.3µg/g pre-hydrolysis and 9.0 to 18.7µg/g after hydrolysis. Lunasin concentrations ranged from 8.5 to 71.0µg/g pre-hydrolysis and 4.0 to 13.2µg/g after hydrolysis. In all products tested, lunasin concentration after PPH significantly correlated with BBI and KTI concentrations. Mutation in two KTI isoforms led to a lower concentration of lunasin after PPH. This is the first report on the potential role of KTI in lunasin stability against PPH and must be considered in designing lunasin-enriched products that could potentially survive digestion after oral ingestion.

Efficacy of slightly acidic electrolyzed water in killing or reducing Escherichia coli O157:H7 on iceberg lettuce and tomatoes under simulated food service operation conditions.

UNLABELLED: The objective of this study was to evaluate the efficacy of slightly acidic electrolyzed (SAEO) water in killing or removing Escherichia coli O157:H7 on iceberg lettuce and tomatoes by washing and chilling treatment simulating protocols used in food service kitchens. Whole lettuce leaves and tomatoes were spot-inoculated with 100 µL of a mixture of 5 strains of E. coli O157:H7. Washing lettuce with SAEO water for 15 s reduced the pathogen by 1.4 to 1.6 log CFU/leaf, but the treatments did not completely inactivate the pathogen in the wash solution. Increasing the washing time to 30 s increased the reductions to 1.7 to 2.3 log CFU/leaf. Sequential washing in SAEO water for 15 s and then chilling in SAEO water for 15 min also increased the reductions to 2.0 to 2.4 log CFU/leaf, and no cell survived in chilling solution after treatment. Washing tomatoes with SAEO water for 8 s reduced E. coli O157:H7 by 5.4 to 6.3 log CFU/tomato. The reductions were increased to 6.6 to 7.6 log CFU/tomato by increasing the washing time to 15 s. Results suggested that application of SAEO water to wash and chill lettuce and tomatoes in food service kitchens could minimize cross-contamination and reduce the risk of E. coli O157:H7 present on the produce. PRACTICAL APPLICATION: SAEO water is equally or slightly better than acidic electrolyzed (AEO) water for inactivation of bacteria on lettuce and tomato surfaces. In addition, SAEO water may have the advantages over AEO water on its stability, no chlorine smell, and low corrosiveness. Therefore, SAEO water may have potential for produce wash to enhance food safety.

Reduction of Escherichia coli O157:H7 on produce by use of electrolyzed water under simulated food service operation conditions.

Treatment of fresh fruits and vegetables with electrolyzed water (EW) has been shown to kill or reduce foodborne pathogens. We evaluated the efficacy of EW in killing Escherichia coli O157:H7 on iceberg lettuce, cabbage, lemons, and tomatoes by using washing and/or chilling treatments simulating those followed in some food service kitchens. Greatest reduction levels on lettuce were achieved by sequentially washing with 14-A (amperage) acidic EW (AcEW) for 15 or 30 s followed by chilling in 16-A AcEW for 15 min. This procedure reduced the pathogen by 2.8 and 3.0 log CFU per leaf, respectively, whereas washing and chilling with tap water reduced the pathogen by 1.9 and 2.4 log CFU per leaf. Washing cabbage leaves for 15 or 30 s with tap water or 14-A AcEW reduced the pathogen by 2.0 and 3.0 log CFU per leaf and 2.5 to 3.0 log CFU per leaf, respectively. The pathogen was reduced by 4.7 log CFU per lemon by washing with 14-A AcEW and 4.1 and 4.5 log CFU per lemon by washing with tap water for 15 or 30 s. A reduction of 5.3 log CFU per lemon was achieved by washing with 14-A alkaline EW for 15 s prior to washing with 14-A AcEW for 15 s. Washing tomatoes with tap water or 14-A AcEW for 15 s reduced the pathogen by 6.4 and 7.9 log CFU per tomato, respectively. Application of AcEW using procedures mimicking food service operations should help minimize cross-contamination and reduce the risk of E. coli O157:H7 being present on produce at the time of consumption.

Seasonal incidence and molecular characterization of Salmonella from dairy cows, calves, and farm environment.

The occurrence patterns and molecular characteristics of Salmonella are important for surveillance and control of the pathogens. Objectives of this study were to determine month-to-month variation and seasonal effects on the occurrence of Salmonella in dairy animals and environments and to characterize selected Salmonella isolates. A total of 7680 animal and environmental samples, collected monthly from a dairy farm, were analyzed for the presence of Salmonella during a 12-month study. Major sources of Salmonella on the dairy farm (% positive) were milking parlor air (62%) and bird droppings (63%) during winter; feeds (50-58%), water (53-67%), calf bedding (63%), soils (60-63%), milking parlor air (60%), and bird droppings (50%) in spring; all animal and environmental samples (40-92%) except milking parlor air (25%) and bulk tank milk (29%) in summer; and feeds (60-71%), cow beddings (59%), cow soils (50%), air (46-71%), and insects (63%) during fall. Salmonella ribotyping indicated that most serovars came from different sources but some might have originated from a common source and transmitted from site to site on the farm. These data provide some important information on key animal and environmental sampling sites needed to initiate on-farm management programs for control of this important foodborne pathogen.

Prevalence of Salmonella in diverse environmental farm samples.

The development of suitable intervention strategies to control Salmonella populations at the farm level requires reliable data on the occurrence and prevalence of the pathogen. Previous studies on Salmonella prevalence have focused on acquiring data from specific farm types and/or selected regions. The purpose of this study was to evaluate the distribution of this pathogen across a variety of farm types and regions in order to generate comparative data from a diverse group of environmental samples. Farm samples (n = 2,496) were collected quarterly from 18 different farms across five states (Tennessee, North Carolina, Alabama, California, and Washington) over a 24-month period. The participating farms included beef and dairy cattle operations, swine production and farrowing facilities, and poultry farms (both broiler chicken and turkey). The samples were analyzed for the presence of Salmonella by means of the U.S. Food and Drug Administration's Bacteriological Analytical Manual methods optimized for farm samples. Salmonella isolates were characterized by automated riboprinting. Salmonella serovars were recovered from 4.7% of all samples. The majority of positive findings were isolated from swine farms (57.3%). The occurrence of Salmonella was lower on dairy farms (17.9%), poultry farms (16.2%), and beef cattle farms (8.5%). The most commonly isolated serovar was Salmonella Anatum (48.4%), which was isolated notably more frequently than the next most common Salmonella serovars, Arizonae (12.1%) and Javiana (8.8%). The results of this study suggest that significant reservoirs of Salmonella populations still exist on swine production facilities and to a lesser extent in other animal production facilities. Data showed that the surrounding farm environment could be an important source of contamination.

Evaluation of methods for recovery of Salmonella from dairy cattle, poultry, and swine farms.

Current official methods for detection and isolation of Salmonella are mostly designed for foods. The objective of this study was to determine optimal methods for detection and isolation of Salmonella from animal and environmental samples of dairy, poultry, and swine farms. Preenrichment in lactose broth versus direct enrichment (no preenrichment) prior to selective enrichment in Rappaport-Vassiliadis, selenite cystine, and tetrathionate incubated at 35 and 42 degrees C and in four differential/selective plating media (brilliant green, bismuth sulfite, Hektoen enteric, and xylose-lysine-tergitol 4 agar base) were evaluated for their ability to recover Salmonella from artificially contaminated samples. The effects of pH adjustments to samples on Salmonella recovery were determined. A pH adjustment of the enrichment broth to 6.8 +/- 0.2 after addition of samples significantly improved recovery of Salmonella. The most effective medium combinations for isolation of Salmonella from farm samples depended on the type of samples. Generalizations of protocols for recovery of Salmonella from farm samples might result in poor recovery, increased recovery time, and increased sample processing costs.