Characterization of metabolites and biomarkers for the probiotic effects of Clostridium cochlearium on high-fat diet-induced obese C57BL/6 mice.

PURPOSE: Probiotic species of butyrate producers have been investigated for the potential in preventing and treating obesity and overweight. However, Clostridium cochlearium has not been linked with any health benefits. We hypothesized that C. cochlearium could be a promising new probiotic with health benefits in improving body weight control and insulin sensitivity. METHODS: Productions of short-chain fatty acids (SCFAs) were characterized for C. cochlearium by NMR and GC-MS analyses. Probiotic effects of C. cochlearium were evaluated through diet-induced obese (DIO) C57BL/6 mice. The influence of C. cochlearium administration on gut SCFAs was measured using GC-MS. LC-MS-based untargeted metabolomic profiling and multivariate analysis were used to assess the serum metabolic alteration, identify biomarkers and pathways in response to the C. cochlearium administration. RESULTS: After 17 weeks of diet intervention, body weight gain of CC group (fed with a high-fat diet supplemented with C. cochlearium) showed a 21.86% reduction from the high-fat diet (HF) control group (P < 0.001), which was specifically reflected on the significantly lowered fat mass (CC vs HF, 17.19 g vs 22.86 g, P < 0.0001) and fat percentage (CC vs HF, 41.25% vs 47.10%, P < 0.0001), and increased lean percentage (CC vs HF, 46.63% vs 43.72%, P < 0.05). C. cochlearium administration significantly reduced fasting blood glucose from week 8 (P < 0.05 or 0.01), and eventually improved insulin sensitivity (HOMA-IR, CC vs HF, 63.77 vs 143.13, P < 0.05). Overall lowered levels of SCFAs were observed in the gut content of CC group. Metabolomic analysis enabled the identification of 53 discriminatory metabolites and 24 altered pathways between CC and HF groups. In particularly, most of the pathway-matched metabolites showed positive correlations with body weight, which included glutamate, phenylalanine, ornithine, PCs, LPCs, AcCas, proline, 5,6-dihydrouracil, pyroglutamic acid, and 1-pyrroline-4-hydroxy-2-carboxylate. CONCLUSIONS: The beneficial effects of C. cochlearium could be related to its ability to restore certain obesity-driven biomarkers and pathways, especially downregulating pathways related to specific amino acids, PCs, LPCs and AcCas. Further research is warranted to investigate related metabolites and metabolic pathways. C. cochlearium may be developed as a promising new probiotic for the prevention or alleviation of obesity and diabetes in human.

Isolation and Structural Characterization of Specific Bacterial ß-Glucuronidase Inhibitors from Noni (Morinda citrifolia) Fruits.

An extract of noni (Morinda citrifolia) fruits has shown potent inhibitory activity on gut bacterial ß-glucuronidase, which could help reduce irinotecan-induced diarrhea. In this study, four bacterial ß-glucuronidase inhibitors were obtained following bioactive assay-guided isolation, including two sesquineolignans, (7S,8S,7'R,8'R)-isoamericanol B (1) and americanol B (2), and two dineolignans, moricitrins A (3) and B (4). Compounds 2-4 are new, and the absolute configuration of compound 1 was determined for the first time. Their chemical structures were elucidated through HRESIMS and NMR spectra, and their absolute configurations were established via the comparison of the experimental and calculated electronic circular dichroism spectra. These compounds showed potent inhibition against gut bacterial ß-glucuronidase with IC50 values in the range 0.62-6.91 µM. The inhibition presented specificity for ß-glucuronidase, as all the compounds showed no or weak effects on digestive enzymes such as α-amylase, α-glucosidase, and lipase, suggesting that their gastrointestinal side effects could be minimized. These specific inhibitors as naturally occurring dietary compounds may be developed as promising candidates to alleviate irinotecan-induced diarrhea.

The Flavonoid Kaempferol Ameliorates Streptozotocin-Induced Diabetes by Suppressing Hepatic Glucose Production.

In diabetes mellitus, the excessive rate of glucose production from the liver is considered a primary contributor for the development of hyperglycemia, in particular, fasting hyperglycemia. In this study, we investigated whether kaempferol, a flavonol present in several medicinal herbs and foods, can be used to ameliorate diabetes in an animal model of insulin deficiency and further explored the mechanism underlying the anti-diabetic effect of this flavonol. We demonstrate that oral administration of kaempferol (50 mg/kg/day) to streptozotocin-induced diabetic mice significantly improved hyperglycemia and reduced the incidence of overt diabetes from 100% to 77.8%. This outcome was accompanied by a reduction in hepatic glucose production and an increase in glucose oxidation in the muscle of the diabetic mice, whereas body weight, calorie intake, body composition, and plasma insulin and glucagon levels were not altered. Consistently, treatment with kaempferol restored hexokinase activity in the liver and skeletal muscle of diabetic mice while suppressed hepatic pyruvate carboxylase activity and gluconeogenesis. These results suggest that kaempferol may exert antidiabetic action via promoting glucose metabolism in skeletal muscle and inhibiting gluconeogenesis in the liver.

Novel Annonaceous acetogenins from Graviola (Annona muricata) fruits with strong anti-proliferative activity.

Five bioactive Annonaceous acetogenins, including three new compounds, annonamuricins A (1), B (2), and C (3), one registered but no spectral data reported compounds, annonamuricin D (4), and one known compound annonacin (5) were isolated from Graviola fruit (Annona muricata) and further determined through bioassay-guided fractionation. All five compounds are C35 Anonnonaceous acetogenins with a mono-tetrahydrofuran ring and four hydroxyls. Their structures were elucidated using spectral methods as well as chemical modification after isolation via chromatographic techniques and HPLC purification. These acetogenins demonstrated potent anti-proliferative activities against human prostate cancer PC-3 cells.

Isolation of three new annonaceous acetogenins from Graviola fruit (Annona muricata) and their anti-proliferation on human prostate cancer cell PC-3.

Bioassay-guided fractionation of the fruit powder of Graviola (Annona muricata) was continued to be conducted and yielded three more novel bioactive compounds: C-35 annonaceous acetogenins, muricins M and N, and C-37 annonaceous acetogenins, muricenin. They all contain a mono-tetrahydrofuran ring and four hydroxyl groups. The structures were elucidated by spectral methods and chemical modification after isolation via open column chromatographic separation and HPLC purification. Especially, murices M and N demonstrated more potent anti-proliferative activities against human prostate cancer PC-3 cells.

A potent antimicrobial compound isolated from Clathria cervicornis.

The sponge of Clathria cervicornis is commonly used in traditional medicine. This study aims to identify the active compound in C. cervicornis and to evaluate its antimicrobial activity. The purified active compound was determined to be crambescidin 800 and was found to be highly active against Acinetobacter baumannii (minimal inhibitory concentration, MIC=2 µg/ml), Klebsiella pneumoniae (MIC=1 µg/ml) and Pseudomonas aeruginosa (MIC=1 µg/ml). A potent antimicrobial compound, crambescidin 800, was isolated in Clathria cervicornis. It is extremely active against three common pathogenic bacteria.

Polybrominated diphenyl ethers with potent and broad spectrum antimicrobial activity from the marine sponge Dysidea.

Three polybrominated diphenyl ethers, 2-(2',4'-dibromophenoxy)-3,5-dibromophenol (1) and 2-(2',4'-dibromophenoxy)-3,4,5-tribromophenol (2) were isolated from the marine sponge Dysidea granulosa; and 2-(2',4'-dibromophenoxy)-4,6-dibromophenol (3) from Dysidea spp. They exhibited potent and broad spectrum in vitro antibacterial activity, especially against methicillin resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA), Escherichia coli O157:H7, and Salmonella. Minimal inhibitory concentration (MIC) was evaluated against 12 clinical and standard strains of Gram positive and negative bacteria. The observed MIC range was 0.1-4.0mg/L against all the Gram positive bacteria and 0.1-16.0mg/L against Gram negative bacteria. 2-(2',4″-Dibromophenoxy)-3,5-dibromophenol showed stronger broad spectrum antibacterial activity than other two compounds. 2-(2',4″-Dibromophenoxy)-3,5-dibromophenol and 2-(2',4'-dibromophenoxy)-4,6-dibromophenol are thermo-stable. The results suggest that 2-(2',4'-dibromophenoxy)-3,5-dibromophenol could be used as a potential lead molecule for anti-MRSA, anti-E. coli O157:H7, and anti-Salmonella for drug development.

Three new anti-proliferative Annonaceous acetogenins with mono-tetrahydrofuran ring from graviola fruit (Annona muricata).

Bioassay-guided fractionation of the fruit powder of graviola (Annona muricata) yielded three novel compounds: muricins J, K, and L. The compounds are all C35 Annonaceous acetogenins with a mono-tetrahydrofuran ring and four hydroxyls. Their structures were elucidated by spectral methods and chemical modification after isolation via chromatographic techniques and HPLC purification. These three acetogenins demonstrated an antiproliferative against human prostate cancer PC-3 cells.

Beneficial effects of a combination of Clostridium cochlearium and Lactobacillus acidophilus on body weight gain, insulin sensitivity, and gut microbiota in high-fat diet-induced obese mice.

OBJECTIVES: Species Lactobacillus acidophilus and butyrate producer Clostridium cochlearium have been shown to have potential antiobesity effects. The aim of this study was to show that the combination of C. cochlearium and L. acidophilus (CC-LA) has beneficial effects on body weight control and glucose homeostasis in high-fat diet-induced obese (DIO) mice. METHODS: In this study, thirty-six 6-wk-old male C57BL/6 mice were randomly assigned to three groups of 12 mice each. The experimental group (CC-LA) was administered with CC-LA mixture and fed ad libitum with a high-fat diet. High-fat diet (HF) control and low-fat diet (LF) control groups were treated with the same dose of sterile water as the CC-LA group. RESULTS: After 17 wk of dietary intervention, the CC-LA group showed 17% less body weight gain than the HF group did (P < 0.01). The CC-LA group also showed significantly reduced incremental area under the curve of oral glucose tolerance test and homeostatic model assessment for insulin resistance compared with the HF group. The results from 16S rRNA sequencing analysis of gut microbiota showed that the CC-LA administration led to overall increased α-diversity indices, and a significant microbial separation from the HF group. The ratio of Firmicutes to Bacteroidetes (F/B) was reduced from 3.30 in the HF group to 1.94 in the CC-LA group. The relative abundances of certain obesity-related taxa were also decreased by CC-LA administration. CONCLUSION: The present study provided evidence that the CC-LA combination reduced obesity and improved glucose metabolism in high-fat diet-treated DIO mice, potentially mediated by the modulation of gut microbiota.

Selective growth inhibition of human breast cancer cells by graviola fruit extract in vitro and in vivo involving downregulation of EGFR expression.

The epidermal growth factor receptor (EGFR) is an oncogene frequently overexpressed in breast cancer (BC), and its overexpression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. This study demonstrated that a graviola fruit extract (GFE) significantly downregulated EGFR gene expression and inhibited the growth of BC cells and xenografts. GFE selectively inhibited the growth of EGFR-overexpressing human BC (MDA-MB-468) cells (IC(50) = 4.8 µg/ml) but had no effect on nontumorigenic human breast epithelial cells (MCF-10A). GFE significantly downregulated EGFR mRNA expression, arrested cell cycle in the G0/G1 phase, and induced apoptosis in MDA-MB-468 cells. In the mouse xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet) significantly reduced the protein expression of EGFR, p-EGFR, and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%, respectively. Overall, dietary GFE inhibited tumor growth, as measured by wet weight, by 32% (P < 0.01). These data showed that dietary GFE induced significant growth inhibition of MDA-MB-468 cells in vitro and in vivo through a mechanism involving the EGFR/ERK signaling pathway, suggesting that GFE may have a protective effect for women against EGFR-overexpressing BC.

Ethyl pyruvate inhibits peroxynitrite-induced DNA damage and hydroxyl radical generation: implications for neuroprotection.

Ethyl pyruvate (EP) has recently been reported to afford protection against neurodegenerative disorders. However, the mechanism underlying EP-mediated neuroprotection remains to be elucidated. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of EP is associated with inhibition of peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite elicited cytotoxicity. Incubation of phiX-174 plasmid DNA with 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time- dependent manner. The presence of EP (0.5-10 mM) was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent fashion. The consumption of oxygen induced by 250 microM SIN-1 was found to be decreased in the presence of EP (0.5-10 mM), indicating that EP might affect the auto-oxidation of SIN-1. It was observed that incubation of the plasmid DNA with authentic peroxynitrite caused significant DNA strand breaks, which could also be dramatically inhibited by EP (0.5-10 mM). EPR spectroscopy in combination with spin-trapping technique using 5,5-dimethylpyrroline-N- oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adducts (DMPO-OH) from authentic peroxynitrite, and that EP at 0.5-10 mM inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that EP can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical generation.

Inhibition of peroxynitrite-mediated DNA strand cleavage and hydroxyl radical formation by aspirin at pharmacologically relevant concentrations: implications for cancer intervention.

Epidemiological studies have suggested that the long-term use of aspirin is associated with a decreased incidence of human malignancies, especially colorectal cancer. Since accumulating evidence indicates that peroxynitrite is critically involved in multistage carcinogenesis, this study was undertaken to investigate the ability of aspirin to inhibit peroxynitrite-mediated DNA damage. Peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1) were used to cause DNA strand breaks in phiX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.25-2mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a significant inhibition of DNA cleavage induced by both peroxynitrite and SIN-1. Moreover, the consumption of oxygen caused by 250 microM SIN-1 was found to be decreased in the presence of aspirin, indicating that aspirin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite, and that aspirin at 0.25-2mM potently diminished the radical adduct formation in a concentration-dependent manner. Taken together, these results demonstrate for the first time that aspirin at pharmacologically relevant concentrations can inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. These results may have implications for cancer intervention by aspirin.

Cruciferous dithiolethione-mediated coordinated induction of total cellular and mitochondrial antioxidants and phase 2 enzymes in human primary cardiomyocytes: cytoprotection against oxidative/electrophilic stress and doxorubicin toxicity.

3H-1,2-dithiole-3-thione (D3T), a cruciferous organosulfur compound, induces cytoprotective enzymes in animal cardiovascular cells. However, it remains unknown if D3T also upregulates antioxidants and phase 2 enzymes in human cardiomyocytes, and protects against cell injury induced by oxidative/electrophilic species as well as doxorubicin. In this study, we found that D3T (10-50 muM) potently induced a series of antioxidants and phase 2 enzymes in primary cultured human cardiomyocytes, including superoxide dismutase (SOD), glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx) glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), aldose reductase (AR), and heme oxygenase (HO). D3T treatment also caused elevation of SOD, GSH, GR, GPx and GST in the isolated mitochondria. We also observed a time-dependent induction by D3T of mRNA expression for Cu,ZnSOD, MnSOD, gamma-glutamylcysteine ligase, GR, GSTA1, GSTM1, NQO1, AR, and HO-1. Pretreatment with D3T conferred concentration-dependent protection against cell injury induced by xanthine oxidase (XO)/xanthine, H(2)O(2), 3-morpholinosydnonimine, 4-hydroxy-2-nonenal, and doxorubicin. Pretreatment with D3T also reduced the formation of intracellular reactive oxygen species by XO/xanthine, H(2)O(2), and doxorubicin. In conclusion, this study demonstrated that D3T potently upregulated many antioxidants and phase 2 enzymes in human cardiomyocytes, which was accompanied by increased resistance to oxidative/electrophilic stress and doxorubicin toxicity.

UHPLC/MS Identifying Potent α-glucosidase Inhibitors of Grape Pomace via Enzyme Immobilized Method.

α-Glucosidases have been a major target in controlling and managing postprandial blood glucose and therefore diabetes treatment. This study aims to further identify and purify active compounds from the most active ethyl acetate fraction collected previously in Tinta Cão grape pomace extract (TCEE) using a newly developed and highly effective immobilization method, including obtaining compounds previously shown to inhibit the enzyme. Purification used crosslinked chitosan beads with α-glucosidases bound to polymer, which acted as immobilized enzyme vehicle to collect inhibitors. Compounds absorbed into the beads were eluded using methanol, where collected fraction was subjected to UHPLC-MS analysis to identify active compounds. Results presented 5 major compounds: viniferifuran (amurensin H), p-coumaroyl-6-O-D-glucopyranoside, p-coumaroyl-6-O-hexoside, (epi)catechin-hexoside, 10-carboxyl-pyranopeonidin 3-O-(6''-O-p-coumaroyl)-glucoside. These findings indicated the particular molecules can be utilized as potent α-glucosidases inhibitors, and may be further tested for postprandial glucose control. PRACTICAL APPLICATION: A potential approach enriched and identified α-glucosidase inhibitors of grape pomace. Set-up of UHPLC/MS detection and identification of active compounds provide qualify assessment in developing grape pomace extract into potent dietary supplement and new drug for diabetes.

Sex Differences and Predictors of Changes in Body Weight and Noncommunicable Diseases in a Random, Newly-Arrived Group of Refugees Followed for Two Years.

We have reported that none of the psychological/mental variables examined predicted the increase in BMI and non-communicable diseases (NCDs) in Iraqi refugees after 1 year resettlement in Michigan. We continuously followed the same cohort of refugees for 2 years (Y2 FU) to further determine the gender difference in predicting of increased BMI and NCDs. Only 20% of the BMI variability could be accounted for by the factors examined. Number of dependent children and depression were positively and stress negatively associated with BMI in male refugees but not in females. Number of dependent children was negatively associated with changes in BMI and in males only. Two-third of the NCD variability was accounted for by gender, BMI, employment status, depression, posttraumatic stress disorders and coping skills. Unmarried, unemployed and with high PTSD scores at Y2 in males were positively and number of dependent children was negatively associated with NCD changes in females. Factors such as dietary patterns and lifestyle may have contributed to the increased BMI and NCDs in these refugees at 2 years post-settlement.

Case study on the soil antibiotic resistome in an urban community garden.

Urban agricultural soils can be an important reservoir of antibiotic resistance, and have great food safety and public health indications. This study investigated antibiotic-resistant bacteria and antibiotic resistance genes in urban agricultural soils using phenotypic and metagenomic tools. In total, 207 soil bacteria were recovered from 41 soil samples collected from an urban agricultural garden in Detroit, MI, USA. The most prevalent antibiotic resistance phenotype demonstrated by Gram-negative bacteria was resistance to ampicillin (94.2%), followed by chloramphenicol (80.0%), cefoxitin (79.5%), gentamicin (78.4%) and ceftriaxone (71.1%). All Gram-positive bacteria were resistant to gentamicin, kanamycin and penicillin. Genes encoding resistance to quinolones, ß-lactams and tetracyclines were the most prevalent and abundant in the soil. qepA and tetA, both encoding efflux pumps, predominated in the quinolone and tetracycline resistance genes tested, respectively. Positive correlation (P<0.05) was identified among groups of antibiotic resistance genes, and between antibiotic resistance genes and metal resistance genes. The data demonstrated a diverse population of antibiotic resistance in urban agricultural soils. Phenotypic determination together with soil metagenomics proved to be a valuable tool to study the nature and extent of antibiotic resistance in the environment.

Antioxidant properties of Fusarium head blight-resistant and -susceptible soft red winter wheat grains grown in Virginia.

Fusarium head blight (FHB) has emerged as a major threat to wheat crops around the world, and it has been hypothesized that wheat antioxidants may play a role against Fusarium infections. The current study aimed to determine antioxidant properties of FHB-resistant wheat grains as compared to susceptible wheat. The wheat samples were collected from a single growing location (Warsaw, VA) and the same growing season. The results showed that both FHB-resistant and -susceptible wheat grains exerted strong radical scavenging activities against DPPH* radical [0.91-1.53 micromol of Trolox equivalents (TE)/g], peroxyl radical (15.5-24.5 micromol of TE/g), and hydroxyl radical (15.7-35.8 micromol of TE/g). Their total phenolic contents ranged from 888 to 1117 microg of gallic acid equivalents (GAE)/g. Five phenolic acids including ferulic, syringic, vanillic, caffeic, and p-coumaric acids were determined in soluble and insoluble fractions of wheat grains, altogether with a range of 219-389 microg/g. On average, the FHB-resistant wheat group showed significantly higher average values in DPPH* and hydroxyl radicals scavenging activities (30 and 41% higher, respectively) than the FHB-susceptible wheat group.

Strategies to promote abundance of Akkermansia muciniphila, an emerging probiotics in the gut, evidence from dietary intervention studies.

Akkermansia muciniphila is a mucin-degrading bacterium commonly found in human gut. A. muciniphila has been inversely associated with obesity, diabetes, inflammation, and metabolic disorders. Due to its highly promising probiotic activities against obesity and diabetes, A. muciniphila has drawn intensive interest for research and development in recent years. A number of human and animal studies have shown that the abundance of A. muciniphila in the gut can be enhanced through dietary interventions. The present review focuses on evidence-based dietary strategies of improving A. muciniphila abundance in the gut by critically appraising up-to-date available human and animal intervention studies on A. muciniphila growth and their impact on risk factors of obesity and diabetes. Their potential mechanisms in promoting A. muciniphila are also discussed along with the discussions of mechanism of action for A. muciniphila to exert probiotic functions.

Antibacterial Activity of 2-(3',5'-Dibromo-2'-methoxyphenoxy)-3,5- dibromophenol Isolated from Phyllospongia papyracea.

A principal active antimicrobial compound, 2-(3',5'-dibromo-2'-methoxyphenoxy)-3,5-dibromophenol, was isolated from the methanol extract of Phyllospongiapapyracea via bioassay-guided fractionation and isolation. The crude extract and the purified compound were assayed to determine the minimal - inhibitory concentration and minimal bactericidal concentration (MBC) using the broth. microdilution method. The purified compound was found to be highly active against Bacillus subtilis and Staphylococcus aureus at MIC=1 µg/mL, Campylobacter jejuni at MIC=2 gg/mL, Pseudomonas aeruginosa at MIC=4 µg/mL; and Streptococcus pneumoniae and Listeria monocytogenes at MIC = 8 µg/mL. The activity of this compound was found to be comparable with antibiotics commonly used to control these species of bacteria. The results establish 2-(3',5'-dibromo-2'-methoxyphenoxy)-3,5-dibromopheno as a potential lead molecule for the development of antibacterial agents.