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Miscellaneous: In-Vitro

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Adjuvant effect of cranberry proanthocyanidin active fraction on antivirulent property of ciprofloxacin against Pseudomonas aeruginosa.

Posted: 
August 22, 2016
Authors: 
Vadekeetil A., Alexandar V., Chhibber S., Harjai K.
Journal: 
Microbial Pathogenesis; 2016. 90:98-103
Abstract: 

Quorum sensing inhibitors (QSIs) act as antivirulent agents since quorum sensing (QS) plays a vital role in regulating pathogenesis of Pseudomonas aeruginosa. However, application of single QSI may not be effective as pathogen is vulnerable to successful mutations. In such conditions, combination of QSIs can be exploited as there can be synergistic or adjuvant action. In the present study, we evaluated the antivirulence efficacy of combination of Vaccinium macrocarpon proanthocyanidin active fraction (PAF) and ciprofloxacin (CIP) at their sub-MICs using standard methods followed by analysis of their mode of action on QS using TLC and molecular docking. There was significant improvement in action of CIP when it was combined with PAF in reducing the QS controlled virulence factors (p<0.05), motilities and biofilm of P. aeruginosa. TLC profiles of QS signals [(Acyl homoserine lactone (AHL) and Pseudomonas quinolone signal (PQS))] indicated that CIP in combination with PAF, besides showing inhibitory action on production of AHLs, also modulated production and inactivation of PQS. Docking scores also supported the observation. We therefore hypothesize that PAF-CIP combination, having improved anti-virulence property; can be exploited as a potent drug pairing against P. aeruginosa.

Antibacterial activity of isolated phenolic compounds from cranberry (Vaccinium macrocarpon) against Escherichia coli.

Posted: 
August 22, 2016
Authors: 
Rodriguez-Perez, C. Quirantes-Pine, R. Uberos, J. Jimenez-Sanchez, C. Pena, A. Segura-Carretero, A.
Journal: 
Food and Function; 2016. 7(3):1564-1573.
Abstract: 

Phenolic compounds from a cranberry extract were isolated in order to assess their contribution to the antibacterial activity against uropathogenic strains of Escherichia coli (UPEC). With this purpose, a total of 25 fractions from a cranberry extract were isolated using semipreparative high performance liquid chromatography (HPLC) and characterized based on the results obtained by reversed-phase HPLC coupled to mass spectrometry detection. Then, the effects on UPEC surface hydrophobicity and biofilm formation of the cranberry extract as well as the purest fractions (a total of 13) were tested. As expected, the whole extract presented a powerful antibacterial activity against UPEC while the selected fractions presented a different behavior. Myricetin and quercitrin significantly decreased (p <0.05) E. coli biofilm formation compared with the control, while dihydroferulic acid glucuronide, procyanidin A dimer, quercetin glucoside, myricetin and prodelphinidin B led to a significant decrease of the surface hydrophobicity compared with the control. The results suggest that apart from proanthocyanidins, other compounds, mainly flavonoids, can act against E. coli biofilm formation and also modify UPEC surface hydrophobicity in vitro, one of the first steps of adhesion.

Cranberry derivatives enhance biofilm formation and transiently impair swarming motility of the uropathogen Proteus mirabilis HI4320.

Posted: 
August 22, 2016
Authors: 
O'May, C. Amzallag, O. Bechir, K. Tufenkji, N.
Journal: 
Can J Microbiol; 2016. 62(6):464-474.
Abstract: 

Proteus mirabilis is a major cause of catheter-associated urinary tract infection (CAUTI), emphasizing that novel strategies for targeting this bacterium are needed. Potential targets are P. mirabilis surface-associated swarming motility and the propensity of these bacteria to form biofilms that may lead to catheter blockage. We previously showed that the addition of cranberry powder (CP) to lysogeny broth (LB) medium resulted in impaired P. mirabilis swarming motility over short time periods (up to 16 h). Herein, we significantly expanded on those findings by exploring (i) the effects of cranberry derivatives on biofilm formation of P. mirabilis, (ii) whether swarming inhibition occurred transiently or over longer periods more relevant to real infections (~3 days), (iii) whether swarming was also blocked by commercially available cranberry juices, (iv) whether CP or cranberry juices exhibited effects under natural urine conditions, and (v) the effects of cranberry on medium pH, which is an indirect indicator of urease activity. At short time scales (24 h), CP and commercially available pure cranberry juice impaired swarming motility and repelled actively swarming bacteria in LB medium. Over longer time periods more representative of infections (~3 days), the capacity of the cranberry material to impair swarming diminished and bacteria would start to migrate across the surface, albeit by exhibiting a different motility phenotype to the regular "bull's-eye" swarming phenotype of P. mirabilis. This bacterium did not swarm on urine agar or LB agar supplemented with urea, suggesting that any potential application of anti-swarming compounds may be better suited to settings external to the urine environment. Anti-swarming effects were confounded by the ability of cranberry products to enhance biofilm formation in both LB and urine conditions. These findings provide key insights into the long-term strategy of targeting P. mirabilis CAUTIs.

Cranberry proanthocyanidins modulate reactive oxygen species in Barrett's and esophageal adenocarcinoma cell lines.

Posted: 
August 22, 2016
Authors: 
Weh, K. M. Aiyer, H. S. Howell, A. B. Kresty, L. A.
Journal: 
Journal of Berry Research; 2016. 6(2):125-136.
Abstract: 

BACKGROUND: We recently reported that a cranberry proanthocyanidin rich extract (C-PAC) induces autophagic cell death in apoptotic resistant esophageal adenocarcinoma (EAC) cells and necrosis in autophagy resistant cells. EAC is characterized by high morbidity and mortality rates supporting development of improved preventive interventions. OBJECTIVE: The current investigation sought to investigate the role of reactive oxygen species (ROS) in the context of C-PAC induced cell death. METHODS: Apanel of human esophageal cell lines of EAC or BE (Barrett's esophagus) origin were treated with C-PAC and assessed for ROS modulation using CellROXReg. Green reagent and the Amplex Red assay to specifically measure hydrogen peroxide levels. RESULTS: C-PAC significantly increased ROS levels in EAC cells, but significantly reduced ROS levels in CP-C BE cells. Increased hydrogen peroxide levels were also detected in C-PAC treated EAC cells and supernatant; however, hydrogen peroxide levels were significantly increased in medium alone, without cells, suggesting that C-PAC interferes or directly acts on the substrate. Hydrogen peroxide levels did not change in C-PAC treated CP-C BE cells. CONCLUSION: These experiments provide additional mechanistic insight regarding C-PAC induced cancer cell death through modulation of ROS. Additional research is warranted to identify specific ROS species associated with C-PAC exposure.

Critical reevaluation of the 4-(dimethylamino)cinnamaldehyde assay: cranberry proanthocyanidin standard is superior to procyanidin A2 dimer for accurate quantification of proanthocyanidins in cranberry products.

Posted: 
August 22, 2016
Authors: 
Krueger, C. G. Chesmore, N. Chen Xin Parker, J. Khoo, C. Marais, J. P. J. Shanmuganayagam, D. Crump, P. Reed, J. D.
Journal: 
Journal of Functional Foods; 2016. 22:13-19.
Abstract: 

The 4-(dimethylamino)cinnamaldehyde (DMAC) assay is currently used to quantify proanthocyanidin (PAC) content in cranberry products. In a multi-operator/multi-day study design, a cranberry proanthocyanidin (c-PAC) standard was compared to procyanidin A2 (ProA2) dimer for accurate quantification of PAC in commercial cranberry juices, lab generated cranberry blends and cranberry powders. The c-PAC standard reflects the structural heterogeneity of cranberry PAC degree of polymerization, hydroxylation pattern and ratios of 'A-type' to 'B-type' interflavanyl bonds. Use of the c-PAC standard to quantify PAC content in cranberry samples resulted in values that were 3.6 times higher than those determined by ProA2. Overall, there was no effect (P>0.05) of operator or day on estimation of PAC concentration. The adoption of c-PAC standard should be considered as an improvement over the use of ProA2 for accurate quantification of cranberry PAC. Improved standardization of bioactive PAC components in functional cranberry foods will aid in establishment of dosage guidelines.

Effect of cranberry (Vaccinium macrocarpon) oligosaccharides on the formation of advanced glycation end-products.

Posted: 
August 22, 2016
Authors: 
Sun J, Liu W, Ma H, Marais JPJ, Khoo C, Dain JA, Rowley DC, Seeram NP
Journal: 
Journal of Berry Research; 2016. 6(2):149-158
Abstract: 

BACKGROUND: The formation and accumulation of advanced glycation end-products (AGEs) are implicated in several chronic human illnesses including type-2 diabetes, renal failure, and neurodegenerative diseases. The cranberry (Vaccinium macrocarpon) fruit has been previously reported to show anti-AGEs effects, attributed primarily to its phenolic constituents. However, there is lack of similar data on the non-phenolic constituents found in the cranberry fruit, in particular, its carbohydrate constituents. Herein, a chemically characterized oligosaccharide-enriched fraction purified from the cranberry fruit was evaluated for its potential anti-AGEs and free radical scavenging effects. OBJECTIVE: The aim of this study was to evaluate the in vitro anti-AGEs and free radical scavenging effects of a chemically characterized oligosaccharide-enriched fraction purified from the North American cranberry (Vaccinium macrocarpon) fruit. METHOD: The cranberry oligosaccharide-enriched fraction was purified from cranberry hull powder and characterized based on spectroscopic and spectrometric (NMR, MALDI-TOF-MS, and HPAEC-PAD) data. The oligosaccharide-enriched fraction was evaluated for its anti-AGEs and free radical scavenging effects by the bovine serum albumin-fructose, and DPPH assays, respectively. RESULTS: Fractionation of cranberry hull material yielded an oligosaccharide-enriched fraction named Cranf1b-CL. The 1H NMR and MALDI-TOF-MS revealed that Cranf1b-CL consists of oligosaccharides ranging primarily from 6-mers to 9-mers. The monosaccharide composition of Cranf1b-CL was arabinose (25%), galactose (5%), glucose (47%) and xylose (23%). In the bovine serum albumin-fructose assay, Cranf1b-CL inhibited AGEs formation in a concentration-dependent manner with comparable activity to the synthetic antiglycating agent, aminoguanidine, used as the positive control (57 vs. 75%; both at 500 micro g/mL). In the DPPH free radical scavenging assay, Cranf1b-CL showed superior activity to the synthetic commercial antioxidant, butylated hydroxytoluene, used as the positive control (IC50=680 vs. 2200 micro g/mL, respectively). CONCLUSION: The in vitro anti-AGEs and free radical scavenging effects of cranberry oligosaccharides support previous data suggesting that these constituents may also contribute to biological effects of the whole fruit beyond its phenolic constituents alone. Also, this is the first study to evaluate a chemically characterized oligosaccharide fraction purified from the North American cranberry fruit for anti-AGEs and free radical scavenging properties.

Effect of glycated albumin and cranberry components on interleukin-6 and matrix metalloproteinase-3 production by human gingival fibroblasts

Posted: 
August 22, 2016
Authors: 
Tipton DA; Hatten AA; Babu JP; Dabbous MKh.
Journal: 
Journal of Periodontal Research. 51(2):228-36
Abstract: 

BACKGROUND AND OBJECTIVE: Gingival fibroblasts have the potential to participate in periodontal inflammation and breakdown, producing interleukin (IL)-6 and matrix metalloproteinase (MMP)-3. Advanced glycation end products (AGEs), formed during diabetic hyperglycemia, might aggravate periodontal inflammation. The cranberry contains anti-inflammatory polyphenols, which inhibit proinflammatory activities of lipopolysaccharide (LPS)- and IL-1beta-stimulated human cells. Little is known of its effects on gingival fibroblast IL-6 or MMP-3 production stimulated by AGEs. The objectives were to determine cranberry effects on IL-6 and MMP-3 production by gingival fibroblasts exposed to the representative AGE, glycated human serum albumin (G-HSA), or LPS +/- G-HSA. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), was derived from cranberry juice. Normal human gingival fibroblasts were incubated with G-HSA or normal HSA or Porphyromonas gingivalis LPS (1 mug/mL) +/- G-HSA, in the presence or absence of preincubation with NDM. IL-6 and MMP-3 were measured by enzyme-linked immunosorbent assay. Data were analyzed using one-way analysis of variance and Scheffe's F procedure. RESULTS: IL-6 production was stimulated by G-HSA or LPS (p

Effects of cranberry components on IL-1beta-stimulated production of IL-6, IL-8 and VEGF by human TMJ synovial fibroblasts.

Posted: 
August 22, 2016
Authors: 
Tipton DA; Christian J; Blumer A.
Journal: 
Archives of Oral Biology. 68:88-96, 2016 Aug
Abstract: 

OBJECTIVE: Osteoarthritis (OA) in the TMJ is characterized by deterioration of articular cartilage and secondary inflammatory changes. Interleukin-1beta (IL-1beta) stimulates IL-6, IL-8, and vascular endothelial growth factor (VEGF) in synovial fluid of TMJ with internal derangement and bony changes. The cranberry (Vaccinium macrocarpon) contains polyphenolic compounds that inhibit production of pro-inflammatory molecules by gingival cells in response to several stimulators. This study examined effects of cranberry components on IL-1beta-stimulated IL-6, IL-8, and VEGF production by human TMJ synovial fibroblast-like cells. DESIGN: Cranberry high molecular weight non-dialyzable material (NDM) was derived from cranberry juice. Human TMJ synovial fibroblast-like cells from joints with degenerative OA and an ankylosed TMJ without degeneration were incubated with IL-1beta (0.001-1nM)+/-NDM (25-250mug/ml) (2h preincubation). Viability was assessed via activity of a mitochondrial enzyme. IL-6, IL-8, and VEGF in culture supernatants were measured by ELISA; NF-kappaB and AP-1 transcription factors were measured in nuclear extracts via binding to specific oligonucleotides. DATA ANALYSIS: ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: NDM did not affect cell viability but inhibited IL-1beta stimulated IL-6, IL-8, and VEGF production in all cell lines (p

Inhibition of herpes simplex type 1 and type 2 infections by Oximacro, a cranberry extract with a high content of A-type proanthocyanidins (PACs-A)

Posted: 
August 22, 2016
Authors: 
Terlizzi ME; Occhipinti A; Luganini A; Maffei ME; Gribaudo G.
Journal: 
Antiviral Research. 132 (pp 154-164),
Abstract: 

In the absence of efficient preventive vaccines, topical microbicides offer an attractive alternative in the prevention of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Because of their recognized anti-adhesive activity against bacterial pathogens, cranberry (Vaccinium macrocarpon Ait.) extracts may represent a natural source of new antiviral microbicides. However, few studies have addressed the applications of cranberry extract as a direct-acting antiviral agent. Here, we report on the ability of the novel cranberry extract Oximacro and its purified A-type proanthocyanidins (PACs-A), to inhibit HSV-1 and HSV-2 replication in vitro. Analysis of the mode of action revealed that Oximacro prevents adsorption of HSV-1 and HSV-2 to target cells. Further mechanistic studies confirmed that Oximacro and its PACs-A target the viral envelope glycoproteins gD and gB, thus resulting in the loss of infectivity of HSV particles. Moreover, Oximacro completely retained its anti-HSV activity even at acidic pHs (3.0 and 4.0) and in the presence of 10% human serum proteins; conditions that mimic the physiological properties of the vagina - a potential therapeutic location for Oximacro. Taken together, these findings indicate Oximacro as an attractive candidate for the development of novel microbicides of natural origin for the prevention of HSV infections.

Adherence Reduction of Campylobacter jejuni and Campylobacter coli Strains to HEp-2 Cells by Mannan Oligosaccharides and a High-Molecular-Weight Component of Cranberry Extract.

Posted: 
March 23, 2016
Authors: 
Ramirez-Hernandez A, Rupnow J, Hutkins RW
Journal: 
J Food Prot 78(8):1496-505
Abstract: 

Campylobacter infections are a leading cause of human bacterial gastroenteritis in the United States and are a major cause of diarrheal disease throughout the world. Colonization and subsequent infection and invasion of Campylobacter require that the bacteria adhere to the surface of host cells. Agents that inhibit adherence could be used prophylactically to reduce Campylobacter carriage and infection. Mannan oligosaccharides (MOS) have been used as a feed supplement in livestock animals to improve performance and to replace growth-promoting antibiotics. However, MOS and other nondigestible oligosaccharides may also prevent pathogen colonization by inhibiting adherence in the gastrointestinal tract. In addition, plant extracts, including those derived from cranberries, have been shown to have antiadherence activity against pathogens. The goal of this study was to assess the ability of MOS and cranberry fractions to serve as antiadherence agents against strains of Campylobacter jejuni and Campylobacter coli. Adherence experiments were performed using HEp-2 cells. Significant reductions in adherence of C. jejuni 29438, C. jejuni 700819, C. jejuni 3329, and C. coli 43485 were observed in the presence of MOS (up to 40 mg/ml) and with a high-molecular-weight fraction of cranberry extract (up to 3 mg/ml). However, none of the tested materials reduced adherence of C. coli BAA-1061. No additive effect in adherence inhibition was observed for an MOS-cranberry blend. These results suggest that both components, MOS and cranberry, could be used to reduce Campylobacter colonization and carriage in livestock animals and potentially limit human exposure to this pathogen.

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