Differences in P-Type and Type 1 Uropathogenic Escherichia coli Urinary Anti-Adhesion Activity of Cranberry Fruit Juice Dry Extract Product and D-Mannose Dietary Supplement.

BACKGROUND: Urinary tract infection (UTI) prevention benefits of cranberry intake are clinically validated, especially for women and children. To ensure the benefits of cranberry dietary supplement products, the anti-adhesion activity (AAA) against uropathogenic bacteria is routinely used in in vitro bioassays to determine the activity in whole product formulations, isolated compounds, and ex vivo bioassays to assess urinary activity following intake. D-mannose is another dietary supplement taken for UTI prevention, based on the anti-adhesion mechanism. OBJECTIVE: Compare the relative AAA of cranberry and D-mannose dietary supplements against the most important bacterial types contributing to the pathogenesis of UTI, and consider how certain components potentially induce in vivo activity. METHODS: The current study used a crossover design to determine ex vivo AAA against both P- and Type 1-fimbriated uropathogenic Escherichia coli of either D-mannose or a cranberry fruit juice dry extract product containing 36 mg of soluble proanthocyanidins (PACs), using bioassays that measure urinary activity following consumption. AAA of extracted cranberry compound fractions and D-mannose were compared in vitro and potential induction mechanisms of urinary AAA explored. RESULTS: The cranberry dietary supplement exhibited both P-type and Type 1 in vitro and ex vivo AAA, while D-mannose only prevented Type 1 adhesion. Cranberry also demonstrated more robust and consistent ex vivo urinary AAA than D-mannose over each 1-week study period at different urine collection time points. The means by which the compounds with in vitro activity in each supplement product could potentially induce the AAA in urines was discussed relative to the data. CONCLUSIONS: Results of the current study provide consumers and healthcare professionals with additional details on the compounds and mechanisms involved in the positive, broad-spectrum AAA of cranberry against both E. coli bacterial types most important in UTIs and uncovers limitations on AAA and effectiveness of D-mannose compared to cranberry.

Improved in vitro Hemagglutination Assays Utilizing P-Type and Type 1 Uropathogenic Escherichia coli to Evaluate Bacterial Anti-Adhesion Activity of Cranberry Products.

Cranberries have a long history of use in the prevention of urinary tract infections. Cranberry products vary in proanthocyanidin content, a compound implicated in preventing the adhesion of uropathogenic Escherichia coli (E. coli) to uroepithelial cells. Testing is routinely done by cranberry product formulators to evaluate in vitro bacterial anti-adhesion bioactivity, shelf-life, and potential efficacy of cranberry products for consumer use to maintain urinary tract health. Hemagglutination assays evaluate the anti-adhesion bioactivity of cranberry products by determining how effectively the products prevent agglutination of specific red blood cells with E. coli expressing P-type and Type 1 fimbriae. The current study sought to improve upon an established anti-adhesion assay method by expanding the number of E. coli strains used to broaden potential in vivo efficacy implications and presenting results using photomicrographic data to improve accuracy and build databases on products that are routinely tested. Different lots of cranberry powder ingredient and two formulated products were tested independently for anti-adhesion activity using the established method and the improved method. Positive harmonization of results on the same samples using rigorous controls was achieved and provides the substantiation needed for the cranberry industry to utilize the improved, rapid in vitro testing method to standardize cranberry products for sufficient anti-adhesion bioactivity and maintain consumer confidence.

Cranberry proanthocyanidins composite electrospun nanofibers as a potential alternative for bacterial entrapment applications.

The interaction between A-type interflavan bonds from cranberry proanthocyanidins (PAC) and surface virulence factors of extra-intestinal pathogenic Escherichia coli (ExPEC) was studied. Electrospun nanofibers (ESNF) were fabricated using PAC and polycaprolactone (PCL) solutions and their physical and chemical properties were characterized. The ability of PAC:PCL composite ESNF to interact with and entrap ExPEC strain 5011 (ExPEC-5011) was evaluated in vitro by plate culturing and when formulated as a biofilter and nanocoating. As a biofilter, the PAC:PCL ESNF exhibited a dose-dependent ability to entrap ExPEC-5011. Images from scanning electron and fluorescent microscopies revealed that ESNF sections with higher amounts of PAC led to higher bacterial entrapment. The effectiveness PAC:PCL ESNF to bind ExPEC when applied as a nanocoating was studied using ESNF-coated polyvinyl chloride intermittent catheter. Results indicate that ExPEC-5011 was entrapped well into the PAC:PCL ESNF coating on the catheter. Overall, our results suggest that incorporating the biomolecule PAC in ESNF is a potential means for applications requiring bacterial entrapment, such as biofunctionalization, biofiltration, and surface coating, among others.

Synthesis of Fluorescent Proanthocyanidin-Cinnamaldehydes Pyrylium Products for Microscopic Detection of Interactions with Extra-Intestinal Pathogenic Escherichia coli.

Synthesis of proanthocyanidin-cinnamaldehydes pyrylium products (PCPP) was achieved by the condensation reaction of proanthocyanidins (PAC) with cinnamaldehyde and four cinnamaldehyde derivatives. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra of PCPP show masses that correspond to (epi)catechin oligomers attached to single, double, or triple moieties of cinnamaldehydes. Synthesized PCPP exhibited fluorescence at higher excitation and emission wavelengths than PAC. Results indicate that PCPP were more bioactive for agglutinating extra-intestinal pathogenic Escherichia coli (ExPEC) compared to PAC. Scanning electron microscopy indicates that PCPP interact with ExPEC surface structures and suggests that PCPP have a higher affinity with the fimbriae-like structures of ExPEC than PAC. Fluorescent microscopy performed on in vitro and in vivo agglutination assays show that PCPP were entrapping ExPEC in a web-like network, thus demonstrating agglutination of ExPEC. This study demonstrated the potential of PCPP to improve our understanding of the temporal and dynamic interactions of PAC in in vitro and in vivo studies.

Cranberry Proanthocyanidins-PANI Nanocomposite for the Detection of Bacteria Associated with Urinary Tract Infections.

Consumption of cranberries is associated with the putative effects of preventing urinary tract infections (UTIs). Cranberry proanthocyanidins (PAC) contain unusual double A-type linkages, which are associated with strong interactions with surface virulence factors found on UTI-causing bacteria such as extra-intestinal pathogenic Escherichia coli (ExPEC), depicting in bacterial agglutination processes. In this work, we demonstrated the efficacy of cranberry PAC (200 µg/mL) to agglutinate ExPEC (5.0 × 108 CFU/mL) in vitro as a selective interaction for the design of functionalized biosensors for potential detection of UTIs. We fabricated functionalized screen-printed electrodes (SPEs) by modifying with PAC-polyaniline (PANI) nanocomposites and tested the effectiveness of the PAC-PANI/SPE biosensor for detecting the presence of ExPEC in aqueous suspensions. Results indicated that the PAC-PANI/SPE was highly sensitive (limit of quantification of 1 CFU/mL of ExPEC), and its response was linear over the concentration range of 1-70,000 CFU/mL, suggesting cranberry PAC-functionalized biosensors are an innovative alternative for the detection and diagnosis of ExPEC-associated UTIs. The biosensor was also highly selective, reproducible, and stable.

Identification of A-Type Proanthocyanidins in Cranberry-Based Foods and Dietary Supplements by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry, First Action Method: 2019.05.

BACKGROUND: Cranberry proanthocyanidins (c-PAC) are oligomeric structures of flavan-3-ol units, which possess A-type interflavan bonds. c-PAC differs from other botanical sources because other PAC mostly have B-type interflavan bonds. Cranberry products used to alleviate and prevent urinary tract infections may suffer from adulteration, where c-PAC are replaced with less expensive botanical sources of PAC that contain B-type interflavan bonds. OBJECTIVE: Identifying the presence of A-type interflavan bonds in cranberry fruit and dietary supplements. METHODS: Thirty-five samples reported to contain A-type PAC (cranberry fruit and cranberry products) and 36 samples reported to contain B-type PAC (other botanical sources) were identified and differentiated using MALDI-TOF MS, deconvolution of overlapping isotope patterns, and principal component analysis (PCA). RESULTS: Our results show that both MALDI-TOF MS and deconvolution of overlapping isotope patterns were able to identify the presence of A-type interflavan bonds with a probability greater than 90% and a confidence of 95%. Deconvolution of MALDI-TOF MS spectra also determined the ratio of A-type to B-type interflavan bonds at each degree of polymerization in cranberry fruit and cranberry products, which is a distinguishing feature of c-PAC in comparison to other botanical sources of PAC. PCA shows clear differences based on the nature of the interflavan bonds. CONCLUSIONS: MALDI-TOF MS, deconvolution of overlapping isotope patterns of MALDI-TOF MS spectra, and PCA allow the identification, estimation, and differentiation of A-type interflavan bonds in cranberry-based foods and dietary supplements among other botanical sources containing mostly B-type interflavan bonds.

Classification of proanthocyanidin profiles using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra data combined with multivariate analysis.

Proanthocyanidin (PAC) profiles of apples (a-PAC), cranberries (c-PAC), and peanut skins (p-PAC) were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Deconvolution of overlapping isotopic patterns indicated that in apples, only 5% of the PAC oligomers contain one or more A-type bonds, whereas in cranberries and peanut skins, 96% of the PAC oligomers contain one or more A-type bonds. MALDI-TOF MS data combined with multivariate analysis, such as principal component analysis (PCA) and linear discriminant analysis (LDA), were used to differentiate and discriminate a-PAC, c-PAC, and p-PAC from one another. Mixtures of c-PAC with either a-PAC or p-PAC at different w/w ratios were evaluated by LDA modeling. The LDA model classified the training, testing, and validation sets with 99.4%, 100%, and 94.2% accuracy. Results suggest that MALDI-TOF MS and multivariate analysis are useful in determining authenticity of PAC from different sources and mixtures of PAC sources.

Inter-Laboratory Validation of 4-(Dimethylamino) Cinnamaldehyde (DMAC) Assay Using Cranberry Proanthocyanidin Standard for Quantification of Soluble Proanthocyanidins in Cranberry Foods and Dietary Supplements, First Action Official MethodSM: 2019.06.

BACKGROUND: Proanthocyanidins (PAC) are oligomers and polymers of flavan-3-ols with putative health benefits. PAC are prevalent in a wide variety of natural products and dietary supplements. OBJECTIVE: An inter-laboratory study was conducted to validate the 4-(dimethylamino)cinnamaldehyde (DMAC) colorimetric assay using a 96-well plate spectrophotometer for the accurate quantification of PAC in cranberry products and to evaluate the comparison of the procyanidin A2 (ProA2) dimer and cranberry PAC (c-PAC) reference standards. METHODS: Four test materials analyzed in this study included cranberry fiber powder, cranberry extract powder, concentrated cranberry juice, and a solution of cranberry PAC (30%, w/v). The samples were homogenized, extracted, sonicated, centrifuged, and analyzed using a 96-well plate spectrophotometer. RESULTS: Linearity for both the ProA2 and c-PAC standards was determined from 4.053 to 50.666 µg/mL and from 13.520 to 135.95 µg/mL, respectively. The relative standard deviation of repeatability (RSDr) values for the four materials analyzed, using both ProA2 and c-PAC standards, met the Standard Method Performance Requirements (SMPR®). Inter-laboratory precision using Horwitz ratio (HorRat) values for the four materials analyzed, using both ProA2 and c-PAC standards, satisfies the acceptance range in Appendix K of the Official Methods of Analysis (2003): Guidelines for Dietary Supplements and Botanicals. The limit of quantification (LOQ) was estimated to be 3.16 µg/mL. CONCLUSIONS: The results produced from this study demonstrate the utility of the c-PAC standard over the ProA2 standard and the advantages of using a 96-well plate spectrophotometer for the accurate quantification of PAC. HIGHLIGHTS: The use of a 96-well plate reader and c-PAC reference standard in the DMAC method improves accuracy and percision for quantification of soluble proanthocyanidins in cranberry foods and dietary supplements.

Effect of birdsfoot trefoil cultivars on exsheathment of Haemonchus contortus in fistulated sheep.

Proanthocyanidin (PAC, condensed tannin) containing forages have well-documented anti-parasitic effects against gastrointestinal nematodes (GIN) of small ruminants. Although extensive research has been conducted on the inhibition of exsheathment of the L3 stage of Haemonchus contortus by in vitro exposure to the extracts of PAC containing plants, only one study has previously attempted to replicate this process in vivo and it was found that consumption of fresh sainfoin slowed the exsheathment rate. No similar studies have explored the effect of feeding condensed tannin forages in the form of hay on in vivo exsheathment of GIN. Another PAC containing forage, birdsfoot trefoil (Lotus corniculatus, BFT), has a large area of adaptation globally and feeding BFT has been shown to reduce fecal egg counts and total worm burdens. However, its effect on the in vivo exsheathment of H. contortus in the rumen is unknown. Recent work from this laboratory showed that BFT populations differ in the ability of aqueous extracts of freeze-dried plants to reduce exsheathment of H. contortus in vitro, and that the reduced exsheathment caused by BFT populations did not directly correlate with PAC content. Therefore, the objective of this study was twofold: 1) to evaluate the ability of birdsfoot trefoil hay to impair ruminal exsheathment of H. contortus in vivo and 2) to measure the difference in exsheathment between three commercially available cultivars of birdsfoot trefoil representing a broad range of in vitro efficacy against H. contortus. Four rumen fistulated ewes were fed three cultivars of birdsfoot trefoil (cv. Bruce, Empire, and Pardee) hay or a control hay (alfalfa/grass hay) in a Latin 4 × 4 design. The effect of consumption of birdsfoot trefoil on the exsheathment of H. contortus larvae in vivo was evaluated. For each exsheathment test, two capsules with 2000 ensheathed third-stage larvae per capsule were placed in the rumen of each ewe for eight hours. Larval containment capsules were made by capping each end of a short piece of Tygon® tubing (ID 9.5 mm, OD 14.3 mm) with an 8 µm NuncTM Cell Culture Insert. Larval exsheathment and motility were examined pre and post rumen exposure. Three exsheathment tests were run per diet cycle. Consumption of BFT hay did not significantly alter larval exsheathment. These results highlight the importance of further in vivo testing on the role of condensed tannins and other plant secondary compounds on larval exsheathment in the rumen.

Antimicrobial proanthocyanidin-chitosan composite nanoparticles loaded with gentamicin.

Cranberry proanthocyanidin-chitosan nanoparticles (PAC-CHT NPs) loaded with antibiotic gentamicin (GEN) (PAC-CHT-GEN NPs) were formulated and characterized according to size, polydispersity (PDI), surface charge, morphology, and encapsulation efficiency (EE). PAC-CHT-GEN NPs were evaluated for their ability to agglutinate E. coli, S. aureus, and P. aeruginosa and their bacteriostatic and bactericidal activity. Results indicate that the PAC-CHT-GEN NPs at 0.5:1.0, 1.0:1.0, and 2.0:1.0 weight ratios formed stable nanoparticles with sizes from 242.9 to 277.4 nm, a PDI from 0.344 to 0.391, and a zeta potential from 34.5 to 38.5 mV, and up to 94% EE. Results indicate that PAC-CHT-GEN NPs have the ability to agglutinate E. coli, S. aureus, and P. aeruginosa. Furthermore, PAC-CHT-GEN NPs exhibited greater bactericidal activity than GEN alone. Results suggested PAC-CHT-GEN NPs form stable, round-shaped, and bioactive nanoparticles with the potential to be use in the treatment of bacterial infections.

Dietary adzuki bean paste dose-dependently reduces visceral fat accumulation in rats fed a normal diet.

The aim of this study was to evaluate the dose-dependent effect of adzuki bean (Vigna angularis) paste (ABP) on visceral fat accumulation in rats. ABP is a rich source of indigestible carbohydrates (18.5%) with fiber and resistant starch (RS) contents of 14.5% and 4.0%, respectively. Animals were fed one of the following diets, control (CON), 30% ABP or 58.9% ABP for 28 days. The daily dietary energy intake was lowered (p < 0.05) and reduced visceral fat accumulation and lower serum lipid levels were observed in ABP fed groups. ABP consumption dose-dependently increased (p < 0.05) the daily fecal lipid and fecal acidic sterol excretions. On the other hand, cecal content and fecal moisture content in the 58.9% ABP group were greater (p < 0.05) than the CON group, while there was no significant difference between the two ABP fed groups. Both 30% and 58.9% ABP diets had significantly (p < 0.05) higher contents of cecal acetic, propionic and n-butyric acids, and lowered cecal pH, independently of the ABP dose. Microbial community data of rats fed ABP diets exhibited higher alpha-diversities than the rats fed CON diet, based on the Shannon Index and the number of observed species index, where the two ABP groups exhibited a similar alpha diversity. The weighted UniFrac-based principal coordinate analysis plot of cecal microbial community data showed that the ABP had a substantial effect on the cecal microbial composition. Furthermore, cecal bacterial 16S rRNA gene sequencing revealed that the ABP supplemented diets decreased the ratio of Firmicutes to Bacteroidetes. These findings suggested that the cecal fermentation of fiber and RS in ABP, might have decreased the energy intake, altered the gut microbiota composition, increased fecal lipid output, and thereby reduced fat accumulation in rats.

Characterization of proanthocyanidin-chitosan interactions in the formulation of composite nanoparticles using surface plasmon resonance.

Chitosan (CHT) interacts with proanthocyanidins (PAC) by a mechanism involving hydrogen bonding and ion-dipole interactions, allowing the spontaneous formation of PAC-CHT composite nanoparticles (PAC-CHT NPs). The interaction between PAC and CHT was characterized by ellipsometry, infrared spectroscopy, and surface plasmon resonance (SPR) to determine the effect of CHT molecular weight (MW), PAC to CHT ratios, and pH on the formulation of PAC-CHT NPs. These parameters also affect the size and morphology of PAC-CHT NPs. Results indicate that CHT MW and pH of the solution impact the interactions of PAC-CHT in two ways: (1) greater CHT MW increases the amount of PAC molecules that attach to the CHT chain, and (2) lower pH of the CHT solutions increases the amount PAC molecules that attach to the CHT chain. Results also show that higher CHT MW, CHT concentration, and pH of the CHT solutions increase the size of PAC-CHT NPs. In contrast, greater PAC concentrations decreases the size of PAC-CHT NPs. This study demonstrates that SPR is a useful technique for measuring the effect of changes in the interaction between PAC and CHT, which in turn affects the size and morphology of PAC-CHT NPs.

Development of a Cranberry Standard for Quantification of Insoluble Cranberry (Vaccinium macrocarpon Ait.) Proanthocyanidins.

Cranberry proanthocyanidins (PACs) can be partitioned into soluble PACs, which are extracted with solvents, and insoluble PACs, which remain associated with fibers and proteins after extraction. Most research on cranberry products only quantifies soluble PACs because proper standards for quantifying insoluble PACs are lacking. In this study, we evaluated the ability of a cranberry PAC (c-PAC) standard, reflective of the structural heterogeneity of PACs found in cranberry fruit, to quantify insoluble PACs by the butanol-hydrochloric acid (BuOH-HCl) method. For the first time, a c-PAC standard enabled conversion of BuOH-HCl absorbance values (550 nm) to a weight (milligram) basis, allowing for quantification of insoluble PACs in cranberries. The use of the c-PAC reference standard for sequential analysis of soluble PACs by the method of 4-(dimethylamino)cinnamaldehyde and insoluble PACs by the method of BuOH-HCl provides analytical tools for the standardization of cranberry-based ingredients.

Composition of Anthocyanins and Proanthocyanidins in Three Tropical Vaccinium Species from Costa Rica.

Total polyphenol content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), and proanthocyanidin (PAC) content were determined in fruit from three tropical Vaccinium species (Vaccinium consanguineum, Vaccinium floribundum, and Vaccinium poasanum) from Costa Rica sampled at three stages of fruit development. Results show that TAC increased as the fruit developed, while TPC, TFC, and PAC content decreased. Anthocyanin profiles were evaluated using electrospray ionization tandem mass spectrometry. Cyanidin and delphinidin glycosides were the predominant anthocyanins for the three tropical Vaccinium species. Proanthocyanidins were characterized using attenuated total reflection Fourier transform infrared spectroscopy, nuclear magnetic resonance, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presence of procyanidin structures with B-type interflavan bonds were observed, but deconvolution of mass spectrometry isotope patterns indicated that PACs with one or more A-type interflavan bonds accounted for more than 74% of the oligomers at each degree of polymerization.

Isolation and Characterization of Blueberry Polyphenolic Components and Their Effects on Gut Barrier Dysfunction.

Highbush blueberries contain anthocyanins and proanthocyanidins that have antimicrobial and anti-inflammatory bioactivities. We isolated and characterized three polyphenolic fractions, a total polyphenol fraction (TPF), an anthocyanin-enriched fraction (AEF), and a proanthocyanidin-enriched fraction (PEF), from freeze-dried blueberry powder and evaluated their effects on an in vitro model of gut barrier dysfunction. High-performance liquid chromatography chromatograms illustrate successful fractionation of the blueberry powder into TPF, AEF, and PEF. AEF contained 21 anthocyanins, and PEF contained proanthocyanidin oligomers of (epi)catechin with primarily B-type interflavan bonds. The model uses a strain of Escherichia coli to disrupt a Caco-2 cell monolayer on Transwell inserts. Barrier function was measured by transepithelial electrical resistance (TEER), a marker of membrane permeability. All fractions were able to restore TEER values after an E. coli challenge when compared to the control, while AEF was able to attenuate the E. coli-induced decrease in TEER in a dose-dependent manner.

Proanthocyanidin-chitosan composite nanoparticles prevent bacterial invasion and colonization of gut epithelial cells by extra-intestinal pathogenic Escherichia coli.

Cranberry proanthocyanidin-chitosan composite nanoparticles (PAC-CHT NPs) were formulated using 2:1, 5:1, 10:1, 15:1 20:1, 25:1, and 30:1 PAC to CHT weight ratio to form round shaped particles. The PAC-CHT NPs were characterized by size, polydispersity, surface charge, morphology, and PAC content. PAC-CHT NPs bioactivity was measured by agglutination of extra-intestinal pathogenic Escherichia coli (ExPEC) and inhibition of gut epithelial cell invasion by ExPEC. Results indicate that by increasing the PAC to CHT ratio 10:1 to 30:1 formed stable nanoparticles with diameters of 122.8 to 618.7 nm, a polydispersity index of approximated 0.4 to 0.5, and a zeta potential of 34.5 to 54.4 mV. PAC-CHT NPs ratio 30:1 agglutinated ExPEC and decreased the ability of ExPEC to invade epithelial cells in a dose-dependent manner. PAC-CHT NPs ratio 10:1 to 30:1 form stable, round-shaped, and bioactive nanoparticles for potential applications in the treatment of ExPEC bacterial infections.

In vitro screening of 51 birdsfoot trefoil (Lotus corniculatus L.; Fabaceae) strains for anti-parasitic effects against Haemonchus contortus.

Secondary plant compounds have shown bioactivity against multi-drug resistant Haemonchus contortus in small ruminants. This study screened 51 strains of birdsfoot trefoil (BFT, Lotus corniculatus) crude aqueous extracts (BFT-AqE) for anti-parasitic activity in vitro against egg hatching, and of those 51 strains, 13 were selected for further testing of motility of first (L1) and third stage (L3) larvae, and exsheathment of L3. Proanthocyanidin content ranged between 1.4 and 63.8 mg PAC g-1 powder across the 51 BFT strains. When tested against egg hatching, 21 of the 51 aqueous extracts had an EC50 of 1-2 mg powder mL-1, 70% of the strains were >90% efficacious at 6 mg powder mL-1 and 11 of the strains were 100% efficacious at 3 mg powder mL-1 BFT-AqE. Across the 13 strains tested against L3, efficacy ranged from 0 to 75% exsheathment inhibition, and 17 to 92% L3 motility inhibition at a concentration of 25 mg powder mL-1 BFT-AqE. There was no correlation between the PAC content of BFT powders and the anti-parasitic activity of aqueous extracts, therefore other secondary compounds may have contributed to the observed anti-parasitic effects. Further testing of BFT using bioactivity-driven fractionation and screening of BFT populations for the identified anti-parasitic compounds is needed.

Anthelmintic efficacy of cranberry vine extracts on ovine Haemonchus contortus.

The discovery that plant secondary compounds, including proanthocyanidins (PAC), suppress gastrointestinal nematode (GIN) infection has provided promise for alternative methods of GIN control in small ruminants. This investigation is the first to examine the anthelmintic potential of cranberry vine (CV) against the GIN Haemonchus contortus. The purpose of this study was to explore the anti-parasitic activity of CV in the form of a specific organic proanthocyanidin extract (CV-PAC) and an aqueous extract (CV-AqE) containing PAC and other compounds. In vitro egg hatching, first (L1) and third (L3) stage larval and adult worm motility and L3 exsheathment were evaluated after a 24-h incubation with CV products. In addition, CV treated worms were observed via scanning electron microscopy, and a preliminary investigation of the efficacy of CV powder against an experimental infection of H. contortus was conducted. The in vivo effect on an experimental infection was determined by administering 21.1 g CV powder to lambs (n = 9 per group) for three consecutive days, and collecting fecal egg count data for four weeks post-treatment. The effect of CV-PAC on egg hatching, L3 motility and exsheathment was limited. However, a substantial effect was observed on motility of post-hatch L1 (EC50 0.3 mg PAC/mL) and adults (EC50 0.2 mg PAC/mL). The CV-AqE showed more effect on egg hatching (EC50 5.3 mg/mL containing 0.6 mg PAC/mL) as well as impacting motility of L1 (EC50 1.5 mg/mL with 0.2 mg PAC/mL) and adults (EC50 3.4 mg/mL with 0.4 mg PAC/mL), but like CV-PAC, did not substantially effect L3 motility or exsheathment. Scanning electron microscopy revealed an accumulation of aggregate on the cuticle around the buccal area of adult worms incubated in CV-AqE and CV-PAC. In the preliminary in vivo study, there was a significant effect of treatment over time (p = .04), although differences in individual weeks were not significant. In summary, both extracts inhibited motility of L1 and adult worms. The higher efficacy of CV-AqE than CV-PAC at levels that contained the same concentrations of PAC tested alone, suggest that other secondary compounds in the CV-AqE contributed to the observed effects on the parasites. This first study of the in vitro and in vivo effects of CV suggest that this readily available plant product may have utility in integrated control of H. contortus and support the need for additional testing to provide further information.

Cranberry proanthocyanidin-chitosan hybrid nanoparticles as a potential inhibitor of extra-intestinal pathogenic Escherichia coli invasion of gut epithelial cells.

Chitosan interacts with proanthocyanidins through hydrogen-bonding, which allows encapsulation and development of stable nanoparticles via ionotropic gelation. Cranberry proanthocyanidins (PAC) are associated with the prevention of urinary tract infections and PAC inhibit invasion of gut epithelial cells by extra-intestinal pathogenic Escherichia coli (ExPEC). We determined the effect of cranberry proanthocyanidin-chitosan hybrid nanoparticles (PAC-CHTNp) on the ExPEC invasion of gut epithelial cells in vitro. PAC-CHTNp were characterized according to size, morphology, and bioactivity. Results showed a decrease in the size of the nanoparticles as the concentration of PAC was increased, indicating that PAC increases cross-linking by hydrogen-bonding on the surface of the chitosan nanoparticles. Nanoparticles were produced with diameters ranging from 367.3 nm to 293.2 nm. Additionally, PAC-CHTNp significantly inhibited the ability of ExPEC to invade the enterocytes by ~80% at 66 µg GAE/mL and by ~92% at 100 µg GAE/mL. Results also indicate that chitosan nanoparticles alone were not significantly different from controls in preventing ExPEC invasion of enterocytes (data not shown) and also there were not significant differences between PAC alone and PAC-CHTNp, suggesting that the new PAC-CHTNp could lead to an increase in the stability of encapsulated PAC, maintain the molecular adhesion of PAC to ExPEC.