Structural characterization of the carbohydrate and protein part of arabinogalactan protein from Basella alba stem and antiadhesive activity of polysaccharides from B. alba against Helicobacter pylori.

BACKGROUND: Increasing drug resistance of Helicobacter pylori has highlighted the search for natural compounds with antiadhesive properties, interrupting the adhesion of H. pylori to stomach epithelia. Basella alba, a plant widely used in Asian traditional medicine, was investigated for its antiadhesive activity against H. pylori. METHODS: B. alba extract FE was prepared by aqueous extraction. Polysaccharides were isolated from FE by ethanol precipitation and arabinogalactan-protein (AGP) was isolated with Yariv reagent. Carbohydrate analyses was performed by standard methods and sequence analysis of the protein part of AGP by LC-MS. In vitro adhesion assay of fluorescent-labelled H. pylori J99 to human AGS cells was performed by flow cytometric analysis. RESULTS: Raw polysaccharides (BA1) were isolated and 9% of BA1 were identified as AGP (53.1% neutral carbohydrates L-arabinose, D-galactose, rhamnose, 5.4% galacturonic acid, 41.5% protein). After deglycosylation of AGP, the protein part (two bands at 15 and 25 kDa in tricine SDS-PAGE) was shown to contain peptides like ribulose-bisphosphate-carboxylase-large-chain. Histological localization within the stem tissue of B. alba revealed that AGP was mainly located at the procambium ring. Functional assays indicated that neither FE nor BA1 had significant influence on viability of AGS cells or on H. pylori. FE inhibited the bacterial adhesion of H. pylori to AGS cells in a dose dependent manner. Best anti-adhesive effect of ~67% was observed with BA1 at 2 mg/mL. CONCLUSION: The data obtained from this study characterize in part the mucilage and isolated polysaccharides of B. alba. As the polysaccharides interact with the bacterial adhesion, a potential uses a supplemental antiadhesive entity against the recurrence of H. pylori after eradication therapy may be discussed.

A prospective whole-mixture approach to assess risk of the food and chemical exposome.

Many widely used chemicals result in ubiquitous human exposure from multiple sources, including diet. Legislation mainly deals with the toxicological evaluation of single substances owing to a methodological and conceptual lack of alternatives, and does so within defined silos subject to over 40 distinct regulations in the EU alone. Furthermore, much of the research and many of the initiatives concerned with the assessment and evaluation of chemical mixtures and their potential effects on human health rely on retrospective analysis. Here we propose an approach for the prospective identification, assessment and regulation of mixtures relevant to human health. We address two distinct aspects of toxicology-which chemicals actually do occur together, and how potential mixture-related health hazards can be predicted-with an adapted concept of the exposome and large-scale hazard screens. The proactive use of the likelihood of co-exposure, together with the new approach of methods-based testing, may be a timely and feasible way of identifying those substances and mixtures where hazards may have been overlooked and regulatory action is needed. Ideally, we would generate co-exposure patterns for specific consumer groups, depending on lifestyle and dietary habits, to assess the specific risk of identified mixtures.

Extract and the quassinoid ailanthone from Ailanthus altissima inhibit nematode reproduction by damaging germ cells and rachis in the model organism Caenorhabditis elegans.

Bark and leaves of Ailanthus altissima (Mill.) Swingle are widely used in European folk medicine to treat intestinal worm infections. The study aimed to rationalize a potential anthelmintic effect of A. altissima extract against the model organism Caenorhabditis elegans. A methanol-water (7:3, v/v) extract of the primary stem bark was tested on L4 larvae of C. elegans for induction of mortality and influence on reproduction. Bioactivity-guided fractionation was performed by chromatography on MCI-gel, preparative HPLC on RP18 stationary phase and fast-centrifugal-partition-chromatography. Structural elucidation of isolated quassinoids was performed by NMR and HR-ESI-MS. The sterilizing effect on C. elegans was investigated by light microscopy and atomic force microscopy of ultra-sections. Different GFP-tagged reporter strains were used to identify involved signaling pathways. A. altissima extract (1 mg/mL) irreversibly inhibited the reproduction of C. elegans L4 larvae. This effect was dependent on the larval stage since L3 larvae and adults were less affected. Bioactivity-guided fractionation revealed the quassinoid ailanthone 1 as the major active compound (IC50 2.47 µM). The extract caused severe damages to germ cells and rachis, which led to none or only poorly developed oocytes. These damages led to activation of the transcription factor DAF-16, which plays a major role in the nematode's response to stress. A regulation via the respective DAF-2/insulin-like signaling pathway was not observed. The current findings support the traditional use of A. altissma in phytotherapy to treat helminth infections and provide a base for standardization of the herbal material.

Shape-controlling effects of hydrohalic and carboxylic acids in TiO2 nanoparticle synthesis.

The ability to synthesize nanoparticles (NPs), here TiO2, of different shapes in a controlled and reproducible way is of high significance for a wide range of fields including catalysis and materials design. Different NP shapes exhibit variations of emerging facets, and processes such as adsorption, diffusion, and catalytic activity are, in general, facet sensitive. Therefore, NP properties, e.g., the reactivity of NPs or the stability of assembled NPs, depend on their shape. We combine computational modeling based on density functional theory with experimental techniques such as transmission electron microscopy, energy-dispersive x-ray spectroscopy, and x-ray powder diffraction to investigate the ability of various adsorbates, including hydrohalic and carboxylic acids, to influence NP shape. This approach allows us to identify mechanisms stabilizing specific surface facets and thus to predict NP shapes using computational model systems and to experimentally characterize the synthesized NPs in detail. Shape-controlled anatase TiO2 NPs are synthesized here in agreement with the calculations in platelet and bi-pyramidal shapes by employing different precursors. The importance of the physical conditions and chemical environment during synthesis, e.g., via competitive adsorption or changes in the chemical potentials, is studied via ab initio thermodynamics, which allows us to set previous and new results in a broader context and to highlight potentials for additional synthesis routes and NP shapes.

Antiadhesive phthalides from Apium graveolens fruits against uropathogenic E. coli.

ETHNOPHARMACOLOGICAL RELEVANCE: Fruits of Apium graveolens (celery) are used traditionally in Persian and European medicine for the treatment of uncomplicated urinary tract infections. AIM OF THE STUDY: The study aimed at identifying potential antiadhesive compounds from celery extracts to provide strategies for improved standardization of the herbal material. MATERIALS AND METHODS: Decoction, hydroalcoholic and acetone extracts were prepared from celery fruits. Bioassay-guided fractionation was performed by Fast Centrifugal Partition Chromatography and preparative HPLC, followed by LC-MS and NMR investigations for structure elucidation. The antiadhesive activity of extracts, fractions and purified compounds was assessed by flow cytometry, evaluating the adhesion of fluorescent-labelled uropathogenic bacteria (UPEC NU14) to T24 bladder cells; mannose served as positive control. Influence of the extract on gene expression of selected adhesins and fitness genes was monitored by qPCR. RESULTS: Concentration-dependent antiadhesive activity was found for the hydroalcoholic and even more for the acetone extract AE (IC50 85 µg/mL) from celery fruits. Bioassay-guided fractionation revealed the presence of the phthalides senkyunolide (1, inactive) and sedanenolide (2, IC50 790 µM). 2 is assessed as the main antiadhesive compound, which accounts for 4.0% in the water extract, for 18% in the hydroethanolic extract and for 71% in AE. Additionally a similar phthalide, Z-ligustilide (5), was shown to exert an IC50 of 611 µM. Furthermore, AE caused a significant upregulation of fimH and sfaG in free floating, non-attached UPEC and significantly down-regulated these genes in adherent bacteria. CONCLUSIONS: Phthalides were identified as the main active compounds in polar and semi-polar extracts, which exert strong antiadhesive activity against uropathogenic E. coli. The current findings support the traditional use in phytotherapy for urinary tract infections and provide a base for standardization of the herbal material.

Antiadhesive hydroalcoholic extract from Apium graveolens fruits prevents bladder and kidney infection against uropathogenic E. coli.

Fruits from Apium graveolens (Celery) are used traditionally in Persian and European medicine for the treatment of uncomplicated urinary tract infections. No data are available on A. graveolens extract on the interplay between uropathogenic E. coli and the eukaryotic host cells and on quorum sensing of the bacteria. The present study aimed to characterize an antiadhesive and anti quorum sensing effect of a characterized A. graveolens extract by specific in vitro assays and to correlate these effects with in vivo data obtained by an animal infection model. Hydroalcoholic extract CSE (EtOH-water, 1:1) from A. graveolens fruits was characterized by UHPLC/+ESI-QTOF-MS and investigated on antiproliferative activity against UPEC (strain NU14) and human T24 bladder cells. Antiadhesive properties of CSE were investigated within two different in vitro adhesion assays. For in vivo studies BALB/c mice were used in an UPEC infection model. The effect of CSE on bacterial load in bladder tissue was monitored within a 4- and 7 days pretreatment (200, 500 mg/kg) of the animals. CSE was dominated by the presence of luteolin-glycosides and related flavons besides furocoumarins. CSE had no cytotoxic effects against UPEC and bladder cells. CSE exerts a dose dependent antiadhesive activity against UPEC strains NU14 and UTI89. CSE inhibited in a concentration-dependent manner bacterial quorum sensing. 4- and 7-day pretreatment of animals with CSE transurethrally infected with UPEC NU14, significantly reduced the bacterial load in bladder tissue. CSE is assessed as an antiadhesive extract for which the traditional use in phytotherapy for UTI is justified.

Chitosan nanoencapsulation of flavonoids enhances their quorum sensing and biofilm formation inhibitory activities against an E.coli Top 10 biosensor.

Phytochemicals have been found to be promising alternatives to conventional antibiotic therapies for the control of bacterial infections, as they may entail less selective pressure and hence reduce the development of resistance. This study involved examining the inhibition of biofilm formation and of quorum sensing (QS), and the cytotoxicity on mammalian cells of two flavonoids, quercetin and baicalein, in free form and associated into chitosan-based nanocapsules. This was done by use of a transformed E. coli Top 10 biosensor strain, while the cytotoxicity was evaluated on MDCK-C7 cells. In free form, application both flavonoids exhibited slight inhibitory activity on the QS response and biofilm formation, a scenario that was improved positively upon encapsulation with chitosan (Mw ∼115,000 g/mol and DA ∼42%). The association efficiency of 99% (quercetin) and 87% (baicalein) was determined, and each formulation had an average diameter of 190 ±â€¯4 and 187 ±â€¯2 nm, and zeta (ζ) potential of +48.1 ±â€¯2.03 and +48.4 ±â€¯3.46 mV, respectively. Both types of systems were stable against aggregation in M9 and MEM media. The in vitro release kinetics data of both flavonoids seemed to be similar with only ∼20% released over the first 5 h, or ∼10% over the first 4 h, respectively, with subsequent sudden release increase up to ∼40% in both cases. The free phytochemicals seemed to be cytotoxic to MDCK-C7 cells at higher doses, however, upon nanoencapsulation, a cytoprotective effect was evidenced. We have gained proof-of-principle of the advantages of encapsulation of two bioactive flavonoids.

Exopolysaccharide-producing Streptococcus thermophilus CRL1190 reduces the inflammatory response caused by Helicobacter pylori.

This work evaluated the ability of the probiotic Streptococcus thermophilus CRL1190 strain and its exopolysaccharides to adhere to gastric mucosa. Probiotic bacteria attachment to the human stomach epithelium was confirmed in human stomach tissue samples and the gastric epithelial cell line AGS. In addition, it was demonstrated that S. thermophilus CRL1190 strain reduced Helicobacter pylori adhesion and attenuated inflammatory response in AGS cells. This is the first demonstration of the capacity of S. thermophilus CRL1190 to adhere to the stomach gastric mucosa, and improve protection against H. pylori through the reduction of its adhesion and the modulation of the inflammatory response. Therefore, S. thermophilus CRL1190 fermented milk is a good candidate for further in vivo studying of the protective effect of functional food against H. pylori infection and gastric inflammatory damage.

Aqueous extract from Orthosiphon stamineus leaves prevents bladder and kidney infection in mice.

BACKGROUND: Extracts from the leaves of Orthosiphon stamineus are used in phytotherapy for treatment of uncomplicated urinary tract infections. PURPOSES: Evaluation of an aqueous extract against infection with uropathogenic Escherichia coli in vivo; investigation of underlying microbiological mechanisms. STUDY DESIGN: In vivo studies in mice and in vitro investigations on cytotoxicity, antiadhesive potential, influence on bacterial gene expression and quorum sensing. METHODS: Extract OWE was prepared by hot water extraction. For in vivo studies BALB/c mice were used in an UPEC infection model. The effect of OWE on bacterial load in bladder/kidney tissue was monitored in pre- and posttreatment. Cytotoxicity of OWE against different UPEC strains, T24 bladder/A498 kidney cells, gene expression analysis, monitoring of phenotypic motility and quorum sensing was investigated by standard methods of microbiology. RESULTS: OWE was quantified (UHPLC) according to the content of rosmarinic acid, cichoric acid, caffeic acid. Three- and 5-day treatment of animals with OWE (750mg/kg) after transurethral infection with UPEC CFT073 reduced the bacterial load in bladder and kidney, similar to norfloxacin. Four- and 7-day pretreatment of mice prior to the infection with UPEC NU14 reduced bacterial bladder colonization. In vitro investigations indicated that OWE (≤2mg/ml) has no cytotoxic or proliferation-inhibiting activity against different UPEC strains as well as against T24 bladder and A498 kidney cells. OWE exerts a dose dependent antiadhesive activity against UPEC strains NU14 and UTI89. OWE reduced gene expression of fimH, but evoked increase of the expression of motility/fitness gene fliC. Increase of bacterial motility on gene level was confirmed by a changed bacterial phenotype by an increased bacterial motility in soft agar assay. OWE inhibited in a concentration-dependent manner bacterial quorum sensing. CONCLUSION: OWE is assessed as a strong antiadhesive plant extract for which the traditional use in phytotherapy for UTI might be justified.

Medicinal plant extracts and plant-derived polyphenols with anthelmintic activity against intestinal nematodes.

Covering: 2001 up to the end of 2016Polyphenols comprise a structurally diverse class of natural products. As the development of new anthelmintic drugs against soil-transmitted helminthiases is an urgent need and polyphenols are widely used in the treatment of nematode infections in traditional medicine and modern science, we summarize the state of knowledge in the period of mainly 2001 up to the end of 2016 on plant extracts with known polyphenolic composition and of defined polyphenols, mainly from the classes of condensed and hydrolysable tannins, flavonoids, and phenylpropanoids. The diverse biological activity against different helminths and the underlying mechanisms are reviewed.

Ethnobotanical survey of traditionally used medicinal plants for infections of skin, gastrointestinal tract, urinary tract and the oral cavity in Borabu sub-county, Nyamira county, Kenya.

ETHNOPHARMACOLOGICAL RELEVANCE: Different communities throughout the world have specialized and profound knowledge on the use medicinal plants for various diseases. However, the detailed information on the respective use may extinct in near future as this knowledge is passed only orally among generations in most of the communities. AIM OF THE STUDY: The present survey aimed to document the use of medicinal plants by traditional healers from the Kisii community, Borabu sub-county in Nyamira county, Kenya, to treat infections of the urinary tract, oral cavity, gastrointestinal system and the skin and to evaluate the social context in which the healers work and practice. MATERIALS AND METHODS: Validated questionnaires were applied to 50 traditional healers in the study region, followed by interviews and structured conversations. Information on the relevant traditionally used medicinal plants and their use were documented, including sampling and identification of voucher specimens. RESULTS: The ethnopharmacological survey revealed 25 medicinal plant species belonging to 19 families. It got evident that most of these species will be extinct in the near future unless appropriate measures are taken, as it turned out difficult to collect some of the wild growing species. Elaeodendron buchananii Loes, Erlangea marginata S. Moore, Acacia gerrardii Benth., Balanites orbicularis Sprague, Solanum renschii Vatke and Orthosiphon hildebrandtii Vatke have not been described before for its medicinal use. Among the 25 species collected from the various regions of Borabu sub-county Urtica dioica L. was the only medicinal plant that was collected from all regions. In contrast Erythrina abyssinica and Rhus natalensis were found in only two regions of the study area. CONCLUSION: The traditional medicinal use of the reported plants for infections should be documented and a great need of awareness from scientists and local government for improved preservation or field cultivation of some species is obvious.

Inhibition of in vitro adhesion and virulence of Porphyromonas gingivalis by aqueous extract and polysaccharides from Rhododendron ferrugineum L. A new way for prophylaxis of periodontitis?

The effect of an aqueous extract from the leaves of Rhododendron ferrugineum (RF) was investigated for its capacity of inhibiting the adhesion of Porphyromonas gingivalis cells to epithelial buccal KB cells. RF was characterized by HPLC (12.1% taxifolin-3-O-ß-l-arabinopyranoside, 1.6% hyperoside, 0.9% isoquercitrin, 1.6% chlorogenic acid and a tannin content of 8.7%). Additionally raw polysaccharides (RPS) were obtained from the leaves of R. ferrugineum by aqueous extraction. RF and RPS interacted in a dose-dependent manner (max. 25% reduction at 1mg/ml each) with the adhesion of P. gingivalis by influencing bacterial outer membrane proteins. On protein level a time- and concentration-dependent inhibition of Arg-gingipain activity by RF was observed, while the Lys-gingipain activity remained unaltered. In addition, RF and RPS inhibited the bacterial hemagglutinin. RF affected the P. gingivalis adhesion also by interacting with KB cells in pre-incubation assays of the eukaryotic host cells, leading to reduced bacterial adhesion of about 75%. Gene expression analysis by RT-PCR indicated significant downregulation for arginine-specific gingipain rgpA by RF, while lysin-specific gingipain kgp and fimbrillinA fimA were strongly upregulated. Moreover, pre-incubation with RF abolished the P. gingivalis induced expression of IL-1ß, IL-6, IL-8 and TNFα in KB cells. Results of this study indicate that an aqueous extract from R. ferrugineum combines cytoprotective and antimicrobial effects by both downregulating the expression of pro-inflammatory genes and inhibiting the adhesion of P. gingivalis. Thus RF may be potential candidate for the development of an adjunctive antimicrobial approach in the prevention of periodontal diseases.

Phytochemical characterization and in vitro wound healing activity of leaf extracts from Combretum mucronatum Schum. & Thonn.: Oligomeric procyanidins as strong inductors of cellular differentiation.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from Combretum mucronatum Schum. & Thonn. are traditionally used for wound healing in Western Africa. Aqueous and hydroalcoholic extracts of the dried leaves recently have been shown to stimulate viability of human keratinocytes and dermal fibroblasts. AIM OF THE STUDY: Phytochemical characterization of the herbal material, development of a validated HPLC methodology for quality control, and pinpointing the underlying pharmacological mechanism under in vitro conditions to understand the impact of C. mucronatum extracts on human skin cells. MATERIALS AND METHODS: Extracts obtained from the leaves from C. mucronatum by using solvents with different polarities (petrol ether, dichloromethane, ethanol-water 50%, water) were investigated concerning phytochemical composition by GC-MS, LC-MS and in part after fractionation and isolation of purified compounds. For quality control of the herbal material an ICH-2 validated UHPLC method was developed for quantification of the lead compounds epicatechin, procyanidin B2, vitexin and isovitexin. In vitro studies were performed using HaCaT keratinocyte cell line, primary keratinocytes and primary skin fibroblasts with determination of viability (MTT assay), cell proliferation (BrdU incorporation ELISA), cell toxicity (LDH release) and keratinocyte differentiation, using involucrin and keratin K10 as differentiation marker (confocal laser scanning microscopy, Western blot). RESULTS: A detailed phytochemical composition analysis of the extracts from the leaves from C. mucronatum was performed (compounds 1-34) and epicatechin, procyanidin B2, vitexin and isovitexin are assessed to be the lead compounds of the polar extract. Quantitative UHPLC investigations indicated mature leaves to have higher polyphenol content in comparison to young leaves. The drying process of the plant material was shown to have great influence on the content of the lead compounds. The aqueous extract (0.1-100µg/mL) did not change cell viability of dermal fibroblasts and keratinocytes but inhibited cellular proliferation rates significantly at 100µg/mL. The extract stimulated cellular differentiation of primary keratinocytes significantly at 1 and 10µg/mL. Procyanidin B2 at 1 and 10µM was shown to be responsible for the induction of this cellular differentiation, while epicatechin, and procyanidins B5, C1 and D1 were inactive. CONCLUSION: The in vitro effects of the aqueous extract on the skin cells rationalized the remedial effect in wound healing and possibly accounts for the reason why this plant may be widely used for this purpose. On the basis of this study extracts from the leaves of C. mucronatum therefore have potential for the use in wound healing.

Isolation and quantification of oligomeric and polymeric procyanidins in leaves and flowers of Hawthorn (Crataegus spp.).

Proanthocyanidins (PAs) constitute a class of polyphenols with flavan-3-ols as monomeric building blocks. These polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of monomeric flavan-3-ols or low molecular oligomers as lead compounds. No reliable analytical methods are available for unambiguous identification of the homologues series of oligo- and polymeric PAs. For Hawthorn leaf and flower (Crataegi folium cum flore) from Crataegus spp. (Rosaceae) a protocol for preparative isolation of oligomeric and polymeric PAs from an acetone-water extract was developed, yielding procyanidin reference clusters with defined degree of polymerization (DP) from 2 to 10 besides a procyanidin-polymer. Identity and purity of these clusters were proven by HPLC, MS and in part NMR studies. For identification and quantification from Hawthorn an ICH-Q2 validated UHPLC method with fluorimetric detection and less than 10min runtime was developed. The method enabled quantification of procyanidin clusters with DP from 2 to 10 besides the polymer fraction. Batch analysis revealed procyanidin contents of about 20 to 45mg/g from a homologues series of oligomeric PAs and about 50% of polymer fraction. Monitoring of procyanidin distribution during seasonal growth of fresh plants of Crataegus monogyna showed more or less constant contents between 20 and 55mg/g dry weight of oligomeric procyanidins during the growing season in the different plant organs with strong accumulation in the flowers and fruits (55mg/g dry weight). From these data it can be speculated that procyanidins serve as part of the plants defense system in the reproductive organs of the plant.

Intestinal formation of trans-crocetin from saffron extract (Crocus sativus L.) and in vitro permeation through intestinal and blood brain barrier.

AIMS: Extracts of saffron (Crocus sativus L.) have traditionally been used against depressions. Recent preclinical and clinical investigations have rationalized this traditional use. Trans-crocetin, a saffron metabolite originating from the crocin apocarotenoids, has been shown to exert strong NMDA receptor affinity and is thought to be responsible for the CNS activity of saffron. Pharmacokinetic properties of the main constituents from saffron have only been described to a limited extent. Therefore the present in vitro study aimed to determine if crocin-1 and trans-crocetin are able to pass the intestinal barrier and to penetrate the blood brain barrier (BBB). Additionally, the intestinal conversion of glycosylated crocins to the lipophilic crocetin had to be investigated. Experiments with Caco-2 cells and two different porcine BBB systems were conducted. Further on, potential intestinal metabolism of saffron extract was investigated by ex vivo experiments with murine intestine. METHODOLOGY: In vitro Caco-2 monolayer cell culture was used for investigation of intestinal permeation of crocin-1 and trans-crocetin. In vitro models of porcine brain capillary endothelial cells (BCEC) and blood cerebrospinal fluid barrier (BCSFB) were used for monitoring permeation characteristics of trans-crocetin through the blood brain barrier (BBB). Intestine tissue and feces homogenates from mice served for metabolism experiments. RESULTS: Crocin-1, even at high concentrations (1000 µM) does not penetrate Caco-2 monolayers in relevant amounts. In contrast, trans-crocetin permeates in a concentration-independent manner (10-114 µM) the intestinal barrier by transcellular passage with about 32% of the substrate being transported within 2 h and a permeation coefficient of Papp 25.7 × 10(-)(6) ± 6.23 × 10(-)(6) cm/s. Trans-crocetin serves as substrate for pGP efflux pump. Trans-crocetin permeates BBB with a slow but constant velocity over a 29 h period (BCEC system: Papp 1.48 × 10(-)(6) ± 0.12 × 10(-)(6) cm/s; BCSFB system Papp 3.85 × 10(-)(6) ± 0.21 × 10(-)(6) cm/s). Conversion of glycosylated crocins from saffron extract to trans-crocetin occurs mainly by intestinal cells, rather than by microbiological fermentation in the colon. CONCLUSION: The here described in vitro studies have shown that crocins from saffron are probably not bioavailable in the systemic compartment after oral application. On the other side the investigations clearly have pointed out that crocins get hydrolyzed in the intestine to the deglycosylated trans-crocetin, which subsequently is absorbed by passive transcellular diffusion to a high extend and within a short time interval over the intestinal barrier. Crocetin will penetrate in a quite slow process the blood brain barrier to reach the CNS. The intestinal deglycosylation of different crocins in the intestine is mainly due to enzymatic processes in the epithelial cells and only to a very minor extent due to deglycosylation by the fecal microbiome. On the other side the fecal bacteria degrade the apocarotenoid backbone to smaller alkyl units, which do not show any more the typical UV absorbance of crocins. As previous studies have shown strong NMDA receptor affinity and channel opening activity of trans-crocetin the use of saffron for CNS disorders seems to be justified from the pharmacokinetic and pharmacodynamic background.

Biophysical analysis of the molecular interactions between polysaccharides and mucin.

Mucoadhesive materials adhere persistently to mucosal surfaces. A mucoadhesive delivery system could therefore facilitate the controlled release of drugs and optimize their bioavailability in mucosal tissues. Polysaccharides are the most versatile class of natural polymers for transmucosal drug delivery. We used microviscosimetry to explore the mucoadhesion of a library of polysaccharide families with diverse structural characteristics as a first step toward the rational design of mucoadhesive polysaccharide-based nanoformulations. Here we show that the magnitude of deviation between the viscosity of mixed polysaccharide-mucin solutions and the corresponding individual stock solutions can indicate underlying molecular interactions. We found that nonlinear monotonic curves predicted a correlation between the magnitude of interaction and the ability of polysaccharide coils to contract in the presence of salt (i.e., chain flexibility). Charge-neutral polysaccharides such as dextran and Streptococcus thermophilus exopolysaccharide did not interact with mucin. Synchrotron small-angle X-ray scattering (SAXS) data supported the previously described structural features of mucin. Furthermore, high-q scattering data (i.e., sensitive to smaller scales) revealed that when mucin is in dilute solution (presumably in an extended conformation) in the presence of low-Mw alginate, its structure resembles that observed at higher concentrations in the absence of alginate. This effect was less pronounced in the case of high-Mw alginate, but the latter influenced the bulk properties of mucin-alginate mixtures (e.g., hydrodynamic radius and relative viscosity) more prominently than its low-Mw counterpart.

Structure of chitosan determines its interactions with mucin.

Synthetic and natural mucoadhesive biomaterials in optimized galenical formulations are potentially useful for the transmucosal delivery of active ingredients to improve their localized and prolonged effects. Chitosans (CS) have potent mucoadhesive characteristics, but the exact mechanisms underpinning such interactions at the molecular level and the role of the specific structural properties of CS remain elusive. In the present study we used a combination of microviscosimetry, zeta potential analysis, isothermal titration calorimetry (ITC) and fluorescence quenching to confirm that the soluble fraction of porcine stomach mucin interacts with CS in water or 0.1 M NaCl (at c < c*; relative viscosity, η(rel), ∼ 2.0 at pH 4.5 and 37 °C) via a heterotypic stoichiometric process significantly influenced by the degree of CS acetylation (DA). We propose that CS-mucin interactions are driven predominantly by electrostatic binding, supported by other forces (e.g., hydrogen bonds and hydrophobic association) and that the DA influences the overall conformation of CS and thus the nature of the resulting complexes. Although the conditions used in this model system are simpler than the typical in vivo environment, the resulting knowledge will enable the rational design of CS-based nanostructured materials for specific transmucosal drug delivery (e.g., for Helicobacter pylori stomach therapy).

Saffron extract and trans-crocetin inhibit glutamatergic synaptic transmission in rat cortical brain slices.

Saffron, the dried stigmata of Crocus sativus L., is used in traditional medicine for a wide range of indications including cramps, asthma, and depression. To investigate the influence of hydro-ethanolic saffron extract (CSE) and trans-crocetin on synaptic transmission, postsynaptic potentials (PSPs) were elicited by focal electrical stimulation and recorded using intracellular placed microelectrodes in pyramidal cells from rat cingulate cortex. CSE (10-200 µg/ml) inhibited evoked PSPs as well as the isolated NMDA and non-NMDA component of PSPs. Glutamate (500 µM) added into the organ bath induced membrane depolarization. CSE decreased glutamate-induced membrane depolarization. Additionally, CSE at 100 µg/ml decreased NMDA (20 µM) and kainate (1 µM)-induced depolarization, whereas AMPA (1 µM)-induced depolarization was not affected. Trans-crocetin (1-50 µM) showed inhibition of evoked PSPs and glutamate-induced membrane depolarization comparable to CSE. Trans-crocetin at 10 µM decreased NMDA (20 µM)-induced membrane depolarization, but did not inhibit the isolated non-NMDA component of PSPs. We conclude that trans-crocetin is involved in the antagonistic effect of CSE on NMDA but not on kainate receptors.

In vitro activity of Cameroonian and Ghanaian medicinal plants on parasitic (Onchocerca ochengi) and free-living (Caenorhabditis elegans) nematodes.

Ethanolic and aqueous extracts of selected medicinal plants from Cameroon and Ghana were assessed for their in vitro anthelmintic activity by using the bovine filarial parasite Onchocerca ochengi and the free living nematode Caenorhabditis elegans, a model organism for research on nematode parasites. Worms were incubated in the presence of different concentrations of extracts and inhibitory effects were monitored at different time points. Among the extracts used in this study, ethanolic extracts of Anogeissus leiocarpus, Khaya senegalensis, Euphorbia hirta and aqueous extracts from Annona senegalensis and Parquetina nigrescens affected the growth and survival of C. elegans and O. ochengi significantly. The mortality was concentration dependent with an LC50 ranging between 0.38 and 4.00 mg/ml for C. elegans (after 72 h) and between 0.08 and 0.55 mg/ml for O. ochengi after a 24 h incubation time. Preliminary phytochemical screenings on these extracts revealed the presence of flavonoids, alkaloids, saponins, carbohydrates and tannins in the extracts. Accordingly, application of A. leiocarpus, K. senegalensis, E. hirta and A. senegalensis extracts could provide alternatives in the control of helminthic infections.