An oil-in-water emulsion containing a combination of ginger extract and synthetic cannabidiol with potent in vitro anti-inflammatory effects alleviates symptoms of atopic dermatitis in a clinical trial.

Atopic dermatitis (AD) is a highly prevalent chronic skin disease. Anti-inflammatory and antipruritic emollients (emollients plus) with excellent cosmetic properties may alleviate AD-related symptoms and reduce the number of exacerbations. To screen for herbal extracts with potent anti-inflammatory and antioxidative potential in human skin cell cultures. Ginger extract and synthetic cannabidiol (CBD) were identified and combined in the cosmetic product BNO 3731, which was evaluated in a randomized clinical trial. Preclinical: anti-inflammatory effects of ginger extract, synthetic CBD and a combination thereof were evaluated in human skin cell cultures by analysing nuclear factor κB activation, release of inflammatory cytokines and endocannabinoid production. Clinical: BNO 3731 was studied in a clinical trial comprising 44 AD patients (adults and children) and compared to a benchmark product over a treatment duration of five days. Symptom severity was evaluated by objective and subjective dermatological assessments as well as physiological skin parameters. Itch intensity was assessed using a numerical rating scale (NRS-11). Preclinical: Ginger extract and synthetic CBD exhibited potent anti-inflammatory and antioxidative effects in vitro which were associated with elevated concentrations of the endocannabinoid, anandamide. Clinical: BNO 3731 significantly alleviated symptoms of AD and improved physiological skin parameters. Itch intensity decreased significantly by 55%, and in 75% of subjects, itch improved ≥2 points on the NRS-11 scale. No adverse events were reported. BNO 3731, containing a unique synergistic combination of ginger extract and synthetic CBD, is an effective and safe treatment option for dry and eczema-prone skin, providing rapid and substantial relief of pruritus.

Phytochemical Profiling, Antioxidant and Tyrosinase Regulatory Activities of Extracts from Herb, Leaf and In Vitro Culture of Achillea millefolium (Yarrow).

Achillea millefolium L. is one of the most known medicinal plants with a broad spectrum of applications in the treatment of inflammation, pain, microbial infections and gastrointestinal disorders. In recent years, the extracts from A. millefolium have also been applied in cosmetics with cleansing, moisturizing, shooting, conditioning and skin-lightening properties. The growing demand for naturally derived active substances, worsening environmental pollution and excessive use of natural resources are causing increased interest in the development of alternative methods for the production of plant-based ingredients. In vitro plant cultures are an eco-friendly tool for continuous production of desired plant metabolites, with increasing applicability in cosmetics and dietary supplements. The purpose of the study was to compare phytochemical composition and antioxidant and tyrosinase inhibitory properties of aqueous and hydroethanolic extracts from A. millefolium obtained from field conditions (AmL and AmH extracts) and in vitro cultures (AmIV extracts). In vitro microshoot cultures of A. millefolium were obtained directly from seeds and harvested following 3 weeks of culture. Extracts prepared in water, 50% ethanol and 96% ethanol were compared for the total polyphenolic content, phytochemical content using the ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-hr-qTOF/MS), antioxidant activity by DPPH scavenging assay and the influence on the activity of mushroom and murine tyrosinases. The phytochemical content of AmIV extracts was significantly different from AmL and AmH extracts. Most of the polyphenolic compounds identified in AmL and AmH extracts were present in AmIV extracts only in trace amounts and the major constituents presented in AmIV extracts were fatty acids. The total content of polyphenols in AmIV exceeded 0.25 mg GAE/g of dried extract, whereas AmL and AmH extracts contained from 0.46 ± 0.01 to 2.63 ± 0.11 mg GAE/g of dried extract, depending on the solvent used. The low content of polyphenols was most likely responsible for the low antioxidant activity of AmIV extracts (IC50 values in DPPH scavenging assay >400 µg/mL) and the lack of tyrosinase inhibitory properties. AmIV extracts increased the activity of mushroom tyrosinase and tyrosinase present in B16F10 murine melanoma cells, whereas AmL and AmH extracts showed significant inhibitory potential. The presented data indicated that microshoot cultures of A. millefolium require further experimental research before they can be implemented as a valuable raw material for the cosmetics industry.

Ambient mass spectrometry and near-infrared spectroscopy - a direct comparison of methods for the quantification of sucralose in e-liquids.

E-liquids have become increasingly popular in society in recent years. A wide variety of flavors and nicotine strengths make it possible for every user to get a product according to their wishes. Many of these e-liquids are marketed with countless different flavors, which are often characterized by a strong and sweet smell. Sweeteners, such as sucralose, are therefore commonly added as sugar substitutes. However, recent studies have shown the potential formation of highly toxic chlorinated compounds. This can be explained by the high temperatures (above 120 °C) within the heating coils and the used basic composition of these liquids. Nevertheless, the legal situation is composed of proposals without clear restrictions, only recommendations for tobacco products. For this reason, a high level of interest lies within the establishment of fast, reliable and cost-effective methods for the detection of sucralose in e-liquids. In this study, a number of 100 commercially available e-liquids was screened for sucralose in order to identify the suitability of ambient mass spectrometry and near-infrared spectroscopy for this application. A highly sensitive high-performance liquid chromatography coupled to a tandem mass spectrometer method was used as reference method. Furthermore, the advantages and limitations of the two mentioned methods are highlighted in order to provide a reliable quantification of sucralose. The results clearly revile the necessity for product quality due to the absence of declaration on many of the used products. Further on, it could be shown, that both methods are suitable for the quantification of sucralose in e-liquids, with beneficial economic and ecological aspects, over classical analytical tools including high-performance liquid chromatography. Clear correlations between the reference and novel developed methods are displayed. In summary, these methods enable an important contribution to ensure consumer protection and elimination of confuse package labelling.

Anti-Inflammatory Extract from Soil Algae Chromochloris zofingiensis Targeting TNFR/NF-κB Signaling at Different Levels.

Inflammatory skin diseases, including atopic dermatitis (AD) and psoriasis, are increasing in populations worldwide. The treatment of patients with AD and other forms of skin inflammation is mainly based on the use of topical corticosteroids or calcineurin inhibitors, which can cause significant side effects with long-term use. Therefore, there is a great need for the development of more effective and less toxic anti-inflammatory agents suitable for the treatment of chronic skin lesions. Here, we screened a number of strains from the ASIB 505 terrestrial algae collection and identified a green algae Chromochloris zofingiensis with pronounced anti-inflammatory properties. We found that a crude nonpolar extract of C. zofingiensis (ID name NAE_2022C), grown upon nitrogen deprivation, acts as a bioactive substance by inhibiting TNFR/NF-κB responses in human skin keratinocyte HaCaT cells. We also found that NAE_2022C suppressed the secretion of pro-inflammatory cytokine tumor necrosis factor α (TNFα) and several Th1- and Th2-related chemokines in a reconstituted human epidermis. The TNFR/NF-κB pathway analysis showed multiple inhibitory effects at different levels and disclosed a direct targeting of IKKß by the extract. Bioassay-guided fractionation followed by high-resolution mass spectrometry detected diacylglyceryl-trimethylhomoserine (DGTS), Lyso-DGTS (LDGTS), 5-phenylvaleric acid, theophylline and oleamide as leading metabolites in the active fraction of NAE_2022C. Further analysis identified betaine lipid DGTS (32:0) as one of the active compounds responsible for the NAE_2022C-mediated NF-κB suppression. Overall, this study presents an approach for the isolation, screening, and identification of anti-inflammatory secondary metabolites produced by soil algae.

Stability evaluation of morphine, hydromorphone, metamizole and esketamine containing analgesic mixtures applied for patient-controlled analgesia in hospice and palliative care.

In this study, different injection solutions containing opioid and nonopioid compounds used for patient-controlled analgesia in hospice and palliative care were evaluated in terms of analyte stability. Investigated injection solutions contained different combinations of morphine, hydromorphone, metamizole and esketamine. For the practical implementation, samples from infusion pumps were daily drawn over a period of 7 days at 22 and 37°C. Quantitative measurements were performed on a high-performance liquid chromatography system with ultraviolet detection applying a validated analytical method. All compounds apart from morphine showed no evident changes in concentration. However, a significant loss of morphine was observed for injection mixtures containing both morphine and metamizole at 37°C. After 7 days, only 72% of the initially measured morphine concentration was measured in the binary and 77% in the ternary mixture. Furthermore, an additional compound was detected that could represent the morphine-metamizole-adduct, "metamorphine". Based on these results, a significantly reduced morphine concentration must be expected after only 3 days if an injection solution mixture containing both morphine and metamizole is administered to a patient at 37°C. Since the analgesic effects of morphine-metamizole adducts have not yet been thoroughly investigated, further clinical studies are necessary before accurate conclusions can be drawn in this regard.

Rapid differentiation and quality control of tobacco products using Direct Analysis in Real Time Mass Spectrometry and Liquid Chromatography Mass Spectrometry.

This work is the first to describe the use of Direct Analysis in Real Time mass spectrometry (DART-MS) for the rapid and cost-effective quality control of cigars. Multivariate analyses (PCA and LDA) on mass spectra led to highly efficient models for the discrimination of tobacco products. In addition to the geographic origin of the cigars, different pretreatments of the tobacco leaves could also be determined. Cross-validations of the models yielded prediction accuracies up to 92.18%. All results were compared using LC-MS as reference method. Multivariate analyses of chromatograms also yielded powerful models with correctness scores up to 88.1%. However, the models generated with LC-MS were neither able to determine the geographic origin of the tobacco leaves nor different pretreatments. In addition to qualitative analysis, the nicotine content of tobacco leaves was also determined via both methods. While precise quantification was not possible with DART-MS, a correlation coefficient of >0.96 was achieved in direct comparison with LC-MS, which allows semi-quantitative statements about the nicotine content without any problems.

Quantification and cytotoxicity of degradation products (chloropropanols) in sucralose containing e-liquids with propylene glycol and glycerol as base.

Electronic cigarettes (e-cigarettes) have gained increasing popularity in recent years, mostly because they are supposed to be less harmful than regular cigarettes. Therefore, it is highly imperative to investigate possible noxious effects to protect the consumers. E-liquids consist of propylene glycol, glycerol, aroma compounds and sweeteners. One of these sweeteners is a chlorinated version of sucrose, namely sucralose. The aim of this work was to investigate degradation products of sucralose in the presence of propylene glycol and glycerol at different temperatures of commercially available e-cigarettes. Chemical analysis and biological tests were simultaneously performed on e-liquid aerosol condensates. The results of the chemical analysis, which was executed by employing GC-MS/GC-FID, demonstrated high amounts of various chloropropanols. The most abundant one is extremely toxic, namely 3-chloropropane-1,2-diol, which can be detected at concentrations ranging up to 10,000 mg/kg. Furthermore, a cytotoxicity investigation of the condensates was performed on HUVEC/Tert2 cells in which metabolic activity was determined by means of resazurin assay. The cellular metabolic activity significantly decreased by treatment with e-liquid aerosol condensate. Due to the results of this study, we advise against the use of sucralose as sweetener in e-liquids.

Innovative Combination of Dispersive Solid Phase Extraction Followed by NIR-Detection and Multivariate Data Analysis for Prediction of Total Polyphenolic Content.

Recently polyphenols attracted great interest in the field of food and nutrition as well as in the pharmaceutical and cosmetics industries due to their health benefits through antioxidative behavior in the human body. However, because of the high number of compounds characterized as phenols and their structural diversity, quantification of polyphenols turns out to be a highly complex task. Although, a wide variety of analytical methods are used for the determination of total polyphenolic content, they are all found to be lacking in a variety of different tasks, such as their limits of detection and quantification, repeatability, accuracy and specificity. For this reason, a novel approach combining the advantages of solid phase purification, near infrared analysis and multivariate data analysis was investigated for the prediction of total polyphenolic content, suitable for a wide range of sample matrices. Dispersive solid phase extraction was performed and optimized using polyvinylpyrrolidone as sorbent, known to selectively bind polyphenols. Near-infrared detection of adsorbed polyphenols was carried out subsequently. Furthermore, the method was in-house validated, examining selectivity, repeatability and accuracy, working range, as well as multivariate limit of detection and limit of quantification, comparing it with two routinely used methods-namely, Folin-Ciocalteu photometric assay and Löwenthal titration. The novel established method was applied for the prediction of total polyphenolic content in tea and wine samples.

C5aR inhibition of nonimmune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2-infected primary human airway epithelia.

BACKGROUND: Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality. OBJECTIVE: Complement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects. METHODS: For this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements. RESULTS: Here, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES. CONCLUSIONS: Crucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.

Quantification of selected aroma compounds in e-cigarette products and toxicity evaluation in HUVEC/Tert2 cells.

During recent years, the number of consumers using so-called e-cigarettes, which are electrical devices to aerosolize a liquid consisting of propylene glycol, glycerol, optional nicotine and flavoring chemicals, has been increasing. Aromas vary from common flavors such as mint to more unusual flavors such as buttermilk or pepperoni pizza. Consumers today can buy e-concentrates that consist of propylene glycol and aroma to blend their own desired flavor at home. Little is known about the composition and concentration of various aroma molecules in the different e-liquids and e-concentrates. In addition, the process of EU-wide regulation is still ongoing. The aim of this research study was to identify and quantify possible undesirable aroma compounds in e-liquids and e-concentrates. Flavoring chemicals such as estragole, benzaldehyde and cinnamaldehyde were quantified. The measurements were carried out on a GC-MS system. The results show the presence of highly concentrated flavoring compounds and limonene oxide in lemon-flavored e-concentrates. In the final step, samples and single-aroma standards were tested for their toxicity to HUVEC/Tert2 cells, where some single-flavoring chemicals such as cinnamic aldehyde revealed significant toxic effects.

Investigation of the evaporation behavior of aroma compounds in e-cigarettes.

The aim of this work was to evaluate the evaporation behavior of certain aroma compounds found in e-liquids. Since an e-liquid is evaporated, the aroma present can reach the lungs and could be absorbed into the body which may have long-term health effects above critical concentrations. Due to a lack in regulations, the sort and concentration of the compounds in sold e-liquids can vary. To capture the aroma compounds in the vapor, a smoking machine was developed. The resulting data represent the amount of aroma reaching the consumers' lungs. The influence of the e-cigarette temperature, ranging from 100 to 315 °C, on the evaporation of benzaldehyde, estragole, and different terpenoids was examined. Additionally, the effect of the liquid base composition on the amount of aroma in the vapor was compared using the analysis of variances. The influence of high temperature, the type of e-cigarette, and the atomizer coil material, which could lead to oxidation of limonene and linalool in the vapor, is shown here.

Comparison survey of EVOO polyphenols and exploration of healthy aging-promoting properties of oleocanthal and oleacein.

Olive oil is widely accepted as a superior edible oil. Great attention has been given lately to olive oil polyphenols which are linked to significant health beneficial effects. Towards a survey of Greek olive oil focusing on polyphenols, representative extra virgin olive oils (EVOOs) from the main producing areas of the country and the same harvesting period have been collected and analyzed. Significant differences and interesting correlations have been identified connecting certain polyphenols namely hydroxytyrosol, tyrosol, oleacein and oleocanthal with specific parameters e.g. geographical origin, production procedure and cultivation practice. Selected EVOOs polyphenol extracts, with different oleacein and oleocanthal levels, as well as isolated oleacein and oleocanthal were bio-evaluated in mammalian cells and as a dietary supplement in the Drosophila in vivo model. We found that oleocanthal and oleacein activated healthy aging-promoting cytoprotective pathways and suppressed oxidative stress in both mammalian cells and in flies.

Poly(N-vinylimidazole/ethylene glycol dimethacrylate) for the purification and isolation of phenolic acids.

In this study we report the novel polymeric resin poly(N-vinyl imidazole/ethylene glycol dimethacrylate) for the purification and isolation of phenolic acids. The monomer to crosslinker ratio and the porogen composition were optimized for isolating phenolic acids diluted in acetonitrile at normal phase chromatography conditions, first. Acetonitrile serves as polar, aprotic solvent, dissolving phenolic acids but not interrupting interactions with the stationary phase due to the approved Hansen solubility parameters. The optimized resin demonstrated high loading capacities and adsorption abilities particularly for phenolic acids in both, acetonitrile and aqueous solutions. The adsorption behavior of aqueous standards can be attributed to ion exchange effects due to electrostatic interactions between protonated imidazole residues and deprotonated phenolic acids. Furthermore, adsorption experiments and subsequent curve fittings provide information of maximum loading capacities of single standards according to the Langmuir adsorption model. Recovery studies of the optimized polymer in the normal-phase and ion-exchange mode illustrate the powerful isolation properties for phenolic acids and are comparable or even better than typical, commercially available solid phase extraction materials. In order to prove the applicability, a highly complex extract of rosemary leaves was purified by poly(N-vinyl imidazole/ethylene glycol dimethacrylate) and the isolated compounds were identified using UHPLC-qTOF-MS.

Development of erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns for selective enrichment of phosphopeptides.

In this study, a novel method for the highly selective enrichment of phosphopeptides using erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns is presented. Erbium phosphate was synthesized by precipitation from boiling phosphoric acid and incubated overnight in erbium chloride solutions. The resulting powder was embedded in a monolithic poly(glycidyl methacrylate/ethylene dimethacrylate) polymer. The monolith was synthesized in a spin column by radical polymerization. Erbium phosphate demonstrated a high affinity and selectivity for phosphopeptides due to the strong interaction of trivalent erbium ions with the phosphate groups of phosphopeptides. The high selectivity and performance of the designed spin columns were demonstrated by successfully enriching phosphopeptides from tryptically digested protein mixtures containing the model phosphoproteins α- and ß-casein, bovine milk, and human saliva. By the implementation of several washing steps, unspecific components were removed and the enriched phosphopeptides were effectively eluted from the spin columns under alkaline conditions. The selective performance of the presented method was further demonstrated by the enrichment of two synthetic phosphopeptides, which were spiked in tryptically digested and dephosphorylated HeLa cell lysates at low ratios. Finally, the presented approach was compared to conventional phosphopeptide enrichment by titanium oxide and revealed higher recoveries for the erbium phosphate doped monoliths.

Solid-phase extraction of plant thionins employing aluminum silicate based extraction columns.

Thionins belong to a family of cysteine-rich, low-molecular-weight (∼5 KDa) biologically active proteins in the plant kingdom. They display a broad cellular toxicity against a wide range of organisms and eukaryotic cell lines. Thionins protect plants against different pathogens, including bacteria and fungi. A highly selective solid-phase extraction method for plant thionins is reported deploying aluminum silicate (3:2 mullite) powder as a sorbent in extraction columns. Mullite was shown to considerably improve selectivity compared to a previously described zirconium silicate embedded poly(styrene-co-divinylbenzene) monolithic polymer. Due to the presence of aluminum(III), mullite offers electrostatic interactions for the selective isolation of cysteine-rich proteins. In comparison to zirconium(IV) silicate, aluminum(III) silicate showed reduced interactions towards proteins which resulted into superior washings of unspecific compounds while still retaining cysteine-rich thionins. In the presented study, European mistletoe, wheat and barley samples were subjected to solid-phase extraction analysis for isolation of viscotoxins, purothionins and hordothionins, respectively. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy was used for determining the selectivity of the sorbent toward thionins. The selectively retained thionins were quantified by colorimetric detection using the bicinchoninic acid assay. For peptide mass-fingerprint analysis tryptic digests of eluates were examined.

Enrichment and desalting of tryptic protein digests and the protein depletion using boron nitride.

Sample preparation still remains a great challenge in modern bioanalysis and the interest in new efficient solid phase extraction (SPE) materials still remains high. In this work, hexagonal boron nitride (h-BN) is introduced as a new SPE material for the isolation and enrichment of peptides. The h-BN is isoelectronic and structurally similar to graphite. It has remarkable properties including good thermal conductivity, excellent thermal and chemical stability and a better oxidation resistance than graphite. BN attracts increasing interest because of its wide range of applicability. In the present work, the great potential of h-BN, as a new SPE-material, on the enrichment, preconcentration and desalting of tryptic digest of model proteins is demonstrated. A special attention was dedicated to the efficient enrichment of hydrophilic phosphopeptides. Two elution protocols were developed for the enrichment of peptides compatible for subsequent MALDI-MS and ESI-MS analysis. In addition, the recoveries of 5 peptides and 3 phosphopeptides with wide range of pI values utilizing h-BN materials with different surface areas were investigated. 84-106% recovery rate could be achieved using h-BN materials. The results were compared with those obtained using graphite and silica C18 under the same elution conditions, and lower recoveries were obtained. In addition, h-BN was found to have a capability of protein depletion, which is requisite for the peptide profiling.

Downregulation of transcription factor E4F1 in hepatocarcinoma cells: HBV-dependent effects on autophagy, proliferation and metabolism.

The multifunctional E4F1 protein is a cellular target of the E1A adenoviral oncoprotein. Interaction between E4F1 and the hepatitis B virus (HBV) protein HBx has been demonstrated in vitro. In this study, RNA interference has been used to downregulate E4F1 in the hepatocellular carcinoma (HCC) cell line HepG2 (HBV negative) and its derivative, HBV expressing HepG2/2.2.15. Reduction of E4F1 levels induced hepatocyte vacuolation (formation of large cytoplasmic vesicles), increased autophagy and caused mitochondrial defects and metabolism changes in HepG2/2.2.15, but not in HepG2. Moreover, downregulation of E4F1 reduced DNA synthesis with partial cell cycle arrest in G1 in both cell types and this effect was more marked in HepG2/2.2.15 than in HepG2. These effects were partially prevented by RNA interference directed to either HBx or to p53. Coprecipitation and western blot experiments detected complexes between E4F1 and HBx in several HCC cell lines. Although a review of mutation and gene expression public databases did not support that E4F1 is specifically altered in liver cancer, our results suggest that E4F1 may neutralize the capacity of HBx to activate a p53-dependent, metabolic and growth arrest phenotype in liver cells, thus possibly contributing to the viability and proliferation of HBV-infected cells.

MALDI-MS tissue imaging identification of biliverdin reductase B overexpression in prostate cancer.

New biomarkers are needed to improve the specificity of prostate cancer detection and characterisation of individual tumors. In a proteomics profiling approach using MALDI-MS tissue imaging on frozen tissue sections, we identified discriminating masses. Imaging analysis of cancer, non-malignant benign epithelium and stromal areas of 15 prostatectomy specimens in a test and 10 in a validation set identified characteristic m/z peaks for each tissue type, e.g. m/z 10775 for benign epithelial, m/z 6284 and m/z 6657.5 for cancer and m/z 4965 for stromal tissue. A 10-fold cross-validation analysis showed highest discriminatory ability to separate tissue types for m/z 6284 and m/z 6657.5, both overexpressed in cancer, and a multicomponent mass peak cluster at m/z 10775-10797.4 overexpressed in benign epithelial tissue. ROC AUC values for these three masses ranged from 0.85 to 0.95 in the discrimination of malignant and non-malignant tissue. To identify the underlying proteins, prostate whole tissue extract was separated by nano-HPLC and subjected to MALDI TOF/TOF analysis. Proteins in fractions containing discriminatory m/z masses were identified by MS/MS analysis and candidate marker proteins subsequently validated by immunohistochemistry (IHC). Biliverdin reductase B (BLVRB) turned out to be overexpressed in PCa tissue. BIOLOGICAL SIGNIFICANCE: In this study on cryosections of radical prostatectomies of prostate cancer patients, we performed a MALDI-MS tissue imaging analysis and a consecutive protein identification of significant m/z masses by nano-HPLC, MALDI TOF/TOF and MS/MS analysis. We identified BLVRB as a potential biomarker in the discrimination of PCa and benign tissue, also suggesting BVR as a feasible therapeutic target.

Solid-phase extraction method for the isolation of plant thionins from European mistletoe, wheat and barley using zirconium silicate embedded in poly(styrene-co-divinylbenzene) hollow-monoliths.

Thionins are cysteine-rich, biologically active small (∼5 kDa) and basic proteins occurring ubiquitously in the plant kingdom. This study describes an efficient solid-phase extraction (SPE) method for the selective isolation of these pharmacologically active proteins. Hollow-monolithic extraction tips based on poly(styrene-co-divinylbenzene) with embedded zirconium silicate nano-powder were designed, which showed an excellent selectivity for sulphur-rich proteins owing to strong co-ordination between zirconium and the sulphur atoms from the thiol-group of cysteine. The sorbent provides a combination of strong hydrophobic and electrostatic interactions which may help in targeted separation of certain classes of proteins in a complex mixture based upon the binding strength of different proteins. European mistletoe, wheat and barley samples were used for selective isolation of viscotoxins, purothionins and hordothionins, respectively. The enriched fractions were subjected to analysis by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometer to prove the selectivity of the SPE method towards thionins. For peptide mass-fingerprint analysis, tryptic digests of SPE eluates were examined. Reversed-phase high-performance liquid chromatography hyphenated to diode-array detection was employed for the purification of individual isoforms. The developed method was found to be highly specific for the isolation and purification of thionins.

Solid-phase extraction of galloyl- and caffeoylquinic acids from natural sources (Galphimia glauca and Arnicae flos) using pure zirconium silicate and bismuth citrate powders as sorbents inside micro spin columns.

Galloyl- and caffeoylquinic acids are among the most important pharmacological active groups of natural compounds. This study describes a pre-step in isolation of some selected representatives of these groups from biological samples. A selective solid-phase extraction (SPE) method for these compounds may help assign classes and isomer designations within complex mixtures. Pure zirconium silicate and bismuth citrate powders (325 mesh) were employed as two new sorbents for optimized SPE of phenolic acids. These sorbents possess electrostatic interaction sites which accounts for additional interactions for carbon acid moieties as compared to hydrophilic and hydrophobic sorbents alone. Based on this principle, a selective SPE method for 1,3,4,5-tetragalloylquinic acid (an anti-HIV and anti-asthamatic agent) as a starting compound was developed and then deployed upon other phenolic acids with success. The recoveries and selectivities of both sorbents were compared to most commonly applied and commercially available sorbents by using high performance liquid chromatography. The nature of interaction between the carrier sorbent and the acidic target molecules was investigated by studying hydrophilic (silica), hydrophobic (C18), mixed-mode (ionic and hydrophobic: Oasis(®) MAX) and predominantly electrostatic (zirconium silicate) materials. The newly developed zirconium silicate and bismuth citrate stationary phases revealed promising results for the selective extraction of galloyl- and caffeoylquinic acids from natural sources. It was observed that zirconium silicate exhibited maximum recovery and selectivity for tetragalloylquinic acid (84%), chlorogenic acid (82%) and dicaffeoylquinic acid (94%) among all the tested sorbents.