Pipes and Potions: Testing the Efficacy of European Folk Preparation Methods for Anticholinergic Solanaceae Plants.

The present article sought to evaluate the efficiency of various folk preparation methods commonly used in Europe for employing anticholinergic Solanaceae plants. The study aimed to uncover which folk methods were effective for the extraction of the anticholinergic tropane alkaloids of these plants, atropine and scopolamine. The folk extractions that were tested sought to simulate the preparation of teas, cold-water infusions, unguents, tinctures, fortified wines, and smoking. All preparation types and a control were then put through an extraction process to see what amount of the alkaloids had been maintained. These extractions were then analysed using high-performance liquid chromatography (HPLC). Cold- and hot-water preparations, tinctures, and fortified wines all proved to be effective means of extracting atropine and scopolamine from plant material under conditions seen in folk usage. Smoking and the oil-based unguent, however, yielded no alkaloids, suggesting a lack of efficiency for these preparations, a problem with our methodology, or possible chemical changes and losses associated with the preparation procedure.

Scopolia carniolica var. hladnikiana: Alkaloidal Analysis and Potential Taxonomical Implications.

The present research sought to compare the content of hyoscyamine/atropine and scopolamine in Scopolia carniolica and its contested variety, S. carniolica var. hladnikiana, with the aim of investigating differences that may be of taxonomical significance. A multi-phase liquid extraction and high-performance liquid chromatography were used to extract and analyse these alkaloids in different organs from plants collected over two years at three sites. Our results showed that hyoscyamine was almost twice as prevalent as scopolamine across our 87 samples. The differences between organ types were large, but so too were intra-organ differences; differences due to organs proved to be significant for hyoscyamine, while they were only marginally significant for scopolamine. The collection site also proved to have a significant influence, but only on hyoscyamine content. The year of collection and the variety proved to not be significant. Our results support the theory that these two varieties are likely one, a view argued by many others, though more work is needed to draw concrete taxonomical conclusions.

Characterization of Biomolecules with Antibiotic Activity from Endophytic Fungi Phomopsis Species.

Recently, growing interest is devoted to investigation of bioactive secondary metabolites of endophytic fungi. Thus, as an extension to our previous achievements related to antimicrobial potential of endophytic fungi, Phomopsis species isolated from conifer needles was selected as appropriately promising natural source for drug discovery. Its dichloromethane and ethanol extracts considerably inhibited growth of Escherichia coli and Staphylococcus aureus. Moreover, the individual compounds of dichloromethane extract have been separated, collected and purified using semi preparative liquid chromatographic analysis and comprehensively characterized using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). Based on their antimicrobial activity and unique structural characteristics in comparison with well-established drugs from the same therapeutic category, two dominant compounds (Z)-(Z)-2-acetoxyprop-1-en-1-yl-3-(3-((E)-3,4-dihydroxypent-1-en-1-yl)oxiran-2-yl)acrylate (denoted as 325-3) and (Z)-(Z)-2-acetoxyprop-1-en-1-yl 3-(3-((E)-4-hydroxy-3-oxopent-1-en-1-yl)oxiran-2-yl)acrylate (denoted as 325-5) were recognized as valuable leading structures for future discovery of novel antibiotics.

Aggregation of Recombinant Monoclonal Antibodies and Its Role in Potential Immunogenicity.

BACKGROUND: Development of new recombinant biotechnology products has greatly expanded in the field of modern pharmacy and medicine. Since biological recombinant molecules are sensitive, simple or composed proteins, their function is heavily dependent on their structure. In addition to their efficacy, biological medicinal products could show side effects such as immunogenicity. Therefore, detection and characterization of protein structural variants is essential during development and quality control of therapeutic proteins that might trigger immunogenic response in organism. METHODS: This article includes proposed detection and characterization of aggregated, as well as other modified forms of monoclonal antibodies (mAb), by using selected chromatographic and spectrometric methods. Additionally, selected mAb's aggregates and modified structural variants of monoclonal antibodies were subjected to the immature monocyte-derived dendritic cells' (DC) examination experiment for monitoring of activated DC cells in order to determine potential immunogenicity of mAb structural variants. Furthermore, potential innate immunogenic response of peripheral blood mononuclear cells (PBMC) cultures to mAb aggregates was also evaluated by measuring pro-inflammatory cytokine response during early exposure of PBMCs to different mAb samples and by determining the effect of mAb aggregates on PBMC proliferation during long-term cultures. RESULT AND CONCLUSION: All developed and proposed analytical methods and immunological in vitro DC and PBMC assays, could be used as platform for complementary analytical characterization and determination of potential for immunogenicity for all biopharmaceutical products which contain monoclonal antibodies as active pharmaceutical ingredients.

Isolation and Determination of Fomentariol: Novel Potential Antidiabetic Drug from Fungal Material.

Diabetes mellitus is one of the leading world's public health problems. Therefore, it is of a huge interest to develop new antidiabetic drugs. Apart from traditional therapy of diabetes, nowadays, importance is given to natural substances with antidiabetic potential. Fomes fomentarius is a mushroom widely used for different purposes, due to its range of already confirmed activities. Fomentariol is a constituent of Fomes fomentarius, responsible for its antidiabetic potential. In that respect, it is important to develop a method for isolation and quantification of fomentariol from fungal material, which will be simple and efficient. Multistep, complex extraction applied in the previously reported studies was avoided with ethanol, providing rapid single-step extraction. The presence of fomentariol in ethanolic extract was confirmed by high-resolution mass spectrometry. Semipreparative HPLC method was developed and applied for isolation from ethanol extract and purification of the active compound fomentariol. It was a gradient reversed-phase method with a mobile phase consisting of acetonitrile and 0.1% formic acid in water and total run time of 15 minutes. The amount of 6.5 mg of high-purity fomentariol was determined by quantitative NMR with toluene as internal standard. The isolated and determined amount of substance can be further used for the quantitative estimation of activity of fomentariol.

Dextran sulphate sodium colitis in C57BL/6J mice is alleviated by Lactococcus lactis and worsened by the neutralization of Tumor necrosis Factor α.

TNFα has a well-established role in inflammatory bowel disease that affects the gastrointestinal tract and is usually manifested as Crohn's disease or ulcerative colitis. We have compared Lactococcus lactis NZ9000 displaying TNFα-binding affibody with control Lactococcus lactis and with anti-TNFα antibody infliximab for the treatment of mice with dextran sulphate sodium (DSS)-induced colitis. L. lactis NZ9000 alleviated the colitis severity one week after colitis induction with DSS, more effectively when administered in preventive fashion prior to, during and after DSS administration. TNFα-binding L. lactis was less effective than control L. lactis, particularly when TNFα-binding L. lactis was administered in preventive fashion. Similarly, an apparently detrimental effect of TNFα neutralization was observed in mice that were intraperitoneally administered anti-TNFα monoclonal antibody infliximab prior to colitis induction. The highest concentrations of tissue TNFα were observed in groups without DSS colitis that were treated either with TNFα-binding L. lactis or infliximab. To conclude, we have confirmed that L. lactis exerts a protective effect on DSS-induced colitis in mice. Contrary to expectations, but in line with some reports, the neutralization of TNFα aggravated disease symptoms in the acute phase of colitis and increased TNFα concentration in colon tissue of healthy mice. Nevertheless, we have demonstrated that oral administration of bacteria with surface displayed TNFα-binding affibody can interfere significantly with TNFα signaling and mimic the infliximab response in the given animal model of colitis.

Expression of a hepatitis A virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity.

An epidemic shift in Hepatitis A virus (HAV) infection has been observed in recent years in rapidly developing countries, with increasing numbers of severe adult cases which has led to renewed interest in vaccination. Our approach in vaccine development uses recombinant expression of the highly immunogenic HAV antigen VP1-P2a in food-grade lactic acid bacterium Lactococcus lactis and in Escherichia coli. We used genetic constructs that enable nisin-controlled expression of the antigen in L. lactis in three different forms: (a) intracellularly, (b) on the bacterial surface and (c) on the bacterial surface fused with the fragment of the E. coli flagellin molecule that can act as a molecular adjuvant. Expression of the two surface forms of the antigen was achieved in L. lactis, and the resulting antigen-displaying bacteria were administered orally to mice. Half the animals in each of the two groups developed specific IgGs, with titers increasing over time and reaching 1:422 without flagellin and 1:320 with flagellin. A much higher titer 1:25,803 was observed with the parenterally administered antigen, which was purified from E. coli. With the latter, a significant mucosal IgA response was also observed. Despite significant titers, the IgGs elicited with oral or parenteral administration could not prevent HAV from infecting cells in a virus neutralization assay, suggesting that the antibodies cannot recognize viral surface epitopes. Nevertheless, orally administered HAV antigen expressed in L. lactis elicited significant systemic humoral immune response showing the feasibility for development of effective HAV vaccine for mucosal delivery.

Determination of raloxifene and its glucuronides in human urine by liquid chromatography-tandem mass spectrometry assay.

A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-ß-glucuronide (M1), raloxifene-4'-ß-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.

Engineered lactic acid bacterium Lactococcus lactis capable of binding antibodies and tumor necrosis factor alpha.

We have optimized the display of the B domain of staphylococcal protein A on the surface of Lactococcus lactis. The maximum binding capacity was estimated at 0.146 µg of antibody per 108 cells and was sustained at 86% after treatment with simulated gastric juice. A tumor necrosis factor alpha (TNF-α)-binding affibody was also displayed and bound TNF-α, which could be useful in the treatment of inflammatory bowel disease.

Chemoporation using saponins or cholates: an alternative method for transformation of bacterial cells.

A new method for fast transformation of competent bacterial cells has been developed. The transformation is induced with cholic acid analogues or saponins which cause reversible disruption of the bacterial membrane. This method shortens the time of transformation without significant loss of transformation efficiency in comparison to heat shock method and is the first reported chemically-induced transformation. New data about interactions between cholates and biomembranes is revealed that may contribute to better understanding of bacterial transformation.