Anti-Atherosclerotic Properties of Aronia melanocarpa Extracts Influenced by Their Chemical Composition Associated with the Ripening Stage of the Berries.

The high content of bioactive compounds in Aronia melanocarpa fruit offers health benefits. In this study, the anti-atherosclerotic effect of Aronia extracts was assessed. The impact on the level of adhesion molecules and the inflammatory response of human umbilical vein endothelial cells (HUVECs) was shown in relation to the chemical composition and the stage of ripening of the fruits. Samples were collected between May (green, unripe) and October (red, overripe) on two farms in Poland, which differed in climate. The content of chlorogenic acids, anthocyanins, and carbohydrates in the extracts was determined using HPLC-DAD/RI. The surface expression of ICAM-1 and VCAM-1 in HUVECs was determined by flow cytometry. The mRNA levels of VCAM-1, ICAM-1, IL-6, and MCP-1 were assessed using the quantitative real-time PCR method. The farms' geographical location was associated with the quantity of active compounds in berries and their anti-atherosclerotic properties. Confirmed activity for green fruits was linked to their high chlorogenic acid content.

Increasing integrated testing in community settings through interventions for change, including the Spring European Testing Week.

BACKGROUND: Maximising access to testing by targeting more than one infection is effective in identifying new infections in settings or populations. Within the EU funded Joint Action INTEGRATE, this paper examined the feasibility and impact of expanding integrated testing for HIV, hepatitis C (HCV), chlamydia, gonorrhoea and/or syphilis in four community-based pilots through targeted interventions in Croatia, Italy and Poland and the Spring European Testing Week since community settings are key in detecting new infections and reaching key populations. METHODS: Pilots led by local INTEGRATE partners prioritised testing for other infections or key populations. The Croatian pilot expanded testing for men who have sex with men to syphilis, chlamydia and gonorrhoea. Italian partners implemented a HIV and HCV testing/information event at a migrant centre. A second Italian pilot tested migrants for HIV and HCV through outreach and a low-threshold service for people who use drugs. Polish partners tested for HIV, HCV and syphilis among people who inject drugs in unstable housing via a mobile van. Pilots monitored the number of individuals tested for each infection and reactive results. The pilot Spring European Testing Week from 18 to 25 May 2018 was an INTEGRATE-driven initiative to create more testing awareness and opportunities throughout Europe. RESULTS: The Croatian pilot found a high prevalence for each syphilis, chlamydia and gonorrhoea respectively, 2.1%, 12.4% and 6.7%. The Italian migrant centre pilot found low proportions who were previously tested for HIV (24%) or HCV (11%) and the second Italian pilot found an HCV prevalence of 6.2%, with low proportions previously tested for HIV (33%) or HCV (31%). The Polish pilot found rates of being previously tested for HIV, HCV and syphilis at 39%, 37%, and 38%, respectively. Results from the Spring European Testing Week pilot showed it was acceptable with increased integrated testing, from 50% in 2018 to 71% in 2019 in participants. CONCLUSIONS: Results show that integrated testing is feasible and effective in community settings, in reaching key populations and minimising missed testing opportunities, and the pilots made feasible because of the European collaboration and funding. For sustainability and expansion of integrated community testing across Europe, local government investment in legislation, financial and structural support are crucial.

Recommendations for collection and integration of community-based testing and linkage to care data into national surveillance, monitoring and evaluation systems for HIV, viral hepatitis and sexually transmitted infections: results from the INTEGRATE Joint Action.

BACKGROUND: National testing strategy, including monitoring and evaluation, is critical in responding to HIV, sexually transmitted infections, and viral hepatitis. Community-based voluntary counselling and testing contributes to early HIV diagnoses among key populations. Countries providing community-based testing, should integrate some core data on testing and linkage to care in these services into national surveillance and monitoring and evaluation systems. This study aimed to support the integration of community-based voluntary counselling and testing data into respective national surveillance and M&E systems for those infections. METHODS: Preliminary consensus on indicators for the integration of community-based voluntary counselling and testing data into respective national surveillance and monitoring and evaluation systems was reached. Pilot studies were conducted in Estonia, Poland, Serbia, Slovakia, Slovenia and Spain. After pilot activities were implemented, the final consensus on indicators was reached. An analysis of the facilitators and barriers faced during pilot studies was conducted to inform the final recommendations for implementation. RESULTS: The minimum set of six indicators to be integrated into national surveillance and monitoring and evaluation systems were: number of tests, number of clients tested, reactivity rate for tests and clients, positivity (active infection) rates for tests and clients, linkage to care rates for clients with reactive and/or positive test result, proportion of all new diagnoses in a country with first reactive test result at community-based voluntary counselling and testing service. Seven additional indicators were identified. Each indicator should be disaggregated by key population, sex and age group. A list of 10 recommendations for the collection and integration of community-based voluntary counselling and testing data into national surveillance and monitoring and evaluation systems for HIV, sexually transmitted infections and viral hepatitis was identified. CONCLUSIONS: Integration of some community-based voluntary counselling and testing monitoring and evaluation data into national surveillance and monitoring and evaluation systems in all pilot countries was achieved. The recommendations will support such integration in other European countries. European Centre for Prevention and Control of Diseases included questions from the minimum list of indicators into their Dublin Declaration questionnaire 2020 to contribute to evidence based community testing policies in European countries.

Integration of community-based testing data into national HIV surveillance in Poland, Serbia and Slovakia within the framework of INTEGRATE project.

BACKGROUND: Community-based voluntary counselling and testing contributes to early HIV diagnoses among key populations. Testing data from such decentralized services is however often not standardized nor linked to national surveillance systems. This study aimed to support the integration of community testing data into respective national surveillance and monitoring and evaluation systems for those infections. We present results from three national pilots, focused on improved data collection and transfer. METHODS:  Within the Joint Action INTEGRATE different pilot activities were planned and implemented according to the local context. In Slovakia, standardised data collection tools were implemented in three community testing services. The data generated was used to calculate the proposed indicators. In Poland, positive test results from the community testing database were linked to the national case-based surveillance database using confirmatory test number, to improve the completeness of behavioural data in the national database. In Serbia, voluntary counselling and testing forms were improved enabling identification of community-based testing. A system to generate unique client identifiers was initiated in the National registry of HIV cases to monitor linkage to care. RESULTS: All three sites were able to estimate most of the agreed indicators. In Slovakia during the study period 675 people were tested for HIV, 410 for hepatitis C and 457 for syphilis, with reactivity rates of 0.4, 2.5 and 1.8%, respectively. For HIV, 66.7% of reactive cases were confirmed and linked to care. In Poland, 28.9% of the community testing sites' records were linked to the national surveillance database (and accounted for 14.3% of all new diagnoses registered here during 2017-2018). Reactivity rate ranged between 1.9% and 2.1%. In Serbia, 80 persons were tested at community sites, from which two had a reactive HIV test result. By linking unique client identifiers from voluntary counselling and testing and National Registry of HIV cases databases, linkage to care within a two-month period was observed for one of two people with reactive HIV test result. CONCLUSIONS:  Pilot activities in the three countries demonstrate that integration of community-based testing data into surveillance systems is feasible and can help improve national surveillance data by providing key information.

Spirostanol Sapogenins and Saponins from Convallaria majalis L. Structural Characterization by 2D NMR, Theoretical GIAO DFT Calculations and Molecular Modeling.

Two new spirostanol sapogenins (5ß-spirost-25(27)-en-1ß,2ß,3ß,5ß-tetrol 3 and its 25,27-dihydro derivative, (25S)-spirostan-1ß,2ß,3ß,5ß-tetrol 4) and four new saponins were isolated from the roots and rhizomes of Convallaria majalis L. together with known sapogenins (isolated from Liliaceae): 5ß-spirost-25(27)-en-1ß,3ß-diol 1, (25S)-spirostan-1ß,3ß-diol 2, 5ß-spirost-25(27)-en-1ß,3ß,4ß,5ß-tetrol 5, (25S)-spirostan-1ß,3ß,4ß,5ß-tetrol 6, 5ß-spirost-25(27)-en-1ß,2ß,3ß,4ß,5ß-pentol 7 and (25S)-spirostan-1ß,2ß,3ß,4ß,5ß-pentol 8. New steroidal saponins were found to be pentahydroxy 5-O-glycosides; 5ß-spirost-25(27)-en-1ß,2ß,3ß,4ß,5ß-pentol 5-O-ß-galactopyranoside 9, 5ß-spirost-25(27)-en-1ß,2ß,3ß,4ß,5ß-pentol 5-O-ß-arabinonoside 11, 5ß-(25S)-spirostan-1ß,2ß,3ß,4ß,5ß-pentol 5-O-galactoside 10 and 5ß-(25S)-spirostan-1ß,2ß,3ß,4ß,5ß-pentol 5-O-arabinoside 12 were isolated for the first time. The structures of those compounds were determined by NMR spectroscopy, including 2D COSY, HMBC, HSQC, NOESY, ROESY experiments, theoretical calculations of shielding constants by GIAO DFT, and mass spectrometry (FAB/LSI HR MS). An attempt was made to test biological activity, particularly as potential chemotherapeutic agents, using in silico methods. A set of 12 compounds was docked to the PDB structures of HER2 receptor and tubulin. The results indicated that diols have a higher affinity to the analyzed targets than tetrols and pentols. Two compounds (25S)-spirosten-1ß,3ß-diol 1 and 5ß-spirost-25(27)-en-1ß,2ß,3ß,4ß,5ß-pentol 5-O-galactoside 9 were selected for further evaluation of biological activity.

Aronia melanocarpa Fruits as a Rich Dietary Source of Chlorogenic Acids and Anthocyanins: 1H-NMR, HPLC-DAD, and Chemometric Studies.

Aronia melanocarpa (Michx.) Elliott's (chokeberry) besides anthocyanins contains significant amounts of hydroxycinnamic acids: Chlorogenic and its isomer neochlorogenic acid. They exhibit antioxidant, anti-inflammatory, antidiabetic, and antibacterial activities, thus they can have a significant impact on the health-promoting properties of Aronia. The aim of our research was to determine the changes in the content of chlorogenic acids (CGAs) and anthocyanins during fruit development and ripening, with a particular emphasis on acids. Aronia fruit samples were collected from July to October on two organic farms in Poland. The chemical composition of the extracts was determined by NMR spectroscopy and HPLC-DAD. 1H-NMR and HPLC data were analyzed using chemometric analysis and multivariate statistics (PCA). The results showed that the content of chlorogenic acids and anthocyanins changes during ripening and depends on the time of harvest and the region of cultivation. A correlation between the time of CGAs reduction and the appearance of anthocyanins was also noticed. The result of our research was also a database in the form of NMR parameters, which allows analysis of the metabolite profile and tracking of its changes. The 1H-NMR spectrum showing anthocyanin and CGA resonance can be considered the Aronia berry fingerprint.

Hepatitis C services at harm reduction centres in the European Union: a 28-country survey.

BACKGROUND: In the context of the WHO's 2016 Viral Hepatitis Strategy and the introduction of treatment that can cure more than 95% of cases with hepatitis C virus (HCV) infection, the European Joint Action on HIV and Co-infection Prevention and Harm Reduction (HA-REACT) undertook a study in the member states of the European Union (EU). It aimed to determine service providers' understanding of the current services in their respective countries and the barriers experienced by PWID in accessing HCV testing, care and treatment services in their country. METHODS: In 2017, 38 purposively selected harm reduction service providers completed a 26-item English-language online survey addressing the availability, accessibility and funding of HCV services at harm reduction centres. HCV-related data and reported findings were extracted by country or by responding organization. RESULTS: Responses were received from all EU member states. Respondents from 23 countries reported that HCV tests are offered by harm reduction services in their countries, and eight countries reported that addiction specialists in their countries are able to prescribe HCV therapy. Almost half of the respondents (45%) said that their respective organizations had established referral systems with centres providing HCV treatment. CONCLUSIONS: Not all EU member states have harm reduction services that provide HCV tests, and many do not have established referral systems with treatment providers. Moreover, the inability of addiction specialists to prescribe HCV treatment points to missed opportunities to make treatment more accessible. Further, discrepancies were noted between the available HCV services and stakeholders' knowledge about their availability.

¹H and ¹³C NMR-based sugar profiling with chemometric analysis and antioxidant activity of herbhoneys and honeys.

BACKGROUND: Herbhoneys, relatively new bee products, are expected to have interesting medicinal properties. However, there is still a lack of data concerning their composition and antioxidant properties. ¹H and ¹³C NMR spectroscopy coupled with chemometric analysis (PCA and PLS-DA) and antioxidant assays (DPPH-ESR and ORAC-FL) were used to study 25 samples of Polish herbhoneys and honeys. RESULTS: Antioxidant activity varied among the samples. The best properties were exhibited by cocoa and instant coffee herbhoneys. The contents of total polyphenols and total carotenoids in the studied samples were found to be 70-1340 mg GAE kg⁻¹ and 0-28.05 mg kg⁻¹ respectively. No significant differences between herbhoney and honey samples were found in their sugar profiles. The PCA of ¹³C NMR spectra of the samples in DMSO-d6 resulted in sample clustering due to sucrose content. CONCLUSION: Herbhoneys have similar antioxidant properties to traditional honeys, being therefore of equal nutritional value. There was a noticeable influence of the extract concentration on the observed antioxidant effect. For samples with high antioxidant activity, polyphenols were responsible for the observed effect. Sample clustering due to sucrose content in the NMR-PCA study allowed effortless detection of adulteration.

Solid-state tautomerism in 2-carboxyindan-1,3-dione.

The structure of 2-carboxyindan-1,3-dione was investigated using a combination of quantum-chemical calculations and solid-state NMR and IR spectroscopy. Due to poor solubility of the compound in different solvents, no single crystals could be obtained. Two dimeric structures formed from the tautomers of 2-carboxyindan-1,3-dione are likely to coexist in the solid state. The dimers interconvert via intramolecular proton transfer in one of the tautomeric forms constituting the dimers. The energy barrier of the intramolecular proton transfer reaction is calculated as 5.82 kcal mol(-1) at the MP2/6-31++G level of theory.

1H and 13C NMR characteristics of ß-blockers.

The ß-blockers are important drugs and decades of clinical experience proved their high medical status. However, to the best of our knowledge, there is no complete assignment of (1)H and (13)C NMR resonances of popular representatives: acebutolol, alpenolol, pindolol, timolol and propranolol and the published NMR data on carvedilol and atenolol are incorrect. Therefore, (1)H and (13)C NMR spectroscopy was applied for the characterization of a series of ß-adrenolytics: carvedilol (1), pindolol (2), alprenolol (3), acebutolol (4), atenolol (5), propranolol (6) and timolol (7). Two-dimensional NMR experiments (COSY, HMQC, HMBC, NOESY) allowed the unequivocal assignment of (1)H and (13)C spectra for solution (DMSO-d(6) ). Salts and bases can be easily distinguished based on (13)C chemical shifts which are within 65.0-65.5 ppm (OC2) and 46.9-47.0 (NC3) for hydrochlorides and larger, ca. 68.4 ppm (OC2) and 50.3-52.6 (NC3) for bases. NMR data of 1-7 should be included in pharmacopoeias.

NMR and gas chromatography studies of lyophilized human brain tumors.

1H HR MAS (high resolution magic angle spinning) NMR is a promising tool in the in vitro characterisation of brain tumors. Brain tissue samples were collected from patients with intracranial tumors during surgery, frozen and lyophilized. The analysis of solid samples was performed by high-speed rotation (33 kHz) H MAS NMR. The most intense resonances arise from lipids (methylene mid-chain and methyl carbons of fatty acids). 1H MAS spectra obtained from the same histological type of brain tumors are similar; spectra of glioblastomas were different from those of meningiomas. 1H and 13C NMR solution spectra of lipid extracts confirmed the presence of aliphatic chains fatty acids and cholesterol as major constituents. Biochemical information on liberated fatty acid composition was obtained by gas-chromatography (GC). The glioma content of the linoleic acid (18:2n6) was found to be greater, whereas the level of docosahexaenoic acid (22:6n3) was significantly reduced.

Structural studies of two capsaicinoids: dihydrocapsaicin and nonivamide. 13C and 15N MAS NMR supported by genetic algorithm and GIAO DFT calculations.

Capsaicinoids are alkaloid type capsaicin analogs with prospective pharmacological activity. However their solid state conformations have not been studied yet. As part of the study, cross polarization (CP) magic angle spinning (MAS) solid state 13C and 15N NMR spectra of dihydrocapsaicin (DHCAP) and nonivamide (NVA) were recorded. Solid state chemical shifts differ from their solution counterparts; remarkable differences occur for carbons C2', C6' and C7' linked to C1' in DHCAP and with methylene carbons C4-C8 in NVA. The doubling of some resonances in the spectra of solid NVA indicates that there are two molecules in the crystallographic asymmetric unit. DFT GIAO calculations of shielding constants were performed for several geometric isomers, including molecules with different orientations of aliphatic chain with respect to aromatic ring. Low-energy conformers were found by genetic algorithm methodology. Comparison of experimental 13C chemical shifts with theoretical (GIAO DFT) shielding parameters was helpful in predicting the most reliable geometry in the solid state. Cross polarization time constants T CP and relaxation times in the rotating frame T H 1ρ were obtained from variable-contact cross-polarization experiments. T H 1ρ are longer in the order: NVA < CAP < DHCAP.

(13)C CP MAS NMR and crystal structure of methyl glycopyranosides.

The X-ray diffraction analysis, (13)C CP MAS NMR spectra and powder X-ray diffraction patterns were obtained for selected methyl glycosides: alpha- and beta-d-lyxopyranosides (1, 2), alpha- and beta-l-arabinopyranosides (3, 4), alpha- and beta-d-xylopyranosides (5, 6) and beta-d-ribopyranoside (7) and the results were confirmed by GIAO DFT calculations of shielding constants. In X-ray diffraction analysis of 1 and 2, a characteristic shortening and lengthening of selected bonds was observed in molecules of 1 due to anomeric effect and, in crystal lattice of 1 and 2, hydrogen bonds of different patterns were present. Also, an additional intramolecular hydrogen bond with the participation of ring oxygen atom was observed in 1. The observed differences in chemical shifts between solid state and solution come from conformational effects and formation of various intermolecular hydrogen bonds. The changes in chemical shifts originating from intermolecular hydrogen bonds were smaller in magnitude than conformational effects. Furthermore, the powder X-ray diffraction (PXRD) performed for 4, 5 and 7 revealed that 7 existed as a mixture of two polymorphs, and one of them probably consisted of two non-equivalent molecules.

Solid-state NMR studies and DFT calculations of flavonoids: baicalein, baicalin and wogonoside.

Three flavonoids of pharmaceutical importance-baicalein, baicalin, and wogonoside-were isolated from a Chinese medicinal plant Scutellaria baicalensis Georgi and studied by 13C NMR in solution and solid state. Two-dimensional (2D) NMR spectroscopy in the liquid phase and dipolar dephasing (DD) experiments in magic-angle spinning (MAS) spectra enabled the assignment of 13C resonances. The cross-polarization (CP) time constants T(CH) and relaxation times T(H) (1rho) were obtained from the variable-contact time experiments. The principal elements of the 13C chemical shift tensor were determined in the spectra recorded under slow sample spinning (2 kHz) using phase-adjusted spinning sideband (PASS)-2D NMR technique, and were verified by density functional theory gauge-independent atomic orbital (DFT GIAO) calculations of shielding constants. Analysis of the 13C delta(ii) and comparison with shielding parameters calculated for different conformers of compounds 1-3 enabled the selection of the most reliable geometry in the solid phase. In all three compounds, an intramolecular hydrogen bond C5--OH...=C4 is formed; the existence of baicalein and baicalin with 'anticlockwise' orientation of OH groups is more probable.

Identification and analysis of drugs in the solid state by 13C CPMAS NMR: suxamethonium chloride and hydrocortisonum (Corhydron).

Cross-polarization (CP) magic angle spinning (MAS) 13C NMR spectroscopy has become a routine tool in pharmacy, employed to identify and characterize drugs in the solid phase. 13C CPMAS NMR spectra were recorded for solid hydrocortisone 21-hemisuccinate and suxamethonium chloride. White crystalline substances, such as these two drugs, can be easily distinguished; and solid-state 13NMR spectra of remarkably good quality are obtained in less than half an hour. 13C CPMAS chemical shifts for solid suxamethonium chloride and hydrocortisone sodium hemisuccinate are given, as well as cross-polarization kinetic parameters for suxamethonium chloride.

Antioxidant activity of extracts and flavonoids from Bidens tripartita.

Extracts from herb and flowers of Bidens tripartita L. (Asteraceae), obtained using solvents of different polarity, were studied for their radical scavenging effects. Antioxidant activities of pure flavonoids: flavanomarein (isookanin 7-O-glucoside), cynaroside (luteolin 7-O-glucoside) and luteolin, which had been isolated from this plant, were also evaluated. Radical-scavenging activity was measured by electron paramagnetic resonance (EPR) spectroscopy using stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The content of flavonoids in flower heads is half of that found in the herb; however, the extract from flowers showed that the antioxidant activity was almost two times higher there. Some extracts (n-BuOH fraction) showed long lasting radical scavenging activity and the EPR spectra were recorded in time to follow the reaction kinetics. Scavenging of DPPH showed second-order kinetics at the beginning of the assay period and later the first-order one. Different kinetics suggested the presence of polymerized and/or less active antioxidants with different scavenging mechanisms for particular polyphenolic compounds. Bur-marigold extracts are a potential source of natural antioxidants that may be used in pharmaceutical or food industry.

Spectroscopic and structural studies of the diosmin monohydrate and anhydrous diosmin.

Diosmin, a flavone glycoside frequently used in therapy of various veins diseases in monohydrate form, exhibits poor solubility in water and low bioavailability. Due to the fact that the anhydrous forms of drugs generally have better bioavailability than the corresponding hydrates, the aim of this study was to analyze the conversion of diosmin monohydrate (DSNM) to anhydrous diosmin (DSNA) that occurs upon heating. The mechanism of this transformation was examined as well as advanced structural studies of each form were performed using 13C CP/MAS SSNMR, DSC, FT-IR and PXRD techniques. Spectroscopic findings were supported by CASTEP-DFT calculations of NMR and IR parameters. The pathway of reversible transformation was specified as follows: DSNM upon heating for 24h at temperature up to 110°C losses non-crystalline water and converts into metastable form (DSNM*) that turns into DSNA during heating at temperature 140°C for next 24h. Under room temperature DSNA spontaneously absorbs moisture from air and turns into a DSNM within 72h. The detailed analysis of CP kinetic parameters (T1ρI) revealed presence of metastable, intermediate form of diosmin (DSNM*) and allowed its characterization. The results are essential for further studies comparing dissolution and bioavailability of DSNM and DSNA. The study provided an understanding of the conversion pathway of the diosmin monohydrate into its anhydrate form when it is exposed to increased temperature.

[The role of oxidative stress in the etiopathogenesis of rheumatoid arthritis]. / Rola stresu oksydacyjnego w etiopatogenezie reumatoidalnego zapalenia stawów.

Numerous scientific investigations confirmed the occurrence of oxidative stress in rheumatoid arthritis patients. There is evidence demonstrating elevated levels of oxidative stress markers and oxidative damage caused by reactive oxygen species (ROS) to lipids, proteins, sugars, and DNA, as well as a significant decrease in total antioxidant capacity, which protects the organism against ROS activity. Extensive ROS production can significantly accelerate the process of articular cartilage damage. It is believed that many disease-modifying anti-rheumatic drugs (DMARDs) affect oxidative stress, although there has been insufficient research to confirm such a relationship.

¹³C solid-state NMR analysis of the most common pharmaceutical excipients used in solid drug formulations Part II: CP kinetics and relaxation analysis.

Excipients used in the solid drug formulations differ in their NMR relaxation and (13)C cross-polarization (CP) kinetics parameters. Therefore, experimental parameters like contact time of cross-polarization and repetition time have a major impact on the registered solid state NMR spectra and in consequence on the results of the NMR analysis. In this work the CP kinetics and relaxation of the most common pharmaceutical excipients: anhydrous α-lactose, α-lactose monohydrate, mannitol, sucrose, sorbitol, sodium starch glycolate type A and B, starch of different origin, microcrystalline cellulose, hypromellose, ethylcellulose, methylcellulose, hydroxyethylcellulose, sodium alginate, magnesium stearate, sodium laurilsulfate and Kollidon(®) were analyzed. The studied excipients differ significantly in their optimum repetition time (from 5 s to 1200 s) and T(1ρ)(I) parameters (from 2 ms to 73 ms). The practical use of those differences in the excipients composition analysis was demonstrated on the example of commercially available tablets containing indapamide as an API. The information presented in this article will help to choose the correct acquisition parameters and also will save the time and effort needed for their optimization in the NMR analysis of the solid drug formulations.