Cardiovascular pathology in patients with superinvasive opisthorchiasis is characterized by severe changes in haemodynamics and myocardial metabolism, impaired automatism, excitability, and conduction of the heart muscle. An analysis of 578 cases  recorded in healthcare facilities treating opisthorchiasis patients with a hyperendemic focus was carried out. We identified a set of cardiac changes in patients with hypereosinophilic syndrome associated with superinvasive opisthorchiasis infection, classified the pathological processes in accordance with ICD-10, and described their pathogenesis. Cardiovascular pathology in patients with superinvasive opisthorchiasis (SO) is characterized by severe changes in haemodynamics and myocardial metabolism, impaired automatism, excitability, and conduction of the heart muscle. Significant, direct correlations between pain syndrome incidence and the duration of invasion, the frequency of re-infections, and stress fluctuations of the host have been found –3.9/L) [Hypereosinophilia is a pathological condition resulting from hypereosinophilic syndrome (HES), in which the number of eosinophilic leukocytes in peripheral blood is greater than 15% (1.5 × 109/L) –5. Hyper9/L) .The most detailed description of the myocardial state in different types of HES in SO was achieved by physicians, who revealed the development of severe forms of myocarditis and described cases of sudden cardiac death (SCD) in patients with SO-associated myocarditis , 7. In aCurrently, HES as the focus of opisthorchiasis is less common; however, HES as a complication of SO is underrepresented in the literature. There is a need to look for other risk factors for SCD and to develop measures for the primary prevention of cardiac death in patients with tissue helminthiases.n=217) and females (n=361) were included, with a female to male ratio of 1.6 : 1. A total of 110 hearts as well as livers . The preparations were stained with haematoxylin and eosin and van Gieson's stain using the Selye and Slinchenko methods. The degree of cellular infiltration and sclerotic processes was recorded by determining the index of the area occupied by cellular elements and fibrous structures (%), as well as their proportion in the composition of infiltrates. The stained preparations were subjected to light-optical analysis, and the images of histological preparations, taken with a microscope and Canon EOS 5D digital camera, were saved on a computer. Using the UTHSCSA Image Tool for Windows 3.0 program, the area of metabolic myocardial necrosis was determined. Mathematical and statistical processing of the data was performed on a personal computer using Statistica 6.1 software (Statsoft®).An analysis of 578 cases  recorded in healthcare facilities treating opisthorchiasis patients with a hyperendemic focus was carried out. The average age of patients was 49.7 ± 3.4 years, and the average age of those who died was 52.3 ± 2.9 years. Males n=27 and femCardiac pain (52.42%), rhythm disturbance (72.31%), palpitation (28.20%), and expansion of cardiac borders to the left (44.29%) were the most common pathologies in patients with SO; ECG changes were detected in 26.47% of patients, and mitral valve prolapse was detected in 1/3 of the individuals examined. Microscopic examination of those who died from accidental causes and complications associated with SO revealed the following changes in heart membranes: fuchsinophilic dystrophy of cardiomyocytes and their apoptosis with no leukocyte environment around the dead elements and diffuse cardiosclerosis. These changes are attributed to the group of lesions associated with hypoxia due to Botkin syndrome, i.e., systematic, short vasospasm of the heart vessels.The most pronounced pathology observed in eosinophilic myocarditis was the scattering and contamination of cardiomyocytes with opisthorchis exometabolites. Subsequently, an aggression of eosinophilic leukocytes followed by myocardial cell death led to the formation of extensive foci of deparenchymatization of the muscle membrane and perivascular, cystoid, and diffuse cardiosclerosis. The direction of collagen fibres coincided with the parasite's exometabolite dispersal Figures –6. In soO. felineus metabolites and contaminated cardiomyocytes), which is denoted as EETs (eosinophil extracellular traps). The mechanism is similar to that of NETs (neutrophil extracellular traps), in which leukocytes (neutrophilic and/or eosinophilic) release a meshwork of DNA fibres and granule proteins.In the definitive stage of eosinophilic leukocyte aggression, fragments of nuclei, cytoplasm, and cytolemma were observed, suggesting the “suicide” of eosinophils during contact with antigens (p < 0.01 − 0.001). Moreover, in HES, with the development of sudden cardiac death, a significant increase in the area of metabolic myocardial necrosis was observed (p < 0.001).Statistical processing of the data on infiltrate cellular composition showed a significant increase in leukocytes and a decrease in endotheliocytes (angiogenesis depression), fibroblasts, and fibrocytes  in SCD associated with HES patients compared to that in those who died by violence in combination with HES (Sudden cardiac death (SCD) is a topical health issue in Russia and abroad. The most frequent (more than 80%) cause of sudden cardiac death is coronary heart disease, while ventricular fibrillation (including reperfusion)  plays a Thus, pathological changes in the heart in SO are explained by coronary blood supply disturbances of a reflex type similar to those of Botkin syndrome. These changes were accompanied by paroxysmal cardialgia, resulting in diffuse cardiosclerosis. Cholecystocoronary syndrome was most prominent in patients with various forms of cholecystitis (chronic SO complication).O. felineus exometabolites are found in all organs, including the intestines, lungs, skin, and gums. Clinical signs of HES depend on the localization (target organs) of eosinophilic leukocytes and O. felineus metabolites; thus, they can either manifest as general, nonspecific reactions, or, for example, if the appendix is affected, the symptoms of acute appendicitis are evident, while infiltration in the kidneys is accompanied only by pain and does not result in organ insufficiency. Thanatogenetic mechanisms of sudden cardiac death in HES involve the development of ventricular fibrillation. Another mechanism of SCD, thromboembolic syndrome due to a parietal thrombus in the setting of eosinophilic pancarditis, was observed in one case.Eosinophilic infiltrates in HES are consistently found in the kidneys , gums F, and mucα-myosin) [Hypereosinophilic syndrome in SO is caused by opisthorchis exometabolites that contaminate cardiomyocytes; the latter subsequently becomes autoantigens and targets for eosinophilic leukocytes. In addition to the cellular immune response mentioned above, there is a humoural component (antibodies to myocardium -myosin) .Blood hypereosinophilia in opisthorchiasis correlates with tissue hypereosinophilia. A high level of IL-5 and hyperproduction of IgE and IgG are involved in the mechanisms of hypereosinophilia .Prevention of SO complications involves the exclusion of primary and recurrent infections, elimination of chronic inflammation, and/or surgeries in cases of gallbladder inflammation. These measures, along with cholecystectomy, also prevent choleperitonitis. Antiparasitic therapy plays a key role in the prevention of heart pathologies and sudden cardiac death. Dehelminthization should be conducted during the chronic phase of the disease. Antiparasitic drugs used in the acute phase and at the peak of HES are complicated by increased eosinophilia, including tissue infiltration, the exacerbation of eosinophilic myocarditis, and vasculitis, thus aggravating SCD. Blood tests and autopsy material analyses showed that even with eosinophilia in over 10% of all white blood cells, dehelminthization caused an increase in eosinophils by 123%–185%, with the development of tissue eosinophilia and eosinophilic myocarditis. Treatment of patients with HES (Loffler's syndrome), which is consistently encountered in SO, is rather challenging and requires an individualized approach.In superinvasive opisthorchiasis, hypereosinophilic syndrome should be referred to as a symptomatic HES, in which eosinophilia refers to a reactive, nonclonal, unlimited impairment of target organs with an infectious parasitic agent. Heart diseases in superinvasive opisthorchiasis should be attributed to secondary cardiomyopathies characterized as dilated, noninflammatory , and metabolic (metabolic necrosis). Inflammatory cardiomyopathies develop with HES and eosinophilic myocarditis, which, in turn, are induced by the contamination of cardiomyocytes by opisthorchis metabolites, turning them into target cells for antigens. Similar mechanisms for alteration, exudative, and proliferative processes in the heart are observed in other helminthic infections –14.


The Aim of this research is to analyze how the socio-demographic characteristics of users of Complementary and Alternative Medicine (CAM) in Serbia influence and impact their consumption of OTC drugs. Respondents and methods: The study employed the third edition of the National Health Survey of the Republic of Serbia, published in 2013, as a data source covering the Serbian population. The sample comprised of 550 interviewed individuals who had been applying a variety of CAM treatments over the previous 12 months. Their socio-demographic characteristics were used as independent variables impacting the consumption of OTC drugs over the previous 2-week period, representing the dependent variable.Results: Two thirds (65.3%) of the CAM users consumed OTC drugs at their own discretion, without recommendation by a physician or a relevant prescription. Users of OTC drugs were most often females whose ages ranged within the average interval of 49.16 ± 16.02, whose education level was to secondary school diploma, who were married and employed, lived in urban areas, mostly Belgrade, belonged to the middle-income group, and followed relevant headlines via public information channels . Comparison of the results revealed, on the one hand, that 2/3 of respondents who had used and 1/3 of those who did not consume OTC drugs had undergone fecal occult blood tests over the past year (p < 0.05) and, on the other hand, that those respondents had been less frequently hospitalized in the previous year (p = 0.05). In addition, the same responders were found to access available health care services more frequently than did others (p < 0.05).Conclusion: Since, according to the statistics, it is highly likely that respondents who were CAM- and OTC drug-users would be less frequently hospitalized and not use medical leave, these results provide a strong indication that this phenomenon should be investigated in more depth. Moreover, the areas to be considered when defining strategies for determining patient treatments should also include the influence of socio-demographic factors on the patient's consciousness that would enable easier understanding of the proper usage of OTC drugs. Complementary and Alternative Medicine (CAM) has attracted global interest in both developed and underdeveloped countries . During first reaction to a minor health condition . The questions and indicators used in the questionnaires were standardized to those adopted by the European Union, in addition to using indicators standardized to those in the “Health For All” database of the WHO or recommended to become EU indicators , 30.Surveys were conducted as a cross-sectional study. The population presented in the research included adults aged 19 and over. Individuals living in care institutions  and in Kosovo and Metohija were not included in the research. The research used a national representation sample, which is a stratified two-phase sample without repetition. The sample frame in the research included all the households listed in censuse to 2011. In order to obtain a random sample, two techniques were used: stratification and multiple-phase sampling. Stratification was conducted in such a way that each of four geographical areas  represented one main stratum in the sample. Each stratum was divided into cities and other regions. The total number of strata was eight. Two-phase sampling included local communities, selected on the basis of probability proportional to their size, and households as units of the second phase selected on the basis of the linear sampling method with a random start and uniform selection steps. All respondents that had used CAM  over the previous 12 months and were living in the Republic of Serbia were extracted from this database, totaling 550.Individuals living in care institutions  and in Kosovo and Metohija were not included in the research. The principles of ICH Good Clinical Practice were followed strictly, and approval was obtained from the Ethics Committee of the Republic of Serbia. The research used data from the Third National Health Survey of Serbia, sponsored by the Ministry of Health's Batut Institute, which provided data to the faculty for the purpose of additional research. The Ethical Standards for Healthcare Research are aligned with the International Medical Association Declaration of Helsinki and legislation specific to the laws of Serbia. Aiming to align with GDPR policies to preserve the discretion of the respondent data collected, all steps were adopted that are stipulated by the law on the protection of personal data , the Official Statistics Law , and Directive 95/46/EC of the European Parliament on the protection of individuals with regard to the processing of personal data and on the free movement of such data .t-test was used for continuous data. Factor analysis was applied as the multivariant technique. The following variables were analyzed through factor analysis: sex, age, education, region, accommodation, work status, well-being index, health self-evaluation, commitment to being healthy, record of chronic disease, medical leave, hospitalization, visits to day-care hospitals, home medical care and Emergency Room Assistance in the past 12 months, consumption of OTC drugs over the previous 2 weeks, risky behavior, routine checks and screening for colon cancer, and health care non-utilization . All analyses were performed using SPSS, version 19.Continuous variables were presented as mean ± standard deviation, and categorical variables as frequency (n) and percentage (%). The Chi-square test, representing the univariant method, was used for categorial data, and the Students' In Serbia, two-thirds (65.3%) of CAM users consume OTC drugs at their own discretion, neither due to a physician's recommendation nor through prescription. OTC drug consumers are most frequently females with an average age of 49.16 ± 16.02, possessing a high-school diploma, married and employed, most frequently living in urban areas, mostly Belgrade, categorized as middle-income as per the “wellbeing index,” and who follow health topics via publicly available information .p < 0.05) and that OTC consumers are, on average, 4.5 years older than those that do not use OTC drugs .By comparing the basic socio-demographic characteristics of respondents that consume and that do no consume OTC drugs, it is evident that the consumers are mostly females , while this practice is rarest among respondents living in the Belgrade region .There was no statistically significant difference in the use of analgesics with respect to gender, educational attainment, region and place of residence, self-evaluation of health status, presence of chronic health disorders, and long-term illness. On the other hand, individuals falling into the middle-income category more often consume analgesics at their own discretion  and that those respondents had less frequently been hospitalized over the previous 12 months   .One-third of OTC drug users who were not able to utilize the required healthcare services, which could be due to a number of factors, such as waiting lists, financial insolvency, or perhaps the distance to the required healthcare institution, were employed. Our research, as well as numerous other studies, showed that fear of losing an existing job could be one of the reasons why employed individuals consume OTC drugs .OTC drug users most often rated their own health as good or average, but despite risky behavior, they had generally never undergone screening for colon cancer. Of those respondents, 56% had chronic health disorders and most often utilized the required form of health care. Most OTC users did not take sick leave and had less frequently been hospitalized or used a day hospital, ambulance, or home treatment in the previous 12 months than had others .Most common drugs, herbal supplements, and/or vitamins that the respondents were taking on their own, without prior consultation with a physician, were bought in pharmacies and are presented in p < 0.01) isolated six factors that explain 53.79% of the variance: Health Condition (17.43% of the variance), Financial Status (10.16% of the variance), Age (7.89% of the variance), OTC Drugs (7.42% of the variance), Risk (6.21% of the variance), and Health Care Utilization (5.94% of the variance).Principal component analysis .The health condition factor groups together the variables health self-evaluation, the existence of chronic disease, and limits to daily activities due to the disease, showing that individuals that suffer from chronic diseases that prevent them from undertaking daily activities evaluate their health as bad. The health condition factor included in the factor analysis also implied the presence of chronic disease . There wSimilarly to our research, other studies have shown that the use of certain drugs reduces the rate of hospitalization in adults . AntiplaThe financial status factor groups five variables: region, accommodation, well-being index, education, and health care non-utilization. This factor shows that individuals living in urban areas in the Vojvodina area, possessing a higher/high degree of education, and classified as very rich as per the well-being index, have never managed to access required health care services due to long waiting lists.The age factor groups the variables age and employment status and shows that the oldest respondents were retired.The OTC drugs factor groups four variables: sex, awareness about health topics, OTC drug use, and hospitalization in the previous 12 months. This factor shows that women who were aware of topics related to health and that had consumed OTC drugs over the previous 12 months had not been hospitalized.The emergency assistance factor, which groups variables related to using emergency assistance in the previous 12 months and risky behavior, showed that already ill patients had been using emergency medical services during the previous 12 months.The factor explaining health care utilization groups variables such as the selection of a private practice physician and being a day-patient over the previous 12 months. This shows that respondents who had selected a private practice physician were hospitalized fewer times for additional diagnosis/treatment (“Day patient”) .Analysis of female respondents aware of health topics via public information systemsp < 0.05). Every third female in the Vojvodina region had consumed OTC drugs over the previous 2 weeks, while every fourth female from the Sumadija and West Serbia regions had not . OTC drugs had been consumed by 56.7% of females following health topics in addition to having risky behavior. Simultaneously, 39.7%, though well-informed on health issues and acting riskily, had not consumed such drugs . Further analysis of the female respondents that consumed OTC drugs and followed health-related topics via public information systems  shows that these respondents had statistically significantly been hospitalized fewer times over the previous year when compared to respondents not using OTC drugs . Although those female respondents had not accessed any health care services, there was no significant statistical difference in the usage of OTC drugs among respondents following health topics.The majority of females (71.1%) that followed health topics via public information systems had consumed OTC drugs over the past 2 weeks. Most commonly, they had a high-school diploma. However, 37.2% of females characterized as having a higher/high education level had also used such drugs, while 26.1% of those at the high-school education level had not . OTC drugs were mostly used by males living in the Belgrade region (38.6%) and less so by males from the regions of Sumadija and West Serbia (8.8%) . No statistically significant difference was found between OTC usage and risky behavior (p > 0.05). Among the observed males, no statistically significant difference was found between the use of OTC drugs and hospitalization over the previous 12 months. Of male respondents, 47.8% had neither utilized health care services nor used OTC drugs.Every second respondent (55.3%) following health topics had consumed OTC drugs over the previous 2-week period. The users of OTC drugs were mostly high-school graduates. No significant statistical difference was found among users of OTC drugs that followed health topics and had different levels of education  of CAM users consume OTC drugs at their own discretion, without either a recommendation from a physician or a prescription. These are mostly females of an average age of 49.16 ± 16.02 with a high-school diploma, married and employed, most frequently living in urban areas, mostly Belgrade, classified as of the middle-income class, and following health topics via public information systems . Most studies that have been undertaken significantly predict that CAM users will be females and interpret this to be the result of their having more frequent communication with their friends and families about their health condition and having been given diagnoses that turned out to be a significant source of information , 35–43. Our data analyses show that the predominant OTC drug users were females with a high-school diploma and also 37.2% of females with a higher/high education diploma, though 19.2% of such females did not use OTC drugs. Globally, the significant predictors in the majority of studies conducted to date point to CAM users being characterized by a high income and vocations requiring a high level of education. The explanation made for this may lie either in the fact that they were financially able to use CAM or in the fact that CAM users are highly educated and hence question the efficiency of conventional medicine , 42–45. Worldwide, health condition and health perception, among selected socio-demographic factors, have been found to be significant in making a decision regarding whether an individual would use CAM and OTC, especially when the condition or the individual's perception of their health was described as bad , 36, 42.In Serbia, according to the Medicines and Medical Devices Act, the advertisement of medicines and medical devices is prohibited, that is, the information that is advertised on a medicinal product or medical device must be true and scientifically proven, cannot mislead the professional and general public, and must be in accordance with the Regulation on the Advertising of Medicines and Medical Devices . HoweverThanks to the research undertaken in Serbia (January 2010–July 2011), pioneering efforts have been made that have resulted in an understanding of the weaknesses in the perceptions of the current and future health workforce toward CAM. It included a questionnaire based on the existing CAM Health Belief Questionnaire (CHBQ), using the Likert scale, where 797 health students and practitioners were interviewed. Students of Dentistry (54.65 ± 6.07) were better informed on CAM therapies than students of Medical Science (50.26 ± 7.92) and students of Pharmacy Science (51.16 ± 7.10). Physicians achieved better results than university professors (55.12 ± 6.55 vs. 50.29 ± 9.50). Furthermore, all of the respondents gave priority to the use of vitamins over any other CAM therapy. In general, consciousness of the use of CAM is increasing in the Balkans (Systematic literature review (42 studies) has found that the prevalence of CAM use ranged from 8 to 90% in patients with prostate cancer . A systeThe analysis of socio-demographic factors and their influence on the proper selection of OTC drugs should become a very significant part of the processes conducted in order to define strategies for determining the treatments for particular patients. The research statistics presented here strongly support OTC drugs correlates with an absence of hospitalization, absence of medical leaves, and absence of day-care hospital, emergency assistance, or home medical care usage in the previous 12 months. Furthermore, wider research focusing on the aforementioned factors should enable the improvement of treatment strategies for enhancing patients' well-being.In addition, although global research indicates growing trends, the actual evidence for the use of CAM methods in Serbia is very poor. In order to enable a comparison of health care service utilization and CAM methods, it would be necessary to take a different approach. This new path would rely on the Ministry of Health of the Republic of Serbia taking a leading role, due to their better understanding of this matter.https://ec.europa.eu/health/indicators_data/indicators_el; https://ec.europa.eu/health/indicators_data/echi_en; http://www.healthpowerhouse.com.Publicly available datasets were analyzed in this study. This data can be found here: ML-S and MJ jointly designed the study and defined the research questions. NM and NR did most of the data mining and extraction, purification of files for missing data and artifacts, and statistical analysis. NM, NR, and ML-S contributed to table and figure creation and interpretation of data. ML-S, MJ, and NR drafted the working version manuscript. All authors contributed to the final version to the extent of important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chiral natural product molecules are generally assumed to be biosynthesized in an enantiomerically pure or enriched fashion. Nevertheless, a significant amount of racemates or enantiomerically enriched mixtures has been reported from natural sources. This number is estimated to be even larger since the enantiomeric purity of secondary metabolites is rarely checked in the natural product isolation pipeline. This latter fact may have drastic effects on the evaluation of the biological activity of chiral natural products. A second bottleneck is the determination of their absolute configurations. Despite the widespread use of optical rotation and electronic circular dichroism, most of the stereochemical assignments are based on empirical correlations with similar compounds reported in the literature. As an alternative, the combination of vibrational circular dichroism and quantum chemical calculations has emerged as a powerful and reliable tool for both conformational and configurational analysis of natural products, even for those lacking UV-Vis chromophores. In this review, we aim to provide the reader with a critical overview of the occurrence of enantiomeric mixtures of secondary metabolites in nature as well the best practices for their detection, enantioselective separation using liquid chromatography, and determination of absolute configuration by means of vibrational circular dichroism and density functional theory calculations. Secondary metabolites play a significant role in drug discovery and development processes , as theyOn examining the chiral drug market, Calcaterra and D’Acquarica  noticed Journal of Natural Products in 2017. Out of 268 papers describing chiral molecules, only 31 (11.6%) checked the enantiomeric purity of their isolated compounds. Even when the assignment of the AC of a given compound is intended, its enantiomeric composition is generally overlooked. In this context, the search for new chemical entities should always include evaluations of the stereoisomeric composition of the target compounds, when applicable. In the absence of such evidence, the relationship between biological activity and structure may be seriously compromised. Regarding natural products, it is commonly taken for granted that chiral secondary metabolites are biosynthesized in an enantiomerically pure form ,6,7,8,9.S)-gossypol (P form) and (R)-gossypol (M form) .,23.1) ant active .Chelidonium majus identified as enantiomeric enriched mixtures by combined analysis of their NMR data, electronic circular dichroism (ECD) spectra, calculations of specific rotations, and enantioselective chromatography profiles furan neolignans consisting of four diastereoisomers and their four enantiomers were isolated from biaceae) . Their AAs exemplified, enantiomeric mixtures of secondary metabolites are not uncommon in Nature and their occurrence is generally underestimated. In the past, natural product chemists used to rely on polarimetry to draw conclusions about either enantiomeric purity or AC of isolated chiral secondary metabolites . Even toonly from differences observed in the measurements of a small optical rotation conducted on minute quantities of material, often of unknown chemical or enantiopurity”.An emblematic example of the problems associated with AC assignments based on optical rotation (OR) values involves the case of the marine sesquiterpene frondosin B . Many atH)-furanones differ from each other by the presence of a single methylene unit and, despite having the same AC, they have opposite signs of OR. Finally, the case of the of the chiral furanones sotolon and the maple furanone  34] ill ill34] iAs demonstrated, it is extremely important to measure stereoisomeric composition of natural products and properly determine their AC. Thus, in the next sections the reader will be provided with a tutorial of the best practices for the efficient method development for chiral separations using LC as well as for the determination of AC using VCD and DFT calculations. The following tutorial sections represent general guidelines from the author’s perspective with selected examples and are not intended to exhaustively review the literature.The study of natural products generally involves the following steps: extraction, fractionation, isolation, structural elucidation, and in the case of chiral molecules, enantiomeric purity and AC determinations . In the e.r.) and e.e. will be employed when non-chiroptical (chromatographic or NMR) and chiroptical methods are used, respectively. However, for published work, the terms are reported as present in the original publications.Different definitions of enantiomeric proportion are used depending on how the mixture of enantiomers is quantified . Herein,Chiral stationaries phases (CSPs) have revolutionized the way that enantiomeric composition is measured; with LC being the most used technique ,36,37. CA number of reviews cover the main aspects relating to the type and classification of the chiral selectors used for preparation of the CSPs ,41,42,43The main focus of enantiomer separation is placed on the CSPs to be used; this can be noticed through the years, since the 1980’s, when the commercial columns became available. The progress in this respect has been amazing and nowadays a wide variety of CSPs are available to be used in different elution modes ,43,46.Regarding the molecular bases for the chiral recognition, depending on the selector, the main interactions involved are hydrogen bonding, π–π, dipole–dipole, steric hindrance and inclusion complexes ,47. For Unfortunately, there is no universal chiral selector for designing the enantioselective separation of an unknown enantiomeric mixture. Moreover, not only is the chiral selector important but also the support used for preparing the phases, which contributes to the retention behavior by non-stereoselective adsorption . The mode.r. is not a trivial task and, in spite of the knowledge that some classes of compounds are well resolved in a given CSP [Hence, finding the CSP at the correct elution mode for measuring iven CSP ,49, the iven CSP ,51. The iven CSP ,51; but iven CSP ,53.e.r. determinations [To meet this end, chiral screening technologies have been pursued using either traditional columns (5 μm fully porous particles) ,56,57,58inations ,26,62,63inations . Polysaccharide-based CSPst-butyl ether cannot be used due to the solubility of the chiral selectors in these solvents [The amylose and cellulose-based CSPs are by far the most used and are usually the first choice, including their use in supercritical fluid chromatography. They were first introduced back in 1984 ,66 and ssolvents ,69. To osolvents ,56,67. Wsolvents ,70.tris-3,5-dimethyl-phenylcarbamate derivative is the most used in either coated or immobilized forms [® IB-3 column [Datura stramonium and Brugmansia arborea seeds, and contaminated buckwheat, using a Chiralpak AY3 column and an LC-QqQ-MS system. By exploring different pH and temperature conditions, the racemization of pure (−)-hyoscyamine to atropine in Solanaceae samples has also been investigated. The racemization occurs rapidly at high temperature and basic pH (pH 9.0). At high temperature (80 °C) and pH 5, the racemization also occurs, but slowly [® IC was used to enantioselectively separate eight pairs of norlignan, neolignan, and lignan enantiomeric mixtures from Acorus tatarinowii with e.e. in the order of 66%, 71%, 63%, 60%, 0%, 38%, 48% and 75% [Amylose and cellulose offer complementary chiral recognition ability and the ed forms ,71. The ed forms . It is ied forms . Another3 column . (+) andt slowly . A Chira and 75% .Macrocyclic antibiotics CSPs®) are able to separate diverse classes of compounds. The changes in interaction according to the elution mode make them a wise choice [e.r. Docking calculations for the free energy were in agreement with the order of elution of the enantiomers. The calculations also showed that the macrocyclic antibiotic CSPs exhibit binding patterns in accordance with the shape and surface of each chiral selector [Macrocyclic antibiotics CSPs , with vae choice . Xanthonselector .Aspergillus similanensis KUFA 0013, were separated using a Chirobiotic T (teicoplanin) column under reversed-phase elution conditions. The comparison with standard amino acids allowed the elucidation of the isolated cyclohexapeptide as cyclo  [Teicoplanin-based chiral stationary phases are well known for their ability to enantionresolve underivatized amino acids . The hydic acid) .Pirkle type CSPsS,S)-Whelk-O1 and l-phenylglycine CSPs under multimodal elution conditions at normal, polar organic, and reversed-phase modes [S,S)-Whelk-O1 CSP, with α ranging from 1.91 to 7.55 and RS ranging from 6.71 to 24.16. The polar organic elution mode afforded the best resolution at a shorter time [The evolution of the Pirkle type CSP has been elegantly reviewed  and showse modes . In thister time . This tyter time ,81.Cyclodextrin-based CSPsCyclodextrins are the only type of CSPs used in gas chromatography ,40,43. IAs an example of innovative application, the enantiomeric composition of hesperetin in human urine has been determined by LC using a 100 m I.D. capillary column, packed with phenyl-carbamate-propyl-β-cyclodextrin at reversed-phase conditions, in order to evaluate its metabolism and elimination properties .It is acknowledged that the development of efficient conditions for enantiomeric separation should start with the screening of CSPs of complementary chiral discrimination ,60. IdeaNowadays, a number of LC systems can offer multi-solvent blending and column management options, which allow the use of 6–12 columns  for automated method development. Supercritical fluid is an excellent approach to find the right conditions for enantiomeric separation. As with LC, it also requires selecting a series of parameters such as CSP and organic modifiers. The new series of SFC systems are able to automatically screen a large number of columns and various mixture ratios of four types of modifiers, which facilitate the efforts required. It should be noticed though, that in day-to-day operations of natural product laboratories these systems are not usual. Cost considerations, availability of equipment, and know-how play important roles in the selection process”. Accordingly, we should pinpoint some important issues and general recommendations for LC separation of an enantiomeric mixture:Polysaccharide-based CSPs are the ones with the broadest enantiomeric discrimination abilities and are complementary to the macrocyclic antibiotics CSPs. For their screening, it is important to explore multimodal elution, thus, one must be aware of solvent miscibility. 100% ethanol (EtOH) is an excellent choice for changing from normal to polar or reverse-phase modes. As a rule, one should mind the instructions provided by the manufacturer.tris-3,5-dimethylphenylcarbamate derivative CSPs are the ones with the highest success rates [For the screening of polysaccharide-based CSPs, it is better to start by exploring the normal elution mode with different proportions of alkane (hexane or heptane) to EtOH or 2-propanol. The amylose- and cellulose-ss rates . Initialss rates . With thThe polar organic mode also gives high selectivity and it is usually a good choice for multi-milligram separations. It is important to give time for equilibration, especially for the coated polysaccharide-based CSPs. To avoid immiscibility problems with the eluents, one should go first to 100% EtOH. Methanol (MeOH) or acetonitrile (ACN) are the solvents of choice, or mixtures of them ,84.Reversed elution mode should also be explored. This is usually done by using acetonitrile or methanol as modifiers in water. The use of buffer solutions is necessary only if the analytes require .® CSPs at polar organic mode for nonionizable enantiomers, or at the polar ionic mode for the ionizable ones. In the first case, ACN is usually used with a small amount of a protic organic solvent, while in the polar ionic mode acid or base is added to MeOH [It is recommended to start the screening with the Chirobiotic to MeOH . The man® CSPs operate well under the reversed mode with MeOH, ACN, and THF as modifiers. The use of an aqueous buffer solution, such as ammonium acetate, is a good selection. The pH can affect drastically the enantioselectivity and should therefore be explored.The Chirobiotic® CSPs under normal elution is also important [The normal elution mode is carried out with alkane and 2-propanol or ethanol. The use of acidic or basic additives for Chirobioticmportant .S,S)-Whelk-O1 CSP in the screening should also be incentivized. The highest selectivity is usually achieved under normal elution conditions but it can be explored as well under polar and reversed-phase conditions [The use of , discern 0.1% e.e. differences or better for samples with high specific rotations [e.e., this should not be performed based on single measurements, even if this is a common practice in natural product chemistry. In reporting the isolation of eight enantiomeric mixtures from Acorus tatarinowii, the authors call attention to the fact that many enantiomeric mixtures are inadvertently considered as enantiomerically pure molecular entities due to the obtained optical rotations and/or Cotton effects in their ECD spectra [Chiroptical methods, which arise from the differential interaction of a chiral non-racemic sample with left- and right-circularly polarized radiation, can also be used for the determination of c excess ,88,89,90otations . Althoug spectra .Instead, it is recommended that calibration curves be constructed to detect a given compound in the presence of different concentrations of its enantiomer. Single measurements of any chiroptical property may lead to erroneous conclusions regarding enantiomeric purity and, possibly, AC. kα radiation, has enabled confident assignment of absolute structure even for hydrocarbons [The determination of ACs can be achieved using several methods, including X-ray crystallography, NMR methods, chemical correlation method, and stereocontrolled total organic synthesis. Even though X-ray crystallography was used in the first assignment of the AC of a chiral compound , and is ocarbons . While vocarbons . Finallyocarbons , and higab initio [As an alternative, chiroptical methods have faced a renewed interest in their use for determining molecular stereochemistry . This evb initio  calculatb initio .Following Nafie’s proposal at the Pharmacopeial Forum in 2013 , the UniDespite the advances and advantages described above, as well as the steady growth in the number of secondary metabolites studied by VOA methods in the last decades ,102, VCDAmong all the methods used to determine the AC of chiral molecules, VCD appears as an excellent tool for the analysis of chiral natural products. This technique has many advantages over other methods widely used since there is no need of single crystals, derivatizations, or ultraviolet-visible (UV-Vis) chromophores. Although non-empirical methods  as well −1), with an optimum absorbance (A) of approximately 0.5. Absorbance values should neither be lower than 0.1 nor higher than 1.0 absorption units. These conditions may require that different regions of the spectrum be measured separately to achieve an appropriate level of absorbance that results in a reliable VCD spectrum. The reliability of a VCD measurement is often assessed by checking the VCD noise level spectrum.The first steps for the measurement of good quality IR and VCD spectra include the selection of solvent, sample concentration, and path length necessary to obtain an IR spectrum in the mid-IR region  or by subtracting the VCD spectrum of the corresponding racemate. In case neither of the above is available, subtraction of the VCD spectrum of the solvent must be carried out, with the best results being obtained in this case using a dual-PEM system.IR, VCD, and noise spectra are usually collected with a spectral resolution in the range of 4–8 cmIR/VCD spectral calculations include initially the construction of a three-dimensional input structure, whose AC is arbitrarily chosen and based on the relative configuration determined from NMR and/or X-ray experimental data. Subsequently, a thorough conformational analysis using molecular mechanics, molecular dynamics or semi-empirical methods, is carried out. The conformers identified are filtered out according to an energy criterion , and subjected to further geometry optimization steps using DFT (commonly B3LYP/6-31G(d)). Dipole and rotational strengths, which are proportional to IR and VCD intensities, respectively, are then calculated at the same level of theory used for geometry optimization. The set of dipole or rotational strengths are finally converted to a full IR or VCD spectrum to be compared with experiment generally assuming Lorentzian band shapes. −1 is considered for comparison with experimental spectra measured at 4 cm−1 resolution. Higher levels of theory, such as B3PW91/cc-pVTZ, may require a scale factor of 0.98 [After the IR and VCD spectra of the contributing conformers have been calculated, they are weighted by their fractional Boltzmann population and summed to produce the final calculated IR and VCD spectra. For best comparison with the experiment, the calculated frequencies are uniformly scaled to compensate for the anharmonicity of the observed frequencies. For DFT (B3LYP/6-31G(d)), a scale factor of 0.97 is typically used for the frequencies, and a bandwidth of 6 cm of 0.98 . Both obCurrently, the method for calculating VCD is available in commercial software such as GAUSSIAN 16, TURBOMOLE 7.2, among others. Most VCD calculations are carried out at the DFT level using the hybrid functionals B3LYP or B3PW91 combined with the 6-31G(d) basis set, considered the minimum basis set for VCD. At least for small molecules, a larger basis set such as cc-pVDZ, cc-pVTZ, TZ2P, and TZVP provide better results compared with 6-31G(d). For larger molecules, however, it has been found that the triple basis set TZVP does not provide practical advantages over 6-31G(d) as far as the accuracy-to-time ratio is concerned . When chSolvent effects on vibrational rotational strengths are typically small, especially for non-polar solvents. However, solvent effects are supposed to affect the optimized geometries and the conformer population in solution. These effects can be accounted for by using different approaches, such as the inclusion of the polarizable continuum model (PCM), the conductor-like screening model (COSMO), or even the inclusion of explicit solvent molecules. Explicit solvation is particularly useful when solute-solvent interactions influence both the conformational population and frequencies/intensities of certain vibrational modes . InclusiAs experimental IR and VCD data are commonly recorded in deuterated solvents, isotopic exchange is also expected to occur when hydroxylated molecules are analyzed in protic solvents . Therefo2 [Finally, when the comparison between calculated and observed data is performed to assign AC, the visual correlation may not be enough for an unambiguous assignment. To assess the level of agreement between calculated and measured VCD spectra, some approaches can be used. The first one consists of extracting dipole and rotational strengths from experimental data and plotting it against the calculated ones. This method offers the possibility of calculating statistical measures, such as the correlation coefficient R2 . A secon2  uses com2  that pro2 .Chirality is ubiquitous in Nature and it is reflected, at molecular level, in the fascinating structural complexity of many secondary metabolites. As demonstrated in this review, however, not always is the same enantiomer/stereoisomer biosynthesized by a given species nor is it produced necessarily in an enantiomerically pure/enriched fashion. These latter facts are commonly overlooked by natural product chemists, even when they attempt to determine the AC of the isolated compounds. A common protocol to determine enantiomeric purity and, sometimes AC, of naturally occurring compounds still involve OR measurements. As discussed in the literature , the pur


Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique to change cortical excitability. Its effects are shown for cognitive processing, and behavior in the motor and perceptual domains. However, evidence of tDCS effects in the perceptual domain particularly for auditory processing is rare. Therefore, and in the context of disturbances in auditory processing in psychiatric populations, e.g., in patients with auditory verbal hallucinations, we aimed to investigate the potential modulatory effect of tDCS on the excitability of left posterior temporal cortex in detail. We included 24 healthy participants in a crossover design, applying sham and anodal stimulation in two measurement sessions 1 week apart. Electroencephalography (EEG) was recorded while participants listened to tones before, during, and after stimulation. Amplitudes and latencies of P50, N100, and P200 auditory-evoked potentials (AEP) were compared between anodal and sham stimulation, and between time points before, during, and after tDCS. In contrast to previous studies, results demonstrate no significant differences between stimulation types or time points for any of the investigated AEP amplitudes or latencies. Furthermore, a topographical analysis did not show any topographical differences during peak time periods of the investigated AEP for stimulation types and time points besides a habituation effect. Thus, our results suggest that tDCS modulation of excitability of the left posterior temporal cortex, targeting the auditory cortex, does not have any effect on AEP. This is particularly interesting in the context of tDCS as a potential treatment for changed electrophysiological parameters and symptoms of psychiatric diseases, e.g., lower N100 or auditory verbal hallucinations in schizophrenia. Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique to change cortical excitability . For tDCBesides effects on cognition  and behaTaken together, only one study investigated the impact of tDCS on excitability of the AC in a systematic manner , and besafter stimulation, we here extended this protocol by assessing AEP at three time points before, during, and after stimulation . Concerning power, a sample size of 21 would be required to achieve a power of 0.8 with an effect size of 0.275 that we estimated based on prior work and α of 0.05. All participants were right-handed  between 18 and 64 years (t-handed  and did The study was approved by the Ethics Committee of Canton Bern (KEK-BE-2016-01741) and conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent.Measurements took place in a crossover design on 2 days with 1 week in between to avoid carry-over effects of stimulation  for Matlab environment . Pure sinusoidal tones of 200 ms duration and 1000 Hz frequency were presented using Panasonic Technics SB-CS6 loudspeakers  with an intensity of 65 dB and inter-stimulus intervals were jittered between 900 and 1100 ms.Stimulus tones were generated using the cogent 2000 toolbox (version 1.32RRID:SCR_009443) and software , filtered between 0.016 Hz and 1000 Hz with a sampling rate of 2500 Hz. Fifty-six passive Ag/Cl EEG electrodes were mounted on the scalp according to the international 10–20 EEG system with CPz as reference .Stimulation was applied using a battery driven constant current stimulator  with the active 5 cm × 7 cm rubber electrode positioned over TP7 and P7 according to the international 10–20 EEG system, and the reference 5 cm × 5 cm rubber electrode over Fp2, AF4, and AF8 Figure . Impedan2) for Matlab environment (version R2017a). To remove eye blink-related artifact, we performed an independent component analysis (ICA) for recordings with stimulator on and off separately after band pass filtering to 1–30 Hz  for Matlab environment after downsampling the averaged data to 200 Hz  and a topographical consistency test (TCT) were conducted with the Ragu toolbox (version March 07 2018o 200 Hz . The PCARRID:SCR_002865). A repeated-measures analysis of variance (ANOVA) with factors stimulation  and time point  was conducted for P50, N100, and P200 amplitudes and latencies where data was normally distributed. Otherwise, non-parametric Wilcoxon Rank Sum tests with Bonferroni-Holm correction for multiple testing were calculated. All tests were accompanied by effect size calculations. In addition, we investigated effects of stimulation and time point on mean values in peak intervals by calculating repeated-measures ANOVAs with factors stimulation  and time point  for the three AEP. Furthermore, we were interested in potential responder/non-responder patterns in the data. We therefore calculated the differences of post-minus pre measurements in the anodal stimulation condition for all ERP amplitudes and latencies to account for changes of stimulation over time. Correlating these variables, we aimed to have a measure of global response to stimulation in one direction (excitation or suppression). Level of significance for all calculations was set to 0.05.Further analyses of amplitude and latency data were performed with SPSS  . The TANGrand Averages of AEP for all conditions are presented in Figure 1 = 0.006, tested separately for amplitudes and latencies) and all show low effect sizes. In addition, the repeated-measures ANOVA on mean values in the peak interval did not show any interaction effect for factors stimulation and time point , nor an effect of factor stimulation , or time point .Mean amplitudes and latencies with standard deviations of P50 are reported for every condition in Table 1 = 0.006, tested separately for amplitudes and latencies) and all show low effect sizes. In addition, the repeated-measures ANOVA on mean values in the peak interval did not show any interaction effect for factors stimulation and time point , nor an effect of factor stimulation , or time point .Table 1 = 0.006, tested separately for amplitudes and latencies Table F = 0.96, p = 0.39], nor an effect of factor stimulation , or time point .For P200, mean amplitudes and latencies with standard deviations are shown for every condition in Table 1 = 0.003), none of the phi coefficients showed a significant correlation of the variables (p > 0.08).As amplitudes and latencies were not normally distributed, we calculated the phi coefficients for all difference variables. With Bonferonni-Holm correction for multiple comparisons  were found for the main factor of stimulation.In the current study, we applied 20 min of anodal and sham tDCS over the left posterior temporal cortex in a double-blind crossover design. We measured AEP before, during, and after stimulation. In contrast to our hypotheses, we did not find any differences in P50 amplitudes for anodal and sham stimulation, precisely for neither of the investigated AEP amplitudes or latencies. Furthermore, no effects of tDCS were evident when comparing AEP amplitudes or latencies before, during, and after stimulation. In addition, the topographical analyses did not indicate topographical differences for the investigated conditions.Particularly interesting about our results is the fact that we found no difference of AEP amplitudes and latencies for the different measurement time points before, during, and after stimulation. As earlier studies reported effects of anodal tDCS on AEP after stimulation , we alsoAn interesting result of the TANOVA was the main effect of time point for the time interval of 240–305 ms. For the grand averages, the amplitudes of the AEP in this particular time interval occurred to be different Figure . To miniThere is substantial discrepancy among the results of our study and previous studies, and the difference in results could be explained by differences in stimulation protocols and study design. In the next paragraphs, we will discuss in more detail three parameters that crucially affect tDCS effects, i.e., montage, stimulation intensity and duration, as well as stimulation protocol, i.e., the conditions to be compared.First, one difference in study protocols between our and previous studies is the tDCS electrode montage. Second, there is a high variability in stimulation durations and intensities among studies, which make a direct comparison of results difficult. Last, the study designs, i.e., the stimulation conditions compared, differ across studies. Our results indicate a need of method optimization, i.e., to test and compare different stimulation parameters to gain an overview of parameters that are effective. Thus, further studies considering other auditory tasks, electrodes montages, application of cathodal stimulation, or variation of other stimulation parameters are warranted to investigate potentially specific or subtle effects of tDCS we might have missed with our design. Particularly for the application of tDCS in auditory processing or psychiatric symptoms .LM, YM, and TD designed the study. KK, MG, and YM conceptualized and performed data analysis. KK collected the data and wrote the first draft of the manuscript. All authors contributed to discussion about interpretation of the results, revised, and approved the final version of the submitted manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Mild cognitive impairment (MCI) is characterized by cognition impairment that does not interfere with the usual activities of daily living. It is considered to be a transitional stage between normal aging and dementia. No treatment is available for MCI. Methods: This retrospective cohort study included 55 patients  with a diagnosis of amnestic MCI who attended the Center for Cognitive Disorder and Dementia of the IRCCS Centro Neurolesi Bonino Pulejo  between January and December of 2017. As we aimed to evaluate the effect of cocoa polyphenols on cognition, the study population was separated into two groups depending on the change in their Mini-Mental State Examination (MMSE) score at a one-year follow-up. Results: Compared to G2 , the rate of polyphenol intake was significantly higher in patients without a worsening in cognition  . By subdividing G1 patients based on whether they improved or were stable at follow-up, we found that 46.2% of those who had improved were treated with polyphenols. Conclusions: Dietary supplementation of cocoa flavonoids seems to reduce the progression of MCI to dementia. Further prospective studies with larger sample volumes are required to confirm these promising findings. Mild cognitive impairment (MCI) is frequent with aging . MCI wasThe rate of MCI-to-dementia conversion depends on the diagnostic criteria employed and the clinical setting  . MCI preNeuropsychological and neuroimaging investigations contribute to differentiating between dementia and MCI diagnoses ,17,18,19This retrospective cohort study included patients with a diagnosis of aMCI who attended the Center for Cognitive Disorder and Dementia of the IRCCS Centro Neurolesi Bonino Pulejo  between January and December of 2017. Usually, after the diagnosis of aMCI  patients are evaluated every six months for at least two years, and are administered the Mini-Mental State Examination (MMSE) to detect cognitive changes. The MMSE measures orientation, immediate and short-term memory, and language functioning, among other things . SpecifiIn order to detect possible risk factors or protectors, during their first visit an extensive personal and medical history is obtained and patients verbally report on their habits regarding the consumption of beverages and food containing polyphenols .Adherence to the study was evaluated through information collected at a follow-up visit (by searching on the available database).®, is composed of cocoa polyphenols , Bacopa monnieri (100 mg/sachet), group B vitamins, vitamin E, and folic acid.Patients with MCI can be treated with nutraceuticals, and only a few of them with cholinesterase inhibitors. One of the most commonly used nutraceuticals, Mexenion®.To evaluate the role of polyphenols in MCI-to-dementia conversion, we selected patients who had been diagnosed with aMCI within the last year (according to DSM-V criteria) and were treated with MexenionThe exclusion criteria were: (i) a diagnosis of dementia; (ii) an age of over 75 years; (iii) the presence of depression and other psychiatric illnesses; (iv) the use of drugs or other nutraceuticals acting on cognitive deficit; (v) the use of antidepressants or other psychoactive drugs.® on cognition, the study population was divided into two groups depending on the change in MMSE score at the one-year follow-up. Thus, the first group (G1) included patients whose MMSE score was stable or increased , whereas the second group (G2) included patients whose MMSE score decreased at least 3 points since the diagnosis.To evaluate the effect of MexenionThe Hospital Research Ethical Committee of the IRCCS Neurolesi approved this study (IRCCSME.CE 13/2018), stating that obtaining informed consent was not necessary due to the retrospective design of the study and the fact that all participants were anonymized.2 test with continuity correction was used to assess the statistical differences in proportions, whereas the unpaired student’s t-test was used to compare continuous variables. To assess the association between the consumption of Mexenion® and the slowing or reversing of cognitive decline, we used the Fisher’s exact test, because our sample size was small, and we reported odds ratios (OR) and the associated 95% confidence intervals (CI). We also performed a multiple logistic regression to adjust for patient characteristics  by using the chi-square goodness of fit test and used the Receiver Operating Characteristic (ROC) curve as a post hoc test.Statistical analysis was performed using Stata 14.1 software, with the level of significance  set to 0.05. Continuous variable data were expressed as means ± SD, whereas categorical variables were expressed as frequencies and percentages. The χOf the 109 patients initially screened, 54 were excluded . Finally, 55 patients  aged 56 to 75 years were included in this study.2 = 13.79, df = 1, p-value < 0.001). Indeed, the average change in MMSE score after one year was 1.77 ± 1.68 in G1 and −4.19 ± 1.87 in G2, which is a statistically significant difference . Similarly, we observed significant differences between the score changes of patients in G1 and G2 for AM  and RAVLT (p < 0.001 for RAVLI and p = 0.003 for RAVLR). Moreover, the two groups were homogenous in their dietary intake of coffee and wine , as well as in their daily portions of fruit and vegetables. A significant difference was found only in green tea intake: patients in G2 consumed more green tea per day than those in G1 (p = 0.01).The clinical-demographical characteristics of the sample are reported in ®, whereas about half of the remaining 53.8% were treated with the nutraceutical and were stable . Moreover, 44% of patients who did not take Mexenion® were stable and 56% were worse at follow-up. The results of Fisher’s exact test showed that Mexenion® is a potential protective factor for slowing or reversing cognitive decline  . The results of the logistic model showed that Mexenion® intake was the only variable having a significant protective effect on the outcome, and the Mantel-Haenszel OR increased from 16.44, CI = , to 17.43, CI = , when controlled by demographical characteristics. The χ2 goodness of fit test did not reject the hypothesis of correctness of our model . Finally, the area under the ROC curve was 0.85, indicating that the logistic model was well fitted  . Indeed,l fitted .MCI is a well-defined clinical condition that is considered to be a prodromal stage of AD. Therefore, although the presence of a spontaneous recovery has been recently described in about 20% of cases, the good management of subjects with MCI could reduce its progression to dementia . ®, whereas, on the contrary, all patients showing an improvement at follow-up had been treated.The results of our study led us to reject the hypothesis of casualty between nutraceutical intake and the reduction of disease progression. Indeed, 87.5% of the patients with a worsening in cognitive functions did not take MexenionNo pharmacological treatment is officially available for MCI because the physiopathological basis of neurodegeneration is not completely understood. However, the presence of neurofibrillary tangles, τ-complexes, and β-amyloid plaques have been identified as hallmarks of degenerative processes in the brain. Thus, three different pathways  are considered to be the causative agents of neurodegeneration . Many authors agree with the hypothesis that neurodegeneration is caused by a triggering of the inflammatory cascade. In particular, it has been demonstrated that an increased level of pro-inflammatory cytokines could alter the blood–brain barrier integrity, leading to a subsequent transmigration of leukocytes inside the brain. The presence of leukocytes activates the chronic oxidative stress cascade, leading to synaptic and neuronal dysfunction ,30.Growing evidence shows reduced sensitivity of insulin receptors in many areas of the brain, especially the hippocampus, cerebellum, and hypothalamus. The reduced activity of these areas seems to be associated with neural loss and the typical memory impairment of neurodegeneration ,32. Fina® is a promising therapeutic approach to reducing age-related cognitive impairment. Indeed, 33.3% of the patients who consumed the nutraceutical were stable at the one-year follow-up, and 60% saw improvements in their cognitive functions (as per MMSE) with regard to attention and memory. From the results of our study we can argue that dietary supplementation with MexenionDesideri et al. carried out the first dietary intervention study on elderly subjects with MCI, demonstrating that the regular intake of cocoa flavonoids might improve cognitive function as well as blood pressure and insulin resistance . We retrPolyphenols are phytochemicals  with antioxidant properties. More than 8000 polyphenols (mostly flavonoids) have been identified, mainly in tea, wine, chocolates, fruits, vegetables, and extra virgin olive oil.Even though polyphenols have in vitro antioxidative activity, the in vivo activity could be reduced by the accessibility of the compound to the brain. It is worth noting that dietary polyphenols do not necessarily have the best bioavailability profile due to environmental, food-related, and host-related factors, as well as the polyphenol chemical structure and interactions with other compounds ,36. AlthPolyphenols have multiple potential pharmacological effects on the slowing of neurodegenerative processes. First, the innate scavenger activity of flavonoids is important in the reduction of chronic oxidative stress that leads to neural loss. In addition, some polyphenols, such as catechins, may prevent neurodegeneration by inducing different signaling pathways , especiaB. monnieri, should also be taken into consideration [All of these molecular actions, which are associated with the concomitant positive effect of polyphenols on endothelial function, are responsible for neuronal plasticity  . Nonethederation .B. monnieri that could improve cognitive performance include steroidal and triterpenoidal saponins. Few in vivo data are available on the bioavailability, metabolism, and pharmacokinetics of these compounds. Therefore, there is no conclusive evidence for the mechanism of action of B. monnieri on cognition, reduction of β-amyloid levels , brain stress hormones, neuroinflammation factors, restoration of cholinergic and GABAergic transmission, anti-oxidant and neuroprotection factor release, and the increase of 5-HT levels and cerebral blood flow [The neuroprotective compounds extracted from ood flow .Our study presents some limitations. First, a prospective cohort study would allow us to directly use the neuropsychological test scores as the outcome variable, and to enroll more patients in order to reduce the information bias. However, the retrospective design of the study was the most appropriate in the presence of the existing data, and the dichotomization of the MMSE score to select patients should overcome the information bias. Indeed, both the goodness of fit test and the ROC curve indicated a good fitting of our logistic model, which included the main confounders such as age and gender.Secondly, the study design does not allow for the adequate consideration of factors which evaluate adherence to cocoa consumption , as the prospective study does .We investigated patients affected by aMCI, a subtype of MCI that is more likely to convert into AD. Notably, in the amnestic subtype of MCI some genetic markers, including ApoE, APP, presenilin 1, presenilin 2, and tau protein, may already be present, therefore predicting the possible future development of AD . Other iIn conclusion, dietary supplementation of cocoa flavonoids seems to reduce MCI-to-dementia conversion. Since there is growing evidence of a worldwide association between aging and the risk of dementia, the World Health Organization expects that only good management of dementia risk factors, such as MCI, could reduce the incidence and prevalence of people with dementia.


Previous studies have suggested that the effects of medical interventions tend to diminish over time. We investigated whether the effects of blood pressure lowering treatment on all-cause mortality and stroke have diminished over time.We conducted meta-regression analyses. We extracted the target trials from two recently published comprehensive systematic reviews and meta-analyses. Adopted variables were relative risk (RR) of all-cause mortality and stroke, trial start year, mean age, sample size, baseline systolic blood pressure (SBP), difference in attained SBP reduction between intervention groups and control groups (SBP difference), and regional dummies. We implemented single meta-regressions, in which the dependent variable was the log of RR and the explanatory variable was each of other adopted variables. We also conducted multiple meta-regressions, in which the dependent variable was the log of RR and explanatory variables were all of other adopted variables. Our variable of greatest interest was trial start year.The included reviews assessed 85 trials with a total of 343,126 participants. Although trial start year was positively associated with the log of RR in the results of single meta-regressions, it lost significance in multiple meta-regressions for both all-cause mortality and stroke.The effects of blood pressure lowering treatment on all-cause mortality and stroke have not diminished over time.The online version of this article (10.1186/s40885-018-0101-9) contains supplementary material, which is available to authorized users. Previous studies suggest that the effects of medical interventions tend to diminish over time –7. For eGiven this, we investigated whether the effects of blood pressure lowering treatments on all-cause mortality and stroke have diminished over time. Numerous clinical trials and meta-analyses indicated that blood pressure lowering treatments lessen serious cardiovascular events and all-cause mortality , 13. To Therefore, the objective of this study was to examine whether the effects of blood pressure lowering treatment on all-cause mortality and stroke have diminished over time.Although there are several systematic reviews and meta-analyses regarding the effects of blood pressure lowering treatment on cardiovascular diseases and mortality, the most recent and comprehensive we are aware of are Ettehad et al. , which wThe Ettehad paper included a whole range of randomized controlled trials on blood pressure lowering treatment. The authors searched MEDLINE from January 1, 1966 till July 7, 2015. They also searched the medical literature and identified trials till November 9, 2015. Their search strategy was compatible to that of Law et al.  and usedThe Brunström and Carlberg paper identified previous systematic reviews on the effect of blood pressure lowering from PubMed, the Cochrane Database of Systematic Reviews, and the Database of Abstracts of Reviews of Effect. They identified trials by scrutinizing reference lists from relevant reviews. They additionally searched PubMed and Cochrane Central Register for Controlled Trials in order to find trials after November 1, 2015 up till February 2017. The Brunström and Carlberg paper included mostly the same type of trials as the Ettehad paper, except for the following points. Unlike the Ettehad paper, the Brunström and Carlberg paper excluded trials in the acute phase after myocardial infarction from meta-analysis. They also excluded trials they deemed to be at high risk of bias. Unlike the Ettehad paper, which required a minimum of 1000 patient-years of follow-up for each trial group, the Brunström and Carlberg paper required a minimum of 1000 patient-years of follow-up for the whole study sample. We constructed our dataset for meta-analysis and meta-regression by adopting the trials included in these two papers. We used the names of included trials as used in the Ettehad paper and/or the Brunström and Carlberg paper.One of the authors (YS) extracted the following data from each trial: trial start year, sample size of trial , number of events , mean age at baseline, mean systolic blood pressure (SBP) at baseline, difference in SBP reduction between the two groups, SBP reduction from the baseline in each group, attained SBP in each group, and the region in which each trial was carried out.As defined in both the Ettehad paper and the Brunström and Carlberg paper, stroke included fatal and non-fatal stroke and excluded transient ischemic attack (TIA). In addition to data on stroke as a whole, we aimed to extract data on its subtypes  from each trial and, where possible, performed analyses based on the subtypes. In the process of data extraction, we found that several papers covered only fatal stroke or non-fatal stroke. For these papers, we recorded the cases shown in each paper. We also found that several papers included TIA in the number of cases, and exclusion of TIA was not possible. In these cases, we extracted the number of cases including TIA.As the Ettehad paper showed the number of events  in its appendix, we compared our extraction results with those of the Ettehad paper. When there were differences, we explored the causes of the differences. This exploration revealed that some data were different from those in the Ettehad paper. Regarding all-cause mortality, there were three differences compared with the Ettehad paper . In PREClinicalTrials.gov. When these did not work, we emailed the corresponding or first author of the original papers.Regarding the variable to identify when each trial was carried out, we used the data of trial start year. To extract the trial start year, we first checked the papers for each trial. When we could not find the trial start year, we checked N = 2127) and assumed both groups included 1064 participants.Differences in the attained SBP reduction between intervention groups and control groups  were determined in the following way. We retrieved three patterns of figures for SBP difference: (1) difference in SBP reduction between the two groups, (2) SBP reduction from the baseline in each group, and (3) attained SBP in each group. We prioritized (1) when reported. If (1) was not reported, we calculated the difference from (2). If neither (1) nor (2) were reported, we calculated the difference from (3). We used mean figures of the whole measurements when reported. When plural measurements were reported and mean figures were not reported, we calculated the mean of plural measurements. When only graphs of SBP transition were shown, we calculated the SBP levels using an online tool . RegardiThe regions in which trials were carried out were categorized as follows: Asia, Europe, North America, Oceania, South America, and mixed regions. Trials carried out in two or more regions were included in the mixed region category. Turkey and Israel were included in Europe.The statistical analyses in this study were carried out in the following two steps. First, we conducted single meta-regressions, in which the dependent variable was the log RR of all-cause mortality or stroke and the explanatory variable was each of the extracted variables, namely trial start year, sample size, age, baseline SBP, SBP difference, and region . RR was calculated by dividing the risk of outcome events of intervention groups by the risk of outcome events of control groups. Risk of outcome events of each group was calculated by dividing the number of participants experiencing outcome events by the number of participants. With regard to the variable of region, there were several variables for regional dummies in the model, but we included it in the table of single meta-regression because they all belonged to the regional category.Second, we conducted multiple meta-regressions, in which the dependent variable was the log RR and the explanatory variables were all of the abovementioned variables . We judged whether the effects of blood pressure lowering treatment on all-cause mortality and stroke have diminished over time from Model 2M and Model 2S. Diminishing was confirmed only when the variable of trial start year was significant at the 5% level.When there was no event in either group in a trial, we added 0.5 to the number of events and the number of non-events in both groups before performing the meta-regressions. All analyses were performed using STATA 15 . For meta-regressions, we used the “metareg” command , which eFor trials with three or more arms, we followed the classification performed in the Ettehad paper or the Brunström and Carlberg paper. The two papers defined the control group in the ONTARGET study  differenThe list of all trials in our study is shown in Additional file A total of 85 trials were selected, representing 343,126 participants. Among these, there were 82 trials for all-cause mortality and 74 trials for stroke. Summary statistics of the trials are shown in Table In Table p = 0.004) in the case of no adjustment for other explanatory variables. For other explanatory variables, RR significantly grew with the rise in sample size and decreased with the enlarged SBP difference. RR was significantly lower in trials carried out in North America and Asia than in mixed region trials.The results of single meta-regressions (Model 1M in Table p = 0.078) after adjusting for other explanatory variables. Similarly, associations of log RR of all-cause mortality with sample size, SBP difference, and regional dummies for North America lost significance after adjusting for other explanatory variables. Trials in Asia still had a significantly lower log RR than mixed region trials after adjusting for other explanatory variables; RR of all-cause mortality of trials in Asia was 16% lower than mixed region trials (exp(b) = 0.84; 95% CI 0.73, 0.97; p = 0.020) after adjusting for other explanatory variables  = 1.05; 95% CI 0.99, 1.10; As the trial start year variable was not significant at the 5% level in multiple meta-regression, we concluded that the effects of blood pressure lowering treatment on all-cause mortality have not diminished over time.p <  0.001) in the case of no adjustment for other explanatory variables. For other explanatory variables, RR significantly climbed with the rise in sample size and fell with the growing baseline SBP and SBP difference. RR was significantly lower in trials carried out in North America, Europe, and Asia than in mixed region trials.In Table p = 0.154) after adjusting for other explanatory variables. Similarly, associations of log RR of stroke with sample size, baseline SBP, and regional dummies for North America and Europe lost significance after adjusting for other explanatory variables. SBP difference still had a significant association with log RR; for every 10 mmHg increase in SBP difference, RR of stroke decreased significantly by 19% (exp(b) = 0.81; 95% CI 0.69, 0.94; p = 0.008) after adjusting for other explanatory variables. Trials in Asia still had a significantly lower log RR than mixed region trials after adjusting for other explanatory variables; RR of stroke for trials in Asia was 17% lower than mixed region trials (exp(b) = 0.83; 95% CI 0.70, 0.99; p = 0.042) after adjusting for other explanatory variables  = 1.07; 95% CI 0.97, 1.19; As the trial start year variable was not significant at the 5% level in multiple meta-regression, we concluded that the effects of blood pressure lowering treatment on stroke have not diminished over time.Regarding analyses by stroke subtypes, out of the 74 trials we covered, we found only five trials , 27–30 wIn this study, we investigated whether the effects of blood pressure lowering treatment on all-cause mortality and stroke have diminished over time by performing meta-regressions in which log RRs of the outcome events were the dependent variable and trial start year was the explanatory variable of greatest interest. Although trial start year was positively associated with log RR in the results of single meta-regressions, it lost significance in multiple meta-regressions which adjusted for mean age, sample size, baseline SBP, SBP difference, and regional dummies for both all-cause mortality and stroke. We concluded that the effects of blood pressure lowering treatment on all-cause mortality and stroke have not diminished over time from the results of the multiple meta-regressions.ClinicalTrials.gov website and emailing the corresponding authors, we were still unable to identify the trial start year for three trials. This may have caused some bias and/or lessened statistical power.In this study, we used the data of trial start year instead of year of publication as the variable to identify when each trial was performed. One problem in using data of trial start year was that some studies do not report this variable in published papers. Although we did our best to identify trial start year by checking the In this study, SBP difference (difference in attained SBP reduction between intervention groups and control groups) was not associated with risk reduction of all-cause mortality in multiple meta-regression. Although Ettehad et al.  indicateIn this study, trials in Asia showed significantly lower RR compared with mixed region trials in both all-cause mortality and stroke. Of the 11 trials carried out in Asia, six were from China, four from Japan, and one from Hong Kong and Japan. The reasons why such higher effects are shown in Asia need to be explored in future studies.2 for multiple regressions was not large enough , suggesting the possibility of other important factors which would substantially change the results of this paper.There are several limitations to this study. First, we included the trials covered by two recent comprehensive systematic reviews. Additional trials not covered by the included systematic reviews may alter these findings. Second, the definition of stroke varied among trials. Variation in definition may have caused biases in our meta-regression on stroke. Third, since most trials included participants with comorbidities, the trials covered in this study might be heterogeneous. Fourth, we assumed a linear relationship between log RRs and trial start year. This assumption may be questioned, and the analysis could be improved by better reflecting the behavior of the data when constructing the model. Fifth, adjusted RThe effects of blood pressure lowering treatment on all-cause mortality and stroke have not diminished over time.Additional file 1:Table S1. Summary of Included Trials. (DOCX 31 kb)Additional file 2:Table S2. Summary of Outcome Events on All-cause Mortality. (DOCX 28 kb)Additional file 3:Table S3. Summary of Outcome Events on Stroke. (DOCX 28 kb)


The internet is becoming the main source of health-related information. We aimed to investigate data regarding heartburn-related searches made by Google users from Australia, Canada, Germany, Poland, the United Kingdom, and the United States. We retrospectively analyzed data from Google Ads Keywords Planner. We extracted search volumes of keywords associated with “heartburn” for June 2015 to May 2019. The data were generated in the respective primary language. The number of searches per 1000 Google-user years was as follows: 177.4 , 178.1 (Canada), 123.8 (Germany), 199.7 (Poland), 152.5 (United Kingdom), and 194.5 (United States). The users were particularly interested in treatment (19.0 to 41.3%), diet (4.8 to 10.7%), symptoms (2.6 to 13.1%), and causes (3.7 to 10.0%). In all countries except Germany, the number of heartburn-related queries significantly increased over the analyzed period. For Canada, Germany, Poland, and the United Kingdom, query numbers were significantly lowest in summer; there was no significant seasonal trend for Australia and the United States. The number of heartburn-related queries has increased over the past four years, and a seasonal pattern may exist in certain regions. The trends in heartburn-related searches may reflect the scale of the complaint, and should be verified through future epidemiological studies.  Clostridum difficile infection [Heartburn can occur incidentally or may be a chronic symptom of gastroesophageal reflux disease (GERD) . This confection ,8,9. Thenfection , the aginfection , and a snfection .Limited access to public health-care has caused many individuals to perceive the Internet as an attractive alternative to real-life health-care professionals ,11. FurtThe approach of investigating data from an electronic medium for epidemiological studies is called infodemiology (information epidemiology) . InfodemGoogle is the most popular search engine among Internet users in Western countries . The “KeConsidering the above, in the present study, we aimed to retrospectively investigate frequency, yearly and seasonal trends of heartburn-related searches made by users from Australia (AU), Canada (CA), Germany (DE), Poland (PL), the United Kingdom (UK), and the United States (US).For this infodemiological study, we extracted research volumes for the term “heartburn”  and generated keywords for the period of June 2015 to May 2019 using the Google Ads Keywords Planner (ads.google.com/aw/keywordplanner). The tool allows to analyze search volumes of terms from the last 48 months. The data were generated separately for each region (chosen language): AU (English), CA (English), DE (German), PL (Polish), UK (English), US (English); search network: Google. The considered regions were countries. A screenshot of the search engine keywords is presented in Additionally, we distinguished keywords that are related to questions regarding heartburn ; its causes, symptoms, and treatment ; relation to diet; and patient groups. Keyword “categories” (based on the character of each keyword) were created. A keyword  can be attributed to multiple categories , and a keyword category may also comprise multiple keywords . In this manuscript, we only present categories of keywords that represent at least 1% of the total number of heartburn-related queries in each country. An illustration of the data collection and manipulation process is presented in p values of < 0.05 were considered to indicate significant difference. The considered seasons were: spring , summer , fall , and winter , and the analyzed years were June 2015–May 2016 (“first”), June 2016–May 2017 (“second”), June 2017–May 2018 (“third”), and June 2018–May 2019 (“fourth”). We visualized the time course of all queries related to heartburn in the chosen countries, generating representative figures using the ggplot2 and ggthemes2 packages of R 3.5.1  [For all countries, we calculated the arithmetical mean of the sum of monthly search volume for each keyword category and all keywords. The data was presented as the total number of queries during the analyzed period for each keyword category and all keywords. Additionally, we divided the total search volume of each category by the total number of searches in the analyzed period, and separately by the number of Google users in each country, as declared by the Keywords Planner. Additionally, we presented the mean Google search engine market share in investigated countries in the years 2015–2019 . SpecifiAustria) . Trends Austria) . The Keywords Planner generated 953 keywords for the English term “heartburn,” 1344 for the Polish term “zgaga,” and 1411 for the German term “Sodbrennen.” After evaluation of the list of generated keywords, 894, 1244, and 1388, respectively, of the terms were included in the final investigation. We presented the most popular keywords in each country in We visualized the number of heartburn-related searches conducted during the analyzed period for all chosen countries . A comparison of the total number of queries in terms of years and seasons is presented in For all analyzed countries, we compared the number of searches relating to the heartburn-related keyword categories in terms of years and seasons . We consIn this infodemiology study, we found that the number of heartburn-related searches has increased over the last four years and may present a seasonal pattern in six Western countries. Infodemiology is a relatively new discipline of science, where web-based data are assessed for various determinants and distributions of information with an aim to inform the public and create tools useful for various policymakers . InfodemTo the best of our knowledge, this is the first infodemiology study to use Google Ads to assess Internet users’ interest in heartburn-related information. Moreover, this is the first study to compare Google Ads data between different languages and different countries. We have chosen to use Google Ads Keyword Planner, which provides estimates of absolute search volume. In the context of heartburn, this tool can be used as a surrogate for the population-level burden of this condition ,23. ThisTo control for any possible influence caused by the differing number of Google users in the analyzed countries, we expressed interest as the number of searches per 1000 Google-user years. Dent et al. estimated that the weekly incidence of GERD symptoms is five per 1000 person-years ; thus, oOur data showed that Google users from DE are particularly interested in the treatment of heartburn. However, after examining the total number of queries related to heartburn treatment, we found that, for DE, the number of queries per 1000 Google user years was similar to that of PL . As mentWe observed a seasonal variation in heartburn-related queries for CA, DE, PL, and UK; moreover, we observed a similar pattern for keyword categories: interest in heartburn-related information was lowest during warm months. Similarly, Hassid et al. reported modest seasonality in Google searches regarding diarrhea and vomiting in the US . We alsoIn all analyzed countries except DE, we observed, over the period examined, a significant growth in the number of queries associated with heartburn. In DE, we merely found a tendency in this regard. This may be caused by a high seasonal variation of searches in this country. Nevertheless, our results may reflect the growing prevalence of GERD in the Western world . The higOlivera et al. previously reported that a substantial group of patients do not consult physicians about their heartburn . For theThe simplicity of interacting with search engines and the comfort of avoiding the need to disclose embarrassing ailments may explain why “Dr. Google” is becoming a major consultant for Internet users . For theEvaluating data sourced from Google Ads Keywords Planner has several limitations. First, we could only collect data pertaining to the last four years . Second,In conclusion, interest in queries associated with heartburn has increased in the past four years in the countries analyzed and, in certain regions, a seasonal pattern may exist. The observed trends in heartburn-related searches may reflect the scale of the complaint, and should be verified through future epidemiological studies. These findings show that health-care providers should provide reliable information online to prevent patients from accessing information of unknown reliability.