Optimization and Validation of Procyanidins Extraction and Phytochemical Profiling of Seven Herbal Matrices of Nutraceutical Interest.

Several medicinal herbal plants are extensively used as sources of bioactive compounds with beneficial effects on human health. This study assessed the procyanidin and polyphenol profiles together with the antioxidant potential of seven herbal medical matrices. To achieve this aim, procyanidin extraction from grape pomace was optimized and validated by monitoring monomeric-trimeric procyanidins. The proposed quantification method was applied to the seven medical herbs, and it proved to be a very efficient protocol for procyanidin-rich extracts analysis. In addition, the Paullinia cupana Kunth. seed was identified as a very rich source of procyanidins (about 5 mg/g dry matrix of each dimeric and about 3 mg/g dry matrix trimeric) with high antioxidant properties. The polyphenolic profile was assessed by HPLC-HESI-MS/MS analysis. The in vitro antioxidant activity was evaluated by DPPH assay to explore the antioxidant properties of the extracts, which were substantially higher in Peumus boldus Molina leaves extracts (935.23 ± 169 µmol of Trolox equivalent/g of dry weight) concerning the other matrices. Moreover, a high Pearson coefficient value was observed between the total flavonoid content (TFC) and DPPH in comparison with the total polyphenol content (TPC) and DPPH, indicating flavonoids as the principal bioactive with antioxidant activity in the extracts.

Effects of Food-Derived Antioxidant Compounds on In Vitro Heavy Metal Intestinal Bioaccessibility.

Environmental contamination by heavy metals (HMs) has emerged as a significant global issue in recent decades. Among natural substances, food-deriving polyphenols have found a valuable application in chelating therapy, partially limited by their low water solubility. Thus, three different hydroalcoholic extracts titrated in quercetin (QE), ellagic acid (EA), and curcumin (CUR) were formulated using maltodextrins as carriers, achieving a powder with a valuable water solubility (MQE 91.3 ± 1.2%, MEA 93.4 ± 2.1, and MCUR 89.3 ± 2%). Overcoming the problem of water solubility, such formulations were tested in an in vitro simulated gastrointestinal digestion experiment conducted on a water sample with standardized concentrations of the principal HMs. Our results indicate that regarding the nonessential HMs investigated (Pb, Cd, As, Sb, and Hg), MQE has been shown to be the most effective in increasing the HMs' non-bioaccessible concentration, resulting in concentration increases in Cd of 68.3%, in As of 51.9%, in Hg of 58.9%, in Pb of 271.4, and in Sb of 111.2% (vs control, p < 0.001) in non-bioaccessible fractions. Regarding the essential HMs, MEA has shown the greatest capability to increase their intestinal bioaccessibility, resulting in +68.5%, +61.1, and +22.3% (vs control, p < 0.001) increases in Cu, Zn, and Fe, respectively. Finally, considering the strong relation between the antiradical and chelating activities, the radical scavenging potentials of the formulations was assayed in DPPH and ABTS assays.

Unveiling the modulation of Pseudomonas aeruginosa virulence and biofilm formation by selective histone deacetylase 6 inhibitors.

Bacterial infections represent a key public health issue due to the occurrence of multidrug-resistant bacteria. Recently, the amount of data supporting the dynamic control of epigenetic pathways by environmental cues has triggered research efforts toward the clarification of their role in microbial infections. Among protein post-translational modifications, reversible acetylation is the most implicated in the feedback to environmental stimuli and in cellular homeostasis. Accordingly, the latest studies identified the histone deacetylase 6 (HDAC6) enzyme as a crucial player in the complex molecular machinery underlying bacterial clearance or killing. A very important milestone for the elucidation of the consequence of HDAC6 activity in bacterial infections is herein described, unveiling for the first time the role of a potent HDAC6 inhibitor in interfering with biofilm formation and modulating virulence factors of P. aeruginosa. We demonstrated that compound F2F-2020202 affected the production of some important virulence factors in P. aeruginosa, namely pyocyanin and rhamnolipids, clearly impairing its ability to form biofilm. Furthermore, evidence of possible QS involvement is supported by differential regulation of specific genes, namely RhlI, phAz1, and qsrO. The data herein obtained also complement and in part explain our previous results with selective HDAC6 inhibitors able to reduce inflammation and bacterial load in chronic infection models recapitulating the cystic fibrosis (CF) phenotype. This study fosters future in-depth investigation to allow the complete elucidation of the molecular mechanisms underlying HDAC6's role in bacterial infections.

Agri-Food Waste Recycling for Healthy Remedies: Biomedical Potential of Nutraceuticals from Unripe Tomatoes (Solanum lycopersicum L.).

Unripe tomatoes represent an agri-food waste resulting from industrial by-processing products of tomatoes, yielding products with a high content of bioactive compounds with potential nutraceutical properties. The food-matrix biological properties are attributed to the high steroidal glycoalkaloid (SGA) content. Among them, α-tomatine is the main SGA reported in unripe green tomatoes. This review provides an overview of the main chemical and pharmacological features of α-tomatine and green tomato extracts. The extraction processes and methods employed in SGA identification and the quantification are discussed. Special attention was given to the methods used in α-tomatine qualitative and quantitative analyses, including the extraction procedures and the clean-up methods applied in the analysis of Solanum lycopersicum L. extracts. Finally, the health-beneficial properties and the pharmacokinetics and toxicological aspects of SGAs and α-tomatine-containing extracts are considered in depth. In particular, the relevant results of the main in vivo and in vitro studies reporting the therapeutic properties and the mechanisms of action were described in detail.

Phytochemical investigation and antioxidant properties of unripe tomato cultivars (Solanum lycopersicum L.).

Unripe tomatoes are among the main waste produced during tomato cultivation and processing. In this study, unripe tomatoes from seven different Italian cultivars have been investigated to evaluate their nutraceutical potential. Phytochemical investigation allowed shedding light on the identification of seventy-five bioactive compounds. The highest amount of polyphenolic and glycoalkaloids along with the high level of antioxidant activities was found in the Datterini tomatoes variety. The peculiarity of this variety is the high chlorogenic acid content, being ten times higher compared to the other cultivars examined. Moreover, the total α-tomatine amount has been found substantially higher (34.699 ± 1.101 mg/g dry weight) with respect to the other tomato varieties analyzed. Furthermore, the cultivars metabolomic profiles were investigated with the PCA approach. Based on Datterini cultivar's metabolomic profile, its waste-recovery could represent a good option for further added value products in pharmaceutical and nutraceutical areas with a high α-tomatine content.

Natural Chalcones for the Management of Obesity Disease.

In the last decade, the incidence of obesity has increased dramatically worldwide, reaching a dangerous pandemic spread. This condition has serious public health implications as it significantly increases the risk of chronic diseases such as type 2 diabetes, fatty liver, hypertension, heart attack, and stroke. The treatment of obesity is therefore the greatest health challenge of our time. Conventional therapeutic treatment of obesity is based on the use of various synthetic molecules belonging to the class of appetite suppressants, lipase inhibitors, hormones, metabolic regulators, and inhibitors of intestinal peptide receptors. The long-term use of these molecules is generally limited by various side effects and tolerance. For this reason, the search for natural alternatives to treat obesity is a current research goal. This review therefore examined the anti-obesity potential of natural chalcones based on available evidence from in vitro and animal studies. In particular, the results of the main in vitro studies describing the principal molecular therapeutic targets and the mechanism of action of the different chalcones investigated were described. In addition, the results of the most relevant animal studies were reported. Undoubtedly, future clinical studies are urgently needed to confirm and validate the potential of natural chalcones in the clinical prophylaxis of obesity.

The Urine Metabolome of R6/2 and zQ175DN Huntington's Disease Mouse Models.

Huntington's disease (HD) is caused by the expansion of a polyglutamine (polyQ)-encoding tract in exon 1 of the huntingtin gene to greater than 35 CAG repeats. It typically has a disease course lasting 15-20 years, and there are currently no disease-modifying therapies available. Thus, there is a need for faithful mouse models of HD to use in preclinical studies of disease mechanisms, target validation, and therapeutic compound testing. A large variety of mouse models of HD were generated, none of which fully recapitulate human disease, complicating the selection of appropriate models for preclinical studies. Here, we present the urinary liquid chromatography-high-resolution mass spectrometry analysis employed to identify metabolic alterations in transgenic R6/2 and zQ175DN knock-in mice. In R6/2 mice, the perturbation of the corticosterone metabolism and the accumulation of pyrraline, indicative of the development of insulin resistance and the impairment of pheromone excretion, were observed. Differently from R6/2, zQ175DN mice showed the accumulation of oxidative stress metabolites. Both genotypes showed alterations in the tryptophan metabolism. This approach aims to improve our understanding of the molecular mechanisms involved in HD neuropathology, facilitating the selection of appropriate mouse models for preclinical studies. It also aims to identify potential biomarkers specific to HD.

Photo-Flow Technology for Chemical Rearrangements: A Powerful Tool to Generate Pharmaceutically Relevant Compounds.

In recent years, photochemistry has increasingly emerged as an enabling methodology in both academia and the pharmaceutical industry. Long photolysis times and the gradual reduction of light penetration remained for many years unsolved issues for photochemical rearrangements, triggering the generation of highly reactive species in an uncontrolled fashion and causing the formation of multiple side products. The emergence of continuous-flow chemistry significantly helped to overcome these issues, thus prompting the implementation of photo-flow-based approaches for the generation of pharmaceutically relevant substructures. This Technology Note highlights the benefits of flow chemistry for photochemical rearrangements, including Wolff, Favorskii, Beckmann, Fries, and Claisen rearrangements. We showcase recent advances for photo-rearrangements in continuous flow applied to the synthesis of privileged scaffolds and active pharmaceutical ingredients.

Broad-spectrum coronavirus 3C-like protease peptidomimetic inhibitors effectively block SARS-CoV-2 replication in cells: Design, synthesis, biological evaluation, and X-ray structure determination.

Despite the approval of vaccines, monoclonal antibodies and restrictions during the pandemic, the demand for new efficacious and safe antivirals is compelling to boost the therapeutic arsenal against the COVID-19. The viral 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for replication with high homology in the active site across CoVs and variants showing an almost unique specificity for Leu-Gln as P2-P1 residues, allowing the development of broad-spectrum inhibitors. The design, synthesis, biological activity, and cocrystal structural information of newly conceived peptidomimetic covalent reversible inhibitors are herein described. The inhibitors display an aldehyde warhead, a Gln mimetic at P1 and modified P2-P3 residues. Particularly, functionalized proline residues were inserted at P2 to stabilize the ß-turn like bioactive conformation, modulating the affinity. The most potent compounds displayed low/sub-nM potency against the 3CLpro of SARS-CoV-2 and MERS-CoV and inhibited viral replication of three human CoVs, i.e. SARS-CoV-2, MERS-CoV, and HCoV 229 in different cell lines. Particularly, derivative 12 exhibited nM-low µM antiviral activity depending on the virus, and the highest selectivity index. Some compounds were co-crystallized with SARS-CoV-2 3CLpro validating our design. Altogether, these results foster future work toward broad-spectrum 3CLpro inhibitors to challenge CoVs related pandemics.

Rejuvenating the [1, 2, 3]-triazolo [1,5-a]quinoxalin-4(5H)-one scaffold: Synthesis and derivatization in a sustainable guise and preliminary antimicrobial evaluation.

The [1,2,3]-triazolo [1,5-a] quinoxalin-4(5H)-one scaffold and its analogues triazole-fused heterocyclic compounds are relevant structural templates in both natural and synthetic biologically active compounds. However, their medicinal chemistry applications are often limited due to the lack of synthetic protocols combining straightforward generation of the central core while also allowing extensive decoration activity for drug discovery purposes. Herein, we report a "refreshed" synthesis of the [1,2,3]-triazolo [1,5-a]quinoxalin-4(5H)-one core, encompassing the use of eco-compatible catalysts and reaction conditions. We have also performed a sustainable and extensive derivatization campaign at both the endocyclic amide nitrogen and the ester functionality, comprehensively exploring the reaction scope and overcoming some of the previously reported difficulties in introducing functional groups on this structural template. Finally, we unveiled a preliminary biological investigation for the newly generated chemical entities. Our assessment of the compounds on different bacterial species (two S. aureus strains, three P. aeruginosa strains, K. pneumonia), and two fungal C. albicans strains, as well as the evaluation of their activity on S. epidermidis biofilm formation, foster further optimization for the retrieved hit compounds 9, 14, and 20.

Validation of an LC-MS/MS Method for the Determination of Abscisic Acid Concentration in a Real-World Setting.

One of the most relevant aspects in evaluating the impact of natural bioactive compounds on human health is the assessment of their bioavailability. In this regard, abscisic acid (ABA) has attracted particular interest as a plant-derived molecule mainly involved in the regulation of plant physiology. Remarkably, ABA was also found in mammals as an endogenous hormone involved in the upstream control of glucose homeostasis, as evidenced by its increase after glucose load. The present work focused on the development and validation of a method for the determination of ABA in biological samples through liquid-liquid extraction (LLE), followed by liquid mass spectrometry (LC-MS) of the extract. To test method suitability, this optimized and validated method was applied to a pilot study on eight healthy volunteers' serum levels to evaluate ABA concentration after consumption of a standardized test meal (STM) and the administration of an ABA-rich nutraceutical product. The results obtained could meet the demands of clinical laboratories to determine the response to a glucose-containing meal in terms of ABA concentration. Interestingly, the detection of this endogenous hormone in such a real-world setting could represent a useful tool to investigate the occurrence of impaired ABA release in dysglycemic individuals and to monitor its eventual improvement in response to chronic nutraceutical supplementation.

Continuous-Flow Technology for Chemical Rearrangements: A Powerful Tool to Generate Pharmaceutically Relevant Compounds.

The efficacy, safety, and scale-up of several chemical rearrangements remain unsolved problems due to the associated handling of hazardous, toxic, and pollutant chemicals and high-risk intermediates. For many years batch processes have been considered the only possibility to drive these reactions, but continuous-flow technology has emerged, for both academic laboratories and pharmaceutical companies, as a powerful tool for easy, controlled, and safer chemistry protocols, helping to minimize the formation of side products and increase reaction yields. This Technology Note summarizes recently reported chemical rearrangements using continuous-flow approaches, with a focus on Curtius, Hofmann, and Schmidt reactions. Flow protocols, general advantages and safety aspects, and reaction scope for the generation of both privileged scaffolds and active pharmaceutical ingredients will be showcased.

Nirmatrelvir treatment of SARS-CoV-2-infected mice blunts antiviral adaptive immune responses.

Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir-an orally available inhibitor of the 3-chymotrypsin-like cysteine protease-has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using mouse models of SARS-CoV-2 infection, we show that nirmatrelvir administration blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.

Optimization of Ursolic Acid Extraction in Oil from Annurca Apple to Obtain Oleolytes with Potential Cosmeceutical Application.

Ursolic acid (UA) is a plant-derived molecule with relevant anti-aging activity, which makes this molecule a potential functional active ingredient in cosmetic formulations. The main objectives of this study were to optimize the UA extraction process from Annurca apple (AA) with sunflower oil as a lyophilic food-grade solvent using Response Surface Methodology (RSM) to determine the potential cosmetic application of the obtained extract. The results of RSM analysis showed a maximum UA yield of 784.40 ± 7.579 (µg/mL) obtained under the following optimized conditions: sunflower oil as extraction solvent, 68.85 °C as extraction temperature, and 63 h as extraction time. The HPLC-DAD-HESI-MS/MS analysis performed on the extract obtained under these conditions, named Optimized Annurca Apple Oleolyte (OAAO), led to the identification of twenty-three phenolic and terpenoid molecules and the quantification of eight of them. To explore the biological properties of OAAO, the in vitro antioxidant activity was evaluated by DPPH, ABTS, and FRAP assays, resulting in 16.63 ± 0.22, 5.90 ± 0.49, and 21.72 ± 0.68 µmol Trolox equivalent/g extract, respectively. Moreover, the permeation study has shown that OAAO may be considered a safe and functional ingredient in potential cosmetic formulations.

Easy access to α-ketoamides as SARS-CoV-2 and MERS Mpro inhibitors via the PADAM oxidation route.

SARS-CoV-2 caused worldwide the current outbreak called COVID-19. Despite multiple countermeasures implemented, there is an urgent global need for new potent and efficient antiviral drugs against this pathogen. In this context, the main protease (Mpro) of SARS-CoV-2 is an essential viral enzyme and plays a pivotal role in viral replication and transcription. Its specific cleavage of polypeptides after a glutamine residue has been considered as a key element to design novel antiviral drugs. Herein, we reported the design, synthesis and structure-activity relationships of novel α-ketoamides as covalent reversible inhibitors of Mpro, exploiting the PADAM oxidation route. The reported compounds showed µM to nM activities in enzymatic and in the antiviral cell-based assays against SARS-CoV-2 Mpro. In order to assess inhibitors' binding mode, two co-crystal structures of SARS-CoV-2 Mpro in complex with our inhibitors were solved, which confirmed the covalent binding of the keto amide moiety to the catalytic Cys145 residue of Mpro. Finally, in order to interrogate potential broad-spectrum properties, we assessed a selection of compounds against MERS Mpro where they showed nM inhibitory potency, thus highlighting their potential as broad-spectrum coronavirus inhibitors.

A Food-Grade Method for Enhancing the Levels of Low Molecular Weight Proanthocyanidins with Potentially High Intestinal Bioavailability.

Proanthocyanidins (PACs) are a group of bioactive molecules found in a variety of plants and foods. Their bioavailability depends on their molecular size, with monomers and dimers being more bioavailable than those that have a higher polymerization degree. This study aimed to develop a method to convert high-molecular-weight PACs to low-molecular-weight ones in a grape seed extract (GSE) from Vitis vinifera L. Therefore, GSE was subjected to alkaline treatment (ATGSE), and its difference in chemical composition, compared to GSE, was evaluated using a molecular networking (MN) approach based on results obtained from HPLC-ESI HRMS/MS characterization analysis. The network analysis mainly noted the PAC cluster with about 142 PAC compounds identified. In particular, the obtained results showed a higher content of monomeric and dimeric PACs in ATGSE compared to GSE, with 58% and 49% monomers and 31% and 24% dimers, respectively. Conversely, trimeric (9%), polymeric (4%), and galloylated PACs (14%) were more abundant in GSE than in ATGSE (6%, 1%, and 4%, respectively). Moreover, in vitro antioxidant and anti-inflammatory activities were investigated, showing the high beneficial potential of both extracts. In conclusion, ATGSE could represent an innovative natural matrix rich in bioavailable and bioaccessible PACs for nutraceutical applications with potential beneficial properties.

Optimization of Phlorizin Extraction from Annurca Apple Tree Leaves Using Response Surface Methodology.

Phlorizin is a plant-derived molecule with relevant anti-diabetic activity, making this compound a potential functional component in nutraceutical formulations for the management of glycemia. It is noteworthy that promising sources for the extraction of phlorizin include apple tree leaves, a by-product of apple fruit production. The main aim of this study was to optimize the extraction process of phlorizin from Annurca apple tree leaves (AALs) using response surface methodology (RSM), and to determine the potential nutraceutical application of the obtained extract. The results of the RSM analysis indicate a maximum phlorizin yield of 126.89 ± 7.579 (mg/g DW) obtained under the following optimized conditions: MeOH/H2O, 80:20 + 1% HCOOH as the extraction solvent; 37.7 °C as the extraction temperature; and 170 min as the time of extraction. The HPLC-DAD-HESI-MS/MS analysis performed on the extract obtained under such conditions, named optimized Annurca apple leaves extract (OAALE), led to the identification of twenty-three phenolic molecules, with fifteen of them quantified. To explore the nutraceutical potential of OAALE, the in vitro antioxidant activity was evaluated by DPPH, ABTS, and FRAP assays, resulting in 21.17 ± 2.30, 38.85 ± 0.69, and 34.14 ± 3.8 µmol Trolox equivalent/g of extract, respectively. Moreover, the IC50 of 0.330 mg/mL obtained from the advanced glycation end-product inhibition assay, further supported the antidiabetic potential of OAALE.

Efficacy of selective histone deacetylase 6 inhibition in mouse models of Pseudomonas aeruginosa infection: A new glimpse for reducing inflammation and infection in cystic fibrosis.

The latest studies identified the histone deacetylase (HDAC) class of enzymes as strategic components of the complex molecular machinery underlying inflammation in cystic fibrosis (CF). Compelling new support has been provided for HDAC6 isoform as a key player in the generation of the dysregulated proinflammatory phenotype in CF, as well as in the immune response to the persistent bacterial infection accompanying CF patients. We herein provide in vivo proof-of-concept (PoC) of the efficacy of selective HDAC6 inhibition in contrasting the pro-inflammatory phenotype in a mouse model of chronic P. aeruginosa respiratory infection. Upon careful selection and in-house re-profiling (in vitro and cell-based assessment of acetylated tubulin level through Western blot analysis) of three potent and selective HDAC6 inhibitors as putative candidates for the PoC, we engaged the best performing compound 2 for pre-clinical studies. Compound 2 demonstrated no toxicity and robust anti-inflammatory profile in a mouse model of chronic P. aeruginosa respiratory infection upon repeated aerosol administration. A significant reduction of leukocyte recruitment in the airways, in particular neutrophils, was observed in compound 2-treated mice in comparison with the vehicle; moreover, quantitative immunoassays confirmed a significant reduction of chemokines and cytokines in lung homogenate. This effect was also associated with a modest reduced bacterial load after compound 2-treatment in mice compared to the vehicle. Our study is of particular significance since it demonstrates for the first time the utility of selective drug-like HDAC6 inhibitors in a relevant in vivo model of chronic P. aeruginosa infection, thus supporting their potential application for reverting CF phenotype.

Beneficial Contribution to Glucose Homeostasis by an Agro-Food Waste Product Rich in Abscisic Acid: Results from a Randomized Controlled Trial.

The control of glucose homeostasis represents the primary goal for the prevention and management of diabetes and prediabetes. In recent decades, the hypoglycemic hormone abscisic acid (ABA) has attracted considerable interest in the scientific literature. In this regard, the high ABA concentration in immature fruits led us to consider these food matrices as candidates for diabetes control. Therefore, the beneficial efficacy of a nutraceutical formulation based on thinned nectarines (TNs) rich in ABA was tested through a three-month, three-arm, parallel-group, randomized controlled trial (RCT) conducted on sixty-one patients with type 2 diabetes (T2D). After 3 months, both the treatments with low doses of TN (500 mg 3 times/day) and high doses of TN (750 mg 3 times/day) showed a significant reduction in glycemic parameters compared to baseline. Treatment with low doses of TN showed a greater insulin-sparing effect (fasting plasma insulin, FPI: −29.2%, p < 0.05 vs. baseline) compared to the high-dose group (FPI: −16.5%, p < 0.05 vs. baseline). Moreover, a significant correlation between glycemia and ABA plasmatic levels was observed for both intervention groups at baseline and after 3 months. Overall, our data reasonably support TN as a promising and innovative nutraceutical product able to contribute to the management of glucose homeostasis.

Cytopathic SARS-CoV-2 screening on VERO-E6 cells in a large-scale repurposing effort.

Worldwide, there are intensive efforts to identify repurposed drugs as potential therapies against SARS-CoV-2 infection and the associated COVID-19 disease. To date, the anti-inflammatory drug dexamethasone and (to a lesser extent) the RNA-polymerase inhibitor remdesivir have been shown to be effective in reducing mortality and patient time to recovery, respectively, in patients. Here, we report the results of a phenotypic screening campaign within an EU-funded project (H2020-EXSCALATE4COV) aimed at extending the repertoire of anti-COVID therapeutics through repurposing of available compounds and highlighting compounds with new mechanisms of action against viral infection. We screened 8702 molecules from different repurposing libraries, to reveal 110 compounds with an anti-cytopathic IC50 < 20 µM. From this group, 18 with a safety index greater than 2 are also marketed drugs, making them suitable for further study as potential therapies against COVID-19. Our result supports the idea that a systematic approach to repurposing is a valid strategy to accelerate the necessary drug discovery process.