Hazelnut (Corylus avellana l.) shells extract: Phenolic composition, antioxidant effect and cytotoxic activity on human cancer cell lines

Hazelnut shells, a by-product of the kernel industry processing, are reported to contain high amount of polyphenols. However, studies on the chemical composition and potential effects on human health are lacking. A methanol hazelnut shells extract was prepared and dried. Our investigation allowed the isolation and characterization of different classes of phenolic compounds, including neolignans, and a diarylheptanoid, which contribute to a high total polyphenol content (193.8 ± 3.6 mg of gallic acid equivalents (GAE)/g of extract). Neolignans, lawsonicin and cedrusin, a cyclic diarylheptanoid, carpinontriol B, and two phenol derivatives, C-veratroylglycol, and β-hydroxypropiovanillone, were the main components of the extract (0.71%-2.93%, w/w). The biological assays suggested that the extract could be useful as a functional ingredient in food technology and pharmaceutical industry showing an in vitro scavenging activity against the radical 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) (EC50 = 31.7 µg/mL with respect to α-tocopherol EC50 = 10.1 µg/mL), and an inhibitory effect on the growth of human cancer cell lines A375, SK-Mel-28 and HeLa (IC50 = 584, 459, and 526 µg/mL, respectively). The expression of cleaved forms of caspase-3 and poly(ADP-ribose) polymerase-1 (PARP-1) suggested that the extract induced apoptosis through caspase-3 activation in both human malignant melanoma (SK-Mel-28) and human cervical cancer (HeLa) cell lines. The cytotoxic activity relies on the presence of the neolignans (balanophonin), and phenol derivatives (gallic acid), showing a pro-apoptotic effect on the tested cell lines, and the neolignan, cedrusin, with a cytotoxic effect on A375 and HeLa cells

Flavonoid microparticles by spray-drying: Influence of enhancersof the dissolution rate on properties and stability

Naringenin (Nn) and Quercetin (Q) have numerous health benefits particularly due to their antioxidant properties. However, their low solubility, bioavailability and stability limit their use as components for functional foods, nutraceuticals and pharmaceutical agents. In this research, Nn- and Q-microparticles were produced by a spray-drying process using a combination of cellulose acetate phthalate (CAP) as coating gastroresistant polymer and swelling or surfactant agents as enhancers of dissolution rate. Raw materials and microparticles produced were all characterized by particle size analysis, differential scanning calorimetry, X-ray diffraction, and imaged by electron and fluorescence microscopy. During 12 months, storage stability was evaluated by analyzing drug content, HPLC and DSC profiles, as well as antioxidant activity (DPPH test). In vitro dissolution tests, using a pH-change method, were carried out to investigate the influence of formulative parameters on flavonoid release from the microparticles. Presence of a combination of CAP and surfactants or swelling agents in the formulations produced microparticles with good resistance at low pH of the gastric fluid and complete flavonoid release in the intestinal environment. The spray-drying technique and the process conditions selected have given satisfying encapsulation efficiency and product yield. The microencapsulation have improved the technological characteristics of the powders such as morphology and size, have given long-lasting storage stability and have preserved the antioxidant properties

Flavonoids and flavonoid-rich natural extracts inhibit cytokine release in cystic fibrosis bronchial epithelial cells by regulating NF-kB pathway

Cystic fibrosis (CF) is a genetic life-shortening condition in Caucasians. Despite being a multi-organ disease, CF is classically diagnosed by symptoms of acute/chronic respiratory disease, with persistent pulmonary infections (Amaral, 2015). CF is caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) protein. Inheritance of mutant CFTR alleles results in surface liquid depletion and defective mucociliary clearance leading to pulmonary failure. Defects in CFTR perturb the regulation of many intracellular signaling pathways including the NF-kB pathway causing excessive production of pro-inflammatory mediators. Current CF therapies are directed to delay CF lung damage by restoring CFTR function and controlling abnormal inflammation. However, only few anti-inflammatory drugs are effective for CF treatments (mainly oral corticosteroids and ibuprofen), these drugs have limited beneficial effects in presence of considerable side effects. Flavonoids have been reported as promising anti-inflammatory drugs and some of them seem to act as CFTR direct activators (Amaral, 2015). From this respect, herbal remedies or plant bioactive molecules may be of great interest. To this aim, we tested the anti-inflammatory activity of apolar extracts from the roots of three Peonia species (Paeoniaceae family), namely P. rockii P. ostii and P. lactiflora, on CFTR ΔF508/ΔF508 CuFi1 cells and normal counterpart. The effects of the extracts on intrinsic as well as TNFα-induced inflammation were evaluated by determining IL-8, IL-6 and RANTES production. Further- more, to study the direct effect of the extracts on NF-kB activation, Human Embryonic Kidney cells were used in transient transfection of NF-kB reporter plasmid and NF-kB activity and cytokine productions were also evaluated. Results showed a significant anti-inflammatory activity of all three Peonia extracts with the P. lactiflora being the most effective. Furthermore, we also tested the anti-inflammatory potential of the pure flavonoid naringin in the same model systems. We found that naringin was able to reduce cytokine release through inhibiting the key enzymes of the NF-kB and MAPK/ ERK pathways. Interestingly, preliminary results on spray dried pharmaceutical formulations of this molecule, show that naringin co-sprayed with leucine improves pharmacological activity of the flavonoid neat raw drug

Aerodynamic Properties and Drug Solubility of Dry Powders Prepared by Spray Drying: Clarithromycin Versus its Hydrochloride Salt

Aerodynamic Properties and Drug Solubility of Dry Powders Prepared by Spray Drying: Clarithromycin Versus its Hydrochloride Salt. Russo P, Manniello M.D., Simonetti A., Petrone A.M., Porta A., Del Gaudio P., Aquino R.P. Department of Pharmacy (DIFARMA), University of Salerno, Fisciano (SA) – Italy; ([email protected]). Introduction and Objectives. The antibiotic therapy for a direct administration to the lung in cystic fibrosis patients has to provide suitable drug availability, possibly in the lower respiratory tract characterized by the presence of thick secretions. Apart from deposition, systemic or local pharmacological activity of an inhalation product depends on drug dissolution into the biological fluids lining the lung. Therefore, one of the crucial step in the therapeutic management of the respiratory disease is the drug solubilization in this site of action. Clarithromycin (CLA; fig.1) is a broad spectrum and a well know macrolide antibiotic usually prescribed particularly for the treatment of respiratory infections, interestingly showing an additional anti-inflammatory effect (Pukhalsky et al., 2004); CLA is characterized by a very poor water solubility (0.33 mg/L). One of the common strategy, for increasing drug solubility in aqueous medium is represented by the production of dry powders in amorphous form using the spray drying technique (Yonemochi et al., 1999). Moreover, CLA has a dimethylamino group, which can be salified for solubility/dissolution improvement (fig.1). Hence, the aim of the present study was to obtain respirable powders of clarithromycin, while improving drug aqueous solubility. Powders were produced with CLA or CLA hydrochloride and characterized in terms of drug content, aerodynamic properties and drug solubility. Finally, in order to assess the effect of the spray-drying process on the antibiotic activity of the engineered particles, microbiological tests were performed. Materials and Methods. Several batches of micronized particles were prepared by spray drying different feed solutions; critical process parameters were solvent composition (isopropyl alcohol/water ratio), drug concentration and pH of the liquid feeds (table 1). Saturated solubility measurements were carried out keeping an excess amount of CLA raw material (RM), CLA spray-dried suspension and hydrochloride spray-dried in phosphate buffer (0.05 M, pH 6.75) at 37°C for 72h. After filtration, the solubility was measured by HPLC method and expressed in mg/ml. The results were reported as mean of three measurements and standard deviation. Particle size distribution of Raw Material and engineered particles was determined using a light-scattering laser granulometer, while particle morphology was assessed by scanning electron microscopy (SEM). The in vitro deposition of the micronized powders was evaluate by means of a Single-Stage Glass Impinger (SSGI; apparatus A; European Pharmacopoeia 8.0), using a proper device for the aerosolization. The antibacterial assay was carried out in MHB by microdilution method using 96-well microtiter plates. Briefly, 200 µl of 1X107 CFU/ml of P. aeruginosa ATCC 27853 were incubated at 37°C with different concentration of drug (4, 6 and 8 µg/ml). Results and Discussion. Morphology and aerodynamic properties of spray-dried particles were strongly dependent on organic solvent concentration as well as on pH of the liquid feeds processed, both influencing drug solubility. Adding clarithromycin to hydroalcoholic mixtures, alkaline feeds (pH~10.5; #1, #2, tab.1) in form of suspensions were obtained.   The resulting spray-dried powders showed good process yield, but unsatisfactory aerodynamic properties, due to a high particle size (tab. 1) and the presence of residual drug crystals mixed to spherical particles (fig.2; #2). With the aim to obtain clear feeds in form of solutions, we tested clarithromycin in its hydrochloride salt form, obtained lowering pH values of feed solutions (pH 6.5). Micronized salified powders showed higher process yield and very interesting FPF values, thanks to smaller and wrinkled particles (fig.2 #4, #6; tab.1). Moreover, water solubility of spray-dried powders was strongly influenced by clarithromycin form. Powders obtained from alkaline feed suspensions showed lower solubility in a phosphate buffer 0.05 M, pH 6.75 (fig.3, # 1). A substantial increase in drug solubility was obtained, at the same conditions, with powders dried from feed solution containing clarithromycin hydrochloride (fig.3 #9-#5). To verify the ability of the produced formulations to was performed. Three different drug concentrations were tested in a multi-well plate. Clarithromycin raw material (RM, line C, figure 4) and #1 (line D, figure 4) showed a lower activity against P. aeruginosa growth compared to #4, #5, #6, containing the hydrochloride form. This different behavior against P. aeruginosa may be due to the lower CLA solubility; at higher concentration, clarithromycin precipitates in RM and #1 wells, becoming unavailable for antibiotic purpose. Conclusions. Clarithromycin inhalable powders containing the drug in its hydrochloride form showed good aerodynamic properties and higher water solubility. Thanks to a fine-tuning of the process parameters and liquid feed composition, no excipients were necessary to obtain respirable powders. The spray drying process of CLA hydrochloride not only preserved antimicrobial activity, but also, increasing drug solubility, improved drug efficacy against P. aeruginosa. References. Pukhalsky, A. L., Shmarina, G. V., Kapranov, N. I., Kokarovtseva, S. N., Pukhalskaya, D., & Kashirskaja, N. J. (2004). Anti-inflammatory and immunomodulating effects of clarithromycin in patients with cystic fibrosis lung disease. Mediators Inflamm, 13(2), 111-117. doi: 10.1080/09629350410001688495 Yonemochi, E., Kitahara, S., Maeda, S., Yamamura, S., Oguchi, T., & Yamamoto, K. (1999). Physicochemical properties of amorphous clarithromycin obtained by grinding and spray drying. Eur J Pharm Sci, 7(4), 331-338

Nanospray Drying as a Novel Technique for the Manufacturing of Inhalable NSAID Powders

The aim of this research was to evaluate the potential of the nanospray drier as a novel apparatus for the manufacturing of a dry powder for inhalation containing ketoprofen lysinate, a nonsteroidal anti-inflammatory drug able to control the inflammation in cystic fibrosis patients. We produced several ketoprofen lysinate and leucine powder batches by means of nanospray dryer, studying the influence of process parameters on yield, particle properties (size distribution and morphology), and, mainly, aerodynamic properties of powders. Micronized particles were prepared from different hydroalcoholic solutions (alcohol content from 0 to 30% v/v) using ketoprofen in its lysine salt form and leucine as dispersibility enhancer in different ratios (from 5 to 15% w/w) with a total solid concentration ranging from 1 to 7% w/v. Results indicated that the spray head equipped with a 7 µm nozzle produced powders too big to be inhaled. The reduction of nozzle size from 7 to 4 µm led to smaller particles suitable for inhalation but, at the same time, caused a dramatic increase in process time. The selection of process variables, together with the nozzle pretreatment with a surfactant solution, allowed us to obtain a free flowing powder with satisfying aerosol performance, confirming the usefulness of the nanospray drier in the production of powder for inhalation

A new cineol derivative, polyphenols and norterpenoids from Saharan myrtle tea (Myrtus nivellei): Isolation, structure determination, quantitative determination and antioxidant activity

Abstract The phytochemical profile of decoction and infusion, obtained from the dried leaves of M. nivellei, consumed as tea in Saharan region, was characterized by UHPLC-PDA-HRMS. Fourteen compounds were characterized and, to confirm the proposed structures a preparative procedure followed by NMR spectroscopy was applied. Compound 3 (2-hydroxy-1,8-cineole disaccharide) was a never reported whereas a bycyclic monoterpenoid glucoside (2), two ionol glucosides (1 and 12), a tri-galloylquinic acid (4), two flavonol glycosides (5 and 9), and a tetra-galloylglucose (7), were reported in Myrtus spp. for the first time. Five flavonol O-glycosides (6, 8, 10–11, and 14) togheter a flavonol (13) were also identified. Quantitative determination of phenolic constituents from decoction and infusion has been performed by HPLC-UV-PDA. The phenolic content was found to be 150.5 and 102.6 mg/g in decoction and infusion corresponding to 73.8 and 23.6 mg/100 mL of a single tea cup, respectively. Myricetin 3-O-β-d-(6″-galloyl)glucopyranoside (5), isomyricitrin (6) and myricitrin (8) were the compounds present in the highest concentration. The free-radical scavenging activities of teas and isolated compounds was measured by the DPPH assay and compared with the values of other commonly used herbal teas (green and black teas). Decoction displayed higher potency in scavenging free-radicals than the infusion and green and black teas

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570