J-filter: An experiment to simplify and isolate specific signals in 1 H NMR spectra of complex mixtures based on scalar coupling constants.

One-dimensional selective NMR experiments relying on a J-filter element are proposed to isolate specific signals in crowded 1 H spectral regions. The J-filter allows the edition or filtering of signals in a region of interest of the spectrum by exploiting the specific values of their 1 H-1 H coupling constants and certain parameters of protons coupled to them that appear in less congested parts of the spectrum (chemical shifts and coupling constants). The new experiments permitted the isolation of specific peaks of phytosterol components in a sample obtained from a liquid nutraceutical recommended for lowering blood cholesterol levels in regions with complete overlap in the 1 H spectrum.

Metabolomic Profile, Plasmatic Levels of Losartan and EXP3174, Blood Pressure Control in Hypertensive Patients and Their Correlation with COVID-19.

Systemic arterial hypertension (SAH) is one of the most prevalent chronic diseases worldwide and is related to serious health complications. It has been pointed out as a major risk factor for COVID-19. This study aimed to determine the impact of COVID-19 on the metabolomic profile, the correlation with the plasmatic levels of losartan and its active metabolite (EXP3174), biochemical markers, and blood pressure (BP) control in hypertensive patients. 1H NMR metabolomic profiles of hypertensive and normotensive patients with and without previous COVID-19 diagnosis were identified. Plasmatic levels of LOS and EXP3174 were correlated with BP, biochemical markers, and the metabolomic fingerprint of the groups. Biomarkers linked to important aspects of SAH and COVID-19 were identified, such as glucose, glutamine, arginine, creatinine, alanine, choline, erythritol, homogentisate, 0-tyrosine, and 2-hydroxybutyrate. Those metabolites are indicative of metabolic alterations, kidney damage, pulmonary dysfunction, and persistent inflammation, which can be found in both diseases. Some hypertensive patients did not reach the therapeutic levels of LOS and EXP3174, while the BP control was also limited among the normotensive patients with previous COVID-19 diagnoses. Metabolomics proved to be an important tool for assessing the effectiveness of losartan pharmacotherapy and the damage caused by SAH and COVID-19 in hypertensive patients.

Metabolomic Profile and Its Correlation with the Plasmatic Levels of Losartan, EXP3174 and Blood Pressure Control in Hypertensive and Chronic Kidney Disease Patients.

Systemic arterial hypertension (SAH) is one of the most prevalent chronic diseases worldwide and, when dysregulated, may cause serious complications. Losartan (LOS) blocks relevant physiological aspects of hypertension, acting mainly on the reduction of peripheral vascular resistance. Complications of hypertension include nephropathy, in which diagnosis is based on the observation of functional or structural renal dysfunction. Therefore, blood pressure control is essential to attenuate the progression of chronic kidney disease (CKD). In this study, 1H NMR metabolomics were used to differentiate hypertensive and chronic renal patients. Plasmatic levels of LOS and EXP3174, obtained by liquid chromatography coupled with mass-mass spectroscopy, were correlated with blood pressure control, biochemical markers and the metabolomic fingerprint of the groups. Some biomarkers have been correlated with key aspects of hypertension and CKD progression. For instance, higher levels of trigonelline, urea and fumaric acid were found as characteristic markers of kidney failure. In the hypertensive group, the urea levels found could indicate the onset of kidney damage when associated with uncontrolled blood pressure. In this sense, the results point to a new approach to identify CKD in early stages and may contribute to improving pharmacotherapy and reducing morbidity and mortality associated with hypertension and CKD.

Zein Nanoparticles Containing Arginine-Based Surfactants: Physicochemical Characterization and Effect on the Biological Properties.

Cationic surfactants carry antimicrobial activity, based on their interaction and disruption of cell membranes. Nonetheless, their intrinsic toxicity limits their applicability. To overcome this issue, a feasible strategy consists of using solid nanoparticles to improve their delivery. The zein nanoparticles were loaded with four cationic arginine-based surfactants: one single chain Nα-lauroyl-arginine (LAM) and three Gemini surfactants Nα Nω-Bis (Nα-lauroyl-arginine) α, ω-diamide) (C3(LA)2, C6(LA)2 and C9(LA)2). Blank and loaded zein nanoparticles were characterized in terms of size, polydispersity and zeta potential. Furthermore, the antimicrobial activity against bacteria and yeasts and the hemolytic activity were investigated and compared to the surfactants in a solution. Nanoparticles were found to be monodisperse, presenting a size of between 180-341 nm, a pdI of <0.2 and a positive zeta potential of between +13 and +53 mV, remaining stable over 365 days. The nanoencapsulation maintained the antimicrobial activity as unaltered, while the extensive hemolytic activity found for the surfactants in a solution was reduced drastically. Nuclear Magnetic Ressonance (NMR), molecular docking and monolayer findings indicated that zein entraps the surfactants, interfering in the surfactant-membrane interactions. Accordingly, the nanoepcasulation of arginine surfactants improved their selectivity, while the cationic charges were free to attack and destroy bacteria and fungi; the aliphatic chains were not available to disrupt the cellular membranes.

Zein Nanoparticles Containing Arginine-Phenylalanine-Based Surfactants: Stability, Antimicrobial and Hemolytic Activity.

Although cationic surfactants have a remarkable antimicrobial activity, they present an intrinsic toxicity that discourages their usage. In this work novel zein nanoparticles loaded with arginine-phenylalanine-based surfactants are presented. The nanoparticles were loaded with two single polar head (LAM and PNHC12) and two with double amino acid polar head surfactants, arginine-phenylalanine (C12PAM, PANHC12). The formulations were characterized and their stability checked up to 365 days. Furthermore, the antimicrobial and hemolytic activities were investigated. Finally, NMR and molecular docking studies were carried out to elucidate the possible interaction mechanisms of surfactant-zein. The nanoparticles were obtained with satisfactory size, zeta potential and dispersibility. The surfactants containing arginine-phenylalanine residues were found to be more stable. The nanoencapsulation maintained the antimicrobial activities unaltered in comparison to the surfactants' solutions. These results are in agreement with the NMR and docking findings, suggesting that zein interacts with the surfactants by the aromatic rings of phenylalanine. As a result, the cationic charges and part of the aliphatic chains are freely available to attack the bacteria and fungi, while not available to disrupt the cellular membranes. This approach opens new possibilities for using cationic surfactants and benefits from their extraordinary antimicrobial responses for several applications.

Silk fibroin/cholinium gallate-based architectures as therapeutic tools.

The combination of natural resources with biologically active biocompatible ionic liquids (Bio-IL) is presented as a combinatorial approach for developing tools to manage inflammatory diseases. Innovative biomedical solutions were constructed combining silk fibroin (SF) and Ch[Gallate], a Bio-IL with antioxidant and anti-inflammatory features, as freeze-dried 3D-based sponges. An evaluation of the effect of the Ch[Gallate] concentration (≤3% w/v) on the SF/Ch[Gallate] sponges was studied. Structural changes observed on the sponges revealed that the Ch[Gallate] presence positively affected the ß-sheet formation while not influencing the silk native structure, which was suggested by the FTIR and solid-state NMR results, respectively. Also, it was possible to modulate their mechanical properties, antioxidant activity and stability/degradation in an aqueous environment, by changing the Ch[Gallate] concentration. The architectures showed high water uptake ability and a weight loss that follows the controlled Ch[Gallate] release rate studied for 7 days. Furthermore, the sponges supported human adipose stem cells growth and proliferation, up to 7 days. TNF-α, IL-6 (pro-inflammatory) and IL-10 (anti-inflammatory) release quantification from a human monocyte cell line revealed a decrease in the pro-inflammatory cytokines concentrations in samples containing Ch[Gallate]. These outcomes encourage the use of the developed architectures as tissue engineering solutions, potentially targeting inflammation processes. STATEMENT OF SIGNIFICANCE: Combining natural resources with active biocompatible ionic liquids (Bio-IL) is herein presented as a combinatorial approach for the development of tools to manage inflammatory diseases. We propose using silk fibroin (SF), a natural protein, with cholinium gallate, a Bio-IL, with antioxidant and anti-inflammatory properties, to construct 3D-porous sponges through a sustainable methodology. The morphological features, swelling, and stability of the architectures were controlled by Bio-IL content in the matrices. The sponges were able to support human adipose stem cells growth and proliferation, and their therapeutic effect was proved by the blockage of TNF-α from activated and differentiated THP-1 monocytes. We believe that these bio-friendly and bioactive SF/Bio-IL-based sponges are effective for targeting pathologies with associated inflammatory processes.

Design, optimization, and in vitro characterization of idebenone-loaded PLGA microspheres for LHON treatment.

Biodegradable poly(lactic-co-glycolic acid) microspheres (PLGA MSs) are attractive delivery systems for site-specific maintained release of therapeutic active substances into the intravitreal chamber. The design, development, and characterization of idebenone-loaded PLGA microspheres by means of an oil-in-water emulsion/solvent evaporation method enabled the obtention of appropriate production yield, encapsulation efficiency and loading values. MSs revealed spherical shape, with a size range of 10-25 µm and a smooth and non-porous surface. Fourier-transform infrared spectroscopy (FTIR) spectra demonstrated no chemical interactions between idebenone and polymers. Solid-state nuclear magnetic resonance (NMR), X-ray diffractometry, differential scanning calorimetry (DSC) and thermogravimetry (TGA) analyses indicated that microencapsulation led to drug amorphization. In vitro release profiles were fitted to a biexponential kinetic profile. Idebenone-loaded PLGA MSs showed no cytotoxic effects in an organotypic tissue model. Results suggest that PLGA MSs could be an alternative intraocular system for long-term idebenone administration, showing potential therapeutic advantages as a new therapeutic approach to the Leber's Hereditary Optic Neuropathy (LHON) treatment by intravitreal administration.

Antifungal Combination Eye Drops for Fungal Keratitis Treatment.

Fungal keratitis (FK) is a corneal mycotic infection that can lead to vision loss. Furthermore, the severity of FK is aggravated by the emergence of resistant fungal species. There is currently only one FDA-approved formulation for FK treatment forcing hospital pharmacy departments to reformulate intravenous drug preparations with unknown ocular bioavailability and toxicity. In the present study, natamycin/voriconazole formulations were developed and characterized to improve natamycin solubility, permanence, and safety. The solubility of natamycin was studied in the presence of two cyclodextrins: HPßCD and HPγCD. The HPßCD was chosen based on the solubility results. Natamycin/cyclodextrin (HPßCD) inclusion complexes characterization and a competition study between natamycin and voriconazole were conducted by NMR (Nuclear Magnetic Resonance). Based on these results, several eye drops with different polymer compositions were developed and subsequently characterized. Permeability studies suggested that the formulations improved the passage of natamycin through the cornea compared to the commercial formulation Natacyn®. The ocular safety of the formulations was determined by BCOP and HET-CAM. The antifungal activity assay demonstrated the ability of our formulations to inhibit the in vitro growth of different fungal species. All these results concluded that the formulations developed in the present study could significantly improve the treatment of FK.

Targeted 1H NMR metabolomics and immunological phenotyping of human fresh blood and serum samples discriminate between healthy individuals and inflammatory bowel disease patients treated with anti-TNF.

Inflammatory bowel disease is a multifactorial etiology, associated with environmental factors that can trigger both debut and relapses. A high level of tumor necrosis factor-α in the gut is the main consequence of immune system imbalance. The aim of treatment is to restore gut homeostasis. In this study, fresh blood and serum samples were used to identify biomarkers and to discriminate between Crohn's disease and ulcerative colitis patients under remission treated with anti-TNF. Metabolomics based on Nuclear Magnetic Resonance spectroscopy (NMR) was used to detect unique biomarkers for each class of patients. Blood T lymphocyte repertories were characterized, as well as cytokine and transcription factor profiling, to complement the metabolomics data. Higher levels of homoserine-methionine and isobutyrate were identified as biomarkers of Crohn's disease with ileocolic localization. For ulcerative colitis, lower levels of creatine-creatinine, proline, and tryptophan were found that reflect a deficit in the absorption of essential amino acids in the gut. T lymphocyte phenotyping and its functional profiling revealed that the overall inflammation was lower in Crohn's disease patients than in those with ulcerative colitis. These results demonstrated that NMR metabolomics could be introduced as a high-throughput evaluation method in routine clinical practice to stratify both types of patients related to their pathology. KEY MESSAGES: NMR metabolomics is a non-invasive tool that could be implemented in the normal clinical practice for IBD to assess beneficial effect of the treatment. NMR metabolomics is a useful tool for precision medicine, in order to sew a specific treatment to a specific group of patients. Finding predictors of response to IFX would be desirable to select patients affected by IBD. Immunological status of inflammations correlates with NMR metabolomics biomarkers.

In situ forming and mucoadhesive ophthalmic voriconazole/HPßCD hydrogels for the treatment of fungal keratitis.

Fungal keratitis is a severe infectious corneal disease. At present, no voriconazole ophthalmic formulations are approved by the FDA or EMA. This lack of therapeutic options leads to the reformulation of intravenous voriconazole preparations (VFEND®) by the hospital pharmacy departments to prepare the appropriate ophthalmic formulations (pharmacy compounding). However, the limited residence time of these formulations leads to an intensive treatment posology that may increase the occurrence of side effects. In the present study, two different hydrogels were developed and characterized in order to improve the voriconazole's ophthalmic solubility, permanence, and security. Voriconazole-cyclodextrin (HPßCD or HPÉ£CD) inclusion complexes in aqueous solutions were characterized by NMR and molecular modeling. Complexes were formed by encapsulation of voriconazole into the cyclodextrin's internal cavity which considerably increases its water solubility. Ocular safety was proven by ocular irritation studies. Permeability studies suggest both hydrogels have good corneal permeability. Furthermore, in vivo ocular permanence study by PET/CT showed a longer permanence time on the ocular surface (t1/2 = 58.91 ± 13.4 min and 96.28 ± 49.11 min for VZHAH and VZISH 0.65 respectively) compared to the voriconazole control formulation (VFEND® t1/2 = 32.27 ± 15.56 min). Results suggest these formulations are a good alternative for the treatment of fungal keratitis.

Development and Characterization of a Tacrolimus/Hydroxypropyl-ß-Cyclodextrin Eye Drop.

Uveitis is a vision inflammatory disorder with a high prevalence in developing countries. Currently, marketed treatments remain limited and reformulation is usually performed to obtain a tacrolimus eye drop as a therapeutic alternative in corticosteroid-refractory eye disease. The aim of this work was to develop a mucoadhesive, non-toxic and stable topical ophthalmic formulation that can be safely prepared in hospital pharmacy departments. Four different ophthalmic formulations were prepared based on the tacrolimus/hydroxypropyl-ß-cyclodextrin (HPßCD) inclusion complexes' formation. Phase solubility diagrams, Nuclear Magnetic Resonance (NMR) and molecular modeling studies showed the formation of 1:1 and 1:2 tacrolimus/HPßCD inclusion complexes, being possible to obtain a 0.02% (w/v) tacrolimus concentration by using 40% (w/v) HPßCD aqueous solutions. Formulations also showed good ophthalmic properties in terms of pH, osmolality and safety. Stability studies proved these formulations to be stable for at least 3 months in refrigeration. Ex vivo bioadhesion and in vivo ocular permanence showed good mucoadhesive properties with higher ocular permanence compared to the reference pharmacy compounding used in clinical settings (t1/2 of 86.2 min for the eyedrop elaborated with 40% (w/v) HPßCD and Liquifilm® versus 46.3 min for the reference formulation). Thus, these novel eye drops present high potential as a safe alternative for uveitis treatment, as well as a versatile composition to include new drugs intended for topical ophthalmic administration.

Unveiling an NMR-Invisible Fraction of Polymers in Solution by Saturation Transfer Difference.

The observation of signals in solution NMR requires nuclei with sufficiently large transverse relaxation times (T2). Otherwise, broad signals embedded in the baseline afford an invisible fraction of nuclei (IF). Based on the STD (saturation transfer difference) sequence, IF-STD is presented as a quick tool to unveil IF in the 1H NMR spectra of polymers. The saturation of a polymer in a region of the NMR spectrum with IF (very short 1H T2) results in an efficient propagation of the magnetization by spin diffusion through the network of protons to a visible-invisible interphase with larger 1H T2 (STDon). Subtracting this spectrum from one recorded without saturation (STDoff) produces a difference spectrum (STDoff-on), with the nuclei at the visible-invisible interphase, that confirms the presence of an IF. Analysis of a wide collection of polymers by IF-STD reveals IF more common than previously thought, with relevant IF figures when STD > 0.4% at 750 MHz. A fundamental property of the IF-STD experiment is that the signal is generated within a single state comprising polymer domains with different dynamics, as opposed to several states in exchange with different degrees of aggregation. Contrary to a reductionist visible-invisible dichotomy, our results confirm a continuous distribution of nuclei with diverse dynamics. Since nuclei observed (edited) by IF-STD at the visible-invisible interphase are in close spatial proximity to the IF (tunable with the saturation time), they emerge as a privileged platform from which gaining an insight into the IF itself.

Toxicological and genotoxic evaluation of anacardic acid loaded-zein nanoparticles in mice.

Anacardic acid extracted from cashew nut shells of Anacardium occidentale L has demonstrated important biological activities, such as antibacterial activity against the cariogenic specie Streptococcus mutans. Zein nanoparticles containing anacardic acid (9.375 µg/mL) were evaluated in terms of toxicity and genotoxicity in vivo. The subacute toxicity assay was used to evaluate the cumulative effects of the oral administration of nanoencapsulated anacardic acid at 2.25 and 112.5 µg/kg for 7 days in mice, simulating a mouth rinse short-term clinical course treatment. Blank zein nanoparticles and saline solution 0.9 % were used as negative controls. Peripheral blood samples were collected to evaluate the genotoxicity in polychromatic erythrocytes using the micronucleus test. The animals were anesthetized, euthanized and the target organs collected, weighed and submitted to histopathological analysis. Liver, kidney and spleen relative weights did not change. Nevertheless, stomach, lung and heart increased the relative weights in the group receiving the highest dose, in which occasional histopathological findings were also identified. Both doses maintained the micronucleus frequency within the normal range and the animals treated with the highest dose presented a discrete weight lost, which could explain the organs' relative weight reductions. Blank and anacardic acid loaded zein nanoparticles were nontoxic when administered repeatedly for 7 days, as no relevant histopathological changes neither genotoxicity were observed. These preparations demonstrated limited toxicity under the conditions used in this study and could become an antibacterial alternative for preventing/treating oral infections in short-term treatments.

In Vitro-In Silico Modeling Approach to Rationally Designed Simple and Versatile Drug Delivery Systems.

Rational design and development of a nanosystem usually relies on empirical approaches as well as a fair degree of serendipity. Understanding how nanosystems behave at the molecular level is of great importance for potential biomedical applications. In this work, we describe a nanosystem composed of two natural compounds, vitamin E and sphingomyelin, prepared by spontaneous emulsification (vitamin E-sphingomyelin nanosystems (VSNs)). Extensive characterization revealed suitable physicochemical properties, very high biocompatibility in vitro and in vivo, and colloidal stability during storage and in biological media, all relevant properties for clinical translation. We have additionally pursued a computational approach to gain an improved understanding of the assembling, structure, dynamics, and drug-loading capacity of VSNs, using both small molecules and biomolecules (resveratrol, curcumin, gemcitabine, and two peptides). The spontaneous formation of compartmentalized VSNs starting from completely disassembled molecules, observed here for the first time, was accurately assessed from the computational molecular dynamics trajectories. We describe here a synergistic in silico/in vitro approach showing the predictive power of computational simulations for VSNs' structural characterization and description of internal interaction mechanisms responsible for the association of bioactive molecules, representing a paradigm shift in the rational design of nanotechnologies as drug delivery systems for advanced personalized medicine.

Hot melt-extrusion improves the properties of cyclodextrin-based poly(pseudo)rotaxanes for transdermal formulation.

This study aimed to investigate whether hot-melt extrusion (HME) processing can modify the interactions between drugs, cyclodextrins and polymers, and in turn alter the microstructure and properties of supramolecular gels. Mixtures composed of amphiphilic polymer (Soluplus), cyclodextrin (HPßCD or αCD), plasticizer (PEG400 or PEG6000) and colloidal silicon dioxide were processed by HME. Carvedilol (CAR) was added to the formulation aiming its transdermal delivery. Extrudates were characterized by HPLC, XRPD, FTIR, DSC, and solid-state NMR. Gels prepared from extrudates (HME gels) or the corresponding physical mixtures (PM gels) in PBS were analyzed regarding components ordering (NMR, SEM), rheology, and CAR diffusion rate. HME led to the loss of the crystalline lattice of CAR and αCD, without causing any drug degradation. Solid NMR indicated that HME promoted the interaction of α-CD and HPßCD with the other components. HME gels had no coarsely disperse particles in their structure and behaved as weak gels (G' ~ G″). In contrast, PM gels contained drug crystals and showed elastic behavior (G' > G″). In general, HME gels were less viscous than PM ones and led to higher drug flux, especially those prepared using HPßCD. Moreover, the association of HPßCD and PEG6000 provided faster drug flux from supramolecular gels regardless the higher gel viscosity. The results evidenced that HME processing can decisively modify the arrangement of the components in the supramolecuar gels and, consequently, their properties, notably increasing drug release rate.

An experimental and computational NMR study of organometallic nine-membered rings: Trinuclear silver(I) complexes of pyrazolate ligands.

This work reports the calculation of the nuclear magnetic resonance (NMR) chemical shifts of eight trinuclear Ag(I) complexes of pyrazolate ligands using the relativistic program ZORA. The data from the literature concern exclusively 1 H, 13 C, and 19 F nuclei. For this reason, one of the complexes that is derived from 3,5-bis-trifluoromethyl-1H-pyrazole has been studied anew, and the 15 N and 109 Ag chemical shifts determined for the first time in solution. Solid-state NMR data of this compound have been obtained for some nuclei (1 H, 13 C, and 19 F) but not for others (14 N, 15 N, and 109 Ag).

Structure, rheology, and copper-complexation of a hyaluronan-like exopolysaccharide from Vibrio.

MO245 exopolysaccharide (EPS) was produced in laboratory conditions from Vibrio genus microorganism isolated from bacterial mats found in Moorea Island. Its structure consists of a linear tetrasaccharide repeating unit →4)-ß-D-GlcpA-(1→4)-α-D-GalpNAc-(1→3)-ß-D-GlcpNAc-(1→4)-ß-D-GlcpA-(1→ containing covalently-linked 5% of glucose, galactose, and rhamnose, determined by methylation analyses and NMR spectroscopy. The molecular weight, radius of gyration (Rg) and intrinsic viscosity, [η], determined by gel permeation chromatography with light scattering and viscosity detection, were 513 ± 4 kDa (PDI, 1.42 ± 0.01), 6.7 ± 0.3 dl/g and 56 ± 0.3 nm respectively. The chelation of the EPS with copper divalent ions leads to the instantaneous formation of gels. The structural similitude proposed, based in an equal ratio of GlcA to N-acetylated sugars and in the same type of glyosidic linkages present in the repeating unit (alternated 1→3 and 1→4 linkages), is translated into analogous physicochemical properties: MO245 EPS is a flexible polyelectrolyte, with scaling exponents similar to that described for HA. This similitude opens opportunities in future drug delivery, tissue engineering, and cosmetic applications.

The subdivision behavior of polymeric tablets.

The subdivision behavior of polymeric tablets produced with the well-known polymers Soluplus® (SOL), polyvinyl pyrrolidone co-vinyl acetate (PVPVA) and hydroxypropyl methylcellulose (HPMC) was evaluated in this study. The polymeric tablets were submitted to different post-treatments (aging, thermal and exposure to compressed gaseous carbon dioxide) and its mechanical, spectroscopic and microstructure properties were assessed. SOL tablets showed the best results for tablet subdivision, particularly, the mean mass variation (3.9%) was significantly lower than the other two polymeric tablets (7.2% and 9.1% for PVPVA and HPMC, respectively), and showed better results than common tablets produced from powder matrices (7-14%). SOL tablets were also more sensitive to the different post-treatments applied, which reduced the mass loss and friability from 1.5% and 0.8%, respectively, to values close to zero and without altering their porosity. The thermal treatment of PVPVA tablets, in turn, also led to similar subdivision results, with mass loss of 0.3% and friability of 0.02%. In contrast, the granules of HPMC presented compaction difficulties making its tablets unsuitable for the subdivision process, even after additional post-treatment. Polymeric matrices with uniform internal structure and appropriate mechanical strength are the key to a better adaptation for the tablet subdivision.

Both chemical and crystalline phase configuration influence the molecular features of humic acids in humic-calcium-phosphates fertilizers.

Phosphate-metal-humic complexes are very relevant in nature due to their crucial role in phosphate availability for plants and microorganisms. Synthetic phosphate-calcium-humic acid (HA) complexes have proven to be efficient sources of available phosphorus for crops. However, the current knowledge about their structure and molecular features is very poor. The structural implications of phosphate interaction with humic binding sites through calcium bridges, in both monocalcium phosphate and dicalcium phosphate is investigated by using molecular modeling, 31P-NMR, 1H-NMR and X-ray diffractometry. The conformational changes in the molecular configuration of the humic acid involved in the interaction resulting from the synthetic process is also studied by using HPSEC and synchronous fluorescence. The results obtained allow us to identify the phosphate type in the crystalline phase that is involved in the interaction of humic acid binding sites and the different forms of calcium phosphate. Synchronous fluorescence also shows that whereas the conformational configuration of the HA binding site is only partially affected in the monocalcium phosphate interaction, it changes in the case of dicalcium phosphate showing simpler molecular arrangements. These changes in the molecular conformation of the binding site in HA in solution may influence the biological activity of the humic acid. On the other hand, HPSEC studies show that the humic-calcium-phosphate interaction is accompanied by increases in the humic acid apparent size distribution. This effect is more intense in the case of monocalcium phosphate system probably due the influence of pH.

Biopharmaceutical Activities Related to Ellagic Acid, Chitosan, and Zein and Their Improvement by Association.

Ellagic acid (EA) has demonstrated several biological properties, such as antioxidant, antimicrobial, and enzymatic inhibition. Zein and chitosan (CHI) are natural polymers whose biological potential has also gained attention. Therefore, this paper aimed to evaluate the antimicrobial, antioxidant, anticollagenase, and antielastase properties of EA, zein, and chitosan isolated or in combination. The microdilution method was used to assess the minimum inhibitory and bactericide concentrations. The antioxidant activity was determined using the 2,2-diphenyl-1-picryl-hydrazila free radical scavenging method. The anticollagenase and antielastase activities were evaluated by specific colorimetric tests. EA has shown inhibitory activity against Staphylococcus aureus and Pseudomonas aeruginosa together with an antioxidant IC50 of 0.079 mg/mL. EA also showed significant collagenase and elastase inhibition. Zein has shown antimicrobial and antioxidant activities itself and enhanced sinergically the antioxidant activity and the antimicrobial activity against P. aeruginosa when combined with EA. CHI increased sinergically the inhibitory activity of EA against both bacterial strains, while showed itself an acceptable antimicrobial activity. 1 H saturation transfer-difference nuclear magnetic resonance experiment confirmed the formation of a complex between EA and zein that could be related with the improvement on its biological performance over the individual compounds, while no chemical interaction was detected between CHI and EA. PRACTICAL APPLICATION: The results reinforce the potential of ellagic acid in combination with zein and/or chitosan as an antimicrobial, antienzimatic, and antioxidant agent. Those findings reinforce the use of these substances, protecting this bioactive from degradation and/or improving the functional characteristics and biopharmaceutical properties.