Formulation and Characterization of Ursodeoxycholic Acid Nanosuspension Based on Bottom-Up Technology and Box-Behnken Design Optimization.

BACKGROUND: Ursodeoxycholic acid (UDCA) is a therapeutic agent used for the treatment of cholestatic hepatobiliary diseases in pediatric patients. It is a bile acid that presents high lipophilicity, and it belongs to Class II of the Biopharmaceutical Classification System (BCS), which exhibits low water solubility and high intestinal permeability, which leads to poor oral absorption. The objective of this work was to design and optimize UDCA nanosuspensions by means of the precipitation-ultrasonication method to improve the solubility, dissolution, and oral bioavailability of UDCA. METHODS: A three-level, three-factor Box-Behnken design was used to optimize formulation variables and obtain uniform, small-particle-size UDCA nanosuspensions. The independent variables were: stabilizer percentage (X1), amplitude (X2), and sonication time (X3), and the dependent variable was the particle size (Y1). In the precipitation-ultrasonication method, UDCA was dissolved in acetone:PEG 400 (1:1 v/v) and quickly incorporated into the antisolvent (pre-cooled aqueous dispersion of HPMC E-15 0.3%), by means of intense sonication at 50 W for 5 min, controlling temperature through an ice water bath. The lyophilization efficacy was evaluated by means of a cryoprotective efficacy test, working with 10% maltose at -80 °C. The nanosuspensions were characterized by dynamic light scattering (DLS), X-ray diffraction, and scanning electron microscopy (SEM). The physicochemical stability was determined at 25 °C and 4 °C at 7, 14, 30, and 60 days, and the UDCA content was analyzed via HPLC-UV. An in vitro dissolution assay and an oral bioavailability study were performed in male Wistar rats. RESULTS: A significant impact was achieved in the optimized nanosuspension with 0.3% (stabilizer), 50 W (amplitude), and 5 min (sonication time), with a particle size of 352.4 nm, PDI of 0.11, and zeta potential of -4.30 mV. It presented adequate physicochemical stability throughout the study and the UDCA content was between 90% and 110%. In total, 86% of UDCA was dissolved in the in vitro dissolution test. The relative oral bioavailability was similar without significant statistical differences when comparing the lyophilized nanosuspension and the commercial tablet, the latter presenting a more erratic behavior. The pharmacokinetic parameters of the nanosuspension and the commercial tablet were Tmax (1.0 ± 0.9 h vs. 2.0 ± 0.8 h, respectively), Cmax (0.558 ± 0.118 vs. 0.366 ± 0.113 µM, respectively), ΔCmax (0.309 ± 0.099 vs. 0.232 ± 0.056, respectively), AUC (4.326 ± 0.471 vs. 2.188 ± 0.353 µg/mL.h, respectively, p < 0.02), and IAUC0-24h (2.261 ± 0.187 µg/mL.h vs. 1.924 ± 0.440 µg/mL.h, respectively). CONCLUSIONS: The developed nanosuspension presents an appropriate dosage and administration for pediatric patients. On the other hand, it exhibits an adequate absorption and UDCA oral bioavailability.

The Argentine Mistletoes Ligaria cuneifolia (Ruiz & Pav.) Tiegh (Loranthaceae) and Phoradendron liga (Gillies ex Hook. & Arn.) Eichler (Santalaceae). Thirty Years of Research.

Ligaria cuneifolia (Ruiz & Pav.) Tiegh (Loranthaceae) and Phoradendron liga (Gillies ex Hook. & Arn.) Eichler (Santalaceae) are regarded as Argentine mistletoes based on their similarities with the European counterpart, Viscum album L. (Santalaceae). These two species are the most used medicinal plants to treat high blood pressure in the Argentinian population. To provide scientific grounds to their traditional use and therapeutic potential, they were selected as herbal drug candidates. The main findings would support the anti-hypertensive action, the anticholesterolemic and antioxidant features of L. cuneifolia, and immunomodulatory properties for both species. Quercetin-O-glycosides, galloyl glycosides, and proanthocyanidins are present in L. cuneifolia while P. liga shows C-glycosyl flavones and 3-deoxyproanthocyanidins. This review summarizes the phytochemical characterization, medicinal properties and reveals promising results warranting future efforts for further investigation.

Dynamics of Polyphenol Biosynthesis by Calli Cultures, Suspension Cultures and Wild Specimens of the Medicinal Plant Ligaria cuneifolia (Ruiz & Pav.) Tiegh. (Loranthaceae). Analysis of Their Biological Activity.

Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) is a South American hemiparasitic species with antioxidant, antitumoral, antimicrobial, and antilipidemic activities attributed to its polyphenolic content. We studied the polyphenolic pattern of L. cuneifolia during different phenological stages: flowering, fruiting, and post-fruiting. The highest total phenolic content was found in stems at post-fruiting (214 ± 12.1 mg gallic acid eq·g-1 DW) and fruiting (209 ± 13.7 mg gallic acid eq·g-1 DW), followed by post-fruiting leaves (207 ± 17.5 mg gallic acid eq·g-1 DW). Flavonoids accumulated at higher levels in leaves and hydroxycinnamic acids in leaves at flowering and post-fruiting. The polyphenolic pattern was similar between organs from wild plants and in vitro cultures, although at a significantly lower level in the latter ones. The performance of calli growing under a 16 h photoperiod in a modified White medium with 1-naphthalene acetic acid (2.50 µM) and Kinetin (9.20 µM) was better than in the dark. When calli grew in media only with auxins (IAA, NAA, and 2,4-D, all at 2.50 µM concentration), its growth and polyphenolic content improved. Cell suspensions with 2.50 µM NAA and 9.20 µM KIN grew slowly and produced very small amounts of polyphenols. As for the antioxidant activity, it was detected in all samples (approximately 1000 µmol trolox eq·g-1 DW) except fruits, where a lower value was found (328 µmol trolox eq·g-1 DW). In vitro cultures have the lowest antioxidant activity when compared to methanolic extracts from organs of wild specimens. Finally, antimutagenic or mutagenic activity in wild plants and in vitro culture extracts was not detected by the Ames test.

Pharmaceutical suspensions of ursodeoxycholic acid for pediatric patients: in vitro and in vivo studies.

Ursodeoxycholic acid (UDCA) is used in the oral therapy of hepatobiliary cholestatic diseases. Due to UDCA low aqueous solubility, two pediatric oral suspensions (25 mg/mL) were formulated with a few excipients, suspension A (SA) and suspension B (SB) with a vehicle, including two suspending agents. Physical, chemical and microbiological stability and a rheological study were performed at three different conditions (5 °C ± 3 °C, 25 °C ± 2 °C/60% RH ± 5% RH and 40 °C ± 2 °C/75% RH ± 5% RH) for 120 days. Moreover, dissolution study, content uniformity, related substances, and a study of relative oral bioavailability were also carried out. Both suspensions were physically, chemically and microbiologically stable throughout the study. SA and SB can be stored at 25 °C and 5 °C for at least 120 days whereas SA can be kept at 40 °C for at least 90 days and SB for 120 days. They both met USP specifications for dissolution, content uniformity, and related substances. SA and SB showed an improved relative oral bioavailability compared to the solid dosage form and they both displayed similar relative oral bioavailability with no significant differences between them. The developed suspensions proved to be safe and adequate and they are ideal for pediatric use for their acceptability, accurate dose administration and treatment adherence.

Antioxidant Activity of Flavonoid Rich Fraction of Ligaria cuneifolia (Loranthaceae).

Ligaria cuneifolia (Ruiz & Pav.) Tiegh. (Loranthaceae), the 'Argentine mistletoe', is a hemiparasite species largely used in folk medicine. The aim of this study was to evaluate the antioxidant activity using in vitro, ex vivo, and in vivo methods. A screening of phenolics was performed by UV spectroscopy on different fractions. The antioxidant capacity was evaluated in vitro by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH. ) assay on a crude extract (CE), ethyl acetate fraction (EAF), and aqueous fraction (AF). The results suggest that EAF concentrates the antioxidant capacity and was selected for further analysis. Capillary electrophoresis was employed to monitor the individual antioxidant capacity and the potential contributors to this effect. Ex vivo assays showed an efficient inhibition of tert-butyl hydroperoxide-induced rat liver phospholipid oxidation, as well as rat brain autoxidation, and H2 O2 -induced DNA damage in blood monocytes. In vivo, the topical application of EAF significantly decreased skin chemiluminescence in a mice model.

In vitro anti-inflammatory properties of Smilax campestris aqueous extract in human macrophages, and characterization of its flavonoid profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of Smilax campestris Griseb (Smilacaceae) have been employed in the treatment of several inflammatory diseases as a traditional herbal medicine. However, the cellular and molecular mechanisms involved in the observed effects remain elusive. Macrophages are known to play a central role in inflammatory responses. These cells are activated in response to a diversity of danger signals and produce several mediators of inflammation that eventually regulate the immune response. For all the above mentioned, scientific evidence is required to support the popular use of S. campestris. AIM OF THE STUDY: We aimed to investigate the anti-inflammatory effect of S. campestris aqueous extract (SME) in activated THP-1 human macrophages, on the production of some mediators of inflammation and oxidative stress in order to provide scientific support for its popular use. MATERIALS AND METHODS: The characterization of SME was assessed by HPLC-MS/MS. The production of the pro-inflammatory cytokines and chemokines was evaluated by ELISA. The activity of metalloproteases was evaluated by zymography. The subcellular localization of the NF-κB transcription factor was analysed by Western blot. The superoxide anion and glutathione levels were assessed by flow cytometry. The cytotoxicity induced by SME in THP-1 macrophages was also investigated by the LDH release test. RESULTS: In the present study, we have identified catechin and glycosylated derivatives of quercetin (quercetin-3-O-glucoside, quercetin-3-O-galactoside, rutin and quercetin-3-rhamnoside) as major components of the aqueous SME. We found that SME significantly decreased the production of the pro-inflammatory cytokines tumour necrosis factor (TNF)- α, interleukin (IL)-1ß, IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 and the activity of the metalloproteinase (MMP)-9, in lipopolysaccharide-activated macrophages derived from the monocytic cell line THP-1. Furthermore, SME diminished the expression of NF-κB p65 subunit in the nuclear fraction. In addition, SME decreased the production of superoxide anion in THP-1 macrophages, without altering the levels of reduced glutathione. CONCLUSION: These results suggest that SME exerts its anti-inflammatory effects in human activated macrophages by inhibiting the production of pro-inflammatory cytokines, matrix metalloproteinases and the NF-κB transcription factor pathway along with a reduction of oxidative stress mediators. Moreover, catechin and glycosylated derivatives of were identified by HPLC-MS/MS in SME. Our findings provide scientific support for the traditional use of the S. campestris extracts.

Variación de los perfiles de polifenoles en Ligaria cuneifolia (R. et. P) Tiegh (Loranthaceae), su estudio mediante electroforesis capilar y su relación con la actividad farmacológica. / Polyphenolic profile variation in Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae), capillary electrophoretic analysis and its relationship with the pharmacological activity

Ligaria cuneifolia (R. et P.) Tiegh. ­Loranthaceae­ es una especie hemiparásita que se desarrolla sobre diferentes hospedantes. Es conocida con el nombre vulgar de "liga" o "liguilla". Debido a su similitud morfológica, constituye el sustituto natural del "muérdago europeo", por lo cual es denominado "muérdago criollo". Las drogas vegetales son matrices complejas en las cuales múltiples componentes actúan en forma sinérgica y son responsables de la acción farmacológica. Con el fin de dar sustento científico al uso folclórico de L. cuneifolia se estudiaron distintas formas de obtención de los extractos, se evaluaron diferentes hospedantes y regiones fitogeográficas. Se desarrolló y validó un método de electroforesis capilar para construir fingerprints o perfiles cromatográficos característicos que permitan evaluar los distintos componentes con el fin de estandarizar los extractos. Se efectuó la comparación con otras técnicas cromatográficas, tales como en cromatografía en capa delgada (TLC) y líquida de alta resolución (HPLC). A su vez, se procedió al aislamiento, purificación y análisis estructural de los compuestos de interés por técnicas espectroscópicas y cromatográficas. Se identificaron diez compuestos, de los cuales cuatro son reportados por primera vez en esta especie. La electroforesis capilar probó ser una técnica adecuada para el control de calidad de los extractos y una alternativa atractiva a las técnicas cromatográficas tradicionales.

Ligaria cuneifolia (R. et P.) Tiegh. ­Loranthaceae­ is a hemiparasite plant which grows on different host trees. It is popularly referred to as "liga" or "liguilla". Due to its morphological similarity, it is considered as the natural substitute for the European mistletoe, for which is known as the "Argentine mistletoe". Herbal drugs are complex matrices in which multiple components acting synergistically are responsible for the pharmacological activity. In order to provide scientific support to the popular use of L. cuneifolia, a capillary electrophoretic method was developed and validated to build a chromatographic profile or fingerprint that allows the evaluation of different components for extract standardization. A comparison was made with other chromatographic techniques such as TLC and HPLC. Isolation, purification and structural analysis of compounds were performed by chromatographic and spectroscopic methods. Ten analytes were identified, four of which are reported for the first time in L. cuneifolia. Capillary electrophoresis proved to be an appropriate tool for the quality control of herbal drugs, as well as an attractive alternative to traditional chromatographic techniques.

Advances in drug delivery, gene delivery and therapeutic agents based on dendritic materials.

Dendrimers are synthetic polymers that grow in three dimensions into well-defined structures. Their morphological appearance resembles a number of trees connected by a common point. Dendritic nanoparticles have been studied for a large number of pharmaceutical and biomedical applications including gene and drug delivery, clinical diagnosis and MRI. Despite the application of dendrimers, research is still in its childhood in comparison with liposomes and other nanomaterials. They are now playing a key role in several therapeutic strategies, with dendrimer-based products in clinical trials. The aim of this review is to describe the state-of-the-art of biomedical applications of dendrimers - and dendrimer conjugates - such as drug and gene delivery and antiviral activity.

Development and validation of a capillary electrophoresis method applied to the analysis of l-citrulline in an oral formulation for pediatric use.

A simple and highly sensitive CE-UV method was applied in the determination of l-ctrulline, which was developed from an oral formulation for pediatric use. The novel method was based on the analysis of l-citrulline for direct ultraviolet detection at 198 nm. The BGE consisted of 10 mM sodium tetraborate and 50 mM SDS at pH 9, and the electrophoretic parameters were optimized. The method was validated in terms of specificity, linearity, LOD, LOQ, precision, accuracy, and robustness. The LOD and LOQ obtained were 1.36 and 4.54 µg/mL, respectively. In addition, the method offers higher sensitivity and specificity compared with the results obtained from HPLC method using UV-detectors, in which l-citrulline needs to be derivatizated. Furthermore, low cost and simplicity of the system allowed the rapid and simple quantitation of l-citrulline in the oral formulation for quality control and stability indicated method.

Development of a novel dual CD-MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh.-Loranthaceae-extracts.

The present work deals with the development and validation of a novel dual CD-MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh.-Loranthaceae-extracts. The BGE consisted of 20 mM pH 8.3 borate buffer, 50 mM SDS, a dual CD system based on the combination of 5 mM ß-CD and 2% w/v S-ß-CD, and 10% v/v methanol. The proposed method has been successfully applied to the comparative analysis of extracts from aerial parts and different hosts, geographical areas, and extraction procedures in order to establish the flavonoid fingerprint of L. cuneifolia. The method was validated according to international guidelines. LOD and LOQ, intra and interday precision, and linearity were determined for catechin, epicatechin, procyanidin B2, rutin, quercetin-3-O-glucoside, quercetin-3-O-xyloside, quercetin-3-O-rhamnoside, quercetin-3-O-arabinofuranoside, quercetin-3-O-arabinopyranoside, and quercetin. The CD-MEKC methodology emerges as a suitable alternative to the traditional HPLC for quality control, fingerprinting, and standardization of L. cuneifolia extracts from different sources.

New Micellar Electrokinetic Chromatographic Method for Analyzing Idebenone in Pediatric Formulations.

A novel, simple and reliable method based on micellar electrokinetic chromatography with ultraviolet detection was developed to analyze idebenone in a pediatric formulation. Idebenone is a synthetic short chain benzoquinone that acts as an electron carrier in the mitochondrial electron transport chain facilitating the production of adenosine triphosphate. It can be found in two different redox states that differ in their physiological properties. Idebenone has been investigated as a treatment in several neurological disorders like Friedreich's ataxia, Leber's hereditary optic neuropathy, mitochondrial encephalomyopathies and senile dementia. Accordingly, a micellar electrokinetic chromatography was employed to discriminate both redox forms. The final optimized system was validated in terms of selectivity, linearity (r2 0.992), limit of detection (0.5 µg/mL), limit of quantification (1.8 µg/mL), intra- and inter-day precision (RSD ≤ 2) and accuracy in terms of recovery studies (99.3-100.5%). Robustness was studied following a Plackett-Burman design. Finally, the validated system was applied to the analysis of idebenone in a pediatric formulation.

Novel and highly sensitive mixed-polymeric electrokinetic chromatography system for determination of contaminants and impurities of heparin samples.

A mixed-polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5% w/v polymeric ß-CD, 0.4% w/v tetronic(®) 1107 and 400 mM tris-phosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated-silica capillary of 50 cm of total length and 75 µm id, an applied voltage of -7 kV, a temperature of 30°C and 200 nm UV-detection. The highly sensitive method developed showed low values of LOD, 0.07% w/w (0.07 µg/mL) (OSCS) and 0.1% w/w (0.1 µg/mL) (Der), and values of LOQ 0.2% w/w (0.2 µg/mL) (OSCS) and 0.3% w/w (0.3 µg/mL) (Der) with a concentration level of Hep sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Owing to its simplicity, high sensitivity, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of Hep in raw material and specially in finished products because of the low amounts of Hep sample required.