Supercritical Carbon Dioxide Extraction of Coumarins from the Aerial Parts of Pterocaulon polystachyum.

Pterocaulon polystachyum is a species of pharmacological interest for providing volatile and non-volatile extracts with antifungal and amebicidal properties. The biological activities of non-volatile extracts may be related to the presence of coumarins, a promising group of secondary metabolites. In the present study, leaves and inflorescences previously used for the extraction of essential oils instead of being disposed of were subjected to extraction with supercritical CO2 after pretreatment with microwaves. An experimental design was followed to seek the best extraction condition with the objective function being the maximum total extract. Pressure and temperature were statistically significant factors, and the optimal extraction condition was 240 bar, 60 °C, and pretreatment at 30 °C. The applied mathematical models showed good adherence to the experimental data. The extracts obtained by supercritical CO2 were analyzed and the presence of coumarins was confirmed. The extract investigated for cytotoxicity against bladder tumor cells (T24) exhibited significant reduction in cell viability at concentrations between 6 and 12 µg/mL. The introduction of green technology, supercritical extraction, in the exploration of P. polystachyum as a source of coumarins represents a paradigm shift with regard to previous studies carried out with this species, which used organic solvents. Furthermore, the concept of circular bioeconomy was applied, i.e., the raw material used was the residue of a steam-distillation process. Therefore, the approach used here is in line with the sustainable exploitation of native plants to obtain extracts rich in coumarins with cytotoxic potential against cancer cells.

Optimization of Coumarins Extraction from Pterocaulon balansae by Box-Behnken Design and Anti-Trichomonas vaginalis Activity.

Trichomonas vaginalis causes trichomoniasis, a nonviral sexually transmitted infection with a high prevalence worldwide. Oral metronidazole is the drug of choice for the treatment of this disease, although high levels of T. vaginalis resistance to this agent are well documented in the literature. This study describes the anti-T. vaginalis activity of an optimized coumarin-rich extract from Pterocaulon balansae. Optimization was performed to maximize extraction of total coumarins by means of a 3-level Box-Behnken design, evaluating the effect of three factors: extraction time, plant : solvent ratio, and ethanol concentration. Optimum conditions were found to be 5 h extraction time and a plant : solvent ratio of 1% (w/v) and 60% (v/v) ethanol, which resulted in approximately 30 mg of total coumarins/g of dry plant. The coumarin-enriched extract exhibited a minimum inhibitory concentration of 30 µg/mL and an IC50 of 3.2 µg/mL against T. vaginalis, a low cytotoxicity, and a high selectivity index (18 for vaginal epithelial cells and 16 for erythrocytes). The coumarins permeation/retention profile through porcine vaginal mucosa was evaluated in Franz-type diffusion cells. After 8 h of kinetics, coumarins were detected in the tissue (4.93 µg/g) without detecting them in the receptor compartment. A significant increase of coumarins in the mucosa layers (8.18 µg/g) and receptor compartment (0.26 µg/g) was detected when a T. vaginalis suspension (2 × 105 trophozoites/mL) was previously added onto the mucosa. No alterations were visualized in the stratified squamous non-keratinized epithelium of the porcine vaginal mucosa after contact with the extract. Overall, these results suggest that the P. balansae coumarin-rich extract may have potential as a treatment for trichomoniasis.

Nanoemulsions Containing a Coumarin-Rich Extract from Pterocaulon balansae (Asteraceae) for the Treatment of Ocular Acanthamoeba Keratitis.

This study describes the incorporation of a coumarin-rich extract from Pterocaulon balansae into nanoemulsions intended for the local treatment of ocular keratitis caused by Acanthamoeba. The n-hexane dewaxed extract of P. balansae was characterized by HPLC/PDA and UPLC/MS. The presence of four major coumarins was detected, where 5-methoxy-6,7-methylenedioxycoumarin was selected as a chemical marker. This extract was then incorporated into nanoemulsions composed of medium chain triglycerides and egg-lecithin, through spontaneous emulsification. Such a procedure yielded the formation of monodisperse nanoemulsions in a sub-300-nm range, regardless of the amount of extract incorporated (1.0-5.0 mg/mL). The amoebicidal activity against Acanthamoeba castellanii was both dose-dependent and incubation time-dependent. A reduction of 95% of trophozoite viability was detected after 24 h of incubation with a nanoemulsion at 1.25 mg/mL of coumarins, being a similar effect detected for chlorhexidine. These results suggest a potential of the formulations developed in this study as a new strategy for the treatment of ocular keratitis caused by Acanthamoeba.

Quantification of Coumarins in Aqueous Extract of Pterocaulon balansae (Asteraceae) and Characterization of a New Compound.

Plants from the genus Pterocaulon are popularly used as antifungal and wound-healing agents. Such activities have been related to coumarins, which are abundant in those plants. Coumarins are soluble in organic solvents, such as hexane and dichloromethane, and some of them are also soluble in hot water. Considering that infusion and decoctions of these plants are used in traditional medicine, the aim of this study was to identify and quantify the coumarins in the aqueous extract of Pterocaulon balansae. The aqueous extract was obtained by dynamic maceration and the compounds were characterized by UPLC-UV-MS analysis. A new coumarin and 5-methoxy-6,7-methylenedioxycoumarin, used for validation of the analytical HPLC method were obtained by partition of the aqueous extract with n-hexane. The HPLC method validated was linear, specific, and precise. Seven coumarins were characterized in the aqueous extract in a range of 0.584-54 mg/g of dry plant material. The main compound, 5,6-dimethoxy-7-(3'-methyl-2',3'-dihydroxybutyloxy)coumarin, is described for the first time in P. balansae together with a new compound, 5,6-dimethoxy-7-(2',3'-epoxy-3'-methylbutyloxy)coumarin.

Topical Nanoemulsions as Delivery Systems for Green Extracts of Pterocaulon balansae Aiming at the Treatment of Sporotrichosis.

Coumarins are benzopyrones found in several plant genera, including Pterocaulon (Asteraceae). These compounds represent an important source of new treatments, especially as antimicrobial and antifungal agents. In this study, two coumarin-rich extracts from Pterocaulon balansae using green technologies were obtained through aqueous maceration (AE) and supercritical fluid extraction (SFE). Such extracts were incorporated into nanoemulsions (NAE and NSFE) composed of a medium-chain triglyceride oil core stabilized by phospholipids. The nanoemulsions exhibited droplet sizes between 127 and 162 nm, pH above 5.0, and viscosity of approximately 1.0 cP, properties compatible with the topical route. The coumarins permeation/retention from formulations through ear porcine skin using Franz-type diffusion cells were evaluated. Whatever the extract, coumarins were distributed in skin layers, especially in the dermis in both intact and impaired (tape stripping) skin. In addition, a significant increase in coumarins that reached up to the receptor fluid was observed for impaired skin, with increases of approximately threefold for NAE and fourfold for NSFE. Finally, antifungal activity of nanoemulsions was evaluated according to minimum inhibitory concentrations, and the values were 250 µg/mL for all strains tested. The overall results demonstrated the feasibility of incorporating P. balansae extracts into nanoemulsions and showed a potential alternative for the treatment of sporotrichosis.

Laronidase-loaded liposomes reach the brain and other hard-to-treat organs after noninvasive nasal administration.

Mucopolysaccharidosis type I (MPS I) is caused by a lack of the lysosomal enzyme α-L-iduronidase (IDUA), responsible for the degradation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate, leading to multisystemic signs and symptoms. Enzyme replacement therapy (ERT) is a treatment that consists of weekly intravenous administrations of laronidase, a recombinant version of IDUA. However, ERT has limited access to certain tissues, such as bone, cartilage, and brain, and laronidase fails to trespass the BBB. In this sense, this study reports the development and characterization of laronidase-loaded liposomes for the treatment of MPS I mice. Liposomal complexes were obtained by the thin film formation method followed by microfluidization. The main characterization results showed mean vesicle size of 103.0 ± 3.3 nm, monodisperse populations of vesicles, zeta potential around + 30.0 ± 2.1 mV, and mucoadhesion strength of 5.69 ± 0.14 mN. Treatment of MPS I mice fibroblasts showed significant increase in enzyme activity. Nasal administration of complexes to MPS I mice resulted in significant increase in laronidase activity in the brain cortex, heart, lungs, kidneys, eyes, and serum. The overall results demonstrate the feasibility of nasal administration of laronidase-loaded liposomes to deliver enzyme in difficult-to-reach tissues, circumventing ERT issues and bringing hope as a potential treatment for MPS I.

Recent Patents Concerning the use of Nanotechnology-based Delivery Systems as Skin Penetration Enhancers.

Nanotechnology-based delivery systems have been considered a promising approach for topical application, considering their characteristics of penetration into/across the skin. The present review aimed to evaluate the recent international scenario of patents concerning the use of nanotechnology- based delivery systems as skin penetration enhancers. A survey of recent patent documents was conducted by using the Espacenet patent database including the terms "skin" in the title and "promot* or enhanc* and penetrat* or absorp* or permeat*" and "nano*" with the truncation symbol (*) in the abstract of documents. A total of 110 patents were published from 2008 to 2018, with 94 technologies being considered. The results demonstrated an increase in innovations concerning nanotechnologybased delivery systems as skin penetration enhancers in recent years. Most patent applicants are from China (60.6%) and Korea (21.3%), and companies (68%) were the most prominent owners. The majority of patent applications (76%) were intended for cosmetic purposes; the types of products and nanostructures were also investigated. Overall results demonstrated the increased interest around the world in patenting products involving skin permeation promotion and nanotechnology for pharmaceutical and, mainly, for cosmetics purposes.

The genus Pterocaulon (Asteraceae) - A review on traditional medicinal uses, chemical constituents and biological properties.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of the genus Pterocaulon (Asteraceae) are used in different parts of the world for treating skin and liver diseases, as well as disorders of the respiratory system, among others. AIM OF THE STUDY: This review aims to discuss the present state of the art concerning the ethnobotanical uses, secondary metabolites and biological effects of Pterocaulon species and their chemical components. MATERIALS AND METHODS: The available information on the genus Pterocaulon was gathered from scientific databases (Web of Science, Pubmed, ScienceDirect, Scopus, ChemSpider, SciFinder ACS Publications, Wiley Online Library). Information was also obtained from local publications, M.Sc. and Ph.D. dissertations. All studies on the ethnobotany, phytochemistry, pharmacology and toxicology of the plants until December 2017 were included in this review. RESULTS: Approximately 40 coumarins and 30 flavonoids have been isolated from Pterocaulon species. Coumarins have been considered the chemotaxonomic markers in the genus and the most active components. Pharmacological studies carried out with extracts and isolated compounds revealed in vitro bioactivities that include antifungal, antiviral, and cytotoxicity. Most of the pharmacological investigations were not correlated with traditional uses of the plants. CONCLUSIONS: Pterocaulon species, a rich source of coumarins, have great ethnomedical potential. Nevertheless, further studies into the pharmacological activities are necessary since none of the purported effects of these plants was fully assessed. In-depth research regarding the toxicity are also required to ensure the safety of these medicinal plants.

A novel, simplified and stability-indicating high-throughput ultra-fast liquid chromatography method for the determination of rosmarinic acid in nanoemulsions, porcine skin and nasal mucosa.

Currently, there is an increasing interest on the development of topical formulations containing rosmarinic acid (RA) due to its well-documented antioxidant activity. This study aimed to develop and validate a stability-indicating ultra-fast liquid chromatography (UFLC) method for the determination of RA in nanoemulsions, porcine skin and nasal mucosa intended to be applied in permeation/retention studies and for development of topical nanoemulsions. Chromatographic separation was carried out using a C18 column packed with 2.6 µm particle size in isocratic conditions using as mobile phase water:acetonitrile (83:17, v/v), acidified with 0.1% trifluoracetic acid (v/v), with a total time of analysis of 3.5 min and detection at 330 nm. RA analysis was specific in the presence of both non-biological (blank nanoemulsion and receptor fluid) and biological matrices (porcine ear skin and porcine nasal mucosa). No interference of degradation products of RA was verified after different stress conditions such as acidic, alkaline, oxidative, light exposure (UV-A and UV-C) and thermal demonstrating the method stability-indicating property. The analytical (0.1-10.0 µg·mL-1) and bioanalytical (0.5-10.0 µg·mL-1) linearity was proved by analysis of the calibration curves of RA and no matrix effect was observed. The method was sensitive, precise and accurate, and showed recovery higher than 85%. The method was considered robust as evaluated by a Plackett-Burman experimental design. In the validated conditions, the RA was determined in the nanoemulsions obtained by spontaneous emulsification procedure (1.007 ±â€¯0.040 mg·mL-1), porcine ear skin (1.13 ±â€¯0.19 µg·cm-2) and nasal mucosa (22.46 ±â€¯3.99 µg·cm-2) after retention/permeation studies. Thus, a highly sensitive, simple, fast and stability-indicating method was developed for RA analysis during the development of topical nanoemulsions and bioanalytical assays in complex matrices.

Box-Behnken design optimization of mucoadhesive chitosan-coated nanoemulsions for rosmarinic acid nasal delivery-In vitro studies.

Mucoadhesive chitosan-coated nanoemulsions for rosmarinic acid (RA) nasal delivery were optimized. The optimum ratio between the formulation components that led to minimum droplet size and PDI, and maximal ζ-potential and RA content was obtained using Box-Behnken design (BBD). Optimized conditions were 8.5% oil phase (w/v), 3:10 lecithin to oil phase ratio (w/w), and 0.1% chitosan final concentration (w/v). Physicochemical characterization, mucoadhesion measurement, in vitro release and permeation/retention were performed. Optimized chitosan-coated RA nanoemulsions presented adequate physicochemical characteristics, high mucoadhesive potential, prolonged drug release, and long-lasting permeation time with a higher RA penetration/retention through porcine nasal mucosa. Cell viability and death by necrosis in fibroblasts cells were also evaluated to investigate the formulations safety. Formulations did not induce cytotoxicity following 24 h (3.125-50 µM) or 48 h (3.125-25 µM) of treatments. Overall results demonstrated that optimized chitosan-coated nanoemulsion showed to be a suitable carrier for RA nasal delivery aiming neuroprotective therapies.