DoE-assisted development and validation of a stability-indicating HPLC-DAD method for simultaneous determination of five cannabinoids in Cannabis sativa L. based on analytical quality by design (AQbD) concept.

INTRODUCTION: Medical uses of Cannabis sativa L. have gained interest in recent decades, which highlights the need for defining appropriate quality specifications for Cannabis-based products. However, the complexity of plant matrices and structural similarity between cannabinoids make analytical development a challenging task. Thus, the application of analytical quality by design (AQbD)-driven approaches can favour the development of fit-for-purpose methods. OBJECTIVES: To develop a high-performance liquid chromatography diode array detector (HPLC-DAD) method for simultaneous quantification of cannabidiol, Δ9 -tetrahydrocannabinol, cannabidiolic acid, tetrahydrocannabinolic acid, and cannabinol in C. sativa by applying an AQbD-driven approach. MATERIALS AND METHODS: Critical method attributes (CMA) were established following the analytical target profile. Critical method variables (CMV) were categorised based on risk assessment and literature review. Selected CMV regarding sample preparation and chromatographic conditions were optimised using response surface methodology (RSM). The working point was estimated by multiple response optimisation using Deringer's desirability function. The validity of the optimal conditions was confirmed experimentally. Method validation was performed according to ANVISA and ICH guidelines. Relative response factors (RRFs) were also determined. RESULTS AND DISCUSSION: Baseline resolution of 12 major cannabinoids was achieved in a 35 min chromatographic analysis. All experimental responses obtained during confirmatory analyses were within the prediction intervals (PI95% ). Method's selectivity, linearity (10-100 µg/mL), precision, bias, extraction recovery, and ruggedness were satisfactorily demonstrated. CONCLUSIONS: The application of an AQbD-driven approach allowed for a better understanding of the effects of the ensemble of CMV on the analyte's behaviour, enabling the definition of appropriate conditions to ensure consistent achievement of the intended method's performance.

Glioprotective Effect of Chitosan-Coated Rosmarinic Acid Nanoemulsions Against Lipopolysaccharide-Induced Inflammation and Oxidative Stress in Rat Astrocyte Primary Cultures.

Rosmarinic acid (RA) is a natural polyphenolic compound with a well-documented neuroprotective effect mainly associated with its anti-inflammatory and antioxidant activities. Recently, our research group developed and optimized chitosan-coated RA nanoemulsions (RA CNE) intended to be used for nasal delivery as a new potential neuroprotective therapy. In this sense, the present study aimed to evaluate the protective and/or therapeutic potential of RA CNE in inflammation/oxidative stress induced by LPS (1 µg mL-1) in rat astrocyte primary cultures. In summary, pre-treatment with RA CNE before exposure to LPS (protective protocol) reduced significantly the LPS-induced alterations in astrocyte cell viability, proliferation, and cell death by necrosis, which was not observed in therapeutic protocol. RA CNE protective protocol also enhanced anti-oxidative status by ~ 50% by decreasing oxygen reactive species production and nitric oxide levels and preventing total thiol content decrease. Finally, our results demonstrate the protective effect of RA CNE in migratory activation and GFAP expression of reactive astrocytes. Overall, our findings indicate for the first time the RA CNE glioprotective potential, associated with an increase in cell viability and proliferation, a preventive effect on cellular death by necrosis, migratory ability and hypertrophic reactive astrocytes, and the reparation of astrocyte redox state.

Box-Behnken Design for Extraction Optimization Followed by High Performance Countercurrent Chromatography: Production of a Flavonoid-enriched Fraction from Achyrocline Satureioides.

The biological properties of Achyrocline satureioides have been mostly ascribed to its major flavonoids quercetin (QCT), luteolin (LUT), and 3-O-methylquercetin (3OMQ). The present study aimed to optimize the extraction by dynamic maceration of the major phenolic compounds in order to obtain in a subsequent step a flavonoid-enriched fraction (FEF) using high performance countercurrent chromatography (HPCCC). A 3-level Box-Behnken design (BBD) was applied to maximize the extraction of the substances, using the plant : solvent ratio (X1 ), extraction time (X2 ), and ethanol concentration (X3 ) as factors. One-step HPCCC semipreparative separation with a solvent system composed of hexane : ethyl acetate : methanol : water (0.9 : 0.9 : 0.8 : 1.0, v/v) was employed to obtain the FEF. The second-order polynomial model was able to fit the experimental data adequately. The linear and quadratic terms of X3 were the most significant factors that affected all the responses. The positive linear term of X3 indicated a substantial increase in extraction yield, while the negative quadratic term showed a nonlinear tendency. Linear terms of X1 suggested a tendency to solvent saturation, except for QCT. The terms of X2 did not affect the responses substantially. The HPCCC method was found to be efficient and rapid for separating the FEF with 71% (w/w) flavonoid content. Overall, the developed extraction procedure coupled with HPCCC proved to be efficient for obtaining an enriched fraction with a very high content of flavonoids from A. satureioides.

Bioactive Dimeric Acylphloroglucinols from the Mexican Fern Elaphoglossum paleaceum.

Two new prenylated acylphloroglucinols, paleacenins A (1) and B (2), were isolated from the rhizome n-hexane and chloroform extracts of the fern Elaphoglossum paleaceum. Both compounds were found to possess the same geranylated filicinic acid moiety but have a different phloroglucinol ring substituent. Their structures were determined using 1H and 13C NMR spectroscopic, HRMS, and ECD analysis. The plant extracts and purified compounds were assayed for inhibition of monoamine oxidase (MAO) activity, and the n-hexane and chloroform extracts displayed 25.0% and 26.5% inhibition of MAO-A, respectively, as well as 42.5% and 23.7% inhibition of MAO-B, respectively. Compounds 1 and 2 exhibited IC50 values of 31.0 (1.3) µM for MAO-A and 4.7 (4.4) µM for MAO-B. Paleacenin A (1) showed a higher selective index (SI) toward MAO-B (SIMAO-B/MAO-A 0.1), and paleacenin B (2) exhibited selectivity to MAO-A (SIMAO-B/MAO-A, 3.5). The extracts showed cytotoxicity against a panel of prostate, cervix, breast, and colon cancer cell lines (IC50 values between 1.7 and 10.6 µg/mL); the pure compounds were more active against the prostate, cervix, and colon cancer cell lines. Paleacenins A (1) and B (2), with IC50 values of 46 and 41 µM, respectively, inhibited nitric oxide production by the RAW264.7 murine macrophage model.

Development of analytical method for the quality control of the fruit bark of Punica granatum L. (pomegranate) and antichemotatic activity of extract and rich fraction in punicalagins.

Pomegranate is of current interest owing to the existing potential for industrial uses of fruit peels. This includes its availability as a raw vegetable material, a byproduct that constitutes residue in the use of the species and is recognized as a functional product, and beneficial health properties, as will be demonstrated in the studies cited. Therefore, it is necessary to ensure its effectiveness and safety. Toward this end, the aim of this study was to develop and validate an analytical method for the separation and quantification of total punicalagin present in the bark of the fruit of Punica granatum by HPLC. Purity tests such as water determination and total ashes were also performed. The ability of the extract and enriched fraction of punicalagin to inhibit leukocyte migration in vitro was determined by the Boyden's chamber method. The developed HPLC method demonstrated good separation and quantification of the punicalagin α and ß anomers. The method is efficient and reliable, and can ultimately be used for the analysis of the extract of pomegranate. The crude extract and the fraction of punicalagins significantly inhibited leukocyte migration at concentrations of 1 and 10 µg/mL in relation to the negative control, indicating potential antichemotactic action.

Compatibility study of rosmarinic acid with excipients used in pharmaceutical solid dosage forms using thermal and non-thermal techniques.

Rosmarinic acid (RA) is a phenolic compound that presents well-documented anti-inflammatory, antioxidant and antitumor activities, and based on its pharmacological potential and poor bioavailability, several solid dosage forms have been developed to RA delivery. Therefore, in literature, there are no reports about RA compatibility with excipients. In this regard, the aim of the present study was to evaluate, for the first time, the compatibility of RA with excipients commonly used in solid dosage forms at a 1:1 (RA:excipient) ratio using differential scanning calorimetry (DSC), thermogravimetry (TG), Fourier-transform infrared (FTIR), solid-state nuclear magnetic resonance (ssNMR), and isothermal stress testing (IST) coupled with liquid chromatography (LC). The excipients selected were hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose (MCC), lactose monohydrate (LAC), polyvinylpyrrolidone (PVP), talc (TALC), croscarmellose sodium (CCS), and magnesium stearate (MgSTE). According to DSC results, physical interactions were found between RA and HPMC, LAC, CCS, and MgSTE. The TG analyses confirmed the physical interactions and suggested chemical incompatibility. FTIR revealed physical interaction of RA with TALC and MgSTE and the ssNMR confirmed the physical interaction showed by FTIR and excluded the presence of chemical incompatibility. By IST, the greatest loss of RA content was found to CCS and MgSTE (>15%), demonstrating chemical incompatibilities with RA. High temperatures used in DSC and TG analyses could be responsible for incompatibilities in binary mixtures (BMs) with HPMC and LAC, while temperature above 25 °C and presence of water were factors that promote incompatibilities in BMs with CCS and MgSTE. Overall results demonstrate that RA was compatible with MCC and PVP.

Anticholinesterase activity and identification of huperzine A in three Mexican lycopods: Huperzia cuernavacensis, Huperzia dichotoma and Huperzia linifolia (Lycopodiaceae).

Huperzine A (Hup A), the alkaloid produced by the Chinese medicinal plant Huperzia serrata, has been documented to be a promising agent for the treatment of Alzheimer's disease due to its potent acetylcholinesterase inhibitory (AChEI) activity. The search for anticholinesterase natural products, as well as for alternative sources of Hup A in Mexican lycopods, prompted us to investigate these plants. The action of methanolic and alkaloidal extracts of three Huperzia species (H. cuernavacensis, H. dichotoma, and H. linifolia) was evaluated using an in vitro anticholinesterase activity assay. Also, chromatographic and spectroscopic analyses were employed to detect the presence of Hup A. Methanolic and alkaloidal extracts of H. cuernavacensis showed IC50 =5.32±0.8µg/mL and 0.74±0.05µg/mL; H. dichotoma displayed AChEI with IC50 values =14.11±2.1µg/mL and 0.64±0.09µg/mL; and H. linifolia presented IC50 =158.37±8.7µg/mL and 4.2±1.24µg/mL, respectively, compared to the control Hup A (IC50= 0.16±0.03µg/mL). Hup A was identified in the extracts of H. dichotoma, but it was not detected in the extracts of H. cuernavacensis and H. linifolia by 1H NMR techniques. This study reveals H. dichotoma as a new source of Hup A, and presents H. linifolia and H. cuernavacensis as potential candidates to obtain other anticholinesterase compounds useful in the Alzheimer's disease treatment.

The monoamine oxidase inhibitory activity of essential oils obtained from Eryngium species and their chemical composition.

CONTEXT: Monoamine oxidase (MAO) inhibitors are used in the treatment of depression, anxiety disorders, and the symptomatic treatment of Parkinson's disease. Eryngium, the most representative of the Apiaceae family, is well known for the presence of essential oils (EOs), which have already demonstrated MAO inhibitory potential. OBJECTIVE: The objective of this study is to evaluate the MAO inhibitory capacity of the EOs obtained from Eryngium floribundum Cham. & Schlecht. (EF), E. eriophorum Cham. & Schlecht. (EE), E. nudicaule Lam. (EN), E. horridum Malme (EH), and E. pandanifolium Cham. & Schlecht. (EP). MATERIALS AND METHODS: EOs were obtained from fresh whole plants by hydrodistillation (3 h). Chemical analyses were performed by GC/MS using apolar and polar columns, with oven temperature from 60 to 300 °C at 3 °C/min. The MAO-A and -B activities were evaluated in vitro by an end-point method using kynuramine as the substrate and mitochondrial suspension or human recombinant enzymes as the enzymatic source. DMSO 2%, clorgyline 10(-7) M, and pargyline 10(-6) M were used as controls. RESULTS AND DISCUSSION: EFEO, EEEO, ENEO, EHEO, and EPEO GC/MS analysis showed (E)-caryophyllene (4.9-10.8%), germacrene D (0.6-35.1%), bicyclogermacrene (10.4-17.2), spathulenol (0.4-36.0%), and globulol (1.4-18.6%) as main constituents. None of the EOs inhibited MAO-A activity (4 and 40 µg/mL). However, EHEO inhibited MAO-B activity with an IC50 value of 5.65 µg/mL (1-200 µg/mL). Pentadecane (10 µM), its major constituent (53.5%), did not display significant MAO-B inhibition. CONCLUSION: The study demonstrates the promising application of Eryngium species as a source of potential central nervous system bioactive secondary metabolites, specially related to neurodegenerative disorders.

Beyond organoleptic characteristics: the pharmacological potential of flavonoids and their role in leukocyte migration and in L-selectin and ß2-integrin expression during inflammation.

Flavonoids are compounds responsible for several organoleptic characteristics of plant-derived foods. They are also bioactive compounds with antiinflammatory role. Different mechanisms for this activity have been reported, but their effects on cell migration are not fully understood. In the present study, the role of flavonoids on leukocyte migration in vivo was investigated, using the carrageenan-induced pleurisy model and intravital microscopy in rats. It was found that quercetin (1), rutin (2), flavone (5), apigenin (6) and flavonol (7) reduced cell migration to the pleural cavity and inhibited rolling, adhesion and transmigration. Additionally, flow cytometry assays showed that the in vitro treatment with all compounds (15-60 µM) did not cause cell death and 1 inhibited the cleavage of L-selectin and the ß2-integrin expression, whereas 2 and 7 only inhibited the ß2-integrin expression. Together, data herein presented clearly show the ability of flavonoids to inhibit in vivo neutrophil influx into inflamed tissue, by acting in different mechanisms of neutrophil migration.

Monoamine oxidase inhibition by monoterpene indole alkaloids and fractions obtained from Psychotria suterella and Psychotria laciniata.

Alkaloid fractions of Psychotria suterella (SAE) and Psychotria laciniata (LAE) as well as two monoterpene indole alkaloids (MIAs) isolated from these fractions were evaluated against monoamine oxidases (MAO-A and -B) obtained from rat brain mitochondria. SAE and LAE were analysed by HPLC-PDA and UHPLC/HR-TOF-MS leading to the identification of the compounds 1, 2, 3 and 4, whose identity was confirmed by NMR analyses. Furthermore, SAE and LAE were submitted to the enzymatic assays, showing a strong activity against MAO-A, characterized by IC(50) values of 1.37 ± 1.05 and 2.02 ± 1.08 µg/mL, respectively. Both extracts were also able to inhibit MAO-B, but in higher concentrations. In a next step, SAE and LAE were fractionated by RP-MPLC affording three and four major fractions, respectively. The RP-MPLC fractions were subsequently tested against MAO-A and -B. The RP-MPLC fractions SAE-F3 and LAE-F4 displayed a strong inhibition against MAO-A with IC(50) values of 0.57 ± 1.12 and 1.05 ± 1.15 µg/mL, respectively. The MIAs 1 and 2 also inhibited MAO-A (IC(50) of 50.04 ± 1.09 and 132.5 ± 1.33 µg/mL, respectively) and -B (IC(50) of 306.6 ± 1.40 and 162.8 ± 1.26 µg/mL, respectively), but in higher concentrations when compared with the fractions. This is the first work describing the effects of MIAs found in neotropical species of Psychotria on MAO activity. The results suggest that species belonging to this genus could consist of an interesting source in the search for new MAO inhibitors.

Alkaloid profiling and anticholinesterase activity of South American Lycopodiaceae species.

The alkaloid extracts of four Huperzia and one Lycopodiella species, from Brazilian habitats, were tested for their in vitro anticholinesterase activities. IC(50) values showed a potent acetylcholinesterase inhibition for H. reflexa (0.11 ± 0.05 µg/mL), followed by H. quadrifariata (2.0 ± 0.3 µg/mL), H. acerosa (5.5 ± 0.9 µg/mL), H. heterocarpon (25.6 ± 2.7 µg/mL) and L. cernua (42.6 ± 1.5 µg/mL). A lower inhibition of butyrylcholinesterase was observed for all species with the exception of H. heterocarpon (8.3 ± 0.9 µg/mL), whose alkaloid extract presented a selectivity for pseudocholinesterase. Moreover, the chemical study of the bioactive extracts performed by GC-MS, revealed the presence of a number of Lycopodium alkaloids belonging to the lycopodane, flabellidane and cernuane groups. Surprisingly, the potent acetylcholinesterase inhibitors huperzines A and B were not detected in the extracts, suggesting that other alkaloids may be responsible for such an effect.

Neuroinflammation and hypersensitivity evidenced by the acute and 28-day repeated dose toxicity tests of ostrich oil in mice.

The ostrich oil (OO) has been topically used for decades to treat skin diseases. Its oral use has been encouraged through e-commerce advertising several health benefits to OO without scientific evidence on its safety or effectiveness. This study presents the chromatographic profile of a commercially available OO and its acute and 28-day repeated dose in vivo toxicological profiles. OO anti-inflammatory and antinociceptive effects were also investigated. Omega-9 (ω-9; oleic acid; 34.6%) and -6 (linoleic acid; 14.9%) were detected as OO main constituents. A high single dose of the OO (2 g/kg of ω-9) demonstrated no or low acute toxicity. However, when orally treated with OO (30-300 mg/kg of ω-9) for 28 consecutive days, mice exhibited altered locomotor and exploratory activities, hepatic damage, and increased hindpaw sensitivity accompanied by increased levels of cytokine and brain-derived neurotrophic factor in their spinal cords and brains. Lack of anti-inflammatory or antinociceptive activities was also evidenced in 15-day-OO treated mice. These results indicate that chronic consumption of OO induces hepatic injury, in addition to neuroinflammation and subsequent hypersensitivity and behavioural changes. Thus, there is no evidence to support OO use to treating illness in humans.

Medical cannabis regulation: an overview of models around the world with emphasis on the Brazilian scenario.

Cannabis sativa has accompanied humankind since ancient times, permeating the most diverse aspects of its existence, among which the search for health promotion and well-being stands out. Nevertheless, during the twentieth century, a series of restrictions and controls have been adopted internationally to prevent the abusive use of this species. Despite that, there has been an increased demand for the medical use of cannabis and its derivatives in the last few decades, especially among patients with debilitating conditions for which the existing therapeutic alternatives are limited. Accordingly, several countries have adopted regulatory strategies to allow access to cannabis-based products. This study aimed to overview the existing regulatory frameworks for medical cannabis around the world, focusing on the current Brazilian scenario. In addition to supply and access regulation aspects, some quality-related issues regarding cannabis-based pharmaceutical products were addressed, with emphasis on risks to patients. The literature research was performed between October 2020 and March 2021. According to the retrieved information, by the time the data collection was completed, thirty-six countries had already implemented regulatory frameworks regarding medical cannabis, and sixteen countries had models under development or in the process of implementation. The characteristics of the assessed regulatory strategies vary considerably from country to country, reflecting sociocultural, historical, and political aspects. Among the key aspects that differed between the assessed models, one can highlight the type of cannabis products that are made available and the technical requirements applied to them, as well as the possible access mechanisms. Different supply regulation strategies were also observed regarding cannabis cultivation, production licensing, and distribution mechanisms. In Brazil, an evolution of the regulatory framework has been noticeable since 2015, even though pending points are still to be addressed, among which are the species' cultivation and the access to it for scientific research purposes. Constructing a regulatory model which provides access to good quality cannabis-based medicines that may meet the patient's needs is still a challenge in the coming years, requiring the engagement of various stakeholders, including regulators, members of the academic community, prescribing professionals, and patients.

Two morphotypes versus two chemotypes of Psidium cattleyanum: Chemical and pharmacological comparison and a rational approach for marker selection.

Psidium cattleyanum has two morphotypes: one with yellow fruits and other with red fruits. The leaves are popularly used as anti-inflammatory. However, no distinction is made between the types. Therefore, this study compared chemical and pharmacological data of both morphotypes to select proper biomarkers to ensure P. cattleyanum leaves quality. After extraction optimization by experimental design, 28 samples were analyzed by HPLC. Using Principal Component Analysis, it was possible to detect two chemotypes, unrelated to the color of the fruits. However, the extracts obtained from both chemotypes seemed to play similar anti-inflammatory effect, demonstrated by anti-chemotactic activity. The compounds common to both chemotypes were isolated and identified as hyperoside, miquelianin and quercitrin; these compounds also demonstrated anti-inflammatory potential. Since both chemotypes played similar activity, along with the isolated flavonoids, these flavonoids were selected as biomarkers for quality control of P. cattleyanum leaves. Following ICH guidelines, a HPLC method was validated. In summary, this study demonstrated that hyperoside, miquelianin and quercitrin can be used as biomarkers for quality control of P. cattleyanum leaves and a method was developed and validated to be used interchangeably for both morpho- and chemotypes.

Appraisal of the antichemotactic activity of flavonoids on polymorphonuclear neutrophils.

Flavonoids are polyphenols that are ubiquitous in plants and frequently consumed in the diet. They are suggested to have many beneficial actions on human health, including anti-inflammatory activity. Their properties have been studied in a number of cell types, but little is known about their effects on neutrophil biology. Consequently, we selected 25 flavonoids with different structural features to evaluate their in vitro inhibition of rat polymorphonuclear neutrophil (PMN) chemotaxis, employing a modified Boyden chamber. Migratory activity was measured towards a chemotactic stimulant, formyl-Met-Leu-Phe or lipopolysaccharide. Furthermore, the cytotoxic effect of flavonoids on PMNs was determined by the release of cytosolic lactate dehydrogenase (LDH). Ten flavonoids significantly retarded the migration of PMNs with at least one of the concentrations tested in a range between 0.625 and 100 µM; the best antichemotactic agents were flavone, flavonol, quercetin and rutin. None of the flavanones evaluated presented any significant inhibition of migration in this assay. Our findings indicated that non-hydroxylated flavones possess a better antichemotactic activity when compared to flavones with hydroxy groups. The presence of a sugar moiety in rutin did not produce any increase in this effect, when compared to the respective aglycone analogue. Finally, none of the flavonoids exhibited cell toxicity and for many of these flavonoids this is the first report of the inhibition of PMN chemotaxis.

Pharmacological evaluation of Copaifera multijuga oil in rats.

CONTEXT: Copaiba oil is an oleoresin made up of resin acids and volatile compounds, and it is obtained by tapping the trunks of trees that are members of the Copaifera L. (Leguminoseae) genus and are found in tropical parts of Latin America. OBJECTIVE: This study analyzed the chemical composition of Copaifera multijuga Hayne oil and conducted preclinical trials to investigate anti-inflammatory effects and any action it may have on the central nervous system (CNS) of rats. MATERIALS AND METHODS: The chemical analysis was carried out using gas chromatography with mass spectroscopy. Anti-inflammatory activity was measured by leucocytes mobilization, by chemotaxis assay in Boyden's chamber, and by pleurisy model in rats. CNS effect was determined by plus maze and open-field assays. The statistical test applied was analysis of variance (ANOVA) followed by Tukey's test or ANOVA followed by Duncan's test. RESULTS: The oil was composed of sesquiterpenes with the predominance of ß-caryophyllene (36.0%), followed by α-copaene (18.8%), ß-bisabolene (8.5%), and α-trans-bergamotene (7.0%). Data demonstrated that at 100 and 200 mg/kg doses and at a concentration of 200 µl/ml copaiba essential oil presented anti-inflammatory effects both in vivo and in vitro based on reduced leukocyte migration to the rats' pleural cavity and to the chemotactic agent lipopolysaccharide solution, respectively. During the experiments investigating CNS effects, locomotive and exploratory activities were reduced and the animals' anxiety increased at 100 and 200 mg/kg. CONCLUSION: The results obtained suggest that copaiba oil has an interesting anti-inflammatory effect and important effect on the CNS.

Comparative analysis of Sambucus nigra and Sambucus australis flowers: development and validation of an HPLC method for raw material quantification and preliminary stability study.

This work was designed to develop a simple, effective, and reliable LC system to identify a chemical marker and compare Sambucus nigra L. and Sambucus australis Cham. et Schltdl. flower extracts (American and European elder). Rutin was the main constituent of both species. The developed method showed a linear response in the range of 10 to 45 microg x mL(-1) for rutin and 1.75 to 3.25 microg x mL(-1) for samples of the Sambucus species. Precision was determined and the relative standard deviations were 1.75 % for HSN and 1.28 % for HSA for intraday precision and 1.28 % and 1.51 % for inter-day precision, respectively, while accuracy was 97.9 % for HSN and 99.41 % for HSA. Quantification and detection limits as well as robustness were determined, presenting adequate results. The LC method showed an adequate performance for the separation of flavonoid glycosides in S. nigra and S. australis extracts, since the presence of interference had been previously evaluated. The analysis of thirty different samples of S. NIGRA and S. australis of different origins did not show significant variability among them. An accelerated stability study revealed a significant decrease in the first 30 days reaching 57 % in 90 days for S. australis samples and a total decrease of 25 % in 90 days for S. nigra samples, considering rutin as the chemical marker. These results will contribute to quality control analysis routines of these raw materials in pharmaceutical production facilities.

Chitosan-coated rosmarinic acid nanoemulsion nasal administration protects against LPS-induced memory deficit, neuroinflammation, and oxidative stress in Wistar rats.

Rosmarinic acid (RA) lipid-nanotechnology-based delivery systems associate with mucoadhesive biopolymers for nasal administration has arisen as a new promising neuroprotective therapy for neurodegenerative disorders (ND). We have previously demonstrated the glioprotective effect of chitosan-coated RA nanoemulsions (RA CNE) against lipopolysaccharide (LPS)-induced damage in rat astrocyte primary culture. Here, we further investigate the protective effect of RA CNE nasal administration on LPS-induced memory deficit, neuroinflammation, and oxidative stress in Wistar rats, since these in vivo studies were crucial to understand the impact of developed delivery systems in the RA neuroprotective effects. The animals were treated through nasal route with RA CNE (2 mg·mL-1), free RA (2 mg·mL-1), blank CNE, and saline (control and LPS groups) administrations (n.a., 100 µL per nostril) twice a day (7 a.m./7 p.m.) for six days. On the sixth day, the animals received the last treatments and LPS was intraperitoneally (i.p.) administrated (250 µg·kg-1). Overall results, proved for the first time that the RA CNE nasal administration elicits a neuroprotective effect against LPS-induced damage, which was associated with increased 1.6 times recognition index, decreased 5.0 and 1.9 times in GFAP+ cell count and CD11b expression, respectively, as well as increased 1.7 times SH in cerebellum and decreased 3.9 times TBARS levels in cerebral cortex in comparison with LPS group. RA CNE treatment also facilitates RA bioavailability in the brain, confirmed by RA quantification. Free RA also demonstrates a protective effect in some studied parameters, although no RA was quantified in the brain.

Extracts of marine sponge Polymastia janeirensis induce oxidative cell death through a caspase-9 apoptotic pathway in human U138MG glioma cell line.

We have studied the apoptotic pathway activated in response to marine sponge extracts of Polymastia janeirensis. The effect on intracellular ROS production was also examined. Exposure of U138MG glioma cell line to doses higher than 5 microg/mL has decreased glioma cell viability, with an IC(50) <15 microg/mL for both aqueous and organic extracts. However, extracts at higher doses (50 and 100 microg/mL) have stronger cytotoxic effects, decreasing more than 90% of glioma cell viability. The antioxidant Trolox (100 microM) reversed the cell death percentage induced by extracts at 10 and 25 microg/mL. The type of cell death induced by such high doses was predominantly necrosis, while a high percentage of apoptotic glioma cells was found at 10 microg/mL. Moreover, inhibition of caspase-8 with Z-IETD (a caspase-8 inhibitor) had no effect on the amount of apoptosis induced by 10 microg/mL, but inhibition of caspase-9 with Z-LEHD (a caspase-9 inhibitor) decreased apoptosis. We also observed a dose-dependent increase in ROS production, and similarly to effects observed on viability of glioma cells, and on cell death, higher doses also had more severe effects. Co-treatment with Trolox significantly reduced ROS production by extracts at doses lower than 50 microg/mL. This is a first report demonstrating that marine sponge extracts of P. janeirensis induce oxidative cell death through a caspase-9 apoptotic pathway.

Development, physico-chemical characterization and in-vitro studies of hydrogels containing rosmarinic acid-loaded nanoemulsion for topical application.

OBJECTIVES: Natural sources with antioxidant activity, such as rosmarinic acid (RA), have been considered as an interesting approach for the development of new anti-ageing skin products. In this context, this study aimed to develop hydrogels containing RA-loaded nanoemulsions and evaluate the effect of the addition of Tween® 80 (a nonionic cosurfactant) in formulations intended to be used for topical application. METHODS: Physico-chemical characterization, in-vitro release and skin retention/permeation from hydrogels of RA-loaded nanoemulsions (containing or not Tween® 80) were evaluated. The RA-loaded nanoemulsion safety profiles were also investigated in keratinocytes (HaCaT cells). KEY FINDINGS: It was observed that all formulations presented adequate physico-chemical characterization for topical application. Furthermore, the results also demonstrated that the presence of Tween® 80 decreased the droplet size and polydispersity index of nanoemulsions and hydrogels. An extended RA release was noted for the hydrogels. However, when comparing the hydrogels, a positive effect of the presence of Tween® 80 on RA retention/permeation in total skin was seen. The RA-loaded nanoemulsion safety profiles demonstrated a good tolerability (3.125-100 µm) in HaCaT cells. CONCLUSIONS: The overall results demonstrated that the formulations developed in this study can be considered as a suitable carrier for RA in a topical application targeting new anti-ageing skin care products.