Impact of an educational hands-on project on the antimicrobial, antitumor and anti-inflammatory properties of plants on Portuguese students' awareness, knowledge, and competences

Promoting environmental and health education is crucial to allow students to make conscious decisions based on scientific criteria. The study is based on the outcomes of an Educational Project implemented with Portuguese students and consisted of several activities, exploring pre-existent Scientific Gardens at the School, aiming to investigate the antibacterial, antitumor and anti-inflammatory properties of plant extracts, with posterior incorporation in soaps and creams. A logo and a webpage were also created. The effectiveness of the project was assessed via the application of a questionnaire (pre- and post-test) and observations of the participants in terms of engagement and interaction with all individuals involved in the project. This project increased the knowledge about autochthonous plants and the potential medical properties of the corresponding plant extracts and increased the awareness about the correct design of scientific experiments and the importance of the use of experimental models of disease. The students regarded their experiences as exciting and valuable and believed that the project helped to improve their understanding and increase their interest in these subjects and in science in general. This study emphasizes the importance of raising students’ awareness on the valorization of autochthonous plants and exploitation of their medicinal properties.

The contribution of phenolic acids to the anti-inflammatory activity of mushrooms: Screening in phenolic extracts, individual parent molecules and synthesized glucuronated and methylated derivatives

In the present study, the ethanolic extracts of fourteen edible mushrooms were investigated for their anti-inflammatory potential in LPS (lipopolysaccharide) activated RAW 264.7 macrophages. Furthermore the extracts were chemically characterized in terms of phenolic acids and related compounds. The identified molecules (p-hydroxybenzoic, p-coumaric and cinnamic acids) and their glucuronated and methylated derivatives obtained by chemical synthesis were also evaluated for the same bioactivity, in order to establish structure-activity relationships and to comprehend the effects of in vivo metabolism reactions in the activity of the compounds. The extracts of Pleurotus ostreatus, Macrolepiota procera, Boletus impolitus and Agaricus bisporus revealed the strongest anti-inflammatory potential (EC50 values 96 ± 1 to 190 ± 6 µg/mL, and also the highest concentration of cinnamic acid (656 to 156 µg/g), which was also the individual compound with the highest anti-inflammatory activity. The derivatives of p-coumaric acid revealed the strongest properties, specially the derivative methylated in the carboxylic group (CoA-M1) that exhibited similar activity to the one showed by dexamethaxone used as anti-inflammatory standard; by contrast, the derivatives of p-hydroxybenzoic revealed the lowest inhibition of NO production. All in all, whereas the conjugation reactions change the chemical structure of phenolic acids and may increase or decrease their activity, the glucuronated and methylated derivatives of the studied compounds are still displaying anti-inflammatory activity.

Anti-inflammatory and antinociceptive activities of Bauhinia monandra leaf lectin

A galactose-specific lectin from Bauhinia monandra leaves (BmoLL) have been purified through ammonium sulphate fractionation followed by guar gel affinity chromatography column. This study aimed to evaluate the potential anti-inflammatory and antinociceptive activity of pure BmoLL in mice. Anti-inflammatory activity was evaluated by 1% carrageenan-induced inflammation in mice treated with BmoLL. Acetic acid-induced abdominal writhing and hot plate methods evaluated antinociceptive activity. BmoLL significantly inhibited the carrageenan-induced paw edema by 47% (30 mg/kg) and 60.5% (60 mg/kg); acetylsalicylic acid (ASA, 100 mg/kg) showed inhibition of 70.5%, in comparison to controls. Leukocyte migration, an immune response to the inflammation process, was significantly reduced in presence of BmoLL; in mice treated with \ASA\ the decrease in leukocyte migration was similar to 15 mg/kg of the lectin. BmoLL at doses of 15, 30 and 60 mg/kg significantly reduced the number of animal contortions by 43.1, 50.1 and 71.3%, respectively.BmoLL leukocyte migration was significantly reduced; in mice treated with \ASA\ the decrease in leukocyte migration was similar to 15 mg/kg of the lectin. BmoLL at doses of 15, 30 and 60 mg/kg significantly reduced the number of animal contortions by 43.1, 50.1 and 71.3%, respectively. The lectin (30 and 60 mg/kg) showed a significant effect in the hot plate assay. BmoLL anti-inflammatory and antinociceptive effects were dose-dependent. The search for new and natural compounds, with minimal side effects, to control pain and inflammation, is constantly increasing. BmoLL has great potential as a natural anti-inflamatory product that can be explored for pharmacological purposes.

New self-assembled supramolecular hydrogels based on dehydropeptides

Supramolecular hydrogels rely on small molecules that self-assemble in water as a result of the cooperative effect of several relatively weak intermolecular interactions. Peptide-based low molecular weight hydrogelators have attracted enormous interest owing to the simplicity of small molecules combined with the versatility and biocompatibility of peptides. In this work, naproxen, a well known non-steroidal anti-inflammatory drug, was N-conjugated with various dehydrodipeptides to give aromatic peptide amphiphiles that resist proteolysis. Molecular dynamics simulations were used to obtain insight into the underlying molecular mechanism of self-assembly and to rationalize the design of this type of hydrogelators. The results obtained were in excellent agreement with the experimental observations. Only dehydrodipeptides having at least one aromatic amino acid gave hydrogels. The characterization of the hydrogels was carried out using transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy and also rheological assays.

Micro- and mesoporous structures as drug delivery carriers for salicylic acid

The potential of salicylic acid (SA) encapsulated in porous materials as drug delivery carriers for cancer treatment was studied. Different porous structures, the microporous zeolite NaY, and the mesoporous SBA-15 and MCM-41 were used as hosts for the anti-inflammatory drug. Characterization with different techniques (FTIR, UV/vis, TGA, 1H NMR, and 13C CPMAS NMR) demonstrated the successful loading of SA into the porous hosts. The mesoporous structures showed to be very efficient to encapsulate the SA molecule. The obtained drug delivery systems (DDS) accommodated 0.74 mmol (341 mg/gZEO) in NaY and 1.07 mmol (493 mg/gZEO) to 1.23 mmol (566 mg/gZEO) for SBA-15 and MCM-41, respectively. Interactions between SA molecules and pore structures were identified. A fast and unrestricted liberation of SA at 10 min of the dissolution assay was achieved with 29.3, 46.6, and 50.1 µg/mL of SA from NaY, SBA-15, and MCM-41, respectively, in the in vitro drug release studies (PBS buffer pH 7.4, 37 °C). Kinetic modeling was used to determine the release patterns of the DDS. The porous structures and DDS were evaluated on Hs578T and MDA-MB-468 breast cancer cell lines viability. The porous structures are nontoxic to cancer cells. Cell viability reduction was only observed after the release of SA from MCM- 41 followed by SBA-15 in both breast cancer cell lines.

Marine origin polysaccharides in drug delivery systems

Oceans are a vast source of natural substances. In them, we find various compounds with wide biotechnological and biomedical applicabilities. The exploitation of the sea as a renewable source of biocompounds can have a positive impact on the development of new systems and devices for biomedical applications. Marine polysaccharides are among the most abundant materials in the seas, which contributes to a decrease of the extraction costs, besides their solubility behavior in aqueous solvents and extraction media, and their interaction with other biocompounds. Polysaccharides such as alginate, carrageenan and fucoidan can be extracted from algae, whereas chitosan and hyaluronan can be obtained from animal sources. Most marine polysaccharides have important biological properties such as biocompatibility, biodegradability, and anti-inflammatory activity, as well as adhesive and antimicrobial actions. Moreover, they can be modified in order to allow processing them into various shapes and sizes and may exhibit response dependence to external stimuli, such as pH and temperature. Due to these properties, these biomaterials have been studied as raw material for the construction of carrier devices for drugs, including particles, capsules and hydrogels. The devices are designed to achieve a controlled release of therapeutic agents in an attempt to fight against serious diseases, and to be used in advanced therapies, such as gene delivery or regenerative medicine.

Searching for therapeutic strategies in a mouse modelo of Machado-Joseph disease: targeting proteostases

Tese de Doutoramento em Ciências da Saúde

Synthetic biology approaches to engineer new pathways for the production of plant secondary metabolites

Secondary metabolites from plants are important sources of high-value chemicals, many of them being pharmacologically active. These metabolites are commonly isolated through inefficient extractions from natural biological sources and are often difficult to synthesize chemically. Therefore, their production using engineered organisms has lately attracted an increased attention. Curcuminoids, an example of such metabolites, are produced in Curcuma longa and exhibit anti-cancer and anti-inflammatory activities. Herein we report the construction of an artificial biosynthetic pathway for the curcuminoids production in Escherichia coli. Different 4-coumaroyl-CoA ligases (4CL) and polyketide synthases (diketide-CoA synthase (DCS), curcumin synthase (CURS) and curcuminoid synthase) were tested. The highest curcumin production (70 mg/L) was obtained by feeding ferulic acid and with the Arabidopsis thaliana 4CL1 and C. longa DCS and CURS enzymes. Other curcuminoids (bisdemethoxy- and demethoxycurcumin) were also produced by feeding coumaric acid or a mixture of coumaric and ferulic acids, respectively. Curcuminoids, including curcumin, were also produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase and 4-coumarate 3-hydroxylase were used. Caffeoyl-CoA O-methyltransferase was used to convert caffeoyl-CoA to feruloyl-CoA. This pathway represents an improvement of the curcuminoids heterologous production. The construction of this pathway in another model organism is being considered, as well as the introduction of alternative enzymes.

Lipid based nanocarriers for the delivery of the bioactive compound resveratrol

Dissertação de mestrado em Biofísica e Bionanossistemas