Chemical Composition and Effect against Skin Alterations of Bioactive Extracts Obtained by the Hydrodistillation of Eucalyptus globulus Leaves

Eucalyptus globulus is planted extensively for pulp, paper and wood production. Although bioactive compounds obtained from its biomass are used as cosmetics ingredients, the skin effects were not yet fully explored. In order to fill this gap, this work aimed to study the protective effect against skin damage provided by the essential oil (EO) obtained from the hydrodistillation of Eucalyptus globulus leaves, and by an extract obtained from the hydrodistillation residual water (HRW). The major compound identified in the EO was 1,8-Cineole, and the phenolic acids in the HRW included gallic acid as the main phenolic constituent. Moreover, non-toxic EO and HRW concentrations were shown to have anti-aging skin effects in vitro, decreasing age-related senescence markers, namely β-galactosidase and matrix metalloproteinases activation, as well as collagen type 1 upregulation. In addition, EO and HRW were found to exhibit depigmenting effects by inhibiting tyrosinase and melanin production, along with potent anti-inflammatory properties. Furthermore, the absence of skin irritation and sensitization in cells exposed to EO and HRW revealed the safety of both extracts for topical use. Taken together, these results highlight the beneficial effects of extracts obtained from Eucalyptus globulus biomass for skin aesthetic and health purposes, which should be explored deeply for the prediction of future pharmaceutical and dermocosmetics industrial applications

Terapia fotodinâmica em combinação com quimioterapia : uma opção no osteossarcoma

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de CoimbraO osteossarcoma é o tipo de sarcoma mais comum e corresponde acerca de 45% de todos os cancros ósseos. A toxicidade dos fármacos antineoplásicos convencionalmente utilizados no seu tratamento é muitas vezes limitante da dose aplicada ou até mesmo a razão para a interrupção da quimioterapia, sendo por isso emergente a necessidade de novas terapias e estratégias para o combate do osteossarcoma, particularmente os casos de doença irressecável. Este projeto surge no sentido de responder a esta necessidade, já abordada em trabalhos prévios a esta dissertação, de ampliar a utilização da terapia fotodinâmica em oncologia com a combinação com outras estratégias terapêuticas como a quimioterapia. Neste trabalho, pretendeu-se estudar a terapia fotodinâmica com base no fotossensibilizador BBr2HPP, tendo em conta os conhecimentos já adquiridos por este grupo de investigação nesta área. Relativamente aos estudos in vitro, o ensaio MTT permitiu verificar o efeito citotóxico que três dos fármacos mais usados de quimioterapia têm na inibição da proliferação celular tendo-se verificado que são dependentes da concentração em que estão presentes. A linha tumoral MNNG-HOS, demonstrou ser sensível ao efeito dos três fármacos quimioterapêuticos estudados. Sabendo que determinados fotossensibilizadores exibem uma elevada seletividade para as células tumorais, estudou-se também o efeito da terapia de combinação numa linha não tumoral, a linha humana de fibroblastos HFF1, não se verificando diferenças estatisticamente significativas. A associação da terapia fotodinâmica com a quimioterapia diminuiu significativamente a atividade metabólica das células tumorais, bem como a viabilidade celular, de forma dependente da concentração de fotossensibilizador e da concentração e do tempo de administração, levando a um efeito sinérgico em combinação com a doxorrubicina e a cisplatina. Demonstrou-se que o decréscimo da atividade metabólica, nas células submetidas à combinação entre a PDT e a doxorrubicina, está relacionado com os mecanismos de morte celular, a apoptose e a necrose, associados a disfunção mitocondrial, a produção intracelular de espécies reativas de oxigénio, e a danos no DNA. XIV Paralelamente, nos estudos in vivo verificou-se que a taxa de crescimento tumoral foi inferior quando os ratinhos foram submetidos à combinação entre a PDT e a doxorrubicina. Foi possível também verificar o sucesso da terapia de combinação sem a penalização do bem-estar dos ratinhos, reforçando mais uma vez o potencial terapêutico. Os estudos de histologia vieram confirmar os resultados promissores obtidos in vitro., os quais mostraram haver uma percentagem necrose superior na terapia fotodinâmica e na terapia combinada do que na terapia apenas com doxorrubicina. Assim, foi possível com este projeto verificar que a terapia fotodinâmica em combinação com a quimioterapia constitui uma modalidade terapêutica com efeito sinérgico que poderá fazer diferença no tratamento do osteossarcoma.Osteosarcoma is the most common type of sarcoma and corresponds to about 45% of all bone cancers. The toxicity of antineoplastic drugs conventionally used in their treatment is often limiting or even reason for the interruption of chemotherapy, therefore there is need for new therapies and strategies to fight osteosarcoma, particularly cases of unresectable disease. This project arises in order to respond to this need, already addressed in studies previous to this dissertation, to expand the use of photodynamic therapy in oncology and to combine it with other therapeutic approaches, such as chemotherapy. In this work, we intended to study photodynamic therapy based on BBr2HPP photosensitizer, also taking into account the knowledge already acquired in this area by the research group. Regarding in vitro studies, the MTT assay has shown the cytotoxicity of three of the most commonly used chemotherapy drugs verifying that inhibition of metabolic activity is dependent on the concentration. The tumor cell line MNNG-HOS, was sensitive to the effect of the three chemotherapeutic drugs studied. Knowing that certain photosensitisers exhibit high selectivity for tumor cells, it was also studied whether the combination therapy would affect non-tumor cells, the HFF1 human fibroblasts, and there were no statistically significant differences. The association of photodynamic therapy to chemotherapy significantly decreased the metabolic activity of the tumor cells as well as cell viability, dependently on the concentration of photosensitizer and the concentration and time of administration of the chemotherapy drug, leading to a synergic effect in combination with doxorubicin and cisplatin. It has been shown that the decrease in metabolic activity in cells subjected to the combination of PDT and doxorubicin, is related to the mechanisms of cell death, apoptosis and necrosis associated with mitochondrial dysfunction, intracellular production of reactive oxygen species, and DNA damage. Similarly, in vivo studies showed that tumor growth was lower when mice were subjected to the combination of PDT and doxorubicin. It was also possible to verify the success of the combination therapy without compromising mice wellbeing, reinforcing the therapeutic potential. The histology studies have confirmed the promising results obtained in vitro, having a percentage of higher necrosis in photodynamic therapy and combination therapy than in therapy only with doxorubicin. XVI With this project it was possible to verify that photodynamic therapy in combination with chemotherapy is a treatment modality with synergistic effect that may make a difference in the treatment of osteosarcoma

A Hydrophilic Sulfated Resveratrol Derivative for Topical Application: Sensitization and Anti-Allergic Potential

Resveratrol (RSV), a naturally occurring metabolite, is widely used in skincare products, but its hydrophobicity impairs its own incorporation into cosmetic formulations. RSV-GS is a synthetic hydrophilic sulfated glycosylated derivative inspired by marine natural products that present a lower cytotoxicity than RSV while exhibiting similar levels of bioactivity. Herein, we predict the skin sensitization potential of this new compound using an in vitro approach based on the OECD 442E guideline. Furthermore, the anti-allergic potential of RSV-GS was also disclosed. The monocyte THP-1 cell line was stimulated with RSV and RSV-GS in the presence or absence of the extreme skin allergen 1-fluoro-2,4-dinitrobenzene (DNFB). The results demonstrated that RSV-GS alone (500 µM) evoked a relative fluorescence index (RFI) lower than the thresholds established by the OECD guideline for CD54 (200%) and CD86 (150%), indicating the absence of a skin sensitization potential. Interestingly, in the presence of the skin allergen DNFB, RSV-GS exhibited the ability to rescue the DNFB-induced maturation of THP-1 cells, with RFI values lower than those for RSV, suggesting the potential of RSV-GS to mitigate skin sensitization evoked by allergens and, consequently, allergic contact dermatitis. These results open new avenues for the use of RSV-GS as a safe and anti-allergic active cosmetic ingredient

Novel fluorinated ring-fused chlorins as promising PDT agents against melanoma and esophagus cancer

Investigation of novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins, derived from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, as PDT agents against melanoma and esophagus cancer is disclosed. Diol and diester fluorinated ring-fused chlorins, including derivatives with 2-(2-hydroxyethoxy)ethanamino groups at the phenyl rings, were obtained via a two-step methodology, combining SNAr and [8π + 2π] cycloaddition reactions. The short-chain PEG groups at the para-position of the phenyl rings together with the diol moiety at the fused pyrazole ring promote a red-shift of the Soret band, a decrease of the fluorescence quantum yield and an increase of the singlet oxygen formation quantum yield, improving the photophysical characteristics required to act as a photosensitizer. Introduction of these hydrophilic groups also improves the incorporation of the sensitizers by the cells reaching cellular uptake values of nearly 50% of the initial dose. The rational design led to a photosensitizer with impressive IC50 values, 13 and 27 nM against human melanoma and esophageal carcinoma cell lines, respectively.The authors thank Coimbra Chemistry Centre (CQC), supported by the Portuguese Agency for Scientific Research, “Fundação para a Ciência e a Tecnologia” (FCT) through project UIDB/00313/2020 and UIDP/QUI/00313/2020, co-funded by COMPETE2020-UE. Center for Innovative Biomedicine and Biotechnology (CIBB) is funded by FCT (UID/NEU/04539/2013) and COMPETE-FEDER (POCI-01-0145-FEDER-007440), through the Strategic Project UIDB/04539/2020 and UIDP/04539/2020. Thanks are also due to CIMAGO (Project 06/2019) and FCT, co-funded by the European Regional Development Fund (FEDER) through Portugal 2020/CENTRO 2020 (CENTRO-01-0145-FEDER-000014/MATIS)

Ring-Fused meso-Tetraarylchlorins as Auspicious PDT Sensitizers: Synthesis, Structural Characterization, Photophysics, and Biological Evaluation

Novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused meso-tetraarylchlorins, with different degrees of hydrophilicity (with methyl ester, hydroxymethyl, and carboxylic acid moieties), have been synthesized and their photophysical characterization as well as in vitro photocytotoxicity assessment against human melanoma and esophageal and bladder carcinomas was carried out. An integrated analysis of the photosensitizers' performance, considering the singlet oxygen generation data, cell internalization, and intracellular localization, allowed to establish relevant structure-photoactivity relationships and the rationalization of the observed photocytotoxicity. In the diacid and monoalcohol series, chlorins derived from meso-tetraphenylporphyrin proved to be the most efficient photodynamic therapy agents, showing IC50 values of 68 and 344 nM against A375 cells, respectively. These compounds were less active against OE19 and HT1376 cells, the diacid chlorin with IC50 values still in the nano-molar range, whereas the monohydroxymethyl-chlorin showed significantly higher IC50 values. The lead di(hydroxymethyl)-substituted meso-tetraphenylchlorin confirmed its remarkable photoactivity with IC50 values below 75 nM against the studied cancer cell lines. Subcellular accumulation of this chlorin in the mitochondria, endoplasmic reticulum, and plasma membrane was demonstrated