Limonete, Hipericão-do-Gerês, Cardo-do-Coalho, Fel-da-Terra : Metodologias de controlo de qualidade com base na fracção fenólica : Estudos de acção antioxidante e hepatoprotectora

Dissertação de Doutoramento em Farmacognosia apresentada à Faculdade de Farmácia da Universidade do PortoNeste trabalho foram estudadas quatro espécies medicinais de grande consumo em Portugal: Lippia citriodora (Limonete), Hypericum androsaemum (Hipericão-do-Gerês), Cynara cardunculus (Cardo-do-Coalho) e Centaurium erythraea (Fel-da-Terra).Embora a caracterização química do Limonete e do Fel-da-Terra, definida em Farmacopeias, seja feita através da detecção de compostos terpénicos, foi objectivo deste trabalho propor metodologias para controlo de qualidade de cada uma das quatro espécies, baseadas nos seus perfis fenólicos. Cada espécie foi extraída por diversos solventes e os extractos obtidos foram analisados por CLAP acoplado a um detector de díodos. Para estabelecer o perfil fenólico é proposto o extracto que fornece o maior número de compostos identificados.O extracto etéreo de Limonete é caracterizado pela presença de luteolina, nepetina, hispidulina, jaceosidina, cirsimaritina, cirsilineol e eupatorina. Todas as amostras revelaram um perfil fenólico comum, no qual a hispidulina e a jaceosidina são os compostos maioritários e a nepetina o composto existente em menor quantidade. O extracto metanólico de Hipericão-do-Gerês é caracterizado pela presença dos ácidos 3- e 5-O-cafeoilquínicos, quercetina 3-O-sulfato, quercetina 3-O-galactósido, quercetina 3-O-glucósido, quercetina 3-O-arabinósido, quercetina 3-O-ramnósido, quercetina, kaempferol e de derivados do floroglucinol, que não foram identificados. Contudo, a quantificação dos diversos compostos revelou quatro perfis distintos, facto que pode indicar a existência de diferentes quimiotipos intraespecíficos. O extracto metanólico de Cardo-do-Coalho é caracterizado pela presença dos ácidos 3-, 4, e 5-O-cafeoilquínicos, ácido cafeico, ácidos 1,3-, 3,4-, 3,5-, 1,5- e 4,5-O-dicafeoilquínicos, luteolina 7-O-glucósido, apigenina 7-O-glucósido e luteolina, sendo os ácidos 5-O-cafeoilquínico e 1,5-O-dicafeoilquínico os compostos mais abundantes. No caso do Fel-da-Terra foi necessário proceder ao isolamento e determinaç ..

Phlorotannin Extracts from Fucales Characterized by HPLC-DAD-ESI-MSn: Approaches to Hyaluronidase Inhibitory Capacity and Antioxidant Properties

Purified phlorotannin extracts from four brown seaweeds (Cystoseira nodicaulis (Withering) M. Roberts, Cystoseira tamariscifolia (Hudson) Papenfuss, Cystoseira usneoides (Linnaeus) M. Roberts and Fucus spiralis Linnaeus), were characterized by HPLC-DAD-ESI-MSn . Fucophloroethol, fucodiphloroethol, fucotriphloroethol, 7-phloroeckol, phlorofucofuroeckol and bieckol/dieckol were identified. The antioxidant activity and the hyaluronidase (HAase) inhibitory capacity exhibited by the extracts were also assessed. A correlation between the extracts activity and their chemical composition was established. F. spiralis, the species presenting higher molecular weight phlorotannins, generally displayed the strongest lipid peroxidation inhibitory activity (IC50 = 2.32 mg/mL dry weight) and the strongest HAase inhibitory capacity (IC50 = 0.73 mg/mL dry weight). As for superoxide radical scavenging, C. nodicaulis was the most efficient species (IC50 = 0.93 mg/mL dry weight), followed by F. spiralis (IC50 = 1.30 mg/mL dry weight). These results show that purified phlorotannin extracts have potent capabilities for preventing and slowing down the skin aging process, which is mainly associated with free radical damage and with the reduction of hyaluronic acid concentration, characteristic of the process.info:eu-repo/semantics/publishedVersio

HPLC–DAD of phenolics in bryophytes Lunularia cruciata, Brachytheciastrum velutinum and Kindbergia praelonga

The chemistry of bryophytes is not well known. The available data indicate interesting chemical constitutions of some bryophyte species, i.e., active and new compounds are to be found within bryophytes, especially liverworts. In this study, one liverwort and two moss species were studied: Lunularia cruciata (L.) Dumort, Brachytheciastrum velutinum (Hedw) Ignatov & Huttunen and Kindbergia praelonga (Hedw) Ochyra. The phenolic compositions of these bryophyte species have not hitherto been reported. Their methanolic extracts were analyzed by reversed-phase HPLC, coupled to a diode-array detector (DAD). Luteolin-7-O-glucoside and quercetin were found in the L. cruciata extract. The extract obtained from B. velutinum contained four phenolic acids (4-O-caffeoylquinic, 5-O-caffeoylquinic, caffeic and ellagic acids) and three flavonoids (apigenin-7-O-glucoside, luteolin and apigenin). The K. praelonga extract was characterized by the presence of several phenolic acids and their derivatives (4-O-caffeoylquinic, 5-O-caffeoylquinic, caffeic, p-coumaric, ferulic and ellagic acids, and caffeic and p-coumaric acid derivatives) and three flavonoids (apigenin-7-O-glucoside, luteolin, apigenin and an un-identified flavanone)

Evaluating the in vitro potential of natural extracts to protect lipids from oxidative damage

Lipid peroxidation is a chemical reaction known to have negative impacts on living organisms’ health and on consumer products’ quality and safety. Therefore, it has been the subject of extensive scientific research concerning the possibilities to reduce it, both in vivo and in nonliving organic matrices. It can be started by a variety of oxidants, by both ROS-dependent and -independent pathways, all of them reviewed in this document. Another feature of this reaction is the capacity of lipid peroxyl radicals to react with the non-oxidized lipids, propagating the reaction even in the absence of an external trigger. Due to these specificities of lipid peroxidation, regular antioxidant strategies—although being helpful in controlling oxidative triggers—are not tailored to tackle this challenge. Thus, more suited antioxidant compounds or technologies are required and sought after by researchers, either in the fields of medicine and physiology, or in product development and biotechnology. Despite the existence of several laboratory procedures associated with the study of lipid peroxidation, a methodology to perform bioprospecting of natural products to prevent lipid peroxidation (a Lipid Peroxidation Inhibitory Potential assay, LPIP) is not yet well established. In this review, a critical look into the possibility of testing the capacity of natural products to inhibit lipid peroxidation is presented. In vitro systems used to peroxidize a lipid sample are also reviewed on the basis of lipid substrate origin, and, for each of them, procedural insights, oxidation initiation strategies, and lipid peroxidation extent monitoring are discussed.info:eu-repo/semantics/publishedVersio

Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage

Common sage (Salvia officinalis L., Lamiaceae) is an aromatic and medicinal plant well known for its antioxidant properties. Some in vivo studies have shown the biological antioxidant effects of sage. However, the intracellular antioxidant mechanisms of action are still poorly understood. In this study, we evaluated the cytoprotective effects of two sage extracts (a water and a methanolic extract) against tert-butyl hydroperoxide (t-BHP)-induced toxicity in HepG2 cells. The most abundant phenolic compounds present in the extracts were rosmarinic acid and luteolin-7-glucoside. Both extracts, when co-incubated with the toxicant, protected significantly HepG2 cells against cell death. The methanolic extract, with a higher content of phenolic compounds than the water extract, conferred better protection in this in vitro model of oxidative stress with liver cells. Both extracts, tested in a concentration that protects 80% against cell death (IC80), significantly prevented t-BHP-induced lipid peroxidation and GSH depletion, but not DNA damage assessed by the comet assay. The ability of sage extracts to reduce t-BHP-induced GSH depletion by 62% was probably the most relevant contributor to the observed cytoprotection. A good correlation between the above cellular effects of sage and the effects of their main phenolic compounds was found. When incubated alone for 5 h, sage extracts induced an increase in basal GSH levels of HepG2 cells, which indicates an improvement of the antioxidant potential of the cells. Compounds present in sage extracts other than phenolics may also contribute to this latter effect. Based in these results, it would be of interest to investigate whether sage has protective effects in suitable in vivo models of liver diseases, where it is known that oxidative stress is involved.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/6942/2001, POCI/AGR/62040/200

Toxicity and structure-activity relationship (SAR) of α, β-dehydroamino acids against human cancer cell lines

A library of N-protected dehydroamino acids, namely dehydroalanine, dehydroaminobutyric acid and dehydrophenylalanine derivatives, was screened in three human cancer cell lines [(lung (A549), gastric (AGS) and neuroblastoma (SH-SY5Y)] in order to characterize their toxicological profile and identify new molecules with potential anticancer activity. Results showed N-protected dehydrophenylalanine and dehydroaminobutyric acid derivatives have no or low toxicity for all tested cell lines. The N-protected dehydroalanines exhibit significant toxic effects and the AGS and SH-SY5Y cells were significantly more vulnerable than A549 cells. Four α,β- dehydroalanine derivatives, with IC50 < 62.5 μM, were selected to investigate the pathways by which these compounds promote cell death. All compounds, at their IC50 concentrations, were able to induce apoptosis in both AGS and SH-SY5Y cell lines. In both cell lines, loss of mitochondrial membrane potential (ΔΨm) was found and caspase activity was increased, namely endoplasmic reticulum-resident caspase-4 in AGS cells and caspase-3/7 in SH-SY5Y cells. When evaluated in a non-cancer cell line, the molecules displayed no to low toxicity, thus suggesting some degree of selectivity for cancer cells. The results indicate that α,β-dehydroalanine derivatives can be considered a future resource of compounds able to work as anticancer drugs.This work received financial support from National Funds (FCT/MEC) through Project UID/QUI/50006/2013, co-financed by European Union (FEDER under the Partnership Agreement PT2020); and from Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (project NORTE-01-0145-FEDER 000024).info:eu-repo/semantics/publishedVersio

Do bioactive carotenoids contribute to the color of edible mushrooms?

Carotenoids are biologically active phytochemicals present as micro-components in fruits and vegetables, being responsible for their yellow, orange and red colors. The chromatographic behavior and the UV absorption spectrum provided by HPLC-DAD analysis constitute the clues for their identification. Mushrooms are of increasing importance in modern nutrition and medicine, due to the presence of metabolites with pharmacological potential. In this work, samples of wild and commercial mushroom species (Agaricus bisporus, Amanita caesarea, Amanita rubescens, Boletus edulis, Cantharellus cibarius, Fistulina hepatica, Hydnum rufescens, Hygrophorus agathosmus, Pholiota nameko, Pleurotus ostreatus, Russula cyanoxantha, Suillus bellini, Suillus bovinus, Suillus granulatus, Suillus luteus, Tricholoma equestre and Tricholoma portentosum) were screened by HPLC-DAD for the presence of carotenoids. By applying this methodology to 22 samples, comprising either lyophilized or fresh materials, only β-carotene was found and just in C. cibarius species. The occurrence of this pigment in other three of the analyzed species previously described raises some questions about the methodology used.info:eu-repo/semantics/publishedVersio

Fisetin derivatives exhibit enhanced anti-inflammatory activity and modulation of endoplasmic reticulum stress

Fisetin (FST) is a dietary flavonol that is known to possess multiple relevant bioactivities, raising the question of its potential health benefits and even its use in novel pharmacological approaches. To attain this prospect, some limitations to this molecule, namely its poor bioavailability and solubility, must be addressed. Inflammation and endoplasmic reticulum (ER) stress are often hand in hand in the context of chronic disease. Both are activated upon perceived disturbances in homeostasis but can be deleterious when intensely or chronically activated. We have synthesized a set of FST derivatives trying to improve the biological properties of the parent molecule. These new molecules were tested along with the original compound for their ability to mitigate the activation of these signaling pathways. FST has proven to be effective against the onset of inflammation, reducing NF-κB activation, cytokine release, inflammasome activation and ROS generation, as well as decreasing the activation of the unfolded protein response (UPR). Some of the tested derivatives are also described as new caspase-1 inhibitors, being also capable of reducing pro-inflammatory cytokines and ER stress markers.(undefined

Anti-Inflammatory Effects of 5α,8α-Epidioxycholest-6-en-3β-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis

[Abstract:] Sea hares of "Aplysia" genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3β-ol (EnP(5,8)) from "Aplysia depilans" Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.Portugal.Fundação para a Ciência e Tecnologia; UID/QUI/50006/2019Portugal. Fundação para a Ciência e Tecnologia; PD/BD/113565/201

Do cultivar, geographical location and crop season influence phenolic profile of walnut leaves?

Walnut leaves from nine different cultivars (Arco, Franquette, Hartley, Lara, Marbot, Mayette, Meylannaise, Parisienne and Rego) were studied for their phenolic compounds. Samples were harvested along three consecutive years, at two different geographical locations, in order to evaluate if significant differences in the phenolics composition can be related with genetic, climatic or geographical factors. Nine compounds (3-caffeoylquinic, 3-p-coumaroylquinic and 4-p-coumaroylquinic acids, quercetin 3- galactoside, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3-rhamnoside, a quercetin 3-pentoside derivative and a kaempferol 3-pentoside derivative) were quantified using an HPLC-DAD methodology. The qualitative profiles were identical for all samples, but differences were observed in terms of individual compounds’ contents. Multivariate statistical analysis was carried out, showing that significant differences exist among production years, which can be related to climatic reasons.Direcção Regional de Agricultura da Beira Litoral (DRABL)Instituto Politécnico de Bragança (ESA