Chemical characterization and evaluation of biological activity of Cynara cardunculus extractable compounds

The Mediterranean species Cynara cardunculus L. is recognized in the traditional medicine, for their hepatoprotective and choleretic effects. Biomass of C. cardunculus L. var. altilis (DC), or cultivated cardoon, may be explored not only for the production of energy and pulp fibers, but also for the extraction of bioactive compounds. The chemical characterization of extractable components, namely terpenic and phenolic compounds, may valorize the cultivated cardoon plantation, due to their antioxidant, antitumoral and antimicrobial activities. In this study, the chemical composition of lipophilic and phenolic fractions of C. cardunculus L. var. altilis (DC), cultivated in the south of Portugal (Baixo Alentejo region) was characterized in detail, intending the integral valorization of its biomass. The biological activity of cultivated cardoon extracts was evaluated in terms of antioxidant, human tumor cell antiproliferative and antibacterial effects. Gas chromatography-mass spectrometry (GC-MS) was used for the chemical analysis of lipophilic compounds. Sixty-five lipophilic compounds were identified, from which 1 sesquiterpene lactone and 4 pentacyclic triterpenes were described, for the first time, as cultivated cardoon components, such as: deacylcynaropicrin, acetates of ��- and ��-amyrin, lupenyl acetate and ��-taraxasteryl acetate. Sesquiterpene lactones were the major family of lipophilic components of leaves (���94.5 g/kg), mostly represented by cynaropicrin (���87.4 g/kg). Pentacyclic triterpenes were also detected, in considerably high contents, in the remaining parts of cultivated cardoon, especially in the florets (���27.5 g/kg). Taraxasteryl acetate was the main pentacyclic triterpene (���8.9 g/kg in florets). High pressure liquid chromatography-mass spectrometry (HPLC-MS) was utilized for the chemical analysis of phenolic compounds. Among the identified 28 phenolic compounds, eriodictyol hexoside was reported for the first time as C. cardunculus L. component, and 6 as cultivated cardoon components, namely 1,4-di-O-caffeoylquinic acid, naringenin 7-O-glucoside, naringenin rutinoside, naringenin, luteolin acetylhexoside and apigenin acetylhexoside. The highest content of the identified phenolic compounds was observed in the florets (���12.6 g/kg). Stalks outer part contained the highest hydroxycinnamic acids abundance (���10.3 g/kg), and florets presented the highest flavonoids content (���10.3 g/kg). The antioxidant activity of phenolic fraction was examined through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. Stalks outer part, and receptacles and bracts extracts demonstrated the highest antioxidant effect on DPPH (IC50 of 34.35 ��g/mL and 35.25 ��g/mL, respectively). (cont.) abstract (cont.) The DPPH scavenging effect was linearly correlated with the total contents of hydroxycinnamic acids (r = -0.990). The in vitro antiproliferative activity of cultivated cardoon lipophilic and phenolic extracts was evaluated on a human tumor cells line of triple-negative breast cancer (MDA-MB-231), one of the most refractory human cancers to conventional therapeutics. After 48 h of exposition, leaves lipophilic extract showed higher inhibitory effect (IC50 = 10.39 ��g/mL) than florets lipophilic extract (IC50 = 315.22 ��g/mL), upon MDA-MB-231 cellular viability. Pure compound of cynaropicrin, representative of the main compound identified in leaves lipophilic extract, also prevented the cell proliferation of MDA-MB-231 (IC50 = 17.86 ��M). MDA-MB-231 cells were much more resistant to the 48 h- treatment with phenolic extracts of stalks outer part (IC50 = 3341.20 ��g/mL) and florets (IC50 > 4500 ��g/mL), and also with the pure compound of 1,5-di-O-caffeoylquinic acid (IC50 = 1741.69 ��M). MDA-MB-231 cells were exposed, for 48 h, to the respective IC50 concentrations of leaves lipophilic extract and pure compound of cynaropicrin, in order to understand their ability in modelling cellular responses, and consequently important potentially signaling pathways for the cellular viability decrease. Leaves lipophilic extract increased the caspase-3 enzymatic activity, contrarily to pure compound of cynaropicrin. Additionally, leaves lipophilic extract and pure compound of cynaropicrin caused G2 cell cycle arrest, possibly by upregulating the p21Waf1/Cip1 and the accumulation of phospho-Tyr15-CDK1 and cyclin B1. The inhibitory effects of leaves lipophilic extract and cynaropicrin pure compound, against the MDA-MB-231 cell proliferation, may also be related to the downregulation of phospho-Ser473-Akt. The antibacterial activity of cultivated cardoon lipophilic and phenolic extracts was assessed, for the first time, on two multidrug-resistant bacteria, such as the Gram-negative Pseudomonas aeruginosa PAO1 and the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), two of the main bacteria responsible for health care-associated infections. Accordingly, the minimum inhibitory concentrations (MIC) were determined. Lipophilic and phenolic extracts of florets did not have antibacterial activity on P. aeruginosa PAO1 and MRSA (MIC > 2048 ��g/mL). Leaves lipophilic extract did not prevent the P. aeruginosa PAO1 growth, but pure compound of cynaropicrin was slightly active (MIC = 2048 ��g/mL). Leaves lipophilic extract and pure compound of cynaropicrin blocked MRSA growth (MIC of 1024 and 256 ��g/mL, respectively). The scientific knowledge revealed in this thesis, either by the chemical viewpoint, or by the biological viewpoint, contributes for the valorization of C. cardunculus L. var. altilis (DC) biomass. Cultivated cardoon has potential to be exploited as source of bioactive compounds, in conciliation with other valorization pathways, and Portuguese traditional cheeses manufacturing.

Metabolic signature of lung cancer: a metabolomic study of human tissues and biofluids

This thesis reports the application of metabolomics to human tissues and biofluids (blood plasma and urine) to unveil the metabolic signature of primary lung cancer. In Chapter 1, a brief introduction on lung cancer epidemiology and pathogenesis, together with a review of the main metabolic dysregulations known to be associated with cancer, is presented. The metabolomics approach is also described, addressing the analytical and statistical methods employed, as well as the current state of the art on its application to clinical lung cancer studies. Chapter 2 provides the experimental details of this work, in regard to the subjects enrolled, sample collection and analysis, and data processing. In Chapter 3, the metabolic characterization of intact lung tissues (from 56 patients) by proton High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy is described. After careful assessment of acquisition conditions and thorough spectral assignment (over 50 metabolites identified), the metabolic profiles of tumour and adjacent control tissues were compared through multivariate analysis. The two tissue classes could be discriminated with 97% accuracy, with 13 metabolites significantly accounting for this discrimination: glucose and acetate (depleted in tumours), together with lactate, alanine, glutamate, GSH, taurine, creatine, phosphocholine, glycerophosphocholine, phosphoethanolamine, uracil nucleotides and peptides (increased in tumours). Some of these variations corroborated typical features of cancer metabolism (e.g., upregulated glycolysis and glutaminolysis), while others suggested less known pathways (e.g., antioxidant protection, protein degradation) to play important roles. Another major and novel finding described in this chapter was the dependence of this metabolic signature on tumour histological subtype. While main alterations in adenocarcinomas (AdC) related to phospholipid and protein metabolisms, squamous cell carcinomas (SqCC) were found to have stronger glycolytic and glutaminolytic profiles, making it possible to build a valid classification model to discriminate these two subtypes. Chapter 4 reports the NMR metabolomic study of blood plasma from over 100 patients and near 100 healthy controls, the multivariate model built having afforded a classification rate of 87%. The two groups were found to differ significantly in the levels of lactate, pyruvate, acetoacetate, LDL+VLDL lipoproteins and glycoproteins (increased in patients), together with glutamine, histidine, valine, methanol, HDL lipoproteins and two unassigned compounds (decreased in patients). Interestingly, these variations were detected from initial disease stages and the magnitude of some of them depended on the histological type, although not allowing AdC vs. SqCC discrimination. Moreover, it is shown in this chapter that age mismatch between control and cancer groups could not be ruled out as a possible confounding factor, and exploratory external validation afforded a classification rate of 85%. The NMR profiling of urine from lung cancer patients and healthy controls is presented in Chapter 5. Compared to plasma, the classification model built with urinary profiles resulted in a superior classification rate (97%). After careful assessment of possible bias from gender, age and smoking habits, a set of 19 metabolites was proposed to be cancer-related (out of which 3 were unknowns and 6 were partially identified as N-acetylated metabolites). As for plasma, these variations were detected regardless of disease stage and showed some dependency on histological subtype, the AdC vs. SqCC model built showing modest predictive power. In addition, preliminary external validation of the urine-based classification model afforded 100% sensitivity and 90% specificity, which are exciting results in terms of potential for future clinical application. Chapter 6 describes the analysis of urine from a subset of patients by a different profiling technique, namely, Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Although the identification of discriminant metabolites was very limited, multivariate models showed high classification rate and predictive power, thus reinforcing the value of urine in the context of lung cancer diagnosis. Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the potential of integrated metabolomics of tissues and biofluids to improve current understanding of lung cancer altered metabolism and to reveal new marker profiles with diagnostic value.

Metabonomics of human amniotic fluid for prenatal diagnostics

O trabalho apresentado nesta tese teve como principais objectivos contribuir para o conhecimento da composi����o do l��quido amni��tico humano (LA), colhido no 2�� trimestre de gravidez, assim como investigar poss��veis altera����es na sua composi����o devido �� ocorr��ncia de patologias pr��-natais, recorrendo �� metabon��mica e procurando, assim, definir novos biomarcadores de doen��as da gr��vida e do feto. Ap��s uma introdu����o descrevendo o estado da arte relacionado com este trabalho (Cap��tulo 1) e os princ��pios das metodologias anal��ticas usadas (Cap��tulo 2), seguida de uma descri����o dos aspectos experimentais associados a esta tese (Cap��tulo 3), apresentam-se os resultados da caracteriza����o da composi����o qu��mica do LA (gravidez saud��vel) por espectroscopia de resson��ncia magn��tica nuclear (RMN), assim como da monitoriza����o da sua estabilidade durante o armazenamento e ap��s ciclos de congelamento-descongelamento (Cap��tulo 4). Amostras de LA armazenadas a -20��C registaram altera����es significativas, tornando-se estas menos pronunciadas (mas ainda mensur��veis) a -70��C, temperatura recomendada para o armazenamento de LA. Foram tamb��m observadas altera����es de composi����o ap��s 1-2 ciclos de congelamento-descongelamento (a ter em conta aquando da reutiliza����o de amostras), assim como �� temperatura ambiente (indicando um per��odo m��ximo de 4h para a manipula����o e an��lise de LA). A aquisi����o de espectros de RMN de 1H de alta resolu����o e RMN acoplado (LC-NMR/MS) permitiu a detec����o de 75 compostos no LA do 2�� trimestre, 6 dos quais detectados pela primeira vez no LA. Experi��ncias de difus��o (DOSY) permitiram ainda a caracteriza����o das velocidades de difus��o e massas moleculares m��dias das prote��nas mais abundantes. O Cap��tulo 5 descreve o estudo dos efeitos de malforma����es fetais (FM) e de cromossomopatias (CD) na composi����o do LA do 2�� trimestre de gravidez. A extens��o deste trabalho ao estudo dos efeitos de patologias no LA que ocorrem no 3�� trimestre de gravidez �� descrita no Cap��tulo 6, nomeadamente no que se refere ao parto pr��-termo (PTD), pr��-eclampsia (PE), restri����o do crescimento intra-uterino (IUGR), ruptura prematura de membranas (PROM) e diabetes mellitus gestacional (GDM). Como complemento a estes estudos, realizou-se uma an��lise preliminar da urina materna do 2�� trimestre para o estudo de FM e GDM, descrita no Cap��tulo 7. Para interpreta����o dos dados anal��ticos, obtidos por espectroscopia RMN de 1H, cromatografia l��quida de ultra efici��ncia acoplada a espectrometria de massa (UPLC-MS) e espectroscopia do infravermelho m��dio (MIR), recorreu-se �� an��lise discriminante pelos m��todos dos m��nimos quadrados parciais e o m��todo dos m��nimos quadrados parciais ortogonal (PLS-DA e OPLS-DA) e �� correla����o espectral. Ap��s an��lise por valida����o cruzada de Monte-Carlo (MCCV), os modelos PLS-DA de LA permitiram distinguir as FM dos controlos (sensibilidades 69-85%, especificidades 80-95%, taxas de classifica����o 80-90%), revelando varia����es metab��licas ao n��vel do metabolismo energ��tico, dos metabolismos dos amino��cidos e gl��cidos assim como poss��veis altera����es ao n��vel do funcionamento renal. Observou-se tamb��m um grande impacto das FM no perfil metab��lico da urina materna (medido por UPLC-MS), tendo no entanto sido registados modelos PLS-DA com menor sensibilidade (40-60%), provavelmente devido ao baixo n��mero de amostras e maior variabilidade da composi����o da urina (relativamente ao LA). Foram sugeridos poss��veis marcadores relacionados com a ocorr��ncia de FM, incluindo lactato, glucose, leucina, valina, glutamina, glutamato, glicoprote��nas e conjugados de ��cido glucur��nico e/ou sulfato e compostos end��genos e/ou ex��genos (<1 ���M) (os ��ltimos vis��veis apenas na urina). No LA foram tamb��m observadas varia����es metab��licas devido �� ocorr��ncia de v��rios tipos de cromossomopatias (CD), mas de menor magnitude. Os perfis metab��licos de LA associado a pr��- PTD produziram modelos que, apesar do baixo poder de previs��o, sugeriram altera����es precoces no funcionamento da unidade fetoplacent��ria, hiperglic��mia e stress oxidativo. Os modelos obtidos para os grupos pr��- IUGR pr��- PE, pr��- PROM e pr��-diagn��stico GDM (LA e urina materna) registaram baixo poder de previs��o, indicando o pouco impacto destas condi����es na composi����o do LA e/ou urina do 2�� trimestre. Os resultados obtidos demonstram as potencialidades da an��lise dos perfis metab��licos do LA (e, embora com base em menos estudos, da urina materna) do 2�� trimestre para o desenvolvimento de novos e complementares m��todos de diagn��stico, nomeadamente para FM e PTD.