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- Antioxidant activity and in vitro inhibition of tumor cell growth by leaf extracts from the carob tree (Ceratonia siliqua)Publication . Custódio, Luísa; Fernandes, Eliana; Escapa, Ana Luísa; López-Avilés, Sandra; Fajardo, Alba; Aligué, Rosa; Alberício, Fernando; Romano, AnabelaThe methanol leaf extracts of female cultivars of the carob tree [Ceratonia siliqua L. (Fabaceae)] and of hermaphrodite and male trees were investigated for their contents of phenolic compounds, their in vitro antioxidant activity, measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and linoleic acid system assays, and their in vitro tumor growth inhibition on HeLa cells. The different cultivars and trees showed high levels of phenols, and considerable variations in the amount of these compounds. The extracts showed significant radical scavenging activity (RSA), which was not significantly affected by the gender of the tree. From the female cultivars tested, Galhosa exhibited the highest RSA. Gender significantly affected the antioxidant activity of the extracts measured by the linoleic acid system assay, and males and hermaphrodites showed the highest activities. The extracts displayed a remarkable ability to inhibit tumor cell proliferation, and their bioactivity varied with different cultivars or trees tested. Extracts from male and hermaphrodite trees exhibited higher capacity to inhibit the proliferation of HeLa cells than the female cultivars.
- Analysis of the volatiles emitted by whole flowers and isolated flower organs of the carob tree using HS-SPME-GC/MSPublication . Custódio, Luísa; Serra, Hugo; Nogueira, José Manuel F.; Gonçalves, Sandra; Romano, AnabelaThe volatiles emitted by fresh whole flowers and isolated flower organs of male, female, and hermaphrodite carob trees (Ceratonia siliqua L.; Leguminosae)were analyzed by headspace solid-phase microextraction followed by capillary gas chromatography and mass spectrometry. The headspace of carob flowers is mainly constituted of high amounts of monoterpenes and sesquiterpenes, and more than 25 compounds were identified. The gender and cultivar affected both the qualitative profile and the relative abundances of the volatiles of whole flowers and isolated floral organs. Linalool and its derivatives (cis-linalool furan oxide, 2,2,6-trimethyl-3-keto-6-vinyltetrahydropyran, cis-linalool pyran oxide, and trans-linalool furan oxide), a-pinene, and a-farnesene were the dominant volatiles. Female flowers had a higher diversity of volatile compounds than males and hermaphrodites, but a lower abundance of the major ones. Similarly, the floral scent of female flowers of cv. Mulata had a higher content of volatiles but a lower abundance of the major ones, when compared to cv. Galhosa. In each of the three gender types of flowers, the nectary disks seemed to be the major source of volatiles.
- Antioxidant and cytotoxic activities of carob tree fruit pulps are strongly influenced by Gender and cultivarPublication . Custódio, Luísa; Fernandes, Eliana; Escapa, Ana Luísa; Fajardo, Alba; Aligué, Rosa; Alberício, Fernando; Neng, Nuno Rosa; Nogueira, José Manuel F.; Romano, AnabelaExtracts from fruit pulps of six female cultivars and two hermaphrodite Portuguese carob trees [(Ceratonia siliqua L., Fabaceae)] exhibited strong antioxidant activity and were rich in phenolic compounds. The extracts decreased the viability of different human cancer cell lines on a dose- and time-dependent manner. Gender and cultivar significantly influenced the chemical content and the biological activities of the extracts. Extracts from hermaphrodite trees had a higher content of phenolic compounds, and exhibited higher antioxidant and cytotoxic activities. Among. females, cv. Aida had the highest radical scavenging activity and total content of phenolics, Mulata the highest capacity to inhibit lipid oxidation and Gasparinha the strongest cytotoxic activity on HeLa cells. The decrease in cell viability was associated with apoptosis on HeLa and MDA-MB-231 lines. (+)-Catechin and gallic acid (GA) were the main compounds identified in the extracts, and GA contributed to the antioxidant activity. Our results show that the antioxidant and cytotoxic activities of carob tree fruit pulps are strongly influenced by gender and cultivar, and provide new knowledge about the advantages of hermaphrodite trees over female cultivars, namely, as a source of compounds with biological interest, which may represent an increase of their agronomic interest.
- In vitro morphogenesis in zygotic embryo cultures of carob tree (Ceratonia siliqua L.)Publication . Custódio, Luísa; Romano, AnabelaIn this study, organogenic cultures were induced from zygotic embryos of carob tree. Basal medium consisted of Murashige & Tucker (1969), supplemented with 0.5 or 1 mg L-1 of 2,4-dichlorophenoxyacetic acid alone or combined with 0.5, 1, 2, 3 and 4 mg L-1 of kinetin. Calli initiation occurred in all the hormonal combinations tested. The zygotic embryo cultures differentiated two types of regenerants: somatic embryos (SEs) and shoots, with a maximum frequency of 30 and 100%, respectively. SEs exhibited morphological abnormalities, such as fusion and absence of cotyledons, and showed secondary embryogenesis. The regenerated shoots were rooted, hardened and transferred to the soil.
- Phenolic composition, antioxidant potential and in vitro inhibitory activity of leaves and acorns of Quercus suber on key enzymes relevant for hyperglycemia and Alzheimer's diseasePublication . Custódio, Luísa; Patarra, João; Albericio, F.; Neng, N. D.; Florencio Nogueira, J. M. F.; Romano, AnabelaThis work reports the in vitro antioxidant and inhibitory activities of hexane, methanol and water extracts of cork oak (Quercus suber) against acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), a-amylase and a-glucosidase. The total content of phenolics (TPC), tannins (TTC) and flavonoids (TFC), and the HPLC profile of the main phenolic compounds present in the extracts, were also determined. In the 2,2-dipheny1-1-picrylhydrazyl (DPPH) assay the best results were obtained with the methanol and water extracts from both leaves and acorns (41-49%). In the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) method the highest activity was observed with the methanol and leaf water extracts (63-71%). The most significant effects on cholinesterase activity were obtained with the methanol leaf extract at the concentration of 1 mg/mL, yielding inhibition values of 79 and 80% on AChE and BuChE, respectively. No relevant activity was detected against a-amylase, but samples significantly inhibited a-glucosidase from baker's yeast, and the best results were obtained with the water and methanol leaf extracts with values of 97 and 89%, respectively, higher than the positive controls used (acarbose and glucobay). The methanol leaf extract had the highest TPC (211 mg GAE/g, DW) and TFC (8.2 mg RE/g, DW), whereas the water extracts had the highest tannin levels (87 CE/g, DW). The main compound in the methanol leaf extract was gentisic acid (24.3 mg/g, DW). Our results suggest that leaves and acorns from cork oak contain compounds useful for alleviating symptoms associated with Alzheimer disease and other neurodegenerative ailments as well as diabetes. (C) 2014 Elsevier B.V. All rights reserved.
- Sex and developmental stage of carob flowers affects composition of volatilesPublication . Custódio, Luísa; Nogueira, José Manuel F.; Romano, AnabelaThe aim of this work was to evaluate the volatiles composition in the scent emitted in vivo by the three types of carob tree (Ceratonia siliqua L.) flowers, during the different developmental stages. Additionally, an overview of some morphological aspects of carob flowers was also done. Carob trees may be male, female, hermaphrodite or exhibit polygamous inflorescences. The androecium of male flowers consists of a group of 5-7 stamens, inserted peripherically around the receptacle. The gynoecium of female flowers is apocarpous. Hermaphrodite flowers are a combination of male and female types. During the flower development six stages were identified. The volatile compounds of flowers were analysed by headspace solid-phase microextraction followed by capillary gas chromatography and mass spectrometry detection (HS-SPME-CGC/MSD). More than 25 compounds were identified, and flowers in stages III and IV showed higher abundances. In male and hermaphrodite flowers the total volatile compounds were more abundant in stage III, and in female flowers in stage IV. Linalool and trans linalool oxide were the dominant volatiles and there were remarkable differences in those compounds between different types of flowers and during the developmental stages. The results observed were discussed in terms of their possible implication on pollination efficiency.
- Sapwood of carob tree (Ceratonia siliqua L.) as a potential source of bioactive compoundsPublication . Custódio, Luísa; Escapa, Ana Luísa; Patarra, João; Aligué, Rosa; Alberício, Fernando; Neng, Nuno Rosa; Nogueira, José Manuel F.; Romano, AnabelaMethanol (ME) and hot water extracts (WE) of carob tree sapwood (Ceratonia siliqua L.) exhibited high antioxidant activity and were rich in phenolic compounds, with the main compounds identified by HPLC/DAD as gentisic acid and (-)-epicatechin. The ME displayed a high in vitro antitumor activity against human tumoural cell lines and reduced intracellular ROS production by HeLa cells after treatment with H2O2. (-)-Epicatechin was shown to contribute to the cytotoxic activity of the ME. This is the first report on the biological activity of carob tree sapwood.
- Phytochemical Profile, Antioxidant and Cytotoxic Activities of the Carob Tree (Ceratonia siliqua L.) Germ Flour ExtractsPublication . Custódio, Luísa; Escapa, A. L.; Fernandes, E.; Fajardo, A.; Aligue, R.; Albericio, F.; Neng, N. R.; Nogueira, J. M. F.; Romano, AnabelaThis work aimed to evaluate the phytochemical content and to determine the antioxidant and cytotoxic activities of methanol extracts of the carob tree (Ceratonia siliqua L.) germ flour. The extracts were rich in phenolic compounds, had considerable antioxidant activity, and reduced the viability of cervical (HeLa) cancer cells. The chemical content and the biological activities of the extracts were significantly affected by gender and cultivar. Female cultivar Galhosa had the highest levels of phenolic compounds, and the highest antioxidant activity. Extracts from the hermaphrodite trees and from the female cultivars Galhosa and Costela/Canela exhibited the highest cytotoxic activity. The most abundant compound was theophylline. The phenolic content was correlated to both antioxidant and cytotoxic activities. Our findings provide new knowledge about the health implications of consuming food supplemented with carob germ flour.
- In vitro cytotoxic effects and apoptosis induction by a methanol leaf extract of carob tree (Ceratonia siliqua L.)Publication . Custódio, Luísa; Escapa, Ana Luísa; Fernandes, Eliana; Fajardo, Alba; Aligué, Rosa; Alberício, Fernando; Neng, Nuno Rosa; Nogueira, José Manuel F.; Romano, AnabelaThis research evaluated the in vitro apoptotic inducing properties of a methanol leaf extract of carob tree (Ceratonia siliqua L.) on a human cervical adenocarcinoma cell line (HeLa). The cell viability effect on a prostate (DU-145), breast (MDA-MB-231) and colon cell line (HCT-166) was also assessed. The effect of the extract on reactive oxygen species (ROS) production by HeLa cells was studied, and a phytochemical evaluation was made by high performance liquid chromatography with diode array detection (HPLC). Total mean yield of identified phenols was 261.1 mg/g DW, and (+)-catechin was the major compound (76.8 mg/g DW). The extract decreased cell viability in a dose-and time-dependent manner, and a more pronounced effect on HeLa line was observed. In vitro cytotoxic activity was associated with apoptosis, but not to the increase of ROS production. Among the tested compounds identified in the extract, the highest activity was detected with gallic acid (GA), (+)-catechin and quercetin, with reductions in HeLa cells viability down to 8.0, 11.9 and 27.1%, respectively. This is the first report on the apoptotic activity of a leaf extract of carob tree on a human cancer cell line, suggesting that it may be a potential source of chemopreventive compounds.
- Floral analysis and seasonal dynamics of mineral levels in carob tree leavesPublication . Custódio, Luísa; Correia, P. J.; Martins-Loução, M. A.; Romano, AnabelaThe mineral concentration of flowers and the seasonal fluctuation of macro- [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)] and micronutrients [iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu)] in leaves of male, female, and hermaphroditic carob trees (Ceratonia siliqua L.) were studied. The nutrient dynamics were linked not only to phenological events, but also to the gender of the trees. The females were able to allocate more nutrients to leaves than male trees, even though male flowers were richer in particular elements such as N and Zn. The hermaphrodites supported the development of both inflorescences and fruits with a lower seasonal variation and a lower leaf nutrient concentration, as compared to the other genders, which may indicate a more efficient use of resources. Flowers had, in general, a higher concentration of N, P, and K, and a lower Mn concentration than in leaves. Flowers of the females had a lower nutrient concentration compared to males or hermaphrodites.