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The aim of this study was to determine the chemical profile of the essential oils (EOs) of three Moroccan lavender species (Lavandula pedunculata, LP; Lavandula angustifolia, LA; and Lavandula maroccana, LM) and to investigate, for the first time, the synergistic effect of the optimal mixture of the EOs with conventional antibiotic ciprofloxacin against three pathogenic foodborne bacteria. Gas chromatography/mass spectrometry analysis showed that eucalyptol (39.05%), camphor (24.21%) and borneol (8.29%) were the dominant compounds of LA-EO. LP-EO was characterized by the abundance of camphor (74.51%) and fenchone (27.06%), whereas carvacrol (42.08%), camphor (17.95%) and fenchone (12.05%) were the main constituents of LM-EO. EOs alone or combined showed a remarkable antimicrobial activity against the tested bacteria with minimum inhibitory concentrations (MICs) ranging from 3.53 mg/mL to 15.96 mg/mL. The optimal mixture, calculated using a mixture design, corresponded to 19% LA, 38% LP and 43% LM. All combination of the EOs and the best EO mixture with ciprofloxacin exhibited a total synergism with fractional inhibitory concentration index (FICI) values ranging from 0.27 to 0.37. The best EO mixture showed the highest gain of 128 fold, especially against Salmonella spp., more than that found testing the EOs separately. These findings should be taken into consideration for a possible application in the pharmaceutical and food industries.

Lavandula pedunculata (Mill.) Cav. (LP) is one of lavender species traditionally used in Morocco to prevent or cure diabetes, alone or in the form of polyherbal preparations (PHP). Therefore, the primary objective of this study was to test the antihyperglycemic effect of the aqueous extract of LP, alone and in combination with Punica granatum L. (PG) and Trigonella foenum-graecum L. (FGK). The secondary objective was to explore some mechanisms of action on the digestive functions. The antihyperglycemic effect of the aqueous extract of LP, alone and in combination with PG and FGK, was studied in vivo using an oral glucose tolerance test (OGTT). In addition, LP extract was tested on the activities of some digestive enzymes (pancreatic α-amylase and intestinal α-glucosidase) in vitro and on the intestinal absorption of glucose ex vivo using a short-circuit current (Isc) technique. Acute and chronic oral administration of LP aqueous extract reduced the peak of the glucose concentration (30 min, p < 0.01) and the area under the curve (AUC, p < 0.01). The effect of LP + PG was at the same amplitude to that of the positive control Metformin (MET). LP aqueous extract inhibited the pancreatic α-amylase with an IC50 almost identical to acarbose (0.44 ± 0.05 mg/mL and 0.36 ± 0.02 mg/mL, respectively), as well as the intestinal α-glucosidase, (IC50 = 131 ± 20 µg/mL) and the intestinal glucose absorption (IC50 = 81.28 ± 4.01 µg/mL) in concentration-dependent manners. LP aqueous extract exhibited potent actions on hyperglycemia, with an inhibition on digestive enzymes and glucose absorption. In addition, the combination with PG and FGK enhanced oral glucose tolerance in rats. These findings back up the traditional use of LP in type 2 diabetes treatment and the effectiveness of the alternative and combinative poly-phytotherapy (ACPP).

This work consists of the comparison of the chemical composition, the antioxidant activity, and the antimicrobial effect of two species of the genus Lavandula essential oils (EOs) and extracts (hydroethanolic, aqueous and decocted), that were collected from the region of Ifrane in Morocco "Lavandula angustifolia and Lavandula peduncultata". Gas chromatographic analyzes coupled with mass spectrometry of the studied EOs showed that L. angustifolia contains camphor and linalool as majority compounds with 20,17% and 12,41% respectively, while camphor (41,15%) and fenchone (15,70%) are the major compounds of L. peduncultata. The dosage of hydroethanolic, aqueous and decocted extracts showed that the content of polyphenols and flavonoids is higher in the hydroethanolic extracts of L. pedunculata, while L. angustifolia contains the highest content of tannins. The determination of the antioxidant activity was carried out using FRAP and DPPH methods, the last method revealed that the EO of L.angustifolia is less effective (IC50=7,98 mg/ml) than that of L. pedunculata (EC50=7,47 mg/ml) while, there was no great divergence concerning the IC50 values of the hydroethanolic and decocted extracts of the two species. As for the aqueous extract, the value recorded by L. pedunculata was slightly higher than that of L. angustifolia. Finally, tests of the antimicrobial activity of EOs and extracts of the two species were carried out on six bacteria and five fungal strains whose antimicrobial power was remarkable. As a conclusion, the two Lavenders’extracts can be considered as alternative sources of powerful natural antioxidants, antifungals and antibiotics.

The present study aimed at evaluating the effectiveness of different natural deep eutectic solvents (NADES) on the extraction of phenolic compounds from Lavandula pedunculata subsp. lusitanica (Chaytor) Franco, on the antioxidant activity, and acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase (Tyr) inhibitory capacities. Ten different NADES were used in this research and compared with conventional solvents. Ultrasound-assisted extraction (UAE) for 60 min proved to be the best extraction condition, and proline:lactic acid (1:1) and choline chloride:urea (1:2) extracts showed the highest total phenolic contents (56.00 ± 0.77 mgGAE/gdw) and antioxidant activity [64.35 ± 1.74 mgTE/gdw and 72.13 ± 0.97 mgTE/gdw in 2.2-diphenyl-1- picrylhydrazyl (DPPH) and 2.20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods, respectively]. These extracts also exhibited enzymes inhibitory capacity particularly against Tyr and AChE. Even so, organic acid-based NADES showed to be the best extractants producing extracts with considerable ability to inhibit enzymes. Twenty-four phenolic compounds were identified by HPLC-HRMS, being rosmarinic acid, ferulic acid and salvianolic acid B the major compounds. The results confirmed that the combination of UAE and NADES provide an excellent alternative to organic solvents for sustainable and green extraction, and have huge potential for use in industrial applications involving the extraction of bioactive compounds from plants. Grant SFRH/BD/145243/2019, UIDB/05183/2020 info:eu-repo/semantics/publishedVersion

Some autochthones plant species with aromatic and medicinal properties are able to naturally colonize contaminated soils from mining areas from Iberian Pyrite Belt contributing to their rehabilitation. A study was carried out in order to characterize and valorise an autochthones species, which has adequate ecophysiological behaviours for phytostabilization of mining areas, as new sources of bioactive substances. The main aims of this study were to: i) characterise the phytochemical profile of the bioextracts from shoots of L. pedunculata growing in soils from São Domingos mining area and a control area; and ii) evaluate the influence of potentially hazardous elements (PHEs) accumulated in the shoots on the quality of the bioextracts.Composite samples of soils, developed on mine wastes and/or host rocks, as well as C Lavandula pedunculata shoots were collected in São Domingos mine (Iberian pyrite Belt, SE of Portugal) and in a reference area with non-contaminated soils and the same climatic conditions. Classical characterisation of soils and total concentrations of potentially hazardous elements in soils and plant shoots were determined. The bioextracts from Lavandula pedunculata shoots were obtained by an accelerated solvent extractor, and the compounds were analysed by GC-MS. Extracts were extracted with hexane and major components were quantified.The total concentrations of some potentially hazardous elements (e.g. As, Cu, Pb and Zn) were higher in soils from São Domingos than in reference area. However, soils from São Domingos are considered as contaminated with As, Cu, Pb and Sb for agriculture and residential/parkland uses. Concentrations of the PHEs (excepted Cr and Mn) in the shoots collected in São Domingos mine were higher than in the non-contaminated areaIn the L. pedunculata extracts, obtained in the single extraction with hexane, were identified 34 compounds accounting between 79 and 89 % of the total identified compounds. Camphor was the major component in all extracts but Fenchone, eucalyptol, verbenone, bornyl acetate, borneol and linalool oxide cis also showed considerable amounts. All these compounds present economic interest. Some variation was obtained in the qualitative composition of the L. pedunculata extracts but, in general, it was not clear the differentiation between populations and, consequently, soil contamination level and concentrations of the potentially hazardous elements in shoots. Environmental rehabilitation of mining areas from Iberian Pyrite Belt with this species can provide economic valorisation by the exploration of this plant-based product for fragrance and pharmaceutical industries.Acknowledgment: This research was supported by Portuguese funds, through Fundação para a Ciência e Tecnologia within the scope of the project UID/AGR/04129/202, and Xunta de Galicia (GRC2014/003).

Spontaneous lavender growing in uncultivated fields in Portugal have been used in traditional medicine for internal and external uses. The essential oils (EOs) of Lavandula stoechas subsp. luisieri are characterized by the presence of trans-α-necrodyl acetate and trans-necrodol. These EOs are able to prevent the generation and deposition of neurotoxic β-amyloid peptide in Alzheimer’s disease. The EOs also present antibacterial, anti-fungal, anti-Leishmania, antioxidant, anti-inflammatory and antifeedant effects. In the case of hydrodistillation, the predominant compound of Lavandula viridis EO was 1,8-cineole, nevertheless in the case of supercritical fluid extraction, the main constituent was camphor. In in vitro shoots EOs, 1,8-cineole and α-pinene were the most important compounds. The EOs presented anti-fungal activity particularly against Cryptococcus neoformans and dermatophytes. The antioxidant and anti-protozoal activities of L. viridis EOs were lower than L. stoechas subsp. luisieri EOs, with hydrodistillation being the best method for obtaining samples with higher antioxidant and anti-acetylcholinesterase activities. The presence of fenchone, 1,8-cineole and camphor was a common trace of the Lavandula pedunculata subsp. pedunculata EOs and in in vitro axillary shoots EOs. Lavandula pedunculata subsp. lusitanica EOs were predominantly constituted of fenchone and camphor. The antioxidant activity of L. pedunculata subsp. lusitanica EOs was poorer than other Lavandula EOs from Portugal.

This study aimed to: i) evaluate the accumulation and translocation patterns of potentially hazardous elements into the Lavandula pedunculata and their influence in the concentrations of nutrients; and ii) compare some physiological responses associated with oxidative stress (concentration of chlorophylls (Chla, Chlb and total), carotenoids, and total protein) and several components involved in tolerance mechanisms (concentrations of proline and acid-soluble thiols and total/specific activity of catalase (CAT) and superoxide dismutase (SOD)), in plants growing in soils with a multielemental contamination and non-contaminated. Composite samples of soils, developed on mine wastes and/or host rocks, and L. pedunculata (roots and shoots) were collected in Sao Domingos mine (SE of Portugal) and in a reference area with non-contaminated soils, Corte do Pinto, with the same climatic conditions. Sao Domingos soils had high total concentrations of several hazardous elements (e.g. As and Pb) but their available fractions were small (mainly

In order to restore the plant cover, improve ecosystem services and decrease the environmental risk of two mine wastes (gossan and sulfide-rich wastes), an integrated biotechnology was tested at long-term and greenhouse conditions. This integrated biotechnology involves the natural isolation of sulfide-rich wastes through an alkaline barrier, covered by designed Technosols (both of them derived from mining and agro-industrial wastes) and a plant cover with Lavandula pedunculata and Cistus ladanifer. Technosols allowed significant germination (L. pendunculata: 16-18%; C. ladanifer: 5-11%) and biomass production of both species (g FW/pot; Roots: 16.3-30.9, Shoots: 41.2-76.4 depending on species and Technosol). In the control was reached the lowest germination (3%) and seedlings died after 40 days, so the improvement of the chemical characteristics of the surface layer, i.e. the implementation of the designed Technosols, is essential to ensure good vegetative development. No visual symptoms of nutritional deficiency and phytotoxicity neither element concentrations above hazardous levels for domestic animals intake were observed in those plants species. The alkaline barrier's components stabilise the sulfide-rich wastes by decreasing the oxidation and capillary rise of acid solutions that are rich in metals/metalloids. The limestone gravel showed, in some places, a thin layer of salts from alunite-jarosite group and metal-oxyhydroxides. As an outcome, the biotechnology was efficient and sustainable allowing the combined rehabilitation of both mine wastes at long-term.

We investigated the metabolic profile and biological activities of the essential oil and polar extracts of Lavandula pedunculata subsp. lusitanica (Chaytor) Franco collected in south Portugal. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that oxygen-containing monoterpenes was the principal group of compounds identified in the essential oil. Camphor (40.6%) and fenchone (38.0%) were found as the major constituents. High-performance liquid chromatography with diode array detection (HPLC-DAD) analysis allowed the identification of hydroxycinnamic acids (3-O-caffeoylquinic, 4-O-caffeoylquinic, 5-O-caffeoylquinic and rosmarinic acids) and flavones (luteolin and apigenin) in the polar extracts, with rosmarinic acid being the main compound in most of them. The bioactive compounds from L. pedunculata polar extracts were the most efficient free-radical scavengers, Fe2+ chelators and inhibitors of malondialdehyde production, while the essential oil was the most active against acetylcholinesterase. Our results reveal that the subspecies of L. pedunculata studied is a potential source of active metabolites with a positive effect on human health. © 2013 Elsevier Ltd. All rights reserved.

Mestrado em cooperação com a Universidade de Salamanca As plantas foram usadas pelos nossos ancestrais, não só como alimento, mas também como uma solução terapêutica. Tendo em conta os seus benefícios para a saúde e, de acordo com a Organização Mundial de Saúde (OMS), a sua utilização como ingrediente na industria farmacêutica e alimentar, aumentou significativamente, ao longo do tempo. Os compostos bioativos são um grupo importante presente nas plantas, atuando na prevenção de doenças humanas associadas ao stresse oxidativo. O objetivo deste estudo foi caracterizar a composição fenólica e avaliar a bioatividade de várias amostras de Lavandula pedunculata (Mill.) Cav, cujas sementes são provenientes de diversas populações naturais de diferentes regiões de Portugal, e comparar os seus extratos aquosos e hidroetanólicos. A partir da análise HPLC-DAD-ESI/MSn foram identificados treze compostos, sendo o ácido salvianólico B, o ácido rosmarínico e luteolina-7-O-glucurónido, os principais compostos presentes. O extrato aquoso de L. pedunculata revelou maior potencial antioxidante, que pode estar relacionado com a sua maior concentração em compostos fenólicos; no entanto, o extrato hidroetanólico demostrou maior potencial anti-inflamatório e capacidade anti-proliferativa. Assim, este estudo destaca os efeitos bioativos desta espécie e abre possibilidades para o seu uso em formulações alimentares e farmacêuticas. No entanto, existem potenciais diferenças nestas propriedades de acordo com a origem geográfica do material vegetal, visto que as amostras do Alentejo apresentaram, em geral, melhores resultados para todas as bioatividades, comparativamente com as amostras de Trás-os-Montes. Plants were used by our ancestors, not only as food, but also as a therapeutic solution. Taking into account their health benefits and, according to the World Health Organization (WHO), their use as an ingredient in the pharmaceutical and food industries has increased significantly, over time. Bioactive compounds are an important group present in plants, acting in the prevention of human diseases associated with oxidative stress. The aim of this study was to characterize the phenolic composition and evaluate the bioactivity of several samples of Lavandula pedunculata (Mill.) Cav, whose seeds were provided from various natural populations of different regions of Portugal, and compare aqueous and hydroethanolic extracts. From the HPLC-DAD-ESI/MSn analysis thirteen compounds were identified, being salvianolic acid B, rosmarinic acid and luteolin-7-O-glucuronide, the main compounds present. L. pedunculata aqueous extract revealed a higher antioxidant potential, which could be related to its higher concentration in phenolic compounds; however, the hydroethanolic extract showed a higher anti-inflammatory potential and anti-proliferative capacity. Thus, this study highlights the bioactive effects of this species and opens possibilities for uses in food and pharmaceutical formulations. However, there are potential differences in such properties according to geographical origin of plant material, as in general, the samples from Alentejo presented higher results in all the bioactivities, compared with Trás-os-Montes samples. Este trabalho foi financiado pelo programa FEDER-Interreg España-Portugal através do projeto 0377_Iberphenol_6_E.

In dry-summer seasonal climates, the beginning of the rainy season can prompt germination under different temperatures, depending on altitude. Understanding germination responses to temperature with altitude is important in fire-prone environments for species regenerating after fire from seeds (seeders), particularly under changing climate. Here we investigated the role of temperature in four Mediterranean seeder shrubs from Central Spain. Seeds from 17 sites (285–1,253 m altitude), of two hard-seeded nanophanerophytes (Cistus ladanifer and C. salviifolius) and two soft-seeded chamaephytes (Lavandula pedunculata and Thymus mastichina) were investigated. Intact and heat shock treated seeds were set to germinate under four temperature regimes, including a treatment simulating future warming. GLM with binomial or gamma functions were used to test treatment effects using altitude as a covariate. Altitude was a significant covariate only in L. pedunculata. Temperature did not affect final germination in either Cistus, but it significantly affected T. mastichina, and interacted with altitude in L. pedunculata, whereby the higher the altitude the less it germinated with decreasing temperature. Germination speed (T50) was lower at colder temperatures in all but C. salviifolius that was insensitive to our treatments. Heat shock significantly increased final germination in both Cistus and T. mastichina, but did not interact with temperature or altitude. We conclude that germination response to temperature, including varying sensitivity with altitude, differed among these species; thus, changes in the timing of the onset of the rainy season will diversely affect populations at various altitudes. We discuss our results in a context of changing climate and fire.

The aim of this study was to characterize the phenolic composition and evaluate the bioactivity of several samples of Lavandula pedunculata (Mill.) Cav, and to compare aqueous and hydroethanolic extracts. Plant materials were obtained by growing some accessions (seed samples) of various wild populations from different regions of Portugal conserved at the Portuguese Genebank in Braga. Phenolic compounds were analised by HPLC-DAD-ESI/MSn, antioxidant potential through in vitro assays (DPPH radical scavenging activity, reducing power and inhibition of lipid peroxidation), cytotoxicity on tumor cells (MCF-7, NCI-H460, HeLa and HepG2) and non-tumor (PLP2) cells, anti-inflammatory activity in rat RAW 264.7 macrophages, by the ability to inhibit NO production and antimicrobial potential by the microdilution method with INT dye (iodonitrotetrazolium chloride). Thirteen compounds were identified, being salvianolic acid B, rosmarinic acid and luteolin-7-O-glucuronide, the main compounds present, with values ranging between 44.3⁻582, 50.9⁻550, and 24.36⁻101.5 mg/g extract, respectively. L. pedunculata aqueous extract revealed a higher antioxidant potential (EC50 values between 14 to 530 μg/mL), which could be related to its higher concentration in phenolic compounds; however, the hydroethanolic extract showed a higher anti-inflammatory (lower EC50 values than 124 μg/mL) potential and antiproliferative capacity (lower GI50 values than 34 μg/mL). Thus, this study highlights the bioactive effects of this species and opens up possibilities of uses in food and pharmaceutical formulations. However, there are potential differences in such properties according to geographical origin of plant material, as in general, the samples from Alentejo presented higher results in all the bioactivities, compared with Trás-os-Montes samples. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Program PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros and R. Calhelha contracts. The authors are grateful to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E. info:eu-repo/semantics/publishedVersion

The chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller) Cav., harvested in North and Central Portugal, were investigated. The essential oils were isolated by hydrodistillation and analyzed by GC and GC/MS. The minimal-inhibitory concentration (MIC) and the minimal-lethal concentration (MLC) of the essential oils and of their major constituents were used to evaluate the antifungal activity against different strains of fungi involved in candidosis, dematophytosis, and aspergillosis. The oils were characterized by a high percentage of oxygenated monoterpenes, the main compounds being 1,8-cineole (2.4-55.5%), fenchone (1.3-59.7%), and camphor (3.6-48.0%). Statistical analysis differentiated the essential oils into two main types, one characterized by the predominance of fenchone and the other one by the predominance of 1,8-cineole. Within the 1,8-cineole chemotype, two subgroups were well-defined taking into account the percentages of camphor. A significant antifungal activity of the oils was found against dermatophyte strains. The essential oil with the highest content of camphor was the most active with MIC and MLC values ranging from 0.32-0.64 microl/ml.

International audience; Lavandula pedunculata (Mill.) Cav. subsp. lusitanica, L. stoechas L. subsp. stoechas and L. viridis l'Hér. are three lavender taxa that belong to the botanical section Stoechas and are widely used as aromatherapy, culinary herb or folk medicine in many Mediterranean regions. The analysis of their bioactive volatile constituents revealed the presence of 124 substances, the most abundant being the bicyclic monoterpenes fenchone, camphor and 1,8-cineole that give these three species their respective chemotypes. Most noteworthy was fenchone which, with its reduced form fenchol, made 48% of the total volatile constituents of L. pedunculata whilst present at 2.-% in L. stoechas and undetectable in L. viridis. In order to provide a molecular explanation to the differences in volatile compounds of these 3 species, two monoterpene synthases (monoTPS) and one sesquiterpene synthase (sesquiTPS) were cloned in L. pedunculata and functionally characterized as fenchol synthase (LpFENS), α-pinene synthase (LpPINS) and germacrene A synthase (LpGEAS). The two other lavender species contained a single orthologous gene for each of these three classes of TPS with similar enzyme product specificities. Expression profiles of FENS and PINS genes matched the accumulation profile of the enzyme products unlike GEAS. This study provides one of the rare documented cases of chemotype modification during plant speciation via changes in the level of plant TPS gene expression, and not functionality.

Mestrado em Engenharia Agronómica - Proteção de Plantas - Instituto Superior de Agronomia The Portuguese flora is very rich in aromatic and medicinal plants, among which are Lavandula stoechas subsp. luisieri and L. pedunculata. The present study was aimed to evaluate the potential antimicrobial activity of five plant extracts and essential oils (EO) obtained from these species, with respect to human and plant pathogens. The EO were extracted from dried material by hydrodistillation and the extracts by a solvent system of increasing polarity. To evaluate the activity of samples against human pathogens the microdilution method was used and determined the values of minimum inhibitory concentration (MIC) at different concentrations: 7.5, 15.5, 31, 62.5, 125, 250 and 500 μg/mL. Results were considered positives for MIC values < 100 μg/mL. Lavandula species showed different antimicrobial properties, attributed to differences in the chemical composition of the respective EO and extracts. For phytopathogenic bacteria a similar procedure was followed. Regarding the fungi, the activity of EO and extracts was evaluated by the disc diffusion method on solid medium inoculated with spore suspensions or mycelial discs at sample concentration of 1 mg/mL and 2 mg/mL, respectively. None of the plant pathogens was inhibited. This study tends to confirm the use of EO and plant extracts to control different types of microbial pathogens

This thesis aims the discovery of valuable plant compounds and to provide a deep and detailed scientific perspective of the following Portuguese aromatic plants: Lavandula viridis L’Hér, Lavandula pedunculata subsp. lusitanica (Chaytor) Franco and Thymus lotocephalus G. López & R. Morales. Volatile and phenolic compounds were recovered by different techniques and extraction solvents and their antioxidant and anti-cholinesterase activities were investigated (Chapters 2 and 3). In vitro cultures were used as alternative sources of phytochemicals, and the results were compared with wild plants. Furthermore, the effect of cyclodextrins on the antioxidant activity and storage stability of the essential oils was evaluated (Chapter 4). Finally, the neuroprotective potential (Chapters 5) and the influence of in vitro digestion on the biological activity of L. viridis extracts and rosmarinic acid (Chapter 6) were investigated. The supercritical fluid extraction technique generated the highest extraction yields in comparison with hydrodistillation, but a larger number of components were identified by hydrodistillation. The chemical analysis highlighted the abundance of oxygen-containing monoterpenes in all the essential oils and in almost all supercritical fluid extracts, and allowed the identification of phenolic acids and flavonoids in the polar extracts. The water:ethanol mixture was the most efficient solvent for the extraction of phenolic compounds followed by infusion. The in vitro cultures accumulated much higher contents of rosmarinic acid than wild plants. The polar extracts were the most efficient free radical scavengers, Fe2+ chelators and inhibitors of lipid peroxidation, while the essential oils were the most active against cholinesterases. Cyclodextrins produced a remarkable enhancement on the antioxidant activity and storage stability of the essential oils. L. viridis extracts attenuated the neurotoxicity induced by H2O2 and their biological activity was assured after in vitro digestion. This study provides relevant knowledge about the chemical composition and biological activity of three Portuguese species. A doença de Alzheimer é uma doença neurodegenerativa caracterizada pela deterioração progressiva e irreversível de diversas faculdades cognitivas e que afeta cerca de 35,6 milhões de pessoas em todo o mundo. Até ao momento, a doença permanece sem cura. A terapia disponível concentra-se apenas na atenuação dos sintomas característicos e a complexidade da patogénese constitui um obstáculo para o desenvolvimento de novos fármacos. Para além do défice progressivo e irreversível do sistema colinérgico, são claras as evidências do envolvimento do stresse oxidativo na doença. As plantas produzem compostos com interesse medicinal, e são uma fonte infindável de compostos biologicamente ativos. A Flora Portuguesa é, neste sentido, particularmente interessante pela sua riqueza em espécies aromáticas e medicinais. Esta tese pretende contribuir para a pesquisa de compostos biologicamente ativos a partir das seguintes espécies aromáticas de Portugal: Lavandula viridis L’Hér, Lavandula pedunculata subsp. lusitanica (Chaytor) Franco e Thymus lotocephalus G. López & R. Morales. Estas espécies são usadas como condimentos ou para fins terapêuticos na medicina tradicional, no entanto, o seu potencial biológico ainda não foi totalmente explorado. A primeira fase deste trabalho, descrita nos Capítulos 2 e 3, visou avaliar o efeito das técnicas e solventes de extração no rendimento e atividade biológica das espécies em estudo. Existem várias técnicas disponíveis para a extração de compostos vegetais as quais têm que ser adaptadas aos metabolitos de interesse. Neste trabalho a fração volátil foi obtida por hidrodestilação e extração por fluidos supercríticos, e os compostos fenólicos foram extraídos por maceração, infusão e soxhlet. Para além do material vegetal colhido no habitat natural, foram também utilizadas culturas in vitro de L. viridis e T. lotocephalus. Os resultados indicam que a extração por fluidos supercríticos permitiu maiores rendimentos de extração comparativamente à hidrodestilação, e no que diz respeito aos extratos polares, os rendimentos mais elevados foram obtidos usando água e a mistura água:etanol (1:1) como solventes. Recorrendo às técnicas cromatográficas foi possível atestar que os óleos essenciais e a grande maioria dos extratos supercríticos são predominantemente constituídos por monoterpenos oxigenados. Registaram-se diferenças qualitativas e quantitativas na composição dos óleos essenciais e dos extratos supercríticos obtidos no segundo separador (compostos mais voláteis), tendo-se identificado um maior número de compostos por hidrodestilação. Os extratos polares são ricos em ácidos hidroxicinâmicos (ácidos 3-Ocafeoilquínico, 4-O-cafeoilquínico, 5-O-cafeoilquínico, cafeico e rosmarínico) e flavonoides (luteolina, apigenina e pinocembrina), tendo o ácido rosmarínico sido identificado como o composto maioritário na maioria dos extratos, incluindo os obtidos das culturas in vitro. Comparando os materiais vegetais estudados, as condições usadas in vitro foram as mais favoráveis à acumulação de ácido rosmarínico. Atualmente existe um interesse crescente na utilização de antioxidantes naturais, particularmente de origem vegetal, na prevenção e/ou tratamento de algumas patologias. A implicação do stresse oxidativo no progresso da doença de Alzheimer sugere os antioxidantes naturais como potenciais agentes preventivos. A aplicação destes metabolitos ativos é imensa; também na indústria alimentar, e a potencial toxicidade dos antioxidantes sintéticos estimula a sua substituição por antioxidantes naturais. Neste contexto, as espécies vegetais em estudo foram testadas quanto ao seu potencial antioxidante designadamente a capacidade de (a) captar diferentes radicais (ORAC - oxygen radical antioxidant capacity, DPPH - 2,2-diphenyl- 1-picrylhydrazyl e TEAC - Trolox equivalent antioxidant capacity); (b) quelar o ferro e (c) inibir a peroxidação lipídica em homogeneizados de cérebro de ratinho. Os extratos hidroalcoólicos e infusões foram, geralmente, os sequestradores de radicais mais eficientes (Capítulos 2 e 3). No que diz respeito aos extratos supercríticos, a pressão de extração afetou a capacidade dos extratos colhidos no primeiro separador de captar o radical peroxilo, sendo que a atividade mais elevada se verificou com o incremento da pressão. Os extratos aquosos, hidroalcoólicos e infusões foram os mais ativos na quelação do ferro e inibição da peroxidação lipídica. Ambos os materiais vegetais estudados (selvagem e micropropagado) demonstraram capacidade na quelação de ferro e os extratos etanólicos das culturas in vitro foram os mais eficientes na inibição da peroxidação lipídica. A presença de ácidos hidroxicinâmicos e flavonoides nos extratos polares poderá, em parte, justificar a atividade antioxidante observada. O solvente de extração influenciou significativamente a recuperação de compostos com potencial antioxidante, sendo a mistura água:etanol (1:1) a mais adequada para estas espécies. Os óleos essenciais estão entre os compostos mais valiosos produzidos pelas plantas com aplicações que se estendem às mais diversas áreas. Contudo, a baixa solubilidade em sistemas aquosos e a elevada volatilidade são alguns dos fatores que condicionam a sua aplicação. Ao longo dos anos vários sistemas de encapsulação têm sido desenvolvidos para ultrapassar estas limitações, nomeadamente as ciclodextrinas. Estas são particularmente interessantes pelo caráter anfifílico característico, e foram por isso usadas neste estudo como agentes solubilizantes, concretamente, a β-ciclodextrina e hidroxipropil-β-ciclodextrina. Os resultados apresentados no Capítulo 4 indicam que as ciclodextrinas, nomeadamente a hidroxipropil-β- ciclodextrina, produziram um aumento significativo na atividade antioxidante dos óleos essenciais, provavelmente consequência do aumento da solubilidade de alguns compostos menos polares. Para além do aumento da atividade antioxidante, esta manteve-se estável ao longo de trinta dias. A estratégia mais utilizada atualmente no tratamento da doença de Alzheimer compreende a melhoria da função colinérgica que é clinicamente conseguida através do uso de inibidores colinesterásicos. Embora estes fármacos licenciados sejam eficientes na atenuação dos sintomas, apresentam efeitos secundários e não impedem o progresso da doença. Estas limitações representam simultaneamente um grande desafio na pesquisa de novos fármacos, e foi com este objetivo que neste trabalho as espécies em estudo foram testadas quanto ao potencial anti-colinesterásico (acetilcolinesterase e butirilcolinesterase). Os óleos essenciais apresentaram, geralmente, as inibições mais promissoras, juntamente com o extrato metanólico de L. viridis, que foi também ativo na inibição enzimática in vivo. A atividade anti-colinesterásica dos óleos essenciais poderá refletir a presença de compostos como 1,8- cineol, α-pineno, mirtenal e viridiflorol. No entanto, é de salientar que a atividade biológica de misturas complexas, como os óleos essenciais e extratos, poderá resultar do somatório das atividades individuais dos seus constituintes, e também da presença de efeitos de sinergismo e antagonismo. Relativamente aos extratos supercríticos, apenas os obtidos no primeiro separador apresentaram inicialmente atividade inibitória; contudo, a indicação da presença de falsos positivos sugere que estes resultados poderão estar sobrestimados. L. viridis foi a espécie mais consistente e promissora em termos de atividade biológica. Os compostos bioativos desta espécie são antioxidantes naturais capazes de atuar como sequestradores de radicais livres, agentes quelantes de metais e inibidores da peroxidação lipídica. Além disso, combinam várias formas de prevenção de doenças multifatorais como a doença de Alzheimer, agindo também como inibidores colinesterásicos. Por estas razões, apenas esta espécie foi testada nos ensaios de neuroproteção (Capítulo 5) e digestão in vitro (Capítulo 6). Os extratos de L. viridis e o seu composto predominante, o ácido rosmarínico, protegeram as células A172 dos efeitos negativos gerados pelo peróxido de hidrogénio. Todos os extratos, com exceção do extrato metanólico na concentração mais baixa, e o ácido rosmarínico atenuaram a neurotoxicidade induzida pelo peróxido de hidrogénio. Além disso, o extrato hidroalcoólico, infusão, e o ácido rosmarínico reduziram a acumulação intracelular de espécies reativas de oxigénio. A capacidade de eliminar radicais livres previamente demonstrada explica, em parte, o efeito protetor observado. A presença de compostos com potencial biológico em determinadas espécies, como no caso de L. viridis, por si só não é garantia de que após o seu consumo continuem ativos no momento em que alcançam os tecidos-alvo; é essencial que estes estejam biodisponíveis. Neste sentido, os extratos mais promissores de L. viridis foram submetidos a condições de digestão in vitro, e as atividades antioxidante e anti-colinesterásica dos extratos metabolizados avaliada (Capítulo 6). O ácido rosmarínico puro foi também estudado para averiguar a influência da matriz vegetal na biodisponibilidade dos compostos. Efetivamente, a matriz do extrato metanólico não influenciou a digestão de ácido rosmarínico, ao contrário do hidroalcoólico que protegeu o composto maioritário da digestão ácida. Estes resultados corroboram a influência da matriz vegetal na biodisponibilidade dos seus constituintes. A atividade antioxidante do extrato metanólico e ácido rosmarínico puro foi assegurada depois dos processos digestivos in vitro. No que respeita à atividade anti-colinesterásica dos extratos metabolizados, apenas a inibição da butirilcolinesterase foi afetada. Estes resultados sugerem que os extratos de L. viridis atravessam o trato gastrointestinal sem comprometer grandemente a sua atividade biológica. Esta tese proporcionou um conhecimento mais alargado da composição química e da atividade biológica das espécies L. viridis, L. pedunculata subsp. lusitanica e T. lotocephalus utilizadas tradicionalmente. Todas as espécies, em particular L. viridis, são ricas em antioxidantes e anti-colinesterásicos naturais capazes de participar ativamente na prevenção e/ou tratamento da doença de Alzheimer, ou outras patologias associadas ao stresse oxidativo. Além disso, L. viridis e T. lotocephalus sintetizam compostos bioativos in vitro representando uma alternativa eficiente à obtenção de metabolitos de elevado valor. Universidade do Algarve, Faculdade de Ciências e Tecnologia

In this study the characterization of a total of 60 honey samples with Protected Denomination of Origin (PDO) collected over three harvests (2009–2011, inclusive), from the Northeast of Portugal was carried out based on the presence of pollen, physicochemical and microbiological characteristics. All samples were found to meet the European Legislation, but some didn’t meet the requirements of the PDO specifications. Concerning the floral origin of honey, our results showed the prevalence of rosemary (Lavandula pedunculata) pollen. The microbiological quality of all the analyzed samples was satisfactory, since fecal coliforms, sulfite-reducing clostridia and Salmonella were absent, and molds and yeasts were detected in low counts. Significant differences between the results were studied using one-way analysis of variance (ANOVA), followed by Tukey’s HSD test. The samples were submitted to discriminant function analysis, in order to determine which variables differentiate between two or more naturally occurring groups (Forward Stepwise Analysis). The variables selected were in this order: diastase activity, pH, reducing sugars, free acidity and HMF. The pollen spectrum has perfect discriminatory power. This is the first study in which a honey with PDO was tested, in order to assess its compliance with the PDO book of specifications X.F. would also like to thank the Xunta de Galicia (Isidro Parga Pondal Program for young researchers, Grant No. IPP-020) SI

Relatório Final de Estágio de Iniciação à Investigação Científica / ISA - Centro de Botânica Aplicada à Agricultura In this study we performed a histochemical characterization of glandular trichomes in plantules of Lavandula pedunculata (Miller) Cavanille exposed to three different photoperiodic regimes: 16h light/8h dark (to control), 12h light/12h dark and still 8h light/16h dark. We aim at understanding whether changes in photoperiodic regime can modify the composition of the secretions of the trichomes. To evaluate the differences between the regimes we were carried a battery of fluorescence and histochemical tests. It was found that there are significant changes in the segregations, mainly characterized by reduced production of secretions even quantitatively and qualitatively.

Relatório de Estágio de Iniciação à Investigação Científica /ISA - Centro de Botânica Aplicada à Agricultura The genus Lavandula is known since classic antiquity as aromatic and medicinal. These characteristics are due to compounds synthesized and accumulated in secretory structures. In Portugal, we find several species of the genus including the L. pedunculata species. In this species, it is possible to distinguish between protective and secretory trichomes. With this work we pretend study the ontogeny of the types of trichomes and plantlets along their development, under different photoperiod conditions, 16 hours light/8 hours dark and 12 hours light/12 hours dark, respectively. Cotyledons and leaves from plantlets along different development were collected and used for scanning electron microscopy. The results demonstrated that in the conditions used the rate of germination of this species was low. Also this study showed that there are variations in plant development under different photoperiod in relation to the number of leaves, dimensions, density and types of the trichomes present.

We studied the water relations of 6 shrub and 3 tree species typical of the mediterranean climate region of central Spain to identify differential responses to water stress between and within species, and to determine if free proline concentration in leaves could be used as a water stress indicator. Predawn and midday water potentials (Ψw) on a seasonal basis, relative water content (RWC), leaf mass per area, foliar nitrogen and free proline concentrations were measured. The lowest water potentials were observed at the end of the summer, with recovery to higher water potentials in the fall and winter seasons. Species differed regarding the annual Ψw fluctuation. Thymus zygis, Halimium viscosum, Genista hirsuta and Juniperus oxycedrus exhibited the most negative midday and predawn Ψw (both less than -6 MPa) with a large magnitude of response to changing conditions in soil moisture of the upper horizon of the soil. Lavandula pedunculata and Cistus ladanifer showed a moderate response. Quercus rotundifolia, Quercus faginea and Retama sphaerocarpa showed a modest response. The Ψw of different size individuals of Quercus rotundifolia and Cistus ladanifer were compared. The annual Ψw fluctuation was greater in small individuals as compared to large individuals. In every species, there was an increase in proline concentration of bulk leaf tissues when predawn Ψw dropped below -5 MPa. Small plants of Cistus ladanifer reached lower water potentials and also higher concentration of proline than bigger plants. Proline could possibly be used as a drought stress indicator in every species except Q. rotundifolia. It is suggested that in addition to water stress avoidance due to deep root systems, some mechanisms of water stress tolerance may operate among shrub and tree species of central Spain.

Lavenders belong to the family Labiatae and represent some of the most popular medicinal plants of great economic importance. Their essential oils are important for the perfume, cosmetic, flavouring and pharmaceutical industries. However, despite its popularity, and the long tradition of use, biological properties of the various Lavandula species are not yet been well sustained by scientific or clinical studies and some available data being inconclusive and controversial [1]. Although Lavandula spp. have similar ethnobotanical properties, however, chemical composition and therapeutic uses differ from different species and main composition of essential oils showed differences with species and with the region were they grow [1,2,3]. L. stoechas L. subsps. luisieri (Rozeira) Rozeira. L. pedunculata (Mill.) Cav. and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Alentejo and Algarve. In our work, essential oils from the stems or leaves from wild grown plants of L. luisieri (Alentejo), L. pedunculata (Alentejo) and L. viridis (Algarve), were extracted by hydrodistillation and analyzed by GC-FID. Antimicrobial activity was evaluated by solid diffusion disk assay and minimal inhibitory concentration (MIC) against pathogenic Gram-positive and Gram-negative bacteria and food spoilage fungi.

Essential oil, ethanolic extract and decoction of 10 plant species from interior Portugal were analyzed for their activity towards acetylcholinesterase (AChE) enzyme and their antioxidant activity. Of these, Melissa officinalis, Paronychia argentea, Sanguisorba minor, Hypericum undulatum and Malva silvestris are used in herbal medicine, Laurus nobilis and Mentha suaveolens as condiments, and Salvia officinalis, Lavandula angustifolia and Lavandula pedunculata also as aromatics. Melissa officinalis and Mentha suaveolens showed AChE inhibitory capacity higher then 50% in the essential oil fraction. Laurus nobilis, Hypericum undulatum, and Sanguisorba minor showed a high inhibition value of AChE in the ethanolic fraction, 64% (1 mg ml(-1)) 68% (0.5 mg ml(-1)), and 78% (1 mg ml(-1)), respectively. Higher values of AChE inhibitory activity were found using decoctions of Lavandula pedunculata, Mentha suaveolens and Hypericum undulatum, 68, 69 and 82% (at a concentration of 5mg dry plant ml(-1) of assay), respectively. The free radical scavenger activity was higher for the polar extracts. In the water extracts most of the plants showed values around 90%. When antioxidant activity was measured with the beta-carotene-linoleic acid assay high activity (65-95%) was also found in the water extracts. Hypericum undulatum, Melissa officinalis and Laurus nobilis showed both high AChE inhibitory capacity and antioxidant activity.

The selection of native Lavandula species and their economic exploitation have increased in the last few years. Micropropagation techniques have been used as an alternative for vegetative propagation allowing the multiplication of selected genotypes and chemotypes. Our previous studies showed that the essential oils of Lavandula pedunculata have an important antifungal activity against dermatophyte strains. Therefore, a new line of investigation concerning the in vitro culture of this species is justified. In the present study we compare the morphology of the leaf trichomes and the chemical composition of their essential oils in both field-growing and in vitro propagated plants.

Juvenile plants of Thymus mastichina, Thymus zygis, Lavandula pedunculata, Genista hirsuta, and Cistus ladanifer were transplanted from the field to the greenhouse; the soil used was the same in which they had grown at their natural site. The plants were grown to maturity to determine comparative growth, water status, and mycorrhizal colonization under greenhouse conditions and to determine the influence of drought on the symbiosis. After 24 months, L. pedunculata showed the most developed root system; G. hirsuta showed the lowest root/shoot ratio and the lowest percentage of mycorrhizal colonization. This percentage ranged from 32.0% to 82.4% for G. hirsuta and L. pedunculata, respectively. There was a positive relationship between root biomass and root/shoot ratio with mycorrhization percentage, and a positive response of water potential to that percentage also occurred. After a moderate drought treatment over 4 weeks, root and shoot biomass and root/shoot ratio as well as mycorrhizal colonization decre...