Acne vulgaris, commonly known as acne, is a chronic skin disease of the pilosebaceous unit, that affects millions worldwide, particularly adolescents and young adults. Clinically, acne is characterized by non-inflammatory and inflammatory lesions, predominantly on the face, but also affecting the neck, chest, and upper back. Beyond its signs and symptoms, acne is also associated with a substantial psychological and emotional burden. The pathogenesis of the disease is multifactorial and involves a complex interplay of several biological events or hallmarks. Traditionally, four mechanisms have been associated with its development, namely: increased sebum production, follicular hyperproliferation/hyperkeratinization, colonization by Cutibacterium acnes bacterium (formerly Propionibacterium acnes), and the release of inflammatory mediators. However, recent research has further complicated the disease’s pathophysiology, with new factors such as changes in sebum composition, differential involvement of innate and adaptive immunity, and dysbiosis of the follicular microbiota, being increasingly described by the scientific community. Given its multifactorial nature, different therapeutic strategies are adopted depending on the severity of the disease. Topical treatments, when used alone, are typically reserved for comedonal or non-inflammatory acne, while in combination topical therapy is considered the first-line approach for mild to moderate acne. In more severe cases, systemic treatments are used as first-line options, with isotretinoin being the standard choice despite its severe risks and side effects. These adverse effects associated with classical acne therapies, together with a behavioral shift among consumers who increasingly prefer more “natural” and/or “green” solutions, have driven both the cosmetic and pharmaceutical industries to invest in the research and development of alternatives or adjuvants to conventional treatments that meet these expectations. Plant-derived ingredients have gained particular attention and, among them, essential oils (EOs), which are secondary metabolites from aromatic plants, stand out for their spectrum of bioactive properties and are extensively used across various health fields, including dermatology. Based on this premise, the aim of this doctoral project was to investigate the bioactive potential of preparations from aromatic and medicinal plants produced in Portugal, with a particular focus on their ability to modulate the main hallmarks involved in acne pathophysiology. Two autochthonous Portuguese species, namely Thymus mastichina (L.) L. (Thymus mastichina; TM) and Cistus ladanifer L. (Cistus ladanifer; CL), and the allochthonous hybrid Thymus × citriodorus (Pers.) Schreb. (Thymus × citriodorus; TC) were investigated for their anti-acne potential, through the study of their EOs in addition to their hydrolates, by-products of EO production, thus aligning this work with the principles of circular economy. Initial screenings of the autochthonous taxa revealed that CL EO, rich in α-pinene, exhibited the highest anti-inflammatory potential (EC₅₀ = 0.002% v/v), although with associated cytotoxicity in murine macrophage cell model (RAW 264.7; IC₅₀ = 0.012% v/v). TM preparations, mainly composed by 1,8-cineole, showed balanced anti-inflammatory effects with superior biocompatibility in both murine macrophages and fibroblasts (L929). CL EO showed the highest antimicrobial activity, with minimum inhibitory concentrations (MICs) ranging from 0.06% (v/v) to 2% (v/v) against the tested microorganisms, which included those used in the Challenge test for cosmetics and representatives of skin microbiota. Regarding the preparations from the allochthonous hybrid, TC EO, mainly composed of geraniol, stood out for its antimicrobial activity against acne-associated bacteria (C. acnes, Staphylococcus epidermidis and S. aureus) and for its ability to prevent and disrupt C. acnes biofilms, even at sub-inhibitory concentrations. TC hydrolate retained some bioactive potential, particularly in modulating C. acnes biofilms, still being deprived of relevant antimicrobial activity. Considering the higher anti-acne potential of TC EO, and to address variability concerns relevant to large-scale application of EOs, four TC EOs from different Portuguese regions were compared. Although all shared geraniol as a major compound, significant differences in yield, efficacy, and cytotoxicity were observed, underscoring the need for standardization protocols. Still, all EOs exhibited antimicrobial activity, with particular effectiveness against C. acnes, as evidenced by MICs ranging from 0.016% (v/v) to 0.031% (v/v), corroborating the anti-acne potential of this hybrid. Building upon previous results and considering the anti-acne potential of the studied preparations, their ability to modulate key acne-related events was further explored using more complex in vitro models that better mimic in vivo conditions. These closer-to-disease models further explored the preparations’ ability to modulate sebum production in human sebocytes (SZ95 cell line), C. acnes-induced inflammation and oxidative stress, keratinocyte proliferation, and bacterial adhesion. Additionally, potential antimicrobial interactions between the different preparations along with their cytotoxic and mutagenic potential were also investigated. Regarding anti-acne efficacy, comprising the above-described models, EOs exhibited overall greater bioactivity compared to their corresponding hydrolates. The EOs of TC and CL stood out for their ability to modulate all the studied hallmarks, demonstrating a multitarget potential. Although less potent, the hydrolates still showed relevant activity, particularly the one from CL, displaying multi-target potential, failing only in inhibiting lipase activity. Regarding the combined antimicrobial activity of the preparations against C. acnes, EO–EO combinations showed consistent synergistic effects (FICIs between 0.250 and 0.375), while the presence of additive interactions depended on the specific combinations. As for safety, TC and CL EOs were the least biocompatible upon human keratinocytes and sebocytes, and all preparations were considered non-mutagenic, except for CL EO, which showed a “likely-positive” result in one of the Salmonella typhimurium strains included in the Ames test. Based on the higher overall efficacy of the tested EOs, the final experimental work focused on potential phylotype-selective efficacy of EOs against clinical C. acnes strains, with different virulence patterns. Phylotype IA1 strains (typically associated with acne lesions), exhibited higher lipase activity and biofilm formation than phylotype II strains (more associated with healthy skin). However, not all virulence factors were phylotype-dependent, which supports the multifactorial nature of this disease. TC EO showed the lowest MICs across all strains (MICs between 0.03% v/v and 0.06% v/v) when compared to the other EOs, demonstrating less variation in MIC values between phylotypes. It was also able to reduce both biofilm biomass and metabolic activity from IA1 phylotype strains, being effective at lower concentrations. These results further support the relevance of TC EO for this application, despite its lack of phylotype-dependent selectivity. In conclusion, this work provides evidence for the anti-acne potential of Portuguese plant preparations, particularly TC and CL EOs, supported by their capacity to modulate different pathophysiological mechanisms associated with disease development. This research also contributes to the scientific valorization of Portuguese natural resources and supports their integration into sustainable dermocosmetic formulations aligned with the principles of circular economy.

Background: The last decades have seen an increased awareness by the scientific community of the extent of resistance to conventional antibiotics, particularly with respect to the emerging multidrug-resistant pathogenic microbes. Additionally, natural antioxidants have received significant attention among food professionals and consumers because of their assumed safety and potential therapeutic value. The aim of this work was to assess the antioxidant activities of eight selected commercial essential oils (EOs), together with the evaluation of their antibacterial and anti-quorum sensing properties. Methods: The chemical profiling of the EOs was performed using gas chromatography-mass spectrometry (GC-MS) analysis. The antioxidant properties of the EOs were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and by β-carotene bleaching test. Disc diffusion assays were employed to evaluate the anti-bacterial and anti-quorum sensing activities of the EOs. Results: It was observed that EOs from three Eucalyptus species are rich in eucalyptol. Generally, linalool is abundant in EOs from four Lavandula species. The oil of Cymbopogon citratus is the one with the best capacity to scavenge the DPPH free radicals and presented great antibacterial activity. Conclusions: The geographical origins of the plant species are determinant factors in the EO composition and in the corresponding biological activities.

A utilização de plantas medicinais tem contribuído para a pesquisa em áreas muito diversificadas da saúde humana, nomeadamente, na descoberta de novas substâncias químicas. Em Portugal tem-se registado, nos últimos anos um forte interesse e incremento do cultivo das plantas aromáticas e medicinais, no entanto, são escassos os estudos agronómicos relativos ao seu cultivo nos sistemas de produção do nosso país. A presente dissertação visou alcançar dois objectivos principais: num primeiro campo, localizado em plena “charneca ribatejana” e tendo por base a tecnologia da armação do solo em camalhão elevado e cobertura com filme de plástico negro, praticada na região na cultura do morango, instalou-se um ensaio visando avaliar o efeito da dotação de rega e densidade de plantação na produção de biomassa e rendimento de óleos essenciais em hortelã-pimenta (Mentha x piperita L.); num outro campo, outrora utilizado no cultivo de arroz em canteiros, localizado no vale da ribeira de Muge, Herdade dos Gagos, instalou-se um outro ensaio visando o estudo da adaptação da mesma espécie a este sistema de cultivo. O primeiro campo foi instalado em Paço dos Negros, em solo de textura arenosa, entre Abril e Novembro de 2011. O delineamento experimental foi de parcelas subdivididas, com quatro repetições. Os tratamentos avaliados foram: duas dotações de rega R1 (6 340 m3/ha no total do ciclo) e R2 (3 170 m3/ha) e duas densidades D1 (4,6 plantas/m2) e D2 (3,3 plantas/m2). As variáveis observáveis estudadas foram: a produção de biomassa e o rendimento do óleo essencial. A maior densidade e maior quantidade de água influenciaram positivamente a produção de biomassa (4 705 kg/ha) com um valor médio de 2 317 kg/ha. O teor IV médio em óleos essenciais (1,80% MS), não foi afetado significativamente por nenhum dos fatores. No campo da Herdade dos Gagos, conseguiu-se observar uma boa adaptação da hortelã-pimenta ao sistema de produção em canteiros outrora utilizados no cultivo de arroz (solos hidromórficos) com uma plantação em camalhões sem cobertura de solo, com uma densidade de 8 plantas/m2. Neste campo obteve-se uma produção de 1conseguiu-se observar uma boa adaptação da hortelã-pimenta ao sistema de produção em canteiros outrora utilizados no cultivo de arroz (solos hidromórficos) com uma plantação em camalhões sem cobertura de solo, com uma densidade de 8 plantas/m2. Neste campo obteve-se uma produção de 16 t/ha de matéria verde, 150 dias após a plantação. O teor de óleos essenciais (1,93% MS) foi ligeiramente superior ao do campo de Paço dos Negros.

Atendendo ao crescente interesse por produtos naturais e consequente incremento do comércio de plantas aromáticas e medicinais, revelou-se imperativa a investigação de técnicas de controlo de fraudes neste âmbito. Apesar da importância das plantas medicinais espontâneas, valoriza-se o seu cultivo em modo de produção biológico, respeitando as boas práticas agrícolas de acordo com os requisitos da Organização Mundial de Saúde e valorizando a qualidade, a segurança e a eficácia destes produtos. Estudando diversas plantas, recorreu-se à extracção de óleos essenciais e à cromatografia em camada fina (TLC) para investigar os seus principais constituintes. Executaram-se ainda processos rápidos de detecção de falsificações, através do teste da mancha do papel, da contracção do volume de essência, do ensaio da fucsina, e da análise das plantas em estado pulverizado. Este trabalho é útil e aplicável em qualquer unidade de produção de plantas aromáticas e medicinais para fins industriais, pois recorre a metodologias simples e de baixo custo. A implementação de diversos procedimentos de gestão de qualidade é uma garantia de um sistema de autocontrolo – HACCP (Hazard Analysis Critical Control Points), segundo o Codex Alimentarius, criando processos dinâmicos que incitem a constante melhoria e desenvolvimento tecnológico, evitando contaminações por agentes físicos, químicos e biológicos, falsificações e fraudes.

Cryptococcosis is an opportunistic fungal disease, caused by the yeasts from the Cryptococcus neoformans complex, frequently associated with high rates of morbidity and mortality. Patients infected with the human immunodeficiency virus (HIV), transplanted or in use of immunosuppressive therapies are the main affected. Fluconazole and amphotericin B, the main antifungals applied in the treatment of the disease, are toxic to the patient and, their use for prolonged time, has selected resistant strains making necessary to search for new compounds that shows good antifungal activity and low levels of toxicity. Thus, the essentials oils (EOs) from plants are promising for this purpose because their antimicrobial property has been already reported. This study aimed to evaluate the biological activity of EOs from Cinnamomum zeylanicum, Syzygium aromaticum, Juniperus virginiana, Rosa centifolia, Illicium verum and eugenol on fungal isolates from C. neoformans complex. Tests were carried out to evaluate the fungal activity, interaction with amphotericin B and fluconazole, time kill assay, mechanism of action and cytotoxicity. The EOs of C. zeylanicum and S. aromaticum and its majority compound, eugenol, presented the best antifungal activity with minimum inhibitory concentrations (MICs) ranging from 128-512 μg/mL and minimum fungicidal concentrations (MFCs) from 256-1024 μg/mL. The EO of S. aromaticum has an increase in its antifungal activity when combined with amphotericin B in all the isolates evaluated while the EO of C. zeylanicum and eugenol showed no changes of its activity when associated with drugs. The study of the mechanism of action revealed that C.zeylanicum, S. aromaticum and eugenol, in MIC, inhibited the synthesis of ergosterol and in MFC promoted membrane damage. The hemolytic activity assays showed low toxicity of these compounds. These results demonstrate action of the EOs from C. zeylanicum and S. aromaticum and eugenol against isolates of C. neoformans complex. Thus, these EOs become promising in the search for new drugs to be used in the treatment of cryptococcosis.

The species Syzygium jambos L. Alston - Myrtaceae, popularly known as yellow jambo, is an Asian tree, predominant in tropical and subtropical regions. Studies show the presence of phenolic compounds, flavonoids, tannins and essential oil. In addition to describing an antioxidant and antimicrobial activity in its hydroethanolic extract, more than in some other species so far considered as very promising. This study was aimed at evaluating the influence of environmental factors such as seasonality, temperature, rain precipitation, soil and leaf mineral nutrients on the essential oil composition and levels of phenolic compounds extracted from the leaves of Syzygium jambos. Samples were collected in two distinct regions: in the northeast and southeast of the state of Goiás, and in the cities Nova America and Rio Verde in dry periods in July and in rainy periods in January, both in 2011. The essential oil was extracted by hydrodistillation in a Clevenger device, modified and submitted to analysis by gas chromatography, attached to mass spectrometry (CG/EM). The dosage of the phenol compounds were made by spectrophotometry. The levels of soil and leaves nutrients were analyzed by spectrophotometric methods and atomic absorption. The data were submitted to Pearson correlation analysis, multiple regression and hierarchical clustering. The results showed that environmental variables on the location had greater influence on the chemical variability of essential oils and phenolic compounds obtained from the leaves of S. jambos. Its content and composition also showed different data according to the time of harvest.

Memora nodosa (Silva Manso) Miers is a native savannah plant popularly used for wound cicatrization (leaves and stems) and for treating abdominal pains and scabies (roots). The purpose of this work is to study the morphoanatomy and determine the composition and variability of the constituents of the essential oil obtained from the M. nodosa leaves, as well as its antimicrobial, leishmanicidal, cicatrizing activity and toxicity. Morphologically, M. nodosa is a bush with clumps of straight or curved stems. It has a deep, pivoting root and presents a chandelier shaped structure in the upper part from where the stems sprout. It presents compound, bipinnate, imparipinnate, oppositely crossed leaves. The inflorescence is racemose with golden-yellow flowers. It is characterized by septifrague capsule type fruit with replum and winged seeds. Anatomically the leaves are hypostomatic with predominantly paracytic stomates. The epidermis is uniseriate with a thick cuticle and glandular trichomes throughout the structure of the young leaves. The essential oil from the M. nodosa leaves collected from seven different locations in the Brazilian savannah was analyzed by GC(FID) and GC/MS. Five substances were identified, benzaldehyde and 1- octene-3-ol were the main constituents. Two different classes regarding sampling location and constituents were identified by means of the principal component and grouping analyses. The antimicrobial activity of the ethanolic extracts from leaves, stems, roots and of essential oil from leaves, and the fractions obtained from the roots, were assessed against Grampositive and Gram-negative bacteria, as well as the Candida albicans fungus, using well diffusion test and the agar dilution method. The ethanolic extract from the roots presented small antimicrobial activity while the hexane and dichloromethane fractions obtained from this extract presented MIC 0.39 mg/mL and 0.78 mg/mL against a number of Gram-positive bacteria and the Candida albicans fungus. The essential oil presented MIC 0.78 mg/mL to 1.56 mg/mL. The antimicrobial activity of the essential oil was the result of synergic action between the benzaldehyde, 1-octene-3-ol, 1-octanol, linalool and the mandelonitrile, constituents of the essential oil. Promastigote forms of Leishmania chagasi were used for the leishmanicidal assessment and it was verified that the ethanol extract from the leaves presented an IC50 of 93.2 μg/mL and from the roots presented an IC50 of 95.87 μg/mL. The hexane fraction from the roots presented an IC50 of 13.51μg/mL. A mixture of β-sitosterol and stigmasterol was isolated and identified in the hexane fraction obtained from the ethanol extract from the roots. This mixture was tested against L. chagasi resulting in an IC50 of 69.79 μg/mL. The cicatrizing activity of the ethanol extract from the leaves and roots was assessed on skin wounds in rats, and it be noted reepithelization occurred faster only in the group treated with a 2% aqueous solution of the ethanol extract from the roots. This action may be related to the alantoine isolated in the ethanol extract from the roots. The toxicity assessment of the ethanol extracts from roots, leaves and hexane fraction obtained from M . nodosa roots, tested on rats, mice and Artemia salina proved that this plant is not potentially toxic. In conclusion, according to the tests performed, the best results were obtained with ethanolic extract and it presented antimicrobial, wound cicatrization and leishmanicidal activities. Hexane and dichloromethane fractions obtained from roots extract presented antimicrobial activity against Gram-positive bacteria and the Candida albicans fungus and hexane fraction strong leishmanicidal activity.

His study aimed to evaluate whether the associations between thymol, carvacrol and eugenol on the immature life stages of Amblyomma sculptum have a synergistic effect, by means of in vitro test and under semi field conditions, as well as to evaluate the cost-benefit ratio of formulations. To assess the synergism, the substances were tested in vitro in isolation and associated at concentrations of 0.3125; 0.625; 1.25; 2.5; 5.0 and 10.0 mg/mL on non-fed larvae; and 2.5; 3.75; 5.0; 7.5; 10.0; 15.0 and 20 mg/mL on non-fed nymphs. For the test in semi field conditions, the two associations that obtained the best efficacy in in vitro tests were selected for application on plots of Panicum maximum artificially infested with A. sculptum larvae. In in vitro tests with larvae, the association between thymol and eugenol demonstrated synergistic effect at all tested concentrations; treatments associating thymol + carvacrol and eugenol + carvacrol demonstrated synergistic effect at a concentration of 5.0 mg/mL. In in vitro tests with nymphs, the association between thymol + eugenol showed synergistic effect at a concentration of 5.0 mg/mL; the association between thymol + carvacrol resulted in a synergistic effect at a concentration of 10.0 mg/mL while treatments with the association of carvacrol + eugenol showed moderate synergism at concentrations of 5.0; 7.5 and 10.0 mg/mL. The association of the substances allowed lower use to achieve mortality rates higher than 95% in in vitro tests, resulting in cost reduction. In semi field conditions the efficacy of the associations was 63.2% for thymol + eugenol association and 42.6% for carvacrol + eugenol association. Thus, depending on concentration, synergistic effects of binary associations between thymol, carvacrol and eugenol were observed for immature stages of A. sculptum, in addition to allowing cost reduction with the active principle for formulation development.

Morphoanatomy, trichomes glandular and phytochemistry of Lomatozona artemisiifolia Baker (Asteraceae - Eupatorieae) - a plant of endemic of Cerrado of Goiás - The Asteraceae family is widely distributed, having 24,000 species and genera 1600-1700, constituting one of the largest families of phanerogams. In the Cerrado, among phanerogams, is the second largest family in number of species. Among the numerous species of Asteraceae is present Lomatozona artemisiifolia Baker, belongs to the tribe Eupatorieae. The objective of this study was to describe anatomically aerial vegetative organs of this species, characterize the glandular trichomes that occur in these vegetative organs and inflorescences, determine the composition of the oil and perform the phytochemical study of the species, as there is no work on this species which is endemic to the Cerrado. For anatomical study were used apexes, samples of completely expanded leaves and stems. The phytochemical screening was performed with aerial vegetative organs and oil extraction was performed with fresh plant material (aerial vegetative organs). The epidermis is uniseriate covered with a thin cuticle striated and mesophyll is dorsiventral. The stem has uniseriate epidermis, cortex and with angular collenchyma lamellar secretory ducts associated with the cortical parenchyma, sclerenchyma fibers forming ice outside the phloem bundles are collateral. Is the presence of eight types of glandular trichomes on the leaf and stem. We identified the presence of traces of alkaloids, coumarins, traces of digitalis glycosides, saponins, flavonoids and glycosides. In oil extraction were identificandos 21 compounds, the major compounds are γ-muuroleno about 28.81%, the β-germacrene with 18.23%, 12.48% caryophyllene, δ-elemene 8.61%, 7.55% δ-carene, β-ocimene 5.94%.