Natural products are complex mixtures of compounds with therapeutic effects which are often reported to be due to the synergistic action of multiple and sometimes unknown components. Consequently, standardization of these products is complex and a lack of effective quality control (QC) criteria in most countries has led to marketing of commercial products with questionable quality, safety and efficacy (QSE). The aim of this study was therefore to develop qualitative and quantitative analytical methods for use in the QC of Ginkgo biloba solid oral dosage forms. Initially, a micellar electrokinetic chromatography (MEKC) method was developed for the identification of the flavonol glycosides, rutin and quercitrin as well as 3 flavonol aglycones, quercetin, kaempferol and isorhamnetin in crude extracts of 4 Ginkgo biloba solid oral dosage forms using ultraviolet (UV) detection. A reversed-flow cyclodextrin-modified MEKC method was subsequently developed for the simultaneous determination of the aforementioned flavonols as well as ginkgolide A, B, C, J and bilobalide (all positive markers) in Ginkgo commercial products. A non-aqueous capillary electrophoresis (CE) method was also developed for fingerprinting the presence of ginkgolic acids (negative markers) in Ginkgo biloba leaf extracts, which are purported to be associated with toxic properties. This method was also applied to 2 Ginkgo biloba commercial products. Since the flavonols have strong UV absorbing chromophores, a reversed phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated using photo-diode-array (PDA) detection which was then successfully applied to fingerprint commercially available Ginkgo biloba solid oral dosage forms as well as quantify the relevant flavonol markers present in these extracts. Sample preparation was simple, rapid and cost efficient with minimal clean-up and the employment of a minibore column which requires low mobile phase flow rates contributed to the economy of the method. Unlike the conventional QC approach, samples were not hydrolyzed and direct determination of 2 intact flavonol glycosides, together with the usual aglycone markers was facilitated which provided maximal content information for fingerprint comparisons. On the other hand, terpene trilactones possess poor chromophores and an alternative detection method to UV was required in order to obtain suitable sensitivity. RP-HPLC with evaporative light scattering detection (ELSD) was selected for quantification of these non-volatile constituents in Ginkgo dosage forms and this method was deemed suitable for the routine QC analysis of these positive markers in commercial products. Since approximately 33 flavonoids have been identified in Ginkgo biloba leaf extracts, baseline separation using UV/PDA detection normally requires complex gradient programs and long analysis times. In addition, unequivocal identification of the flavonoids with similar UV spectra and elution times cannot be guaranteed. A liquid chromatographic tandem mass spectrometric (LC-MS-MS) method was therefore developed and validated in order to ensure accurate quantification of the selected flavonol marker compounds in Ginkgo commercial products. LC-MS-MS analysis of Ginkgo extracts revealed, in addition to rutin, the possible presence of other quercetin analogues, quercetin-3-Orhamnoside-7-O-glucoside or quercetin-3-O-glucoside-7-O-rhamnoside, previously unreported in Ginkgo biloba leaf extracts or dosage forms. In terms of evaluating the most suitable analytical method for QC, CE shows exceptional potential in the future analysis of Ginkgo biloba dosage forms while HPLC-PDA and HPLC-ELSD are currently the most affordable and practical instruments for the routine analysis of the flavonols and terpenoids, respectively. LC-MS-MS proved to be pivotal for the accurate identification and quantification of the flavonols due to interference by other flavonoid compounds with similar retention times and UV spectra to the peaks of interest. All quantitative and qualitative results revealed large discrepancies in the marker content between the products regardless of which batch was analysed and product labels disclosed little relevant information. Although currently not required by most regulatory agencies, some of the usual quality criteria applied to orthodox medicines was evaluated. In particular, dissolution analysis, disintegration, tablet hardness and weight uniformity were assessed and revealed similar inconsistencies. This thesis emphasises that implementation of effective QC criteria is long overdue and is essential to ensure consistent product QSE of commercially available Ginkgo biloba solid oral dosage forms.

Esta Dissertação apresenta informações gerais sobre os medicamentos fitoterápicos e os aspectos toxicológicos de uma amostra de dez plantas medicinais comercializadas como medicamentos fitoterápicos no Brasil, pesquisados junto aos principais bancos de dados e fontes públicas de informação. A análise dos medicamentos fitoterápicos cobre as dimensões histórica, econômica e farmacêutica. No tocante aos aspectos toxicológicos, foram avaliados os dados de toxicidade pré-clínica de dez plantas selecionadas, e realizado um levantamento das informações publicadas para Hypericum perforatum e Piper methysticum, relacionadas ao termo toxicidade, segundo três importantes bancos de dados. Adicionalmente, este trabalho aborda as propostas para realização de ensaios de toxicidade pré-clínica para os fitoterápicos segundo a OMS e legislação brasileira, e as normas para avaliação de substâncias químicas segundo a OECD. / This thesis presents general information on herbal medicines and on the toxicological aspects of a sample of ten medicinal plants traded as herbal medicines in Brazil. The survey was carried out in the main databases and public sources of information. The analysis on the herbal medicines encompasses the historical, economic and pharmaceutical dimensions. In what concerns toxicological aspects, data on pre-clinical toxicity were evaluated for Hypericum perforatum and Piper methysticum, these keywords being related to toxicity in three important databases. In addition, this study discusses the proposals for pre-clinical toxicity trials on herbal medicines according to WHO and the Brazilian legislation, and the standards for evaluation of chemical substances according to OECD.

Esta Dissertação apresenta informações gerais sobre os medicamentos fitoterápicos e os aspectos toxicológicos de uma amostra de dez plantas medicinais comercializadas como medicamentos fitoterápicos no Brasil, pesquisados junto aos principais bancos de dados e fontes públicas de informação. A análise dos medicamentos fitoterápicos cobre as dimensões histórica, econômica e farmacêutica. No tocante aos aspectos toxicológicos, foram avaliados os dados de toxicidade pré-clínica de dez plantas selecionadas, e realizado um levantamento das informações publicadas para Hypericum perforatum e Piper methysticum, relacionadas ao termo toxicidade, segundo três importantes bancos de dados. Adicionalmente, este trabalho aborda as propostas para realização de ensaios de toxicidade pré-clínica para os fitoterápicos segundo a OMS e legislação brasileira, e as normas para avaliação de substâncias químicas segundo a OECD. / This thesis presents general information on herbal medicines and on the toxicological aspects of a sample of ten medicinal plants traded as herbal medicines in Brazil. The survey was carried out in the main databases and public sources of information. The analysis on the herbal medicines encompasses the historical, economic and pharmaceutical dimensions. In what concerns toxicological aspects, data on pre-clinical toxicity were evaluated for Hypericum perforatum and Piper methysticum, these keywords being related to toxicity in three important databases. In addition, this study discusses the proposals for pre-clinical toxicity trials on herbal medicines according to WHO and the Brazilian legislation, and the standards for evaluation of chemical substances according to OECD.

A preocupação com a exposição do Homem ao ruído excessivo data dos primórdios da civilização. Com a modernidade, os níveis de ruído têm sido crescentes não só nas atividades laborais, como também nas sociais e recreativas, levando a perda auditiva induzida por níveis de pressão sonora elevada (PAINPS) a ser considerada a segunda maior causa de surdez. Esses fatos nos levaram a realizar estudo experimental buscando avaliar o efeito do nível de pressão sonora elevada e do extrato seco de Ginkgo biloba EGb 761 na cóclea de cobaias albinas, fêmeas e adultas, pela avaliação de eventuais alterações da sua citoarquitetura, por meio de microscopia de luz e pela observação das alterações do epitélio sensorial com microscopia eletrônica de varredura / Human exposition to excessive sound levels has been a cause for preoccupation since the early stages of our civilization. In the Modern Era, sound levels have increased not only at work but also during social and leisure activities, leading the hearing loss induced by high levels of sound pressure to be considered the second most important cause of deafness. Bearing that in mind an experimental work was designed in an attempt to search for the effects of high sound pressure and Ginkgo biloba EGb 761 dry extract to the cochlea of albino adult female guinea pigs with the evaluation of cell architecture alterations by means of light microscopy and also by the evaluation of alterations to the sensory epithelium with the scanning electron microscopy (SEM) array

A preocupação com a exposição do Homem ao ruído excessivo data dos primórdios da civilização. Com a modernidade, os níveis de ruído têm sido crescentes não só nas atividades laborais, como também nas sociais e recreativas, levando a perda auditiva induzida por níveis de pressão sonora elevada (PAINPS) a ser considerada a segunda maior causa de surdez. Esses fatos nos levaram a realizar estudo experimental buscando avaliar o efeito do nível de pressão sonora elevada e do extrato seco de Ginkgo biloba EGb 761 na cóclea de cobaias albinas, fêmeas e adultas, pela avaliação de eventuais alterações da sua citoarquitetura, por meio de microscopia de luz e pela observação das alterações do epitélio sensorial com microscopia eletrônica de varredura / Human exposition to excessive sound levels has been a cause for preoccupation since the early stages of our civilization. In the Modern Era, sound levels have increased not only at work but also during social and leisure activities, leading the hearing loss induced by high levels of sound pressure to be considered the second most important cause of deafness. Bearing that in mind an experimental work was designed in an attempt to search for the effects of high sound pressure and Ginkgo biloba EGb 761 dry extract to the cochlea of albino adult female guinea pigs with the evaluation of cell architecture alterations by means of light microscopy and also by the evaluation of alterations to the sensory epithelium with the scanning electron microscopy (SEM) array

[ES] El correcto desarrollo de tejidos vasculares depende del ajuste preciso entre la proliferación de células vasculares y la diferenciación celular. En Arabidopsis thaliana, la proliferación de células vasculares en el cambium es potenciada por la citoquinina, la síntesi de la cual está promovida por la actividad dependiente de auxina de un heterodímero de factores de transcripción (TF) formado por LONESOME HIGHWAY (LHW) y por TARGET OF MONOPTEROS 5 (TMO5). Como mecanismo de seguridad, las auxinas también activan un módulo inhibidor que implica la inducción precisa de la Termospermina (Tspm) sintasa ACAULIS5 (ACL5) en células vasculares proliferantes por acción conjunta de las auxinas y del TF Class III HD-ZIP (C3HDZ) AtHB8. Entonces, la Tspm permite la traducción de las proteínas SACL de forma celular autónoma, que perjudican la actividad de LHW. Sin embargo, la observación de que estos elementos están presentes en los genomas de todas las plantas terrestres - y no sólo de las plantas vasculares - plantea dos preguntas desde una perspectiva evolutiva: (i) ¿cuál es la función de estos genes en las plantas terrestres no vasculares? y (ii) ¿cuándo se creó el módulo regulador concreto? En esta Tesis, mediante la combinación de análisis filogenéticos, celulares y moleculares con la hepática Marchantia polymorpha, proponemos que la auxina y C3HDZ son reguladores ancestrales de la expresión de ACL5, y que esta conexión se mantiene en las traqueófitas y las briófitas existentes. Por el contrario, la traducción dependiente de Tspm de SACL parece ser específica de las traqueófitas, basado en la aparición de un uORF conservado en la secuencia 5' líder de los tránscritos de SACL y en evidencia experimental basada en ensayos transitorios para la traducción de SACL. De acuerdo con estas observaciones, las funciones de MpACL5 y MpSACL son diferentes en M. polymorpha. MpACL5 se expresa en "notches" apicales y modula la bifurcación de los meristemos. Por otro lado, la expresión de MpSACL está mayoritariamente excluida de los "notches" apicales y su actividad afecta negativamente la producción de gemas y rizoides mediante la interacción con MpRSL1. Finalmente, la hibridación de ARN in situ de ortólogos de C3HDZ, ACL5 y SACL en la gimnosperma Ginkgo biloba, el helecho Ceratopteris richardii y la licófita Selaginella kraussiana indican que la expresión de los tres genes se solapa en los tejidos vasculares. Nuestros resultados sugieren que la función de C3HDZ, ACL5 y SACL ha seguido trayectorias evolutivas divergentes en briófitas y traqueófitas, para controlar, finalmente, diferentes funciones específicas dentro de cada linaje. Sólo en las traqueófitas se formó el módulo regulador y se asoció con la restricción de la proliferación de células vasculares. / [CA] El correcte desenvolupament dels teixits vasculars depèn del precís ajust entre la proliferació de cèl·lules vasculars i la diferenciació cel·lular. En Arabidopsis thaliana, la proliferació de cèl·lules vasculars al càmbium és potenciada per la citoquinina, la síntesi de la qual està promoguda per l'activitat dependent d'auxina d'un heterodímer de factors de transcripció (TF) format per LONESOME HIGHWAY (LHW) i TARGET OF MONOPTEROS 5 (TMO5). Com a mecanisme de seguretat, l'auxina també activa un mòdul inhibidor que implica la inducció precisa de la Termospermina (Tspm) sintasa ACAULIS5 (ACL5) en cèl·lules vasculars proliferants per l'acció conjunta de l'auxina i del TF Class III HD-ZIP (C3HDZ) AtHB8. Llavors, la Tspm permet la traducció de les proteïnes SACL de forma autònoma cel·lular, que perjudiquen l'activitat de LHW. Tanmateix, l'observació de que aquests elements estan presents en els genomes de totes les plantes terrestres - i no només de les plantes vasculars - planteja dues preguntes des d'una perspectiva evolutiva: (i) quina és la funció d'aquests gens en les plantes terrestres no vasculars? i (ii) quan es va crear el mòdul regulador complet? En aquesta Tesi, mitjançant la combinació d'anàlisis filogenètics, cel·lulars i moleculars amb la hepàtica Marchantia polymorpha, proposem que l'auxina i C3HDZ són reguladors ancestrals de l'expressió d'ACL5, i que aquesta connexió es mantén en els traqueòfits i briòfits existents. Per contra, la traducció depenent de Tspm de SACL sembla ser específica dels traqueòfits, basat en l'aparició d'un uORF conservat a la seqüència 5' líder dels trànscrits de SACL i en evidència experimental basada en assajos transitoris per a la traducció de SACL. D'acord amb aquestes observacions, les funcions de MpACL5 i MpSACL són diferents a M. polymorpha. MpACL5 s'expressa en "notch" apicals i modula la bifurcació dels meristems. D'altra banda, l'expressió de MpSACL està majoritàriament exclosa dels "notch" apicals i la seva activitat afecta negativament la producció de gemmes i rizoids mitjançant la interacció amb MpRSL1. Finalment, la hibridació d'ARN in situ d'ortòlegs de C3HDZ, ACL5 i SACL a la gimnosperma Ginkgo biloba, la falguera Ceratopteris richardii i el licòfit Selaginella kraussiana indica que l'expressió dels tres gens es solapa als teixits vasculars. Els nostres resultats suggereixen que la funció de C3HDZ, ACL5 i SACL va seguir trajectòries evolutives divergents en briòfits i traqueòfits, per controlar, finalment, diferents funcions específiques dins de cada llinatge. Només en els traqueòfits es va formar el mòdul regulador i es va associar amb la restricció de la proliferació de cèl·lules vasculars. / [EN] The correct development of vascular tissues depends on the precise adjustment between vascular cell proliferation and cell differentiation. In Arabidopsis thaliana, vascular cell proliferation in the cambium is enhanced by cytokinin, whose synthesis is promoted by the auxin-dependent activity of a transcription factor (TF) heterodimer formed by LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS 5 (TMO5). As a safety mechanism, auxin also deploys a negative feedforward regulatory module which involves the precise induction of the Thermospermine (Tspm) synthase ACAULIS5 (ACL5) in proliferating vascular cells by the joint action of auxin and the class-III HD-ZIP (C3HDZ) AtHB8 TF. Tspm then allows the cell-autonomous translation of the SACL proteins, which impair the activity of LHW. However, the observation that these elements are present in the genomes of all land plants -and not only vascular plants- poses two questions from an evolutionary perspective: (i) what is the function of these genes in non-vascular land plants? and (ii) when was the full regulatory module assembled? In this Thesis, through the combination of phylogenetic, cellular, and molecular genetic analyses with the liverwort Marchantia polymorpha, we propose that auxin and C3HDZ are ancestral regulators of ACL5 expression, and that this connection is maintained in extant tracheophytes and bryophytes. On the contrary, thermospermine-dependent translation of SACL seems to be specific of tracheophytes, based on the appearance of a conserved uORF in the 5' leader sequence of SACL transcripts and on experimental evidence using transient assays for SACL translation. In agreement with these observations, the functions of MpACL5 and MpSACL are different in M. polymorpha. MpACL5 is expressed in apical notches and modulates meristem bifurcation. On the other hand, MpSACL expression is mostly excluded from apical notches and its activity negatively affects gemmae and rhizoid production through the interaction with MpRSL1. Finally, in situ RNA hibridization of C3HDZ, ACL5 and SACL orthologs in the gymnosperm Ginkgo biloba, the fern Ceratopteris richardi and the lycophyte Selaginella kraussiana indicates that the expression of the three genes overlaps in vascular tissues. Our results suggest that the function of C3HDZ, ACL5 and SACL followed divergent evolutionary trajectories in bryophytes and tracheophytes, to ultimately control different lineage-specific functions. Only in tracheophytes was the regulatory module assembled and associated with the restriction of vascular cell proliferation. / Solé Gil, A. (2023). Origin & Evolution of the C3HDZ-ACL5-SACL Regulatory Module in Land Plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196681

The desire to enhance and make ourselves better is not a new one and it has continued to intrigue throughout the ages. Individuals have continued to seek ways to improve and enhance their well-being for example through nutrition, physical exercise, education and so on. Crucial to this improvement of their well-being is improving their ability to remember. Hence, people interested in improving their well-being, are often interested in memory as well. The rationale being that memory is crucial to our well-being. The desire to improve one’s memory then is almost certainly as old as the desire to improve one’s well-being. Traditionally, people have used different means in an attempt to enhance their memories: for example in learning through storytelling, studying, and apprenticeship. In remembering through practices like mnemonics, repetition, singing, and drumming. In retaining, storing and consolidating memories through nutrition and stimulants like coffee to help keep awake; and by external aids like notepads and computers. In forgetting through rituals and rites. Recent scientific advances in biotechnology, nanotechnology, molecular biology, neuroscience, and information technologies, present a wide variety of technologies to enhance many different aspects of human functioning. Thus, some commentators have identified human enhancement as central and one of the most fascinating subject in bioethics in the last two decades. Within, this period, most of the commentators have addressed the Ethical, Social, Legal and Policy (ESLP) issues in human enhancements as a whole as opposed to specific enhancements. However, this is problematic and recently various commentators have found this to be deficient and called for a contextualized case-by-case analysis to human enhancements for example genetic enhancement, moral enhancement, and in my case memory enhancement (ME). The rationale being that the reasons for accepting/rejecting a particular enhancement vary depending on the enhancement itself. Given this enormous variation, moral and legal generalizations about all enhancement processes and technologies are unwise and they should instead be evaluated individually. Taking this as a point of departure, this research will focus specifically on making a case for ME and in doing so assessing the ESLP implications arising from ME. My analysis will draw on the already existing literature for and against enhancement, especially in part two of this thesis; but it will be novel in providing a much more in-depth analysis of ME. From this perspective, I will contribute to the ME debate through two reviews that address the question how we enhance the memory, and through four original papers discussed in part three of this thesis, where I examine and evaluate critically specific ESLP issues that arise with the use of ME. In the conclusion, I will amalgamate all my contribution to the ME debate and suggest the future direction for the ME debate.