Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Bc. Hana Studená Supervisor: Doc. RNDr. Jiřina Spilková, CSc. Consultant: RNDr. Anna Polášková Title of diploma thesis: Quality control of herbal drug Sambuci fructus from cultivated plants The main aim of this thesis was to verify the quality of elderberry fruits of the cultivars Sambo, Sambu, Samdal, Samyl and of the wild elderberry according to tests specified in the Czech Pharmacopoeia 2009 and the Czech Pharmaceutical Codex 1. Loss on drying, total ash, ash insoluble in hydrochloric acid were determined. Another aim of this thesis was to compare certain cultivars of elderberry according to the content of anthocyanins in different parts of the fruits. A new modification of the spectrophotometric analysis using the pH-differential method was applied and its linearity and accuracy verified. Further, stability of the samples under given conditions was studied. A loss of anthocyanins content was observed, which was lower than 10 percent. The highest amount of anthocyanins was found in the cultivars Samyl and Samdal, the lowest amount in Sambu and wild elderberry. Although the determined quantities of anthocyanins differed in various parts of the fruit, the amount of anthocyanins in exocarp was...

Mon travail a consisté essentiellement à proposer une traduction moderne et scientifique de l’herbier du Ps.-Apulée, un texte du IVe siècle qui contient des recettes médicales associées à diverses plantes médicinales. Cet ouvrage a connu un grand succès au Moyen Âge et il en existe de nombreux manuscrits. Néanmoins, il a longtemps été méprisé et considéré comme une simple compilation d’ouvrages grecs. L’édition commentée la plus récente est celle d’Ackermann en 1788 et il n’y a pas de traduction française qui tienne compte l’édition d’E. Howald et H. Sigerist en 1927 et des progrès accomplis depuis dans la connaissance de ce texte. Nous avons donc essayé de traduire le texte de l’Herbier édité par ces deux savants tout en expliquant, critiquant et parfois contestant certains de leurs choix. Nous avons aussi cherché dans l’introduction et dans les notes à situer l’oeuvre dans la tradition médicale en travaillant à la fois sur les sources de l’oeuvre et sur sa postérité et nous avons joint à la traduction en annexes un certain nombre de nos outils de travail. Nous espérons que ce travail permettra de mieux comprendre ce que représentaient les plantes médicinales, « les simples », dans l’Antiquité tardive. / The primary objective of my research was to offer a modern and technical translation of the Herbarius of Pseudo-Apuleius. This document dates back to the 4th century and contains medical prescriptions associated with plants. During the Middle Age, the book was quite successful as numerous copies are available. For a long time, however, it was despised and considered as a simple compilation of Greek works. The most recent edition with commentaries is from Ackermann in 1788 and there is unfortunately no sufficient French translation to fully benefit from the 1927 commentaries of E. Howald and H. Sigerist and from more recents studies on this text. We have therefore attempted to translate the text edited by these two experts and have tried to explain as well as critique and at times dispute their choices. In the Introduction and the notes section, we have been mindful to respect the text’s medical tradition, researching its original sources and its posterity. We hope this research will help better understand the role and importance of medicinal plants, also known as “the simple ones”, in the late antiquity.

<p>This Master Thesis was conducted at Octapharma AB in Stockholm.</p><p>Anticomplementary activity (ACA) is a measure of the product’s abilities to activate the complement system. IgG aggregates are mainly responsible for this activation. Two different performances of a method for determination of ACA in Octagam^® are available. The two performances are based on the reference method for test of ACA in immunoglobulins in the European Pharmacopoeia Commission Guideline 6.0 (chapter 2.6.17). The method is carried out either in test tubes or on microtiter plates. The test tube method can be performed either in a manual manner or modified, being more automated. The latter performance has been applied in this study. The plate method is more automated than both of the tube methods. The plate method and the manual tube method have earlier seemed to result in different outcomes, which was the basis for this thesis.</p><p>The plate method and the modified test tube method have been compared and robustness parameters have been studied in order to see which factors influence on the end result. The adequacy of using Human Biological Reference Preparation (human BRP) as a control for the ACA method in general has also been investigated. Samples of the product are outside the scope of this thesis and have not been investigated.</p><p>According to this study, the plate method and the modified tube method are not comparable with regard to complement titration results and to ACA of the BRP control. A higher precision is gained with the plate method. This in combination with the higher degree of automation makes the plate method advantageous in several aspects. When it comes to the robustness of the ACA method in general, the sheep red blood cells (SRBC) used are critical. Haemolysin dilution and complement activity seem to be critical as well.</p><p>Human BRP is, according to this study more adequate as a reference for the plate method than for the tube method. An In house control is believed to be more representative to the ACA method in general as it is of the same nature as the samples analysed, in contrast to the human BRP.</p>

Malaria is a parasitic disease claiming one million lives worldwide annually. Unfortunately, malaria-endemic countries in need of good quality medicines are also overwhelmed with counterfeit or substandard medicine. This results in treatment inefficacy, resistance towards treatment and death. Counterfeit or substandard quinine sulfate tablets are known to have infiltrated the market, however at this point in time, treatment efficacy of quinine sulfate has fortunately not yet been significantly impaired by resistance, but immediate action is required to prevent it from becoming obsolete. Validated analytical methods with justified specifications are effective in controlling the quality of medicines and to minimise the effect of poor quality medicines. Pharmacopoeia specifies analytical quality control procedures and accompanying specifications to standardise acceptable levels of product quality. Understandably, different monographs of different pharmacopoeias are developed by different independent laboratories and therefore their respective test procedures/specifications for the same FPP may differ from each other. Institutions such as the Pharmacopoeial Dicussion Group (PDG) aim to harmonise pharmacopoeia in order to synchronise final outcomes. This study evaluated the relevancy of differences in analytical procedures, results and specifications for quinine sulfate tablets set by the United States Pharmacopoeia (USP), British Pharmacopoeia (BP) and International Pharmacopoeia (Ph.Int.) in an aim to ensure that these different methods all provide with similar final outcomes and that they be effective in successfully evaluating the quality of quinine sulfate tablets. Four quinine sulfate tablet products were obtained from different manufacturers and were subjected to the tests of all three pharmacopoeia – BP, USP and Ph.Int. The results from identification, assay and related substance testing concluded that the outcomes were the same between the pharmacopoeia despite their differences in techniques/procedures/specifications. The assay, identification and related substances methods and specifications set by each respective monograph were deemed appropriate to evaluate the quality of quinine sulfate tablets. Even with differences in methodology, quantitative techniques and specifications, the USP and BP dissolution methods for quinine sulfate tablets shared the same final outcome at the first stage of dissolution, whereas none of the products achieved a compliant outcome using the Ph.Int. dissolution method. Possible reasons for the poor dissolution (when using the Ph.Int. method) were identified and investigated. Investigation into the solubility of quinine sulfate found the Ph.Int. dissolution method conditions to be too stringent, as the solubility of quinine sulfate in phosphate buffer pH 6.8 (dissolution medium specified by the Ph.Int.) was found to be much less than in acidic media (as proposed by the BP and USP dissolution methods). Several adapted dissolution methods (called developmental studies) were investigated to serve as potential alternatives for the Ph.Int. dissolution method. The developmental studies investigated an alternative dissolution medium, agitation rates (50 rpm, 75 rpm, 100 rpm) and medium volumes (500 ml, 750 ml, 900 ml and 1000 ml). Developmental study 6 was proposed as an alternative dissolution method. Developmental study 6 stipulates the use of the same medium as the original Ph.Int. method, as it was deemed the medium of choice for its discriminatory ability. To address the impaired solubility of quinine sulfate in phosphate buffer, the medium volume and agitation were increased (in reference to the original method) to 900 ml and 100 rpm respectively. The same analytical quantitation technique (UV-Vis spectroscopy) is proposed for Developmental study 6. The newly proposed method provided with final outcomes comparable to that of the USP and BP, however having more discriminatory power than the USP and BP. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Malaria is a parasitic disease claiming one million lives worldwide annually. Unfortunately, malaria-endemic countries in need of good quality medicines are also overwhelmed with counterfeit or substandard medicine. This results in treatment inefficacy, resistance towards treatment and death. Counterfeit or substandard quinine sulfate tablets are known to have infiltrated the market, however at this point in time, treatment efficacy of quinine sulfate has fortunately not yet been significantly impaired by resistance, but immediate action is required to prevent it from becoming obsolete. Validated analytical methods with justified specifications are effective in controlling the quality of medicines and to minimise the effect of poor quality medicines. Pharmacopoeia specifies analytical quality control procedures and accompanying specifications to standardise acceptable levels of product quality. Understandably, different monographs of different pharmacopoeias are developed by different independent laboratories and therefore their respective test procedures/specifications for the same FPP may differ from each other. Institutions such as the Pharmacopoeial Dicussion Group (PDG) aim to harmonise pharmacopoeia in order to synchronise final outcomes. This study evaluated the relevancy of differences in analytical procedures, results and specifications for quinine sulfate tablets set by the United States Pharmacopoeia (USP), British Pharmacopoeia (BP) and International Pharmacopoeia (Ph.Int.) in an aim to ensure that these different methods all provide with similar final outcomes and that they be effective in successfully evaluating the quality of quinine sulfate tablets. Four quinine sulfate tablet products were obtained from different manufacturers and were subjected to the tests of all three pharmacopoeia – BP, USP and Ph.Int. The results from identification, assay and related substance testing concluded that the outcomes were the same between the pharmacopoeia despite their differences in techniques/procedures/specifications. The assay, identification and related substances methods and specifications set by each respective monograph were deemed appropriate to evaluate the quality of quinine sulfate tablets. Even with differences in methodology, quantitative techniques and specifications, the USP and BP dissolution methods for quinine sulfate tablets shared the same final outcome at the first stage of dissolution, whereas none of the products achieved a compliant outcome using the Ph.Int. dissolution method. Possible reasons for the poor dissolution (when using the Ph.Int. method) were identified and investigated. Investigation into the solubility of quinine sulfate found the Ph.Int. dissolution method conditions to be too stringent, as the solubility of quinine sulfate in phosphate buffer pH 6.8 (dissolution medium specified by the Ph.Int.) was found to be much less than in acidic media (as proposed by the BP and USP dissolution methods). Several adapted dissolution methods (called developmental studies) were investigated to serve as potential alternatives for the Ph.Int. dissolution method. The developmental studies investigated an alternative dissolution medium, agitation rates (50 rpm, 75 rpm, 100 rpm) and medium volumes (500 ml, 750 ml, 900 ml and 1000 ml). Developmental study 6 was proposed as an alternative dissolution method. Developmental study 6 stipulates the use of the same medium as the original Ph.Int. method, as it was deemed the medium of choice for its discriminatory ability. To address the impaired solubility of quinine sulfate in phosphate buffer, the medium volume and agitation were increased (in reference to the original method) to 900 ml and 100 rpm respectively. The same analytical quantitation technique (UV-Vis spectroscopy) is proposed for Developmental study 6. The newly proposed method provided with final outcomes comparable to that of the USP and BP, however having more discriminatory power than the USP and BP. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Whole cell biosensors have been extensively used for monitoring toxicity and contamination of compounds in environmental biology and microbial ecology. However, their application in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET) has been limited. According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to construct and develop whole cell microbial biosensors with Pseudomonas aeruginosa ATCC 9027. Constitutive promoters: PlysS, Pspc, Ptat, Plpp and PldcC and the lux-cassette were inserted into plasmid pME4510 and transformed into P. aeruginosa ATCC 9027 cells to produce bioluminescent strains. Plasmids were found to be maintained stably (~50 copies per cell) throughout the growth and death cycle. The novel bioluminescent strains were validated in accordance with the pharmacopoeia using bioluminescence detection and quantification followed by comparison with the traditional plate counting method. The bioluminescent method was found to be accurate, precise and equivalent at a range of 103 – 107 CFU/mL, as compared with plate counting. Recovery of bacterial cells was quantified using bioluminescence; this method proved to be accurate with percentage recoveries between 70-130% for all bioluminescent strains. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. Therefore, the bioluminescent constructs passed/exceeded pharmacopoeial specified criteria for range, limit of detection, accuracy, precision and equivalence. Physiology of the validated bioluminescent strains was studied by assessing the growth and death patterns using constitutive gene expression linked with bacterial replication. Promoter strengths were evaluated at various stages of the growth and death pattern and related to promoter sequences. PlysS, Ptat and Plpp were relatively strong promoters whilst PldcC and Pspc were relatively weak promoters. Relative promoter strength decreased in the order of Plpp>Ptat>PlysS>PldcC>Pspc during the exponential phase whilst Ptat was stronger than Plpp during the stationary phase of growth. Plpp had its highest level of expression during the exponential phase, while Ptat had relatively stable lux expression during the stationary phase. Correlations between relative bioluminescence and CFU at 24 hours were greater than 0.9 indicating a strong relationship for all bioluminescent strains. Reduction in correlation coefficients to approximately 0.6 between relative bioluminescence and CFU and between relative fluorescence and CFU beyond 24 hours indicated that a certain proportion of cells were viable but non-culturable. Tat-pME-lux showed steady bioluminescence compared to CFU count (R>0.9) throughout 28 days of growth. Equivalence analysis showed no significant difference between the bioluminescence and plate count method throughout 28 days of growth for all five bioluminescent strains. Applicability of these novel bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication and bioluminescence as a measure of constitutive gene expression. PET using benzalkonium chloride and benzyl alcohol showed no significant difference between the bioluminescent method and the plate count method. Good correlations between bioluminescence, CFU count and fluorescence were obtained for benzalkonium chloride (BKC) concentrations (R>0.9) between 0.0003% and 0.0025% against strains lysR25, lppR4 and tatH5. Similarly, good correlations (R>0.9) between the three parameters were obtained for benzyl alcohol (BA) concentrations between 0.125% and 2% against strains lysR25, lppR4 and tatH5. The bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003 - 0.02%) and BA (0.25 - 2%) during preservative efficacy tests. These bioluminescent constructs therefore are good candidates for selection for preservative efficacy testing. The bioluminescent method and traditional plate counting method were also found to be equivalent for construct tatH5 at a concentration of 0.125% BA. PET testing with BKC and BA showed that tatH5-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence. Together with the results from growth and death kinetics, where tatH5 showed the greatest constitutive expression, it can be concluded that P. aeruginosa ATCC 9027 tatH5-pMElux is the best construct for testing various antimicrobial agents. This study has shown that according to the pharmacopoeial requirements, the bioluminescent method is more accurate, precise and equivalent to the traditional plate counting method and therefore can be utilised instead of the traditional plate counting method for the purpose of preservative efficacy testing.

This Master Thesis was conducted at Octapharma AB in Stockholm. Anticomplementary activity (ACA) is a measure of the product’s abilities to activate the complement system. IgG aggregates are mainly responsible for this activation. Two different performances of a method for determination of ACA in Octagam® are available. The two performances are based on the reference method for test of ACA in immunoglobulins in the European Pharmacopoeia Commission Guideline 6.0 (chapter 2.6.17). The method is carried out either in test tubes or on microtiter plates. The test tube method can be performed either in a manual manner or modified, being more automated. The latter performance has been applied in this study. The plate method is more automated than both of the tube methods. The plate method and the manual tube method have earlier seemed to result in different outcomes, which was the basis for this thesis. The plate method and the modified test tube method have been compared and robustness parameters have been studied in order to see which factors influence on the end result. The adequacy of using Human Biological Reference Preparation (human BRP) as a control for the ACA method in general has also been investigated. Samples of the product are outside the scope of this thesis and have not been investigated. According to this study, the plate method and the modified tube method are not comparable with regard to complement titration results and to ACA of the BRP control. A higher precision is gained with the plate method. This in combination with the higher degree of automation makes the plate method advantageous in several aspects. When it comes to the robustness of the ACA method in general, the sheep red blood cells (SRBC) used are critical. Haemolysin dilution and complement activity seem to be critical as well. Human BRP is, according to this study more adequate as a reference for the plate method than for the tube method. An In house control is believed to be more representative to the ACA method in general as it is of the same nature as the samples analysed, in contrast to the human BRP.

Dans les trois anciens pays indochinois, le Vietnam, le Cambodge et la RDP Lao, la médecine traditionnelle rencontre un succès grandissant auprès de la population. Conscients de l’importance de la médecine traditionnelle, les trois gouvernements l’ont reconnue et intégrée, chacun différemment, dans leur système de soins de santé national. L’encadrement juridique de cette médecine millénaire diffère d’un pays à l’autre. La législation de la médecine traditionnelle du Cambodge demeure particulièrement faible. De plus, de nombreux problèmes persistent et menacent la préservation et le développement de cet héritage précieux. Avec l’aide de partenaires internationaux, les trois gouvernements aséaniens tentent de renforcer l’encadrement juridique de leurs médecines et de leurs pharmacopées traditionnelles / In the three former Indochinese countries, Vietnam, Cambodia and PDR Lao, traditional medicine success is growing among the population. Recognizing the importance of traditional medicine, the three governments have recognized and integrated it in a different way into the national health care system. The legal framework of this millennial medicine differs from one country to another. Traditional medicine legislation in Cambodia remains particularly weak. Moreover, many problems persist and threaten the preservation and development of this precious heritage. With the help of international partners, the three ASEAN governments are trying to strengthen the legal framework for their traditional medicines and pharmacopoeias.

L'objet de la présente étude est de déterminer quelle place était accordée à la pratique de la médecine et aux professionnels de santé dans les cours princières occidentales du bas Moyen Age, à travers l'exemple de la cour des ducs de Bourgogne Valois, de l'avènement de Philippe Hardi en 1363 à la mort de Marie de Bourgogne en 1482. Dans une première partie sont identifiées les différentes professions médicales représentées à la cour de Bourgogne : physiciens ou médecins, chirurgiens, barbiers, épiciers-apothicaires et sages-femme, ainsi que leurs différentes voies d'accès à la cour ducale. Ces spécialistes se distinguent par la formation qu'ils ont reçue et par leurs compétences particulières qui, réunies, leurs permettent de former une équipe soignante polyvalente. L"organisation de celle-ci au sein de l'hôtel ducal et ses limites seront abordées dans une deuxième partie. Ce personnel médical, gravitant au plus près de la famille ducale, bénéficie de revenus et privilèges notables, afférents à la place qui leur est attribuée à la cour. La troisième partie de cette thèse est consacrée à la pratique médicale proprement dite : les différentes étapes de la prise en charge du patient, nommée "collatio", sont détaillées. Elles visent à établir le diagnostic. Les divers procédés permettant de rétablir l'équilibre humoral sont ensuite décrits : ils prennent la forme, en préventif, de conseils d'hygiène de vie ; en curatif, de traitements physiques et psychologiques, médicamenteux et chirurgicaux. Le rôle fondamental de praticiens de santé au moment des naissances et décès princiers constituent les dernières analyses de ces travaux; Fondés sur un dépouillement de sources comptables essentiellement, mais aussi normatives, didactiques et narratives, ils sont accompagnés d'un catalogue prosopographique reprenant la biographique de chacun des praticiens ayant fréquenté la cour de Bourgogne au cours de la période considérée. / The purpose of this study is to determine what place was given to the practice of medicine ans health professionals in Western princely courts of the late Middle Ages, through the example of the court of dukes of Burgundy Valois, from the accession of Philip the Bold in 1363 to the death of Mary Burgundy in 1482. In the first part are identified the various medical professions represented at the court of Burgundy : physicians, surgeons, barbers, apothecaries ans midwives, and their different access to the ducal court. These spécialists are distinguished by their training and their special sdills which, combined allows them to form e versatile healthcare team. The organization of the latter in the "hôtel", and limitations, will be addressed in the second part. This gravitating medical personnel closer to the ducal family gains income and notable privileges relating to the place assigned to them in court. The third part of this dissetation is devoted to the actual medical practice : the different stages of the parient's care are detailed, namely the collective consultation, to estavlish the diagnosis. The various methods to restore humoral balance are then desceibed : lifestyle advices for the prenventive aspect ; and physical, psychological, medical and surgical treatments for the curative one. The fundamental role of health practitoners at the time of princely births and deaths constitute the latest analysis of this work. Based mainly on accounting sources, but also normative, didactic and narrative, they are accompanied by a prosopographic catalog gathering the biography of each practitioner who attended the court of Burfundy during the period.

Résumé confidentiel / Résumé confidentiel