Digitized by Kansas Correctional Industries / Department: Biochemistry.

Thesis (M.A.) -- Central Connecticut State University, 2009. / Thesis advisor: Cheryl L. Watson. "... in partial fulfillment of the requirements for the degree of Master of Arts in Biomolecular Science." Includes bibliographical references (leaves 38-40). Also available via the World Wide Web.

Os caracóis terrestres são animais capazes de produzir através de glândulas localizadas em toda superfície do seu corpo, uma secreção glicoproteica denominada muco, que dentre outras funções, apresenta poder antibacteriano, que pode auxiliar nos processos de reparação de feridas de origens diversas. Desta forma, o objetivo da presente pesquisa foi o de avaliar macroscópica e histologicamente, os efeitos reparadores do muco dos escargots Achatina fulica e Achatina achatina monochromatica, em lesões provocadas na pele de camundongos da linhagem "hairless"; verificar sua potencialização após a ingestão de uma ração contendo em sua formulação uma planta com propriedades cicatrizantes comprovadas, o Confrei (Symphytum officinale); e analisar bioquimicamente a composição do muco destes caracóis. Foram selecionados caracóis das espécies Achatina fulica (n=30) e Achatina monochromatica monochromatica (n=30), retirado seu muco através de estimulação manual da glândula podal e verificação de seu efeito reparador. Utilizaram-se camundongos da linhagem "hairless" (n=75), submetidos à intervenção cirúrgica e tratados com muco de ambas as espécies de caracóis. As características macroscópicas da lesão foram registradas em protocolo e avaliadas. Fragmentos de pele foram submetidos à biópsia aos 3, 5 e 7 dias de experimento, fixados em Paraformoldeído, processados e incluídos em parafina. Os cortes foram corados com Hematoxilina-Eosina e Tricrômio de Mason. Macroscopicamente, os animais tratados mostraram edemas menores, maior presença de crosta e maior contração das bordas das feridas ao final do experimento, quando comparados ao grupo controle. Histologicamente, os grupos tratados também revelaram melhor reparação da lesão, apresentando edemas menos intensos, grandes quantidades de tecido de granulação e infiltrados inflamatórios. O grupo tratado com muco de caracóis alimentados com ração contendo Symphytum officinale foi o que apresentou melhores resultados. / Land snails are animals able to produce through glands located all over their body surface, a glycoprotein secretion called mucus. This mucus, within other roles, shows an antibacterial power that can help in the healing processes of wounds from several sources. Therefore, the aim of this study was to evaluate through macroscopic and histological techniques the repairing effects of the mucus in the snails Achatina fulica and Achatina achatina monochromatica in lesions intentionally caused to hairless linage mice skin; to investigate mucus potential effect after snails have been fed with a diet formulation containing a plant with proved scar properties, the Comfrey (Symphytum officinale); and to study the biochemical composition of mucus from these snails. Snails of Achatina fulica (n=30) and Achatina monochromatica monochromatica (n=30) species were sorted, the mucus extracted through manual stimulation of the podal gland and the repairing effect studied. Hairless linage mice (n=75) used in this study went through surgery and then treated with mucus from both species of snails. The macroscopic characteristics of the lesion were recorded and assessed. Fragments of skin were taken to biopsy at 3, 5 and 7 days of trial, fixed in paraformaldehyde, processed and embedded in wax. Sections were stained with Hematoxilin-Eosin and Tricromio of Mason. Macroscopically, treated mice showed minor swelling, higher presence of scabs and higher contraction of wound edges at the end of the trial, when compared with control. Histologically, groups that received treatment also showed better lesion repair, presenting less intense swellings, large amount of scar tissue and inflammatory infiltrates. The group treated with mucus from snails that received a diet containing Symphytum officinale presented the best results.

Resumo: O Symphytum officinale (confrei) é uma planta usada desde a antiguidade para o tratamento da consolidação de fraturas. Existem relatos de seu uso in natura , como fitoterápico ou como medicamento homeopático. Recentemente foram relatados casos de intoxicação em humanos e em animais que consumiram a planta e desenvolveram lesões hepáticas graves que foram associadas à presença de alcalóides pirrolizidínicos. Tais fatos levaram à proibição do uso interno da planta. No entanto, não há relato de efeitos adversos associados à sua formulação homeopática. O objetivo deste trabalho foi comparar os efeitos do Symphytum officinale, preparado como medicamento fitoterápico e homeopático, avaliando sua toxicidade hepática e renal. Foram utilizados 42 ratos adultos saudáveis, divididos em três grupos de acordo com os diferentes tratamentos: Symphytum officinale 500mg/kg (F), Symphytum officinale 6CH 2 glóbulos (H) e água como placebo no grupo controle negativo (C), que foram administrados diariamente a cada animal por via oral por gavagem. Os sacrifícios foram realizados 30 e 60 dias após o início do tratamento. A avaliação toxicológica foi feita através de análise bioquímica da atividade enzimática da fosfatase alcalina (FA), aspartato aminotransferase (AST), alanina aminotransferase (ALT), gama glutamil transferase (GGT) e da concentração plasmática de uréia e creatinina. Também foi realizada análise histológica e histomorfométrica de fígado e rim. Os dados foram submetidos aos testes estatísticos de t (Student) e ANOVA 1 fator (5%). Tanto as análises bioquímicas quanto histológicas indicaram discretas alterações hepáticas e evidenciaram ausência de alterações renais. As modificações no perfil de atividade enzimática, na uréia e creatinina e no peso dos animais sugeriram alterações no metabolismo hepático interferindo na síntese... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Symphytum officinale (comfrey) is a plant used over thousands of years to treat bone fractures consolidation. There are reports of its use in natura, as phytotherapic and as homeopathic medication. Recent scientific reports described cases of human and animal intoxication, with development of severe hepatic lesions after consumption of this plant, associated with the presence of pyrrolizidine alkaloids. Because of these facts its internal use was prohibited. However, there are no reports of adverse effects of Symphytum officinale 's homeopathic formulation. The aim of this work was to compare the effects of Symphytum officinale used as phytotherapic and as homeopathic formulations, evaluating its hepatic and renal toxicity. Adult healthy rats (n=42) were divided in 3 groups according to treatment with Symphytum officinale 500mg/kg (F), Symphytum officinale 6CH 2 globules (H), and water as placebo for negative control (C), given daily orally by gavage to each animal. They were sacrificed 30 and 60 days after beginning of treatments. The toxicological evaluation was realized by biochemical analysis of the enzymatic activity of alkaline phosphatase (AF), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) and of creatinine and urea plasmatic concentration. Histological and histomorphometric analysis of liver and kidney was also conducted. Data were submitted to statistical t (Student) and ANOVA 1 factor (5%) tests. Both biochemical and histological analysis showed mild hepatic alterations and no renal alterations. The modifications of the enzymatic activity, of urea and creatinine, and of rats' weight suggested hepatic metabolic alterations interfering on protein synthesis. Histological analysis was compatible with chronic mild hepatitis with development of important fibrosis only in the F group. The phytotherapic formulation used was... (Complete abstract click electronic access below) / Orientador: Adriana Aigotti Haberbeck Bandão / Coorientador: Maria Nadir Gasparoto Mancini / Banca: Rosilene Fernandes da Rocha / Banca: João Ernesto de Carvalho / Mestre

Made available in DSpace on 2014-06-11T19:27:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-21Bitstream added on 2014-06-13T19:15:38Z : No. of bitstreams: 1 lima_ap_me_sjc.pdf: 840973 bytes, checksum: 2f4db3725d3a6941b05476631d72d7de (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O Symphytum officinale (confrei) é uma planta usada desde a antiguidade para o tratamento da consolidação de fraturas. Existem relatos de seu uso in natura , como fitoterápico ou como medicamento homeopático. Recentemente foram relatados casos de intoxicação em humanos e em animais que consumiram a planta e desenvolveram lesões hepáticas graves que foram associadas à presença de alcalóides pirrolizidínicos. Tais fatos levaram à proibição do uso interno da planta. No entanto, não há relato de efeitos adversos associados à sua formulação homeopática. O objetivo deste trabalho foi comparar os efeitos do Symphytum officinale, preparado como medicamento fitoterápico e homeopático, avaliando sua toxicidade hepática e renal. Foram utilizados 42 ratos adultos saudáveis, divididos em três grupos de acordo com os diferentes tratamentos: Symphytum officinale 500mg/kg (F), Symphytum officinale 6CH 2 glóbulos (H) e água como placebo no grupo controle negativo (C), que foram administrados diariamente a cada animal por via oral por gavagem. Os sacrifícios foram realizados 30 e 60 dias após o início do tratamento. A avaliação toxicológica foi feita através de análise bioquímica da atividade enzimática da fosfatase alcalina (FA), aspartato aminotransferase (AST), alanina aminotransferase (ALT), gama glutamil transferase (GGT) e da concentração plasmática de uréia e creatinina. Também foi realizada análise histológica e histomorfométrica de fígado e rim. Os dados foram submetidos aos testes estatísticos de t (Student) e ANOVA 1 fator (5%). Tanto as análises bioquímicas quanto histológicas indicaram discretas alterações hepáticas e evidenciaram ausência de alterações renais. As modificações no perfil de atividade enzimática, na uréia e creatinina e no peso dos animais sugeriram alterações no metabolismo hepático interferindo na síntese... / Symphytum officinale (comfrey) is a plant used over thousands of years to treat bone fractures consolidation. There are reports of its use in natura, as phytotherapic and as homeopathic medication. Recent scientific reports described cases of human and animal intoxication, with development of severe hepatic lesions after consumption of this plant, associated with the presence of pyrrolizidine alkaloids. Because of these facts its internal use was prohibited. However, there are no reports of adverse effects of Symphytum officinale ´s homeopathic formulation. The aim of this work was to compare the effects of Symphytum officinale used as phytotherapic and as homeopathic formulations, evaluating its hepatic and renal toxicity. Adult healthy rats (n=42) were divided in 3 groups according to treatment with Symphytum officinale 500mg/kg (F), Symphytum officinale 6CH 2 globules (H), and water as placebo for negative control (C), given daily orally by gavage to each animal. They were sacrificed 30 and 60 days after beginning of treatments. The toxicological evaluation was realized by biochemical analysis of the enzymatic activity of alkaline phosphatase (AF), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) and of creatinine and urea plasmatic concentration. Histological and histomorphometric analysis of liver and kidney was also conducted. Data were submitted to statistical t (Student) and ANOVA 1 factor (5%) tests. Both biochemical and histological analysis showed mild hepatic alterations and no renal alterations. The modifications of the enzymatic activity, of urea and creatinine, and of rats’ weight suggested hepatic metabolic alterations interfering on protein synthesis. Histological analysis was compatible with chronic mild hepatitis with development of important fibrosis only in the F group. The phytotherapic formulation used was... (Complete abstract click electronic access below)

Os caracóis terrestres são animais capazes de produzir através de glândulas localizadas em toda superfície do seu corpo, uma secreção glicoproteica denominada muco, que dentre outras funções, apresenta poder antibacteriano, que pode auxiliar nos processos de reparação de feridas de origens diversas. Desta forma, o objetivo da presente pesquisa foi o de avaliar macroscópica e histologicamente, os efeitos reparadores do muco dos escargots Achatina fulica e Achatina achatina monochromatica, em lesões provocadas na pele de camundongos da linhagem "hairless"; verificar sua potencialização após a ingestão de uma ração contendo em sua formulação uma planta com propriedades cicatrizantes comprovadas, o Confrei (Symphytum officinale); e analisar bioquimicamente a composição do muco destes caracóis. Foram selecionados caracóis das espécies Achatina fulica (n=30) e Achatina monochromatica monochromatica (n=30), retirado seu muco através de estimulação manual da glândula podal e verificação de seu efeito reparador. Utilizaram-se camundongos da linhagem "hairless" (n=75), submetidos à intervenção cirúrgica e tratados com muco de ambas as espécies de caracóis. As características macroscópicas da lesão foram registradas em protocolo e avaliadas. Fragmentos de pele foram submetidos à biópsia aos 3, 5 e 7 dias de experimento, fixados em Paraformoldeído, processados e incluídos em parafina. Os cortes foram corados com Hematoxilina-Eosina e Tricrômio de Mason. Macroscopicamente, os animais tratados mostraram edemas menores, maior presença de crosta e maior contração das bordas das feridas ao final do experimento, quando comparados ao grupo controle. Histologicamente, os grupos tratados também revelaram melhor reparação da lesão, apresentando edemas menos intensos, grandes quantidades de tecido de granulação e infiltrados inflamatórios. O grupo tratado com muco de caracóis alimentados com ração contendo Symphytum officinale foi o que apresentou melhores resultados. / Land snails are animals able to produce through glands located all over their body surface, a glycoprotein secretion called mucus. This mucus, within other roles, shows an antibacterial power that can help in the healing processes of wounds from several sources. Therefore, the aim of this study was to evaluate through macroscopic and histological techniques the repairing effects of the mucus in the snails Achatina fulica and Achatina achatina monochromatica in lesions intentionally caused to hairless linage mice skin; to investigate mucus potential effect after snails have been fed with a diet formulation containing a plant with proved scar properties, the Comfrey (Symphytum officinale); and to study the biochemical composition of mucus from these snails. Snails of Achatina fulica (n=30) and Achatina monochromatica monochromatica (n=30) species were sorted, the mucus extracted through manual stimulation of the podal gland and the repairing effect studied. Hairless linage mice (n=75) used in this study went through surgery and then treated with mucus from both species of snails. The macroscopic characteristics of the lesion were recorded and assessed. Fragments of skin were taken to biopsy at 3, 5 and 7 days of trial, fixed in paraformaldehyde, processed and embedded in wax. Sections were stained with Hematoxilin-Eosin and Tricromio of Mason. Macroscopically, treated mice showed minor swelling, higher presence of scabs and higher contraction of wound edges at the end of the trial, when compared with control. Histologically, groups that received treatment also showed better lesion repair, presenting less intense swellings, large amount of scar tissue and inflammatory infiltrates. The group treated with mucus from snails that received a diet containing Symphytum officinale presented the best results.

The thesis titled "Biology, seed characteristics and growth establishment of common comfrey (Symphytum officinale L.)" dealt with the evaluation methods of reproduction comfrey and evaluation of the impact of environmental conditions to the grow of comfrey. Further it dealt with germination tests and their statistical evaluation while respecting the specific work with seeds. They were evaluated seed characteristics, which are reflected in the growth of these plants arising both in soil and in a laboratory environment. Literary part gave information about biology, characteristics, morphology, prevention and regulation of comfrey even more its cultivation, nutrition of the grassland and seed characteristics, germination and dormancy. In the final section of work was monitored coverage, botanical images, representation of herbaceous species growing near comfrey. The study also discussed vernalization, size of comfrey leaves, growth and development depending on the content of nutrients (soil fertility) and water in the soil and on the number of cuts to its growth. There were designed a suitable ways of reproduction and growing of comfrey.

Beinwell enthält neben erwünschten Substanzen wie Allantion und Rosmarinsäure auch toxische Pyrrolizidinalkaloide (PA), die die Anwendung limitieren. Ziel der vorliegenden Arbeit war es, die Expression des Gens für Homospermidinsynthase, ein zentrales Enzym der Biosynthese von PA, stark zu verringern. Dafür wurden künstliche microRNA verwendet. Dabei wurden Haarwurzelkulturen mit entsprechenden Transgenen erzeugt. Es wurden Daten zur mRNA-Menge, PA-Gehalt und Allantoingehalt sowie Wachstumsparameter erhoben und zueinander in Beziehung gesetzt.:1. Einleitung 1.1. Gattung Symphytum (Beinwell) 1.2. Pyrrolizidinalkaloide 1.3. Haarwurzeln als Modellsystem und Sekundärstoffproduzenten 1.3.1. Agrobacterium rhizogenes 1.3.2. Haarwurzelkulturen in der Biotechnologie 1.4. Zielstellung 2. Material und Methoden 2.1. Materialien 2.2. Erzeugung und Kultivierung der pflanzlichen Zell- und Organkulturen 2.3. Erzeugung der gentechnisch modifizierten Agrobacterium-Stämme 2.4. Qualitative und quantitative Untersuchungen an Nukleinsäuren 2.4.1. Nukleinsäurepräparation 2.4.2. Gelelektrophorese 2.4.3. Protokolle für die Polymerasekettenreaktion 2.5. Sekundärstoffanalytik 2.5.1. Allantoinbestimmung 2.5.2. Quantifizierung der Pyrrolizidinalkaloide 3. Ergebnisse 3.1. Erzeugung und Untersuchung der Haarwurzelkulturen 3.1.1. Herstellung der Kulturen 3.1.2. Wachstum und Biomasseakkumulation 3.2. Bestimmung der Expression von HSS und DHS mittels qPCR 3.2.1. Methodenetablierung 3.2.2. Expression von HSS und DHS in Haarwurzelkulturen 3.3. Analytik von Sekundärmetaboliten 3.3.1. Bestimmung des Allantoingehalts 3.3.2. Bestimmung des Pyrrolizidinalkaloidgehalts 3.4. Gibt es einen Zusammenhang zwischen HSS-Expression und Alkaloidgehalt? 4. Diskussion 4.1. Erzeugung und Wachstum von Symphytum-Haarwurzelkulturen mit und ohne zusätzliche Transgene 4.2. Produktion von Allantoin in Haarwurzelkulturen 4.3. Beeinflussen die miRNA-Konstrukte die Expression von HSS und DHS? 4.4. Beeinflußt die Regulation der HSS-Expression den PA-Gehalt? 4.5. Beurteilung der PA-Gehalte in Haarwurzelkulturen 4.6. Ausblick 5. Zusammenfassung 6. Verzeichnisse 7. Anhang

This diploma thesis deals with macerates of comfrey (Symphytum officinale) in selected fats (food lard, cosmetic lard and almond oil). Selected fats were first characterized by dry matter content, saponification, acid, iodine number, peroxide value. At the same time, total and free fatty acids were determined using GC-FID. Macerates were prepared by extraction of comfrey roots with selected fats. In prepared macerates the change in acid and peroxide value was monitored, as well as the content of selected bioactive compounds. The content of total phytosterol and total carotenoid content was determined by UV-VIS spectrometry, phytosterols and carotenoids were also analysed using HPLC-DAD. Total phenolic content was measured using Folin-Ciocalteu reagent and antioxidant activity by ABTS assay. The measured properties were then compared with industrially produced comfrey ointment. In macerated fats the increase in acid and peroxide value was determined. Furthermore, an increase in the content of total phytosterols and total carotenoids was observed. Using HPLC-DAD the content of -sitosterol and stigmasterol was determined, but carotenoids were not detected. Of the tocopherols, only DL--tocopherol acetate was detected. During maceration, the content of total phenolic compound in fat increased, which caused a change in antioxidant activity.