Thesis (MSc (Biochemistry))--University of Limpopo (Medunsa Campus), 2009. / Background and Purpose The stem bark, roots and leaves of Sclerocarya birrea (S. birrea), {(A. Rich) Hochst}, subspecies caffra (Sond) Kokwaro are widely used in South Africa and some African countries as folk medicine in the treatment and management of a variety of human ailments, including diabetes mellitus. Although the blood glucose lowering effect of the stem bark extract of S. birrea have been confirmed using experimental animal models of diabetes, there is no clear understanding of the mechanism(s) whereby S. birrea stem bark extracts and/or their components exert their blood glucose lowering effects. The primary aim of the current study was to study the in vitro inhibitory effects of S. birrea stem bark extracts on the activities of selected diabetic related carbohydrate metabolizing enzymes (α-amylase, α-glucosidase and glucose 6-phosphatase). The current study also investigated the acute in vivo effect of S. birrea stem bark acetone extract on postprandial blood glucose levels after oral sucrose loading as well as the effect of S. birrea stem bark aqueous extract on hepatic glucose 6-phosphatase activity. In addition, the long term (21 days) effects of S. birrea stem bark acetone extract on fasting blood glucose levels, plasma insulin levels, plasma triglyceride and body weight in normal and alloxan induced diabetic rats were also investigated. Methods For in vitro studies: Crude hexane, acetone, methanolic and aqueous extracts of the stem bark extract of S. birrea were prepared by means of a sequential solvent extraction procedure and screened for inhibitory activities against human urinary α-amylase, rat pancreatic α-amylase, Bacillus stearothermophilus α-glucosidase and rabbit liver glucose 6-phosphatase using standard procedures for assaying the activities of these enzymes. IC50 values and mode of inhibition of extracts demonstrating appreciable inhibitory activity against α-amylase and α-glucosidase were determined and compared with those of acarbose, a known inhibitor of these two enzymes. The IC50 value and mode of inhibition of extracts demonstrating appreciable inhibitory activity against glucose 6-phosphatase were determined and compared with those of sodium orthovavadate and sodium tungstate, known inhibitors of glucose 6-phosphatase. In vivo studies: In vivo studies were conducted in normal and alloxan induced diabetic male WKY rats. Diabetes was induced in rats that had been fasted for 12 h by a single intraperitoneal injection of 140 mg/kg body weight of alloxan monohydrate freshly dissolved in sterile normal saline. The effect of S. birrea stem bark acetone extract on postprandial blood glucose level was determined in 18 h fasted diabetic and normal rats by administering orally, the plant extract (300 mg/kg) 30 minutes before an oral sucrose loading and measuring postprandial blood glucose levels after sucrose loading by means of a MediSense’s Optimum Xceed Glucometer (MOXG). In addition, rat intestinal dissacharidase (α-glucosidase/sucrase) activity was determined in homogenate of small intestine of rats sacrificed one hour after given orally either plant extract or acarbose. The in vivo effect of S. birrea stem bark extract on glucose 6-phosphatase was determined by measuring the activity of hepatic glucose 6-phosphatase at the end of the study. For the determination of the long term (chronic) effect of S. birrea stem bark crude acetone extract on blood glucose levels, body weight and water intake, alloxan induced diabetic and normal WKY rats were treated daily with S. birrea stem bark crude acetone extract (300 mg/kg) for 21 days. Fasting blood glucose levels and changes in body weight were determined on day 0, 7, 14 and 21 after initiation of treatment by means of a MOXG and gravimetrically respectively. Water intake was determined on the same days that blood glucose levels were determined by measuring the amount of water left overnight by each rat and subtracting this amount from the initial amount water given to each rat. Blood was also collected at the end of the study for the measurement of plasma glucose, triglyceride and insulin levels. Plasma glucose and plasma triglyceride levels were measured using commercially available kits based respectively on the glucose oxidase and the glycerol blanked methods (Beckman Coulter®’s UniCell DXC 800 Synchron® Clinical System). Plasma insulin levels were determined by means of an enzyme linked immunosorbant assay (ELISA) adapted to the Beckman Coulter® Ireland Inc’s UniCell DXI 800 Access® Immunoassay System. Results In vitro studies: The crude methanolic and acetone S. birrea stem bark extracts strongly inhibited both human urinary α-amylase and rat pancreatic α-amylase in a competitive manner. The inhibitory effect of the crude methanolic extract on both enzymes was significantly stronger than acarbose. Hexane and acetone crude extracts of the stem-bark of S. birrea demonstrated the highest percentage inhibition against B. stearothermophilus α-glucosidase. The mode of inhibition of the crude hexane extract on B. stearothermophilus α-glucosidase appeared to be a noncompetitive one. However, the this plant extract appeared to be a less potent inhibitor of α-glucosidase enzyme than acarbose. Rabbit liver glucose 6-phophatase was strongly inhibited by the crude aqueous S, birrea stem bark extract in a competitive manner. In vivo studies: Administration of S birrea stem bark acetone extract 30 min before oral sucrose loading significantly suppressed (P < 0.01) the rise in postprandial blood glucose levels in treated rats compared to control rats. The crude extract also decreased significantly the intestinal disaccharidase activity of experimental rats compared to control rats. These observations suggest that the in vitro inhibitory effects of the crude hexane extract on α-glucosidase enzymes are applicable in vivo Daily, continuous oral treatment of alloxan–induced diabetic and normal WKY rats with S. birrea stem bark extract for 3 weeks resulted in significant reductions in fasting blood glucose levels and water intake of treated diabetic rats compared with diabetic controls. The extract, however, failed to bring about any significant change in the body weight, plasma insulin levels, plasma triglyceride levels and hepatic glucose 6-phosphatase of treated diabetic rats compared to diabetic control rats Conclusions The results of the current study suggest that the observed in vitro inhibitory effect of S. birrea stem bark acetone extract on alpha glucosidase enzymes are applicable in vivo whereas the observed in vitro inhibitory effect of S. birrea stem bark aqueous extract on glucose 6-phosphatase are not applicable in vivo. Furthermore, in the current study S. birrea stem bark acetone extract appears to lower blood glucose levels of alloxan induced diabetic rats without increasing their plasma insulin levels. Thus, it can be concluded on the basis of the current study that S. birrea stem bark acetone and hexane extracts exert their blood glucose lowering effect in alloxan induced diabetic rats in part, through inhibition of intestinal brush border α-glucosidase enzymes.

Submitted by Maryse Santos (maryseeu4@gmail.com) on 2016-09-02T13:41:55Z No. of bitstreams: 1 Dissertação de mestrado- Glauber Palma de Oliveira: 263777 bytes, checksum: 5c6aff5230edc4d6a8f43d052783106b (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-09-16T13:21:03Z (GMT) No. of bitstreams: 1 Dissertação de mestrado- Glauber Palma de Oliveira: 263777 bytes, checksum: 5c6aff5230edc4d6a8f43d052783106b (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-09-16T13:23:47Z (GMT) No. of bitstreams: 1 Dissertação de mestrado- Glauber Palma de Oliveira: 263777 bytes, checksum: 5c6aff5230edc4d6a8f43d052783106b (MD5) / Made available in DSpace on 2016-09-16T13:23:47Z (GMT). No. of bitstreams: 1 Dissertação de mestrado- Glauber Palma de Oliveira: 263777 bytes, checksum: 5c6aff5230edc4d6a8f43d052783106b (MD5) Previous issue date: 2014-02-25 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to evaluate the antimicrobial activity of aqueous extracts of the fruits and stem bark and a formulation orabase of Libidibia ferrea against biofilm microorganisms by agar diffusion method and broth microdilution, and to evaluate toxicity through hemolysis assay and fibroblast cell culture. For this purpose, aqueous extracts were prepared and from these was designed one orabase. The microorganisms used for determination of MICs were Streptococcus salivarius (ATCC 7073), Streptococcus mutans (ATCC 25175), Streptococcus oralis (ATCC 10557), Lactobacillus casei (ATCC 7469) and Candida albicans (40040 INCQS). As a positive control Chlorhexidine 0.12% and as a negative control the vehicle. In cytotoxicity tests the extract of the stem bark, the formulation vehicle, adjuvants and Orabase formulation were tested. In hemolytic activity, as a positive control Triton X-100, and in the activity of cultured cells doxorubicin, drug pattern of cell viability. As negative controls, the carrier and the diluent of the formulation. The results were presented using descriptive statistics. The results showed that both extracts of the fruits as the stem bark showed antimicrobial activity. MICs for the stem bark extract of jucá were 2.62 mg/mL (Streptococcus mutans), 2.25 mg/mL (Streptococcus oralis), 4.12 mg/mL (Lactobacillus casei), 2.62 mg/mL (Candida albicans) and 3.0 mg/mL (Streptococcus salivarius). As for the extract of the fruit MIC’s values were 3.75 mg/mL (Streptococcus mutans), and 3.37 mg/mL (Streptococcus oralis , Candida albicans and Lactobacillus casei). There was no activity for Streptococcus salivarius. Regarding the Orabase formulation MICs were 0.45 mg/mL (Streptococcus mutans), 0.48 mg/mL (Streptococcus oralis), 0.41 mg/ mL (Lactobacillus casei), 0.48 mg/mL (Candida albicans) and 0.48 mg/mL (Streptococcus salivarius ). As for the hemolysis test, both the extract of the stem bark as the formulation and its components did not cause hemolysis in mg / mL. There was an absence of hemoglobin in the supernatant and sedimented erythrocytes. The solutions tested showed no toxicity when tested against fibroblast cells. Based on the results it was concluded that the extract of the stem bark and the orabase formulation of Libidibia ferrea L. showed antimicrobial activity against microorganisms of the biofilm and were not toxic when tested in erythrocytes and cell culture. / O objetivo do presente estudo foi avaliar a atividade antimicrobiana dos extratos aquosos da vagem e da casca do caule e de uma formulação em orabase de Libidibia ferrea frente a microrganismos do biofilme dental através do método de difusão em ágar e microdiluição em caldo; e a toxicidade através do teste de hemólise e cultura de células de fibroblastos. Para tanto, extratos aquosos foram preparados e a partir destes foi formulada uma orabase. Os microrganismos utilizados para a determinação da CIM (Concentração Inibitória Mínima) foram: Streptococcus salivarius (ATCC 7073); Streptococcus mutans (ATCC 25175); Streptococcus oralis (ATCC 10557); Lactobacillus casei (ATCC 7469) e a Candida albicans (INCQS 40040). Como controle positivo a Clorexidina 0,12% e como controle negativo o veículo. Nos testes de citotoxicidade foram testados o extrato da casca do caule, veículo da formulação, seus adjuvantes e a formulação orabase. Na atividade hemolítica, foi utilizado como controle positivo o Triton X-100, e no teste de viabilidade celular a doxorrubicina e como controle negativo, foram utilizados o veículo e o diluente da formulação em ambos os testes. Os resultados obtidos foram apresentados através da estatística descritiva. Os resultados mostraram que tanto os extratos da vagem quanto da casca do caule apresentaram atividade antimicrobiana. As CIMs para o extrato casca do caule de jucá em relação ao microrganismo foram 2,62 mg/mL (Streptococcus mutans), 2,25 mg/mL (Streptococcus oralis), 4,12 mg/mL (Lactobacillus casei), 2,62 mg/mL (Candida albicans) e 3,0 mg/mL (Streptococcus salivarius). Já para o extrato da vagem os valores de CIM foram 3,75 mg/mL (Streptococcus mutans), e 3,37 mg/mL (Streptococcus oralis, Candida albicans e Lactobacillus casei). Não houve atividade para Streptococcus salivarius. Em relação à formulação em orabase as CIMs foram de 0,45 mg/mL (Streptococcus mutans), 0,48 mg/mL (Streptococcus oralis), 0,41 mg/mL (Lactobacillus casei), 0,48 mg/mL (Candida albicans) e 0,48 mg/mL (Streptococcus salivarius). Quanto ao teste de hemólise, tanto o extrato da casca do caule quanto a formulação e seus componentes não causaram hemólise. Verificou-se um sobrenadante com ausência de hemoglobina e eritróticos sedimentados. As soluções testadas não apresentaram toxicidade quando testadas em fibroblastos humanos. Baseado nos resultados foi possível concluir que o extrato da casca do caule e a formulação em orabase de Libidibia ferrea L. apresentaram ação antimicrobiana frente aos microrganismos do biofilme da cavidade bucal e não foram tóxicos quando testados em hemácias e cultura de células.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-06-05T10:40:20Z No. of bitstreams: 2 Dissertação - Débora Cristina da Silva Lima - 2012.pdf: 2824147 bytes, checksum: 65736b430e09552d0474dcec6ed8b516 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-06-05T10:56:30Z (GMT) No. of bitstreams: 2 Dissertação - Débora Cristina da Silva Lima - 2012.pdf: 2824147 bytes, checksum: 65736b430e09552d0474dcec6ed8b516 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-05T10:56:30Z (GMT). No. of bitstreams: 2 Dissertação - Débora Cristina da Silva Lima - 2012.pdf: 2824147 bytes, checksum: 65736b430e09552d0474dcec6ed8b516 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-11-30 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Lafoensia pacari A. St. Hil. (Lythraceae), popularly known in Brazil as “pacari”, is a species native of Cerrado. In folk medicine, the leaves and stem bark are used to treat cancer and as an anti-inflammatory and cicatrizant agent. Due to the wide use of this plant as a therapeutic resource, the present study evaluated the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activities of the ethanolic extract from L. pacari stem bark and leaves using the Ames test and the mouse bone marrow micronucleus test. Our results demonstrated that the ethanolic extract from L. pacari leaves, presented no mutagenic activity in bacteria, but presented a mild genotoxicity in the highest concentration at 24 h in mice bone marrow cells. Exhibited moderate cytotoxic effect on bacteria and a mild cytotoxic action in the highest concentration by micronucleus test. The extract showed antimutagenic activity by Ames test and in the micronucleus test demonstrated antigenotoxic action in all concentrations at 24 h, and in the lowest concentration at 48 h. Showed anticytotoxic effect in all concentrations and times tested. In the evaluation of the genotoxic, cytotoxic, antigenotoxic and anticytotoxic activities of the ethanolic extract from L. pacari stem bark, the results demonstrated that the extract exhibited no mutagenic activity in bacteria, however showed a mild genotoxicity in the highest concentration in mice. Exhibited moderate cytotoxic effect in bacteria and a mild cytotoxicity at 24 h by micronucleus test. Showed strong antimutagenic activity by Ames test, as well as antigenotoxic and anticytotoxic activities in the mouse bone marrow micronucleus test, demonstrating to be a strong protective agent against DNA damage. / Lafoensia pacari A. St-Hil, (Lythraceae) popularmente conhecida no Brasil como pacarí, é uma espécie nativa do Cerrado. Na medicina popular, suas folhas e casca do caule são utilizadas para tratar câncer, como um agente cicatrizante e antiinflamatório. Devido à grande utilização dessa planta como recurso terapêutico, o presente trabalho avaliou as atividades genotóxica, citotóxica, antigenotóxica e anticitotóxica do extrato etanólico das folhas e da casca do caule de L. pacari, utilizando o teste de mutagenicidade de Ames e o teste do micronúcleo em medula óssea de camundongos. Nossos resultados demonstraram que o extrato etanólico das folhas de L. pacarí, não apresentou atividade mutagênica em bactérias, mas apresentou uma leve genotoxicidade na concentração mais elevada em células de medula óssea de camundongos no tempo de 24 h. Exibiu efeito citotóxico moderado em bactérias e uma leve ação citotóxica, na concentração mais elevada no teste de micronúcleo. O extrato apresentou atividade antimutagênica pelo teste de Ames e no teste do micronúcleo demonstrou ação antigenotóxica em todas as concentrações no tempo de 24 h e na concentração mais baixa no tempo de 48 h. Apresentou efeito anticitotóxico em todas as concentrações e tempos testados. Na avaliação das atividades genotóxica, citotóxica, antigenotóxica e anticitotóxica do extrato etanólico da casca do caule de L. pacarí os resultados provaram que este extrato não exibiu atividade mutagênica em bactérias, no entanto apresentou uma genotoxicidade fraca na maior concentração em camundongos. Exibiu um efeito citotóxico moderado em bactérias e uma leve citotoxicidade, somente no tempo de 24 h pelo teste de micronúcleo. Apresentou forte atividade antimutagênica pelo teste Ames, bem como atividade antigenotóxica e anticitotoxica no teste de micronúcleo em medula óssea de camundongos, demonstrando ser um forte agente protetor contra danos no DNA.

The stem bark of the protected tree species, Curtisia dentata (Burm. f.)C.A.Sm., is one of the most popular plant species harvested and traded at traditional medicine markets in South Africa. The overexploitation of C. dentata trees lead to a “Near Threatened” conservation status and the population trend is portrayed as “declining”. In the KwaZulu-Natal Province of South Africa, C. dentata is completely conservation dependent. This study is not based on drug discovery or toxicological studies, but on the concern that the stem bark of C. dentata trees are harvested, prepared into remedies and consumed as traditional medicine without knowledge regarding the chemical compounds in the stem bark, particularly since the chemical composition of C. dentata stem bark was unknown to date. Phytochemical analyses were firstly conducted to determine the chemical composition of C. dentata stem bark using various solvents and various analytical methods, and secondly, to determine how seasons and regional separation of C. dentata trees affect the chemical profiles of C. dentata stem bark from an environmental and nature conservation perspective. Plants are known to contain numerous chemical compounds. Compounds isolated from a particular plant species are therefore not the only compounds present in that species, and although a plant has proven pharmacological properties, they can still cause harm. Previous studies on C. dentata aimed at validating the plant species as a medicinal plant by examining extracts of the leaves, twigs and stem bark’s potentials against known pathogens and selected cancer cells in vitro and in vivo, and its anti-inflammatory, antioxidant and antiverotoxic properties. Four pentacyclic triterpenoids and one steroidal compound were also previously isolated from C. dentata leaves, however, the leaves are not used in traditional medicines, but were suggested as alternative for stem bark as the harvesting of leaves is less destructive. The efficacy of these compounds as therapeutic agents is, however, compromised by their low solubility in water and thus their potential to penetrate permeating biological membranes. Moreover, in vitro toxicity studies distort the picture of its actual potentials on human health as the whole human metabolome and all its processes, including uptake and phase I and phase II biotransformation are not included. In vivo toxicity studies on mammalian animal species may also not present a true picture of a chemical or extract’s toxic effects on humans as animal metabolisms differ from those of humans. The chemical composition of leaves and stem bark may furthermore also be in contrast to some extent, and therefore chemical compounds were also isolated from C. dentata stem bark in this study. Scientific studies on plant-based medicines generally involve the discovery or identification of compounds that may be beneficial, and which can be exploited in future. Chemical compounds in traditional medicines or other plant-based health products which may cause adverse effects are generally ignored. Moreover, scientific studies that consider that some compounds present in plant extracts may derive from contaminants are equally limited. Traditional plant-based medicines are neither standardized nor regulated in South Africa. Users of traditional plant-based traditional medicines therefore consume uncertain dosages of both beneficial and hazardous substances, as well as contaminants simultaneously. Certain chemical compounds are carcinogens or mutagens or have the ability to accumulate in human tissues. Adverse effects may therefore only manifest after several years of use and will subsequently not be connected to the use of a particular traditional plant-based medicine. The goal of the thesis is therefore to provide a holistic portrayal of the full spectrum of chemical compounds in extracts of C. dentata stem bark and to discuss, where literature is available, the effect(s) each chemical compound may have on human health. Moreover, this thesis investigates variations in chemical composition and concentration in individual trees, seasonal variations and variations in composition and concentrations in the stem bark of C. dentata trees from geographically distinct regions. Most unexpected was that not all C. dentata stem bark samples contained chemical compounds with known beneficial potentials at each sampling date, and that chemical compounds may be region-specific and also tree-specific, which confirms that plants produce secondary metabolites according to the needs of each individual plant. Additional insight into the chemical composition and concentration of C. dentata trees is provided by the distribution profiles of amino acids in C. dentata stem bark. Extreme variations within populations and between geographical areas support the need for the cultivation of C. dentata trees to ensure sustainable production of homogenous material for chemical homogeneity. / Environment Science / PhD. (Environment Science)