Cancer has become a major health problem due to its high rate of morbidity and mortality. Severe side effects associated with most of the available anticancer drugs and the development of the drug resistant cells are the major hurdles limiting their application and therapeutic success. Much attention has thus been focused on natural compounds with minimal or no toxicity in humans and having capacity to suppress proliferation and/or induce apoptosis in cancer cells. Therefore, it is imperative to discover and develop new anticancer drugs. Endophytic fungi though underexploited have remained a rich source of structurally novel and biologically active secondary metabolites. That they are a prolific resource for new compounds in drug discovery is evident from a plethora of reported research findings of the recent past. Those of endophytic fungi especially isolated from medicinal plants with known therapeutic value have especially caught the attention of several research groups worldwide for the production of host associated or novel lead molecules. In this backdrop, the focus of my research work has been centered on exploring the endophytic fungal population associated with Catharanthus roseus in search of production of novel anticancer compounds. Twenty endophytic fungi were isolated from different parts of Catharanthus roseus plant. The ethyl acetate (EA) extracts of three week grown liquid cultures of the individual endophytes were assessed in vitro for their cytotoxic activities in HeLa and HepG2 cancer cells using MTT assay. Of all the isolates thus screened, the EA extract from an isolate identified as Chaetomium nigricolor exhibited significant cytotoxicity in the tested cell lines with a better activity profile against HeLa cells. To understand if the liquid culture filtrate or the mycelium harbored the active cytotoxic principle, the C. nigricolor culture filtrate and mycelia was extracted separately using organic solvents with varying polarity namely ethyl acetate, dichloromethane, chloroform and hexane. These extracts were further tested for cytotoxicity induction in HeLa and MCF-7 cells by MTT assay. The ethyl acetate culture filtrate and mycelial extracts showed best cytotoxic activity on HeLa cells compared to other extracts; therefore, the mycelial and culture filtrate ethyl acetate extracts of C. nigricolor were pooled and was used for further work. Hexane culture filtrate extract of C. nigricolor showed best antiproliferative activity against MCF-7 cells. Thus, the compounds with anticancer potential from both ethyl acetate pooled extracts and hexane culture filtrate extract of C. nigricolor were purified using bioassay guided isolation method. This finally resulted in the isolation of two different anticancer compounds, one each from EA pooled extract and Hexane culture filtrate extracts of C. nigricolor named as compound 1 and compound 2, respectively. Based on various analyses including mass spectrometry (MS and MS-MS) and UV-visible, Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, the compounds 1 and 2 were identified as 1, 2 Bis (diethylamino) ethene-1, 2 diol (BDED) and Nonacos-4-enoic acid (NA), respectively An improved understanding of the cellular responses to chemotherapeutic agents in cancer cells and the underlying molecular mechanisms gains particular relevance in the efforts to improve the clinical outcome of chemotherapeutic agents. Arresting the growth of cancer cells in one way or the other and induction of apoptosis in a drug specific manner is the hallmark of almost all the clinically used anticancer drugs. Towards this end, evaluation of the cytotoxic effects of the newly identified compounds on various human cancer cells was carried out. BDED was examined for in vitro cytotoxicity of against a panel of five human cancer cell lines namely HeLa, A-431, A549, COLO 205, and MCF-7. Among the cell lines screened, HeLa cells were most vulnerable to BDED treatment with an IC50 value of 27 μM. This cytotoxicity was later identified as resulting from apoptosis induction by BDED which was scored by the characteristic events of membrane externalization of phosphatidylserine, cytoplasmic shrinkage and chromatin condensation which were observed in a large majority of the BDED-treated cells. Further detailed studies were performed to delineate the cellular mechanisms of BDED-induced apoptosis in HeLa cells. Analysis of cell cycle progression by propidium iodide (PI) staining revealed BDED-induced cell cycle arrest in the G1 phase of cell cycle. The results indicated that BDED induced a significant ROS generation and a significant loss of mitochondrial membrane potential which were abrogated upon pretreatment of the cells with an antioxidant, N-acetyl cysteine (NAC). These observations suggested the probable involvement of the initial events of BDED-induced ROS production in causing cell death via mitochondria-based intrinsic pathway of apoptosis. In addition, the results from the expression profile of pro- and anti- apoptotic proteins by western blot analysis in the BDED-treated HeLa cells further corroborated this hypothesis. These included an abnormally deregulated cellular abundance of BAX 1 and BCL 2 proteins, elevated levels of APAF-1, and activated cleaved species of procaspases 9 and 3. In addition, a pronounced abundance of cleaved PARP protein was observed in these experiments. Thus, the results suggest that BDED induced apoptosis in HeLa cells via ROS mediated mitochondrial dependent pathway. In parallel, the in vitro cytotoxic effects of NA was also studied, the second antiproliferative compound identified from hexane extracts of C. nigricolor using a panel of four human cancer cell lines - A-431, A549, COLO 205, and MCF 7. NA was thus found to be most potent against MCF 7 (breast cancer) cells. PI staining-based viability assays and microscopic observations showed a dose dependent cytotoxicity of NA on MCF 7 cells. Our data reveled an IC 50 of 40 μM for NA The NA-induced apoptosis was confirmed by flow cytometric detection of membrane externalization of phosphatidylserine using Annexin V FITC/PI dual staining. NA was also observed to induce cytotoxicity best at 72 h this could be attributed to the fact that it is a derivative of a Nonocosane-(a naturally prevalent molecule found in several vegetables). Similar to our results from BDED-treated HeLa cells, ROS generation and loss of mitochondrial membrane potential which were abrogated on pretreatment with NAC, were also observed in NA-treated MCF-7cells. Similar to several other fatty acids, NA was also observed to induce cell cycle arrest in the G1 phase of cell cycle. Further, apoptotic signature of an altered expression of anti-apoptotic BCL-2 and pro-apoptotic BAX, APAF-1 and procaspase 9 and 3 and a cleaved PARP were observed in NA-treated MCF 7 cells. In conclusion, the results indicate that BDED and NA have cytotoxic and apoptotic effects on HeLa (cervical cancer) and MCF 7 (breast cancer) cell lines, respectively. This leaves open further avenues to evaluate their potential application as anticancer agents for treatment of human cervical and breast cancers.

OLIVEIRA, Francisco Ítalo Fernandes de. Influência do modo de irrigação na tolerância de plantas ornamentais à salinidade da água. 2016. 103 f. Dissertação (Mestrado em Solos e Nutrição de Plantas)-Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Anderson Silva Pereira (anderson.pereiraaa@gmail.com) on 2017-01-26T16:59:49Z No. of bitstreams: 1 2016_dis_fifoliveira.pdf: 2741362 bytes, checksum: 0461d0245af9c6ece1c9a5111667008f (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-01-26T20:32:19Z (GMT) No. of bitstreams: 1 2016_dis_fifoliveira.pdf: 2741362 bytes, checksum: 0461d0245af9c6ece1c9a5111667008f (MD5) / Made available in DSpace on 2017-01-26T20:32:19Z (GMT). No. of bitstreams: 1 2016_dis_fifoliveira.pdf: 2741362 bytes, checksum: 0461d0245af9c6ece1c9a5111667008f (MD5) Previous issue date: 2016 / World trade of flowers and ornamental plants, of which the most significant branch is cut flowers, followed by the live plants, is an important segment economically to Brazil especially for the Northeast. Although the Northeast region has favorable climatic conditions for the cultivation of flowers and ornamental plants, low quality irrigation water, it is revealed as a problem. The plant species exhibit different mechanisms of tolerance to salt, in some cases, allowing their survival and production. Thus, the research aimed to evaluate the tolerance of four ornamental species to increasing levels of salinity of irrigation water and different modes of supply of water. The research was conducted in a protected environment, in the period from September to November 2015, in the experimental area of the weather station at the University Campus of Pici, the Federal University of Ceará, Fortaleza - Ceará. The treatments were distributed in randomized blocks, arranged in a split plot design with four replications with six levels of salinity of irrigation water to the plots (A1 - 0.6; A2 - 1.2; A3 - 1.8; A4 - 2 4; A5 - 3.0 and A6 - 3.6 dS/m), two modes of irrigation in the subplots (M1 - localized irrigation and M2 - not localized irrigation) and four ornamental species in subsubplots (E1 - Catharanthus roseus; E2 - Allamanda cathartica; E3 - Ixora coccinea and E4 - Duranta erecta), with 48 plants in each block, totaling one hundred ninety-two experimental units. The seedlings of ornamentals were transferred to plastic pots with a volume of 7 liters filled with a gravel layer and substrate, comprising a mixture of sand and earthworm humus, in the ratio 2:1 respectively. They were evaluated biometric, physiological, nutritional and sensorial parameters in plants. The most appropriate variables to classify the species as tolerance to salinity, are related to biomass production. The species are more sensitive to the irrigation method not located than the localized mode. The species that are more tolerant to salinity of irrigation water were Catharanthus roseus and Ixora coccinea. / O comércio mundial de flores e plantas ornamentais, do qual o ramo mais expressivo é o de flores de corte, seguido pelo de plantas vivas, é um segmento importante economicamente para o Brasil, sobretudo, para a região Nordeste. Embora a região Nordeste apresente condições climáticas favoráveis ao cultivo de flores e plantas ornamentais, a água de irrigação de baixa qualidade, revela-se como um problema. As espécies vegetais apresentam diferentes mecanismos de tolerância aos sais, em alguns casos, permitindo sua sobrevivência e produção. Dessa forma, a pesquisa objetivou avaliar a tolerância de quatro espécies ornamentais a níveis crescentes de salinidade da água de irrigação e diferentes modos de fornecimento da água. A pesquisa foi desenvolvida em ambiente protegido, no período de setembro a novembro de 2015, na área experimental da Estação Agrometeorológica, no Campus Universitário do Pici, da Universidade Federal do Ceará, município de Fortaleza – Ceará. Os tratamentos foram distribuídos em blocos casualizados, arranjados em parcelas subsubdivididas, com quatro repetições, com seis níveis de salinidade da água de irrigação nas parcelas (A1 - 0,6; A2 – 1,2; A3 – 1,8; A4 – 2,4; A5 – 3,0 e A6 – 3,6 dS/m), dois modos de irrigação nas subparcelas (M1 - irrigação localizada e M2 - irrigação não localizada) e quatro espécies ornamentais nas subsubparcelas (E1 - Catharanthus roseus; E2 - Allamanda cathartica; E3 - Ixora coccinea e E4 - Duranta erecta), com 48 plantas em cada bloco, totalizando cento e noventa e duas unidades experimentais. As mudas das espécies ornamentais foram transplantadas para vasos de material plástico, com volume de 7 litros, preenchidos com uma camada de brita e com substrato, composto por uma mistura de areia e húmus de minhoca, na proporção de 2:1, respectivamente. Foram avaliados parâmetros biométricos, fisiológicos, nutricionais e sensoriais nas plantas. As variáveis mais adequadas para classificar as espécies, quanto a tolerância a salinidade, são as relacionadas a produção de biomassa. As espécies são mais sensíveis à irrigação pelo modo não localizado do que ao modo localizado. As espécies que se mostraram mais tolerantes a salinidade da água de irrigação foram a Catharanthus roseus e Ixora coccinea.

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2018 / Diabetes mellitus is defined as a group of metabolic disorders characterised by chronic hyperglycaemia due to insufficient production and/or action of insulin and is regarded as one of the major sources of morbidity, mortality and economic burden to the modern society. A large body of scientific evidence support the fact that oxidative stress is elevated in diabetic conditions. Oxidative stress plays a significant role in the development of secondary complications of diabetes including diabetes-linked male sexual dysfunction. The management of sexual dysfunction as a secondary complication of diabetes relies on the management of the underlying diabetic condition. Glycaemic control and increased antioxidant protection are therefore necessary in the management of diabetes-induced oxidative stress male infertility. Pharmacological management of diabetes in form of various antihyperglycaemic, synthetic drugs has improved the outlook of diabetic patients; however, they are expensive, harbour unfavourable adverse effects and some have done little to prevent secondary complications of diabetes including diabetes-induced male sexual dysfunction. In addition to this, access to basic technologies for the management of diabetes mellitus and its secondary complications is still a challenge in low resource areas. Because of these challenges, there is a need to search for alternative remedies such as medication from natural products which are more affordable, well tolerated by the human body and are easily accessible. Medicinal plants are therefore viewed as an easily accessible and potent source of antioxidants capable of scavenging free radicals and fighting diabetes-induced oxidative stress. This study therefore investigated the effects of vindoline; an alkaloid extractable from Cantharanthus roseus in ameliorating diabetes-induced oxidative stress effects in testicular and epididymal tissues using male Wistar rats. Forty-eight (48), 6-week old male Wistar rats weighing between 190-230g with a conventional microbial status were divided into 6 groups, n=8, and used for this research project. Group 1 was the normal control, group 2 comprised non-diabetic rats treated with vindoline, and group 3 was the non-diabetic group of rats treated with glibenclamide- the standard drug for the treatment of diabetes. Group 4 was the diabetic control, group 5 comprised diabetic rats treated with vindoline and group 6 was the diabetic group of rats treated with glibenclamide. Diabetes was induced in group 4, group 5 and group 6 rats by subjecting them to 10% fructose water over a period of 2 weeks and thereafter, administering a single intraperitoneal injection of 40 mg/kg b.w streptozotocin (STZ). Fasting blood glucose levels were measured 72 hours after STZ injection and hyperglycaemia was confirmed where fasting blood glucose levels were more than 18mmol/l. The diabetic control (group 4) had higher fasting blood glucose levels, lower body weights as well as lower testicular and epididymal weights in comparison to the normal control (group 1). Additionally, the extent of lipid peroxidation in testicular and epididymal tissues of the diabetic control (group 4) was higher in comparison to that of the normal control (group 1). The diabetic control had lower testicular and epididymal antioxidant enzyme activities (superoxide dismutase and catalase) and lower oxygen radical absorption capacity (ORAC) in comparison to the normal control. Ferric reducing antioxidant power (FRAP) in testicular and epididymal tissues of the diabetic control (group 4) were not significantly different from those of the normal control (group 1). Treatment of diabetic rats with vindoline (group 5) for 5 weeks significantly reduced fasting blood glucose levels although the extent of reduction could not restore diabetic blood glucose levels to near-normal levels. Overall, treatment of diabetic rats with vindoline was able to minimise testicular oxidative stress as reflected by reduction in testicular malondialdehyde (MDA) levels. Furthermore, results of this study showed an increase in both testicular and epididymal catalase activities, an increase in epididymal SOD, an increase in testicular ORAC as well as an increase in both testicular and epididymal FRAP levels after 5 weeks of treating diabetic rats with vindoline (group 5). Epididymal lipid peroxidation levels, epididymal ORAC levels and testicular SOD levels of diabetic rats treated with vindoline (group 5) were however not significantly different from those of the diabetic control (group 4). Treatment of diabetic rats with vindoline or glibenclamide could not restore total body weights and testicular weights of group 5 and group 6 rats respectively, to near-normal levels. Furthermore, epididymal weights and testicular SOD activity of diabetic rats treated with vindoline (group 5) were not significantly different from those of the normal control (group 1). In conclusion, findings from this study demonstrated that treatment with vindoline could have protective effects against diabetes-induced oxidative stress in both testicular and epididymal tissues of male Wistar rats. Vindoline can therefore be considered a potential agent for the management of diabetes-induced oxidative stress male sexual dysfunction. Further studies with advanced technologies are however recommended to study the possible efficacy of vindoline in ameliorating diabetes-induced oxidative stress male sexual dysfunction. Furthermore, studies on the dose-dependent effects and long-term effects of vindoline administration on male reproductive function as well as the overall safety of treatment with vindoline are necessary.

Catharanthus roseus (Madagascar periwinkle), is a well-known medicinal plant that produces a vast array of terpenoid indole alkaloids (TIAs), including two anticancer compounds vinblastine and vincristine. Industrial scale production of TIAs is hampered by the difficulties of total chemical synthesis of these compounds and the fragmented knowledge on TIA pathway. Transcriptional regulation of the TIA biosynthetic pathway has not been thoroughly investigated in Catharanthus and only a few structural genes have been identified as the targets of two master regulators: the basic helix-loop-helix (bHLH) transcription factor (TF) CrMYC2 and APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), ORCA3. Next generation sequencing (NGS) has been used as a tool to isolate novel genes encoding enzymes and regulators of TIA pathway in Catharanthus. In this dissertation, I have performed the transcriptomic analysis of transgenic Catharanthus hairy roots ectopically expressing a dominant repressive form of CrMYC2 or ORCA3 in order to understand their potential impact on the TIA transcriptional regulatory network and to identify and characterize novel target(s) of these two key TFs. MYC2 acts as regulatory hub involved in diverse aspects of plant growth, development, and specialized metabolite biosynthesis by coordinating the crosstalk among different phytohormone signals. CrMYC2 was initially identified in Catharanthus as a regulator of ORCA3. CrMYC2 transactivates ORCA3 by binding to the T/G-box in jasmonate-responsive element (JRE) of ORCA3 promoter. RNA interference (RNAi) mediated knockdown of CrMYC2 strongly reduced TIA accumulation in Catharanthus cell suspension culture. However, the potential influence of CrMYC2 on the expression of other regulatory and structural genes in the TIA pathway remains poorly understood. Transcriptomic analyses revealed that CrMYC2 plays an essential role in JA-induced gene expression and the differentially expressed genes are involved in diverse aspects of growth and development as well as abiotic and biotic stress responses in Catharanthus. Additionally, the expression of genes related to auxin, ethylene, and abscisic acid signaling cascades were affected in hairy roots with modified CrMYC2 expression, suggesting this TF mediates cross-talk between JA and other phytohormones. Surprisingly, overexpression of CrMYC2 resulted in repressed expression of TIA pathway genes in transgenic hairy roots. Expressions of key activators of indole and iridoid pathway were downregulated whereas expression of repressors were upregulated in CrMYC2 hairy roots. Activators (i.e. CrMYC2 and ORCA3) and repressors (i.e. G-box binding factors; GBFs) have been isolated and characterized for their role in regulation of TIA pathway. However, the interconnection between those regulators and the underlying molecular mechanism has not been throughly studied. I identified (i) the interaction of CrMYC2 with CrGBFs and (ii) how this cross-family transcription factor interactions fine-tunes TIA biosynthesis in Catharanthus. The expression profiles of CrMYC2 and CrGBFs were highly correlated in different tissues and in response JA. Moreover, CrMYC2 interacted with CrGBF1 and CrGBF2 in both yeast and plant cells. CrGBF1 and CrGBF2 could form homo- and hetero-dimer which bound T/G-box elements of TIA pathway gene promoters. In plant cells, CrGBF1 antagonizes the activity of CrMYC2 on target promoters in a dosage dependent-manner. Similarly, CrMYC2 can overcome CrGBF1-mediated repression of target promoters in a dosage dependent manner. ORCA3 is another major regulator of TIA biosynthesis in Catharanthus. The transcriptomic analysis of ORCA3 transgenic hairy roots revealed (i) the effect of ORCA3 on newly identified TIA pathway biosynthetic enzymes; (ii) identify the potential effect of ORCA3 on three biological processes: abiotic stress response, plant secondary metabolic process, and response to hormonal stimulus; and (iii) the identification of potential regulator(s) of TIA biosynthesis using ORCA3 based co-expression analysis.

Catharanthus roseus (Madagascar periwinkle) is the exclusive source of an array of terpenoid indole alkaloids (TIAs) that are used in the treatments of hypertension and certain types of cancer. TIA biosynthesis is under stringent spatiotemporal control and is induced by jasmonate (JA) and fungal elicitors. Tryptamine, derived from the indole branch, and secologanin from the iridoid branch are condensed to form the first TIA, strictosidine. Biosynthesis of TIA is regulated at the transcriptional level and several transcription factors (TFs) regulating the expression of genes encoding key enzymes in the pathway have been isolated and characterized. The JA-responsive APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), ORCA3, and the basic helix-loop-helix (bHLH) factor, CrMYC2, are the key activators of the TIA biosynthesis. Recently, two other TFs, the bHLH IRIDOID SYNTHESIS 1 (BIS1) and BIS2 were also identified as regulators of TIA pathway. Analysis of C. roseus genome sequence has revealed that ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and ORCA5. In plants, physically linked clusters of TFs are less characterized. Moreover, the regulation of TF clusters is relatively unexplored. My research uncovered that the ORCA gene cluster is differentially regulated. ORCA4 and ORCA5, while functionally overlapping with ORCA3, regulate an additional set of TIA pathway genes. ORCA4 or ORCA5 overexpression has resulted in significant increase of TIA accumulation in C. roseus hairy roots. In addition, ORCA5 directly regulates the expression of ORCA4 and indirectly regulates ORCA3, likely via unknown factor(s). Interestingly, ORCA5 also activates the expression of ZCT3, a negative regulator of the TIA pathway. In addition CrMYC2 is capable of activating ORCA3 and co-regulating pathway genes concomitantly with ORCA3. Several lines of evidence suggest that, in addition to the transcriptional control, biosynthesis of TIAs is also controlled at the posttranslational level, such as protein phosphorylation. Available literature indicates that a mitogen-activated protein kinase (MAPK) cascade is involved in this process. Analysis of C. roseus MAP kinome, identified two independent MAPK cascades regulating the indole and iridoid branches of the TIA pathway. We showed that the ORCA cluster and CrMYC2 act downstream of a MAP kinase cascade consisting of CrMAPKK1, CrMAPK3 and CrMAPK6. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulates TIA pathway genes expressions and boosts TIA accumulation. The other cascade, consisting of CrMAPKK6 and CrMAPK13, mostly regulates the iridoid branch of the TIA pathway. Overexpression of CrMAPK13 in C. roseus hairy roots significantly upregulates iridoid pathway genes and boosts tabersonine accumulation. Moreover, we recently identified the third MAPK cascade, consisting of CrMAPKK1 and CrMAPK20, that negatively regulates the indole branch of the TIA pathway. Overexpression of CrMAPK20 in C. roseus hairy roots represses the genes regulated by CrMYC2-ORCAs and reduces catharanthine accumulation. These findings significantly advance our understanding of transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus.

Les transporteurs ABC sont les membres d'une superfamille de protéines qui utilisent l'hydrolyse de l'ATP pour déplacer une large gamme de substrats au travers des membranes biologiques. Les membres de la sous famille ABCC sont généralement caractérisés par un domaine transmembranaire supplémentaire en région N-terminal (TMD0). Dans cette étude, nous avons analysé deux gènes ABCC de plantes : CrABCC1 de Catharanthus roseus et AtABCC13 son orthologue chez Arabidopsis thaliana. L'analyse phylogénétique répartit les ABCC de plantes dans 3 clades distinctes. Les clades I et II sont spécifiques aux plantes tandis que le clade III est le seul associant des ABCC humains et de plantes. Le criblage de la base de données a permis d'identifier 16 séquences ABCC chez Catharanthus roseus parmi lesquelles 2 appartiennent à CrABCC. / ABC transporters are members of a large superfamily of proteins that utilize ATP hydrolysis to translocate a wide range of substrate across biological membranes. Members of C. subfamily (ABCC) are generally structurally characterized by an additional (N-Terminal) transmembrane domain (TMD0). In this study the analysed two plant ABCC : CrABCC1 from Catharanthus roseus and AtABCC13, it's ortholog in Arabidopsis thaliana. Phylogenetic analysis of plant ABCCs separates their protein sequences over three distinct clusters : I and II are plant specific whereas cluster III is the only gathering humain and plant ABCCs. Screening of plant database allowed us to identify 16 different ABCCs sequences in Catharanthus roseus.

Thesis (DPhil (Biomedical Science))--Cape Peninsula University of Technology, 2019. / Diabetes mellitus (DM) is a group of metabolic disorders characterised by persistent high blood glucose levels together with abnormal metabolism of macromolecules. If the hyperglycemia is not controlled, adverse metabolic changes could occur leading to the progressive development of severe complications. Formation of reactive oxygen/nitrogen species and inflammatory responses are principal mechanisms that have been implicated in the development of hyperglycemia-induced tissue damage. The commercially available drugs utilised in the treatment of diabetes have been linked to detrimental side effects hence the need to discover alternative medicines especially from medicinal plants. Catharanthus roseus is both a medicinal and ornamental plant that is traditionally used to treat various diseases. It has been reported to possess antidiabetic, anticancer, antimicrobial and antioxidant properties. The plant has been shown to possess more than 100 monotepernoid indole alkaloids which were linked to the plants’ antihyperglycemic and antioxidant effects. Therefore, this study was carried out to investigate the effect of vindoline; a bioactive compound derived from C. roseus against type 2 diabetes–induced complications. The study also investigated the effects of Catharanthus roseus extracts in RIN-5F cell line. The study was carried out in two parts: viz in vitro and the in vivo assessments. The in vitro study initially investigated the polyphenolic content and antioxidant activities of vindoline and the 3 extracts (methanolic, aqueous and the dichloromethane) of C.roseus. The assays used to evaluate the antioxidant capacity of the extracts include oxygen radical absorbance capacity (ORAC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibitory assay. Among the evaluated extracts, the methanolic extract demonstrated both high total polyphenolic content and antioxidant capacity. The HLPC analysis of the extracts was performed and showed highest concentrations of vindoline in the dichloromethane extract and the aqueous extract exhibited the least. The antioxidant activities of vindoline were determined and compared to a known antioxidant, ascorbic acid. Vindoline revealed stronger ORAC activity than ascorbic acid however the ferric reducing antioxidant power did not show any significant differences (p < 0.05). Insulin secretion studies were performed in a β-cell insulinoma cell line- RIN-5F exposed to different concentrations of glucose (high, low and in the absence of glucose). The studies were carried out to compare the β-cell stimulatory effect of vindoline to the extracts. After performing cytotoxic experiments, concentrations that resulted in about 80% cell viability were used to determine the insulin secretory effects. In cells that exposed to glucotoxicity (50 mM glucose), vindoline showed the highest β-cell stimulatory effect (p < 0.05) when compared to the untreated controls and to the cells that were treated with the methanolic extract. In cells that were exposed to a low glucose concentration, vindoline additionally showed significant β-cell stimulatory effect at p < 0.05 when compared to the aqueous and the methanolic extracts. Thereafter, the intracellular reactive oxygen species assay (ROSA) was performed in glucotoxicity-induced cells after treatment with vindoline and the respective extracts. The results were compared to the untreated control: vindoline, methanolic and the dichloromethane extracts indicated significant reduction in ROS generation (p < 0.05). Further measurement of the release of TNF-α, a pro-inflammatory cytokine in the cells following treatment, the results were not significant among the groups at p < 0.05. The carbohydrate enzymes inhibitory activity of vindoline and extracts of C.roseus (50, 25, 12.5 and 6.125 mg/ml) were measured. The alpha glucosidase inhibitory activities of the extracts at 50 mg/ml resulted in < 30% enzyme inhibition with no significant differences among the groups at p < 0.05. At lower concentrations, the dichloromethane extract exhibited significantly lower inhibitory activities when compared to the methanolic and the aqueous extract (p < 0.05). The alpha amylase inhibitory activity of the methanolic extract was significantly increased at all concentrations; recording the highest enzyme inhibition of approximately 40% (p < 0.5). However, the dichloromethane extract did not show any enzyme inhibitory activity. The enzyme inhibitory activity of vindoline was compared to acarbose-a known standard drug, for both enzymes; vindoline did not show appreciable enzyme inhibition when compared to acarbose (p < 0.05). In vivo studies were performed in a type 2 diabetes (T2DM) rat model in which T2DM was induced in 6 weeks old male Wistar rats by having them drink 10% fructose solution ad libitum for 14 days followed by a single intraperitoneal injection of streptozotocin (STZ 40 mg/kg) in freshly prepared 0.1 M citrate buffer (pH 4.5). Animals were randomly divided into six groups (n=8) and received daily treatments for 6 weeks with the vehicle, vindoline (20 mg/kg) or glibenclamide (5 mg/kg) via oral gavage. The effects of the treatments on blood glucose, insulin, body weight, organ weight, serum biochemical parameters, oxidative status, inflammatory markers and tissue histology were assessed in diabetic and non-diabetic rats. Administration of vindoline significantly (p < 0.05) reduced the fasting blood glucose in diabetic rats by 15% and significantly increased serum insulin levels when compared to the diabetic controls. Vindoline and glibenclamide significantly (p < 0.05) reduced the levels of circulating hepatic enzymes in T2DM; the results were significant when compared to the diabetic controls. Treatment with vindoline significantly improved the hepatic antioxidant status as indicated by increased ORAC, superoxide dismutase and catalase activities, indicative of the protective effect of vindoline in diabetes-induced hepatic injury. Assessment of the levels of pro-inflammatory cytokines in the hepatic tissue indicated remarkable reduction of TNF-ɑ by (-41%) and IL-6 (-28%) in diabetic rats treated with vindoline when compared to the diabetic controls (p < 0.05). The serum lipid profile showed marked increases in the levels of serum lipids (triglycerides, low density lipoproteins, total cholesterol and very low density lipoproteins) in diabetic controls when compared to all treatment groups (p < 0.05). Therefore, vindoline and glibenclamide showed possible protective effects against diabetes-induced cardiovascular disease. Kidney function assessment revealed increased levels of urea and creatinine in the diabetic control group. Vindoline and glibenclamide significantly reduced the urea and creatinine levels in diabetic rats. Vindoline additionally improved the FRAP in diabetic hearts. The SOD activity and ORAC were increased while lipid peroxidation was reduced in the kidneys of diabetic rats treated with vindoline when compared to the diabetic control (p < 0.05). Histopathological assessment in diabetic rats showed severe damage of the liver, kidney and pancreas. Treatment of diabetic rats with vindoline restored the structure of these organs which was indicated by minimum structural changes. The expression of pro-apoptotic marker caspase 9 in response to glucose stress was significantly higher in the diabetic control group when compared to all the treatment groups. Treatment with vindoline showed remarkable reduction of caspase 9 expression in the diabetic rats. In conclusion, persistent high blood glucose levels resulted in free radical induced tissue damage in the type 2 diabetes rat model. Vindoline demonstrated protective effects against diabetes induced hepatic, cardiac, pancreatic and nephritic injuries. In addition, vindoline improved insulin secretion in both in vitro and in vivo setups hence the findings suggest that vindoline could be an important agent that can be considered in the treatment and management of diabetes and diabetic complications.

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semi-synthetic anticancer drugs. Biosynthesis of TIAs is tissue-specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. This study reports the isolation and characterization of the G10H promoter and two WRKY transcription factors regulating TIA biosynthesis. Geraniol 10-hydroxylase (G10H) controls the first committed step in the biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. For functional characterization, fusion constructs of G10H promoter fragments with the GUS reporter gene were generated and expression was analyzed in a tobacco protoplast transient expression assay. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between -191 and -147, -266 and -188, and -318 and -266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings, GUS expression was tissue-specific, restricted to the leaf and actively growing cells around the root tip. GUS expression was not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitors and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade. The majority of TIA pathway gene promoters contain typical W-box elements, which are frequently found to be the binding sites of WRKY transcription factors. CrWRKY1 and CrWRKY2 transcription factors were isolated using a degenerate PCR method. The C. roseus WRKY transcription factor, CrWRKY1 is preferentially expressed in roots and induced by phytohormones, jasmonate, gibberellic acid and ethylene. Overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially tryptophan decarboxylase (TDC), as well as transcriptional repressors ZCT1, ZCT2 and ZCT3. In contrast, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3 and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX-repressor domain to CrWRKY1, resulted in down-regulation of TDC and ZCTs but up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W-box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid and C. roseus protoplast assays. In CrWRKY1 hairy roots, up-regulation of TDC increased TDC activity, tryptamine concentration and resistance to 4-methyl tryptophan inhibition. Compared to control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors, including ORCA3, CrMYC2 and ZCTs, may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants. CrWRKY2 is induced by methyljasmonate induction. In plant, CrWRKY2 expression is mainly found in young leaves and the stem. The stable transformation of CrWRKY2 in C. roseus hairy roots up-regulated many pathway genes, especially the genes in vindoline biosynthesis. The accumulation of vindoline was observed in CrWRKY2 hairy roots.

A vinca (Catharanthus roseus) é uma planta perene, arbustiva, pertencente à família Apocinaceae, cujas folhas e raízes possuem propriedades medicinais. A presença de sintoma de mosaico e deformação foliar em plantas dessa espécie, associados com a presença de partículas alongadas e flexuosas, característica de vírus pertencentes ao gênero Potyvirus, conduziu a estudos complementares para a identificação e caracterização desse vírus. No estudo da gama parcial de hospedeiras foram testadas 28 espécies, envolvendo oito famílias botânicas. Catharanthus roseus e Nicotiana benthamiana apresentaram sintomas de mosaico foliar e Chenopodium amaranticolor e C. quinoa apresentaram lesões locais cloróticas nas folhas inoculadas. A transmissão do vírus com afídeos foi avaliada com as espécies Aphis gossypii, Myzus nicotianae e Toxoptera citricidus. Apenas Aphis gossypii e Myzus nicotianae transmitiram o vírus. O antissoro policlonal produzido contra este potyvirus reagiu com o vírus homólogo e com o Passionfruit woodiness virus (PWV) e Cowpea aphid-borne mosaic virus (CABMV), mas não com o Lettuce mosaic virus (LMV), Papaya ringspot virus - type W (PRSV-W), Potato virus Y (PVY) e Zucchini yellow mosaic virus (ZYMV). O peso molecular da proteína capsidial (CP) foi de aproximadamente 34 kDa. A reação de PCR realizada com os oligonucleotídeos universais de potyvirus e oligonucleotídeos específicos posteriomente confeccionados amplificaram três fragmentos de aproximadamente 0,8, 1,0 e 1,4 Kb, os quais após o seqüênciamento geraram um fragmento de 1654 nucleotídeos (nt) da região 3' terminal do genoma, que inclui parte do gene da replicase viral (Nib), a região codificadora completa do gene da proteína capsidial (CP), seguida de 286 nt da região 3' não traduzida (3'NTR). A identidade da seqüência de nucleotídeos do gene da CP variou de 67,0 a 76,0%, quando comparada com as de outros membros da família Potyviridae. A maior identidade foi com o Omphalodes virus Y (76,0%). A identidade dos aminoácidos deduzidos da proteína capsidial variou de 62,0 a 71,0%, sendo a maior com East Asian Passiflora virus (71%). Para a região não traduzida (3'NTR) a identidade variou de 16,8 a 28,6%. Em conjunto esses dados indicam que este vírus é uma nova espécie dentro do gênero Potyvirus, para o qual se propõe o nome de Vírus do mosaico do Catharanthus (Catharanthus mosaic virus - CatMV). / Catharanthus roseus is known as the common periwinkle or Madagascar periwinkle. It is a perennial, evergreen herb in the family Apocynaceae, which was originally native to the island of Madagascar, although both name and classification are contradictory in some literature. The plants grow up to 80 cm high; have glossy, dark green leaves and bloom during summer. The flowers range from white to hot pink to purple. The species has historically been used in popular medicine to treat a wide assortment of human diseases, as it contains more than 150 useful alkaloids. Plants of C. roseus exhibiting mosaic symptoms followed by malformation of the leaf blades and flower variegation were collected from a garden at the University of São Paulo, School of Agriculture (Piracicaba, State of São Paulo, Brazil). Preliminary electron microscopy exams of negatively stained leaf sap revealed that the symptoms were associated with potyvirus-like particles. The objective of the present work was to obtain further biological, immunological and molecular data to better characterize this species of the genus Potyvirus, family Potyviridae. Of 28 plant species from eight botanical families inoculated mechanically with this potyvirus, only C. roseus and Nicotiana benthamiana developed systemic mosaic, whereas Chenopodium amaranticolor and C. quinoa exhibited only chlorotic local lesions. The virus was transmitted by Aphis gossypii and Myzus nicotianae, but not by Toxoptera citricidus. Polyclonal antiserum raised against this potyvirus reacted with the homologous virus, Passion fruit woodiness virus (PWV) and Cowpea aphid borne mosaic virus (CABMV) in PTA-ELISA. The molecular mass of the coat protein (CP) was approximately 34 kDa. RT-PCR from viral RNA amplified a fragment of approximately 1654 nucleotides (nt) at the 3'-terminal of the viral genome, containing portion of the replicase gene (Nib), the entire CP gene and the 3' untranslated region (3'UTR) (286 nt). When the nucleotide sequence of the CP gene was compared with other members of the Potyviridae family, identities varied from 67.0 to 76.0%. The highest identity was with Omphalodes virus Y. Identity of the deduced amino acid of the CP varied from 62.0 to 71.0%, with the highest for East Asian Passiflora virus. For the 3' UTR, identities varied from 16.8 to 28.6%. The name Catharanthus mosaic virus (CatMV) is proposed for this new potyvirus.

A vinca (Catharanthus roseus) é uma planta perene, arbustiva, pertencente à família Apocinaceae, cujas folhas e raízes possuem propriedades medicinais. A presença de sintoma de mosaico e deformação foliar em plantas dessa espécie, associados com a presença de partículas alongadas e flexuosas, característica de vírus pertencentes ao gênero Potyvirus, conduziu a estudos complementares para a identificação e caracterização desse vírus. No estudo da gama parcial de hospedeiras foram testadas 28 espécies, envolvendo oito famílias botânicas. Catharanthus roseus e Nicotiana benthamiana apresentaram sintomas de mosaico foliar e Chenopodium amaranticolor e C. quinoa apresentaram lesões locais cloróticas nas folhas inoculadas. A transmissão do vírus com afídeos foi avaliada com as espécies Aphis gossypii, Myzus nicotianae e Toxoptera citricidus. Apenas Aphis gossypii e Myzus nicotianae transmitiram o vírus. O antissoro policlonal produzido contra este potyvirus reagiu com o vírus homólogo e com o Passionfruit woodiness virus (PWV) e Cowpea aphid-borne mosaic virus (CABMV), mas não com o Lettuce mosaic virus (LMV), Papaya ringspot virus - type W (PRSV-W), Potato virus Y (PVY) e Zucchini yellow mosaic virus (ZYMV). O peso molecular da proteína capsidial (CP) foi de aproximadamente 34 kDa. A reação de PCR realizada com os oligonucleotídeos universais de potyvirus e oligonucleotídeos específicos posteriomente confeccionados amplificaram três fragmentos de aproximadamente 0,8, 1,0 e 1,4 Kb, os quais após o seqüênciamento geraram um fragmento de 1654 nucleotídeos (nt) da região 3' terminal do genoma, que inclui parte do gene da replicase viral (Nib), a região codificadora completa do gene da proteína capsidial (CP), seguida de 286 nt da região 3' não traduzida (3'NTR). A identidade da seqüência de nucleotídeos do gene da CP variou de 67,0 a 76,0%, quando comparada com as de outros membros da família Potyviridae. A maior identidade foi com o Omphalodes virus Y (76,0%). A identidade dos aminoácidos deduzidos da proteína capsidial variou de 62,0 a 71,0%, sendo a maior com East Asian Passiflora virus (71%). Para a região não traduzida (3'NTR) a identidade variou de 16,8 a 28,6%. Em conjunto esses dados indicam que este vírus é uma nova espécie dentro do gênero Potyvirus, para o qual se propõe o nome de Vírus do mosaico do Catharanthus (Catharanthus mosaic virus - CatMV). / Catharanthus roseus is known as the common periwinkle or Madagascar periwinkle. It is a perennial, evergreen herb in the family Apocynaceae, which was originally native to the island of Madagascar, although both name and classification are contradictory in some literature. The plants grow up to 80 cm high; have glossy, dark green leaves and bloom during summer. The flowers range from white to hot pink to purple. The species has historically been used in popular medicine to treat a wide assortment of human diseases, as it contains more than 150 useful alkaloids. Plants of C. roseus exhibiting mosaic symptoms followed by malformation of the leaf blades and flower variegation were collected from a garden at the University of São Paulo, School of Agriculture (Piracicaba, State of São Paulo, Brazil). Preliminary electron microscopy exams of negatively stained leaf sap revealed that the symptoms were associated with potyvirus-like particles. The objective of the present work was to obtain further biological, immunological and molecular data to better characterize this species of the genus Potyvirus, family Potyviridae. Of 28 plant species from eight botanical families inoculated mechanically with this potyvirus, only C. roseus and Nicotiana benthamiana developed systemic mosaic, whereas Chenopodium amaranticolor and C. quinoa exhibited only chlorotic local lesions. The virus was transmitted by Aphis gossypii and Myzus nicotianae, but not by Toxoptera citricidus. Polyclonal antiserum raised against this potyvirus reacted with the homologous virus, Passion fruit woodiness virus (PWV) and Cowpea aphid borne mosaic virus (CABMV) in PTA-ELISA. The molecular mass of the coat protein (CP) was approximately 34 kDa. RT-PCR from viral RNA amplified a fragment of approximately 1654 nucleotides (nt) at the 3'-terminal of the viral genome, containing portion of the replicase gene (Nib), the entire CP gene and the 3' untranslated region (3'UTR) (286 nt). When the nucleotide sequence of the CP gene was compared with other members of the Potyviridae family, identities varied from 67.0 to 76.0%. The highest identity was with Omphalodes virus Y. Identity of the deduced amino acid of the CP varied from 62.0 to 71.0%, with the highest for East Asian Passiflora virus. For the 3' UTR, identities varied from 16.8 to 28.6%. The name Catharanthus mosaic virus (CatMV) is proposed for this new potyvirus.