11 |
Effects of Developmental Stage, Exogenous Sugar Composition, and Reactive Oxygen Species on Artemisinin and Related Compounds in Artemisia annuaArsenault, Patrick Ryan 27 April 2010 (has links)
Artemisinin (AN), a sesquiterpene, derived from the herb, Artemisia annua is the most widely used anti-malarial compound. Current production is insufficient to meet the growing demand for this important drug. Many experiments have been done to try and deduce what factors may be important to increased yield. Here is is shown that many disparate phenomena known to induce AN production may be linked under the umbrella of reactive oxygen species (ROS). To that end, the metabolite and transcriptional changes associated with the transition from vegetative growth to flowering have been investigated. In addition, the role that exogenous sugars play in modulating these same factors has been explored in young seedlings. Lastly, exposure to DMSO was shown to increase AN production and that it may be linked to ROS. These combined results wered further explored to determine the effects of direct ROS elicitation and subsequent quenching on the production of AN and related metabolites. Information gained here supported a new alternative hypothesis for the role of ROS in AN production, one in which hydrogen peroxide may be controlling the balance of deoxyartemisinin (deoxyAN) and AN.
|
12 |
The Effects of Phytohormones on Growth and Artemisinin Production in Hairy Root Cultures of Artemisia Annua L.McCoy, Mark Christopher 29 May 2003 (has links)
"The in vitro addition of plant growth regulators (i.e. phytohormones) to Agrobacterium transformed hairy root cultures affects morphological and biochemical changes, resulting in altered growth and secondary metabolite accumulation rates in root tissues. Significant increases in both growth and secondary product accumulation have been observed, upon incubation with phytohormones, in some species. Consequently, the use of phytohormones in vitro has received increasing attention as a potential means for increasing those plant secondary products notoriously produced in small quantities. However, currently little is known about the specific effects of phytohormones on growth and secondary metabolism. The Chinese herb Artemisia annua L. produces artemisinin, an effective antimalarial therapeutic. Efforts to increase the amount of artemisinin via chemical synthesis or field-grown crops have met with huge costs and disappointingly low yields, respectively. Agrobacterium transformed hairy root cultures of A. annua (Clone YUT16) produce artemisinin and undergo rapid growth compared to non-transformed, making them a good model system to study secondary metabolite production. Demonstrated herein is the first definitive evidence, by any hairy root species, of a favorable response to exogenous combinatorial hormone application as well as the development of a two-stage culture system alluding to optimal growth and artemisinin production conditions in A. annua hairy roots. Furthermore, analysis of artemisinin and biomass accumulation in A. annua hairy roots in the presence of phytohormones has revealed effective individual as well as combinatorial phytohormone concentrations suitable for increasing single and bulk root growth, and artemisinin production. The effectiveness of an optimal phytohormone combination, with respect to time of addition, its relationship to inoculum size, and its combination with the provision of fresh nutrients and or mechanical stress to the roots is also described resulting in artemisinin yields of up to 0.8 ìg/g F.W. Although the findings contained herein are not yet optimized they do, however, argue for the potential usefulness of a two-stage production scheme using phytohormones to increase plant secondary metabolite production in vitro."
|
13 |
Proteome analysis of glandular trichome from Artemisia annua L.January 2011 (has links)
Wu, Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 56-70). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.Ill / ABSTRACT --- p.V / TABLE OF CONTENTS --- p.IX / LIST OF ABBREVIATIONS --- p.XII / Chapter CHAPTER 1. --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- THE DISEASE OF MALARIA --- p.1 / Chapter 1.1.1 --- Pathogenesis --- p.2 / Chapter 1.1.2 --- The treatment of malaria --- p.4 / Chapter 1.2 --- THE PLANT OF ARTEMISIA ANNUA L --- p.5 / Chapter 1.2.1. --- Horticulture --- p.5 / Chapter 1.2.2. --- Historical Importance --- p.6 / Chapter 1.3 --- ARTEMISININ --- p.7 / Chapter 1.3.1 --- The content and distribution of artemisinin --- p.7 / Chapter 1.3.2 --- The biosynthesis of artemisnin --- p.8 / Chapter 1.4 --- TRICHOMES --- p.13 / Chapter 1.4.1 --- Structure and function of trichomes --- p.13 / Chapter 1.4.2 --- Trichome investigation in A. annua --- p.14 / Chapter 1.5 --- PROTEOMICS --- p.17 / Chapter 1.5.1 --- The basic principle of proteomics --- p.17 / Chapter 1.5.2 --- Two-dimensional gel electrophoresis --- p.18 / Chapter 1.5.3 --- Mass Spectrometry --- p.19 / Chapter 1.5.4 --- Gel-free proteomics --- p.20 / Chapter 1.6 --- OBJECTIVES --- p.21 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- CHEMICALS --- p.23 / Chapter 2.2 --- PLANT MATERIALS --- p.23 / Chapter 2.3 --- ISOLATION OF GLANDULAR TRICHOMES --- p.23 / Chapter 2.4 --- PROTEIN EXTRACTION . --- p.25 / Chapter 2.5 --- Two DIMENSIONAL GEL ELECTROPHORESIS --- p.25 / Chapter 2.6 --- IMAGINE ANALYSIS --- p.26 / Chapter 2.7 --- IN GEL DIGESTION AND PROTEIN IDENTIFICAIOTN BY MASS SPECTROMETRY --- p.27 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSION --- p.29 / Chapter 3.1 --- THE ISOLATION OF GLANDULAR TRICHOMES --- p.29 / Chapter 3.2 --- 2DE PATTERNS OF A. ANNUA LEAVE TRICHOMES AND LEAF TISSUE --- p.32 / Chapter 3.3 --- IDENTIFICATION OF PROTEINS IN GLANDULAR TRICHOMES --- p.34 / Chapter 3.3.1 --- Protein involved in electron transport chain --- p.47 / Chapter 3.3.2 --- Protiens invovled in metabolism --- p.48 / Chapter 3.3.2.1 --- artemisinin biosynthesis --- p.48 / Chapter 3.3.2.2 --- glycolysis --- p.49 / Chapter 3.3.2.3 --- other metabolic enzymes --- p.50 / Chapter 3.3.3 --- Proteins involved in transcription and translation --- p.51 / Chapter 3.3.4 --- Protein involved in proteolysis --- p.51 / Chapter 3.3.5 --- "Detoxificaiton, stress related protein" --- p.52 / Chapter 3.4 --- PERSPECTIVE --- p.53 / Chapter 3.5 --- CONCLUSION --- p.53 / REFERENCES --- p.56
|
14 |
The effects of phytohormones on growth and artemisinin production in hairy root cultures of artemisia annua l.McCoy, Mark Christopher. January 2003 (has links)
Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: Phytohormones. Includes bibliographical references (p.73-79).
|
15 |
Investigation into the effects of Artemisinin in myocardial ischaemia reperfusion injuryBabba, M. A. January 2015 (has links)
Artemisinin is herbal drug with a wide range of biological and physiological function. It is currently administered in the treatment against uncomplicated F.Palcifarum infections. It has also been shown to be cytotoxic against a variety of cancer cells. Despite the promise of many anti cancer drugs, drug induced cardiotoxicity has constantly threatened drug applicability especially in patients with co-morbities. Artemisinin has been shown to be cardioprotective, although the intracellular pathways remain to be elucidated. In this study, isolated perfused rat hearts were subjected to 35 minutes of ischaemia and 120 minutes reperfusion or primary cardiac myocytes subjected to 120 minutes hypoxia and 120 minutes reoxygenation where artemisinin (4.3μM) was administered in presence and absence of the PI3K inhibitor (wortmannin) (0.1μM), p70S6K inhibitor (rapamycin) (0.1μM), non selective nitric oxide synthase inhibitor (L-NAME) (100μM) and inducible nitric oxide synthase inhibitor (aminoguanidine) (100μM). At the end of the experiment, hearts underwent infarct size to risk ratio assessment via tri-phenyltetrazolium chloride staining or western blot analysis for p-Akt and p70S6K. Cardiac myocytes were assessed for either MTT analysis, cleaved-caspase 3 or for eNOS/iNOS or p-BAD activity using flow cytometry. In isolated hearts, artemisinin (0.1μM-100μM) showed a significant dose dependent decrease in infarct size (P<0.01-0.001 vs. I/R control). It was also shown to significantly improve cellular viability (66.5±6.3% vs. 29.3±6.1% in H/R, P<0.01) and decrease the levels of cleaved caspase-3 compared to the H/R control group (17.1±2.0% vs. 26.8±2.0% in H/R, P<0.001). Artemisinin was shown to confer protection via the activation of the PI3K-Akt-p70S6k cell survival pathway and presented an upregulation in p-eNOS and iNOS expression. Furthermore, co-administering artemisinin with doxorubicin showed artemisinin reverses I/R or H/R injury as well as doxorubicin-induced injury via the nitric oxide signalling pathway. Additionally, in HL-60 cells, the co-administration doubled artemisinins cytotoxicity while also implicating the nitric oxide pathway. This is the first study to shows that artemisinin ameliorates doxorubicin mediated cardiac injury whilst enhancing its cytotoxicity in HL-60 in a nitric oxide dependent manner. This study concluded that artemisinin was both anti apoptotic and protective against myocardial I/R injury via the PI3K-Akt-BAD/P70S6K and via the nitric oxide cell survival pathway as well as pro-apoptotic against HL-60 in a nitric oxide dependent manner.
|
16 |
Examining the role of K13 in artemisinin-resistant Plasmodium falciparum malariaStokes, Barbara January 2020 (has links)
Despite the concerted efforts of researchers, policy makers and public health workers worldwide, malaria persists as a significant disease threat for nearly half the world’s population. Recent advances in vector control measures, diagnostics and antimalarial drug therapies have contributed greatly to reducing the incidence of clinical disease, and by extension, the number of deaths attributable to malaria in the past two decades; however, the latter remains high—over 400,000 people die each year from malaria, the vast majority of these being children under the age of five. Our ability to rapidly and effectively treat malaria has been a cornerstone of efforts to control and eradicate this devastating disease. Nonetheless, the constant evolution and spread of drug-resistant forms of the Plasmodium parasites that cause malaria—particularly the most virulent of these, Plasmodium falciparum—have historically greatly hindered these efforts, compromising the efficacy of every previous first-line treatment.
Today, treatment of P. falciparum malaria relies on artemisinin derivatives, an exquisitely potent and fast-acting class of antimalarials that are deployed ubiquitously in artemisinin-based combination therapies, or ACTs. Now, emerging resistance to ACTs threatens to once again reverse the hard-fought advances made in the global fight against malaria. Resistance to artemisinin itself was first documented in western Cambodia and northwest Thailand in 2009 and has continued to spread throughout Southeast Asia at alarming rates. Reports of resistance to ACTs followed soon thereafter. Artemisinin resistance has also emerged de novo in other parts of the world. The major concern is that it will spread to Africa, where the disease burden is highest.
Previous studies have provided compelling evidence that resistance to artemisinin results primarily from specific point mutations in the C-terminal Kelch propeller domain of the P. falciparum protein K13. Here, we have addressed two central aims regarding the role of this protein in mediating resistance to artemisinin. The first was to genetically dissect the contribution of a panel of K13 polymorphisms to artemisinin resistance and parasite fitness as assessed in vitro, with the latter being a key factor impacting the spread of resistance-conferring alleles in high-transmission settings. These experiments were conducted by CRISPR-Cas9-mediated gene editing, which allowed us to successfully engineer K13 mutations into a variety of strain backgrounds, including, for the first time, recently culture-adapted African parasites. These experiments clearly show that there is no genetic obstacle to the acquisition of artemisinin resistance in African parasites; however, they also suggest that fitness costs associated with these mutations may counter-select against the spread of resistance.
The second aim relating to K13 was to investigate the underlying biology of this protein. To this end, we raised monoclonal antibodies to recombinant K13 and generated transgenic lines expressing tagged versions of the protein. Using these tools, we describe the subcellular localization of K13 in wild-type and mutant parasites in the presence and absence of drug pressure, and identify potential K13-associated proteins. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13, suggesting that mutations result in a loss of protein function.
In order to overcome K13-mediated artemisinin resistance, novel therapeutics with distinct modes of action will be required. In our last aim, we characterize inhibitors of a particularly promising new antimalarial drug target, the proteasome. We report that these covalent peptide vinyl sulfone inhibitors are highly potent against genetically diverse parasites, including K13-mutant, artemisinin-resistant lines. Moreover, we observe that parasites do not readily acquire resistance to these compounds, nor do related compounds select for cross-resistance to one another. We also observe strong synergy between artemisinin and related compounds with these inhibitors in both K13 mutant and wild-type parasites. These results highlight the potential for targeting the Plasmodium proteasome as a means of overcoming artemisinin-resistant malaria.
|
17 |
Malarial pathogenesis and interventions in Kelch mediated Artemisinin resistance in Plasmodium falciparumPittala, Keerthana 14 June 2019 (has links)
Malaria, a parasitic disease, was commonly associated with third world countries, with the highest mortality in nations in Sub-Saharan Africa and Asia. But, travel increases the risk of spread to more temperate regions, such as Western Europe and the United States where Malaria has been successfully eradicated. In the past 40 years, with a better understanding of the mosquito vector and the parasite itself, advancements in treatment and containment have been made.
Understanding the parasite as well as its pathogenesis is vital in formulating effective treatments. Following the incidences of Plasmodium falciparum, knowlesi, vivax, malaria, ovale, and less commonly cynomolgi and simium over time as well as region helps to better illuminate the methods of Malarial transmission, interplay with environmental factors, and methods of treatment. While each species of parasite is similar in terms of mode of infection, they differ slightly when considering incubation periods and diagnostic and treatment techniques.
Many drugs have been developed to treat Malaria and include Chloroquine, Primaquine, and derivatives of Artemisinin. While the discovery of these drugs was a significant breakthrough that dramatically reduced incidence and deaths caused by Malaria, improper administration of treatment has led to a recent increase in strains of the parasite which have developed drug resistance to Artemisinin Combination Therapies (ACT’s). Of these species, P. falciparum and P. vivax, the most common causes of malaria, are also so far the only species to have developed drug resistance. The goal of this thesis is to explore popular interventions, both drug and public health based, and how research focus has now shifted to better understanding the mechanism of parasitic drug resistance, specifically linked to mutations found in the Kelch protein in P. Falciparum. The recent findings of Kelch mutations pave the way towards addressing the growing problem of anti-Malarial resistance.
|
18 |
Application of hot melt extrusion for improving bioavailability of artemisinin a thermolabile drugKulkarni, Chaitrali S., Kelly, Adrian L., Gough, Tim, Jadhav, V., Singh, K., Paradkar, Anant R 16 November 2017 (has links)
Yes / Hot melt extrusion has been used to produce a solid dispersion of the thermolabile drug artemisinin. Formulation and process conditions were optimised prior to evaluation of dissolution and biopharmaceutical performance. Soluplus®, a low Tg amphiphilic polymer especially designed for solid dispersions enabled melt extrusion at 110ºC although some drug-polymer incompatibility was observed. Addition of 5% citric acid as a pH modifier was found to suppress the degradation. The area under plasma concentration time curve (AUC0-24hr) and peak plasma concentration (Cmax) were four times higher for the modified solid dispersion compared to that of pure artemisinin. / EPSRC grant no (EP/J003360/1) and UKIERI: UK-India Education and Research Initiative (TPR 26).
|
19 |
Novel formulations of a poorly soluble drug using the extrusion process.Kulkarni, Chaitrali S. January 2013 (has links)
Hot melt extrusion has attracted recent interest from the pharmaceutical industry and academia as an innovative drug delivery technology. This novel technique has been shown to be a viable and robust method for preparing different drug delivery systems including pellets, implants, tablets, capsules and granules. The aim of this research was to understand hot melt extrusion processing and explore its pharmaceutical applications. Two applications of hot melt extrusion (HME) have been investigated to improve the properties of poorly soluble thermolabile drugs; polymeric solid dispersions and solid state polymorphic transformation.
HME is a solvent free, continuous and readily scalable technique which is increasingly being considered as a viable alternative to conventionally used batch techniques. However, the high temperature and shear forces imparted by the extrusion process can limit its applications with heat sensitive active pharmaceutical ingredients (APIs). Artemisinin was selected as a model drug which being thermolabile in nature and possesses processing challenges to processing HME. A low Tg amphiphillic copolymer, Soluplus® was selected as a matrix material. Drug-polymer compatibility was studied using rotational rheometry and thermal characterisation. The drug was found to be completely dissolved within the polymer, although some discolouration of the mixture was observed, indicating degradation of the API. The addition of a small percentage of citric acid to the formulation was found to prevent this degradation by increasing the pH. The dissolution profile of the formulation was approximately five times higher compared to that of the pure drug. The pharmacokinetic study was carried out using Albino rats to calculate bioavailability. The area under plasma concentration time curve (AUC0-24hr) and peak plasma concentration (Cmax) were four times higher for the prepared solid dispersion compared to that of pure artemisinin. Extruded solid dispersions were found to be amorphous in nature and maintained stability for 2 years.
A second route to improving the solubility of poorly soluble APIs was also investigated. It was found that under carefully controlled conditions, high temperature extrusion (HTE) could be used to achieve polymorphic transformation with a number of APIs. This solvent-free continuous process was demonstrated with artemisinin, piracetam, carbamazepine and chlorpropamide. Artemisinin was used as a detailed case study of stability, solvent mediated transformation and mechanism of polymrophic transformation during extrusion, using computational modelling and model shear flows. At high temperature, phase transformation from orthorhombic to triclinic crystals was found to occur via the vapour phase. Under mechanical stress the crystalline structure was disrupted, leading to new surfaces being continuously formed and exposed to high temperatures; thus accelerating the transformation process. Polymorphic transformation during HTE was found to comprise three stages; i) preheating and conveying; ii) vapour phase transformation and size reduction and iii) continuous transformation and agglomeration. The triclinic form showed four times greater dissolution rate as compared to the orthorhombic form. The triclinic form showed two fold increase in bioavailability in Albino rats.
|
20 |
Effects of artificial polyploidy in transformed roots of Artemisia annua L.De Jesus, Larry 24 April 2003 (has links)
In most plant species artificial polyploidy generally enhances the vigor of determinate plant parts and may be favorable where vegetative organs and biomass constitute the economic product. Furthermore, artificial polyploidy has been considered a method of increasing production potential of plants secondary metabolites. However, despite considerable research on polyploid plants, very few cases of polyploid medicinal plants have been reported. Artemisia annua L. synthesizes artemisinin, an antimalarial sesquiterpene lactone. Artemisinin can be synthesized, but it is costly compared to the naturally derived product. Hairy root cultures of Artemisia annua L. (clone YUT16) show rapid growth and produce artemisinin. This culture offers a good model system for studying artemisinin production. Others have shown that tetraploid Artemisia annua L. plants produce more artemisinin/mg DW than diploids. These yields were offset, however, by decreases in biomass productivity. Little is known about how polyploidy may affect growth production of hairy roots. Using colchicine, we have produced four stable tetraploid clones of Artemisia annua L. from YUT16 hairy root clone. Compared to the diploid clone, these tetraploid clones showed major differences in growth and development. Nevertheless, artemisinin yields of these tetraploid clones were 2-5 times higher than the diploid and their production seemed to be by the age of the inoculum. This work will prove useful in furthering our understanding of the effects of artificial polyploidy on the growth and secondary metabolite production of hairy roots.
|
Page generated in 0.0886 seconds