Discovery of Natural Antiprotozoals From
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DISCOVERY OF NATURAL ANTIPROTOZOALS FROM MEDICINAL PLANTS SAUSSUREA COSTUS AND CARICA PAPAYA Inauguraldissertation Zur Erlangung der wuerde eines Doktors der Philosophie Vorgelegt der Philosophisch-Naturwissenschaftlechen Fakultaet Der Universitaet Basel Von Tasqiah Julianti Aus INDONESIEN Basel 2014 Original document stored on the publication server of the University of Basel edoc.unibas.ch This work is licenced under the agreement „Attribution Non-Commercial No Derivatives – 3.0 Switzerland“ (CC BY-NC-ND 3.0 CH). The complete text may be reviewed here: creativecommons.org/licenses/by-nc-nd/3.0/ch/deed.en Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Matthias Hamburger PD. Dr. Olivier Potterat Prof. Dr. Reto Brun Basel, den 25.03.2014 Prof. Dr. Jörg Schibler Dekan Attribution-Noncommercial-No Derivative Works 2.5 Switzerland You are free: to Share — to copy, distribute and transmit the work Under the following conditions: Attribution. 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Quelle: http://creativecommons.org/licenses/by-nc-nd/2.5/ch/deed.en Datum: 3.4.2009 TABLE OF CONTENTS TABLE OF CONTENTS LIST OF FIGURES ……………………………………………………………………………. iii ABBREVIATIONS …………………………………………………………………………… iv SUMMARY ……………………………………………………………………………………. v ZUSAMMENFASSUNG …………………………………………………………………….. vii CHAPTER I AIM OF THE WORK ………………………………………………………………………… 1 CHAPTER II INTRODUCTION 1.1 Tropical diseases ……………………………………………………………………… 2 1.1.1 Human African trypanosomiasis …………………………………………... 3 1.1.2 Malaria ………………………………………………………………………... 5 1.2 Discovery of hits from natural sources ……………………………………………... 9 1.2.1 Antiprotozoal compounds discovery using extract libraries ……………. 12 1.2.2 Ethnopharmacology-based discovery of antiprotozoal compounds……. 13 1.3 Indonesia: traditional medicines and malaria ……………………………………... 16 1.3.1 Traditional medicines ……………………………………………………….. 16 1.3.2 Malaria and ethnomedicines for malaria ………………………………….. 19 References ………………………………………………………………………………… 23 CHAPTER III Antitrypanosomal sesquiterpene lactones from Saussurea costus ………………………… 32 CHAPTER IV HPLC-based activity profiling for antiplasmodial compounds in the traditional Indonesian medicinal plant Carica papaya L. …………………………………….................. 40 i TABLE OF CONTENTS CHAPTER V Quantification of the antiplasmodial alkaloid carpaine in papaya leaves (Carica papaya) …………………………………………………………………………………………... 58 CHAPTER VI CONCLUSION & OUTLOOK …………………………………………………………........ 79 ACKNOWLEDGMENTS …………………………………………………………………..... 82 CURRICULUM VITAE ……………………………………………………………………… 83 ii LIST OF FIGURES LIST OF FIGURES Figure 1 Miniaturized HPLC-based activity profiling approach…………………….. 10 Figure 2 The construction of extracts library …………………………………………... 12 Figure 3 Relief on Borobudur temple about the tradition of drinking jamu .……….. 15 Figure 4 Logo of herbal medicines ……………………………………………………… 16 iii ABBREVIATIONS LIST OF ABBREVIATIONS ACT Artemisinin-based combination therapy CDC Centers for Disease Control and Prevention CFS Cerebrospinal fluid DALY Disability-adjusted life years DNDi Drugs for Neglected Disease initiative ELSD Evaporative light scattering detector ESI-MS Electronspray ionization-mass spectroscopy FLD Flourescence detector HAT Human African trypanosomiasis HPLC High pressure/performance liquid chromatography HR-MS High resolution mass spectroscopy HTS High throughput screening IC50 50% growth inhibitory concentration MMV Medicines for Malaria Venture MS Mass spectroscopy MS/MS Tandem mass spectroscopy NA-DFC National Agency-Drug and Food Control NMR Nuclear magnetic resonance NP Natural product NTD Neglected tropical disease OHT Obat herbal terstandar (standardized herbal medicine) PDA Photo diode array ResNetNPND Research Network Natural Products against Neglected Diseases SAR Structure-activity relationship SI Selectivity index TD Tropical disease UHPLC Ultra high pressure/performance liquid chromatography WHO World Health Organization iv SUMMARY SUMMARY Medicinal plants have been an important source for the discovery of therapeutic agents for infectious diseases. In order to explore their potential an appropriate selection of plant species is important. In our attempt to find hits targeting antiprotozoal diseases, we utilized an extract library setting, and ethnomedicinal information. A library of 1800 plant and fungal extracts was screened for in vitro effects against Trypanosoma brucei rhodesiense STIB 900 strain and Plasmodium falciparum K1 strain. The ethyl acetate extract of Saussurea costus roots, and the methanolic extract of Carica papaya leaves were selected for further studies. HPLC-based activity profiling enabled the localization and identification of the active constituents of these plants extracts. Sensitive hyphenated analytical methods such as HPLC-PDA-ESI-TOF-MS and microprobe NMR were used for structure elucidation of the isolated compounds. X-ray crystallography was used in combination with electronic circular dichroism to determine the absolute configuration of selected compounds. The ethyl acetate extract of S. costus roots potently inhibited the growth of T. b. rhodesiense in vitro. HPLC-based activity profiling led to the identification of four sesquiterpene lactones. Three structurally related sesquiterpene lactones that originated from different sources were also investigated. All compounds exhibited profound activity against T. b. rhodesiense with IC50 values between 0.8 – 21.9 µM. Cytotoxicity was tested on rat myoblast L-6 cells, where IC50 values of 1.6 to 19.4 µM were observed, and provided selectivity indices (SI) between 0.5 and 6.5. The most active compounds in this study were the germacranolides costunolide, parthenolide, and eupatoriopicrin. The leaves of the Indonesian ethnomedicinal plant C. papaya are a known antimalarial remedy. So far, the active principles have not been investigated from a phytochemical and pharmacological point of view. HPLC-based activity profiling of the methanolic extract from C. papaya leaves against P. falciparum led to the discovery of five alkaloids and four flavonol glycosides. All compounds exhibited in vitro antimalarial activity against P. falciparum K1 strain, albeit to varying degrees. Three dimeric alkaloids showed potent activity with IC50 values ranging from 0.2 to 1.8 µM, and SI from 24.2 to 107.5. The isolated flavonol glycosides were less active, with IC50 values between 13.2 – 16.8 µM, and selectivity indices of more v SUMMARY than 9. Lower activity was observed for the two monomeric alkaloids (IC50 ≥ 77 µM). Carpaine (IC50 of 0.2 µM; SI of 107.5) was the most interesting compound in this study and was, hence, selected for further evaluation of its in vivo pharmacological properties using a 4-day suppressive assay on mice. However, only a reduction of parasitemia by 11.9% was observed. With the aid of X-ray crystallography and ECD calculation, the absolute configuration for carpaine was established as 1S,11R,13S,14S,24R,26S. Carpaine represents a new scaffold for anti-plasmodial drugs. An analysis of carpaine content by means of UPLC- MS/MS was pursued with 28 leaf samples from Indonesia and one from India. The carpaine content varied from 0.02 to 0.31%. vi ZUSAMMENFASSUNG ZUSAMMENFASSUNG Medizinalpflanzen, die traditionell zur Behandlung von Infektionskrankheiten eingesetzt werden, spielen bei der Entdeckung neuer Wirkstoffe eine grosse Rolle. Für die Identifizierung neuer Leitstrukturen ist zudem die Auswahl geeigneter Pflanzen von entscheidender Bedeutung. Auf der Suche nach antiprotozoal wirkenden Verbindungen wurden sowohl ethnobotanische Informationen als auch die Ergebnisse eines vorausgegangenen Extraktscreenings verwendet. Insgesamt wurden 1800 Pflanzen- und Pilzextrakte auf ihre in vitro Aktivtät gegen den Trypanosoma brucei rhodesiense STIB 900 Stamm und den Plasmodium falciparum K1 Stamm hin untersucht. Der Ethylacetat-Extrakt aus den Wurzeln von Saussurea costus und der methanolische Extrakt aus den Blättern von Carica papaya wurden für weitere Untersuchungen ausgewählt. Das HPLC-basierte Aktivtätsprofiling ermöglichte die Lokalisierung und Identifizierung der aktiven Extraktkomponenten. Für die Strukturaufklärung