University of Khartoum Graduate College Medical and Health Studies Board

Isolation and Structure Elucidation of Leishmanicidal Alkaloids from Argemone mexicana L. and Nauclea latifolia Smith.

By Omima Mekki Khider Mekki B. Sc. (1999), M. Sc. (2005) (U of K)

A thesis submitted in fulfillment of the requirement for the award of the degree of Doctor of Philosophy in Pharmaceutical Chemistry of University of Khartoum

Supervisor Prof. Sami Ahmed Khalid B. Pharm., M. Pharm., PhD.

2016 DEDICATION

This thesis is dedicated to my lovely family,

To my Parents for their lifelong devotion, patient and prayers

To my dearest Brothers and Sisters for their kind help and prayers

To my respectful supervisor prof. Sami for his kind support

Omima

TABLE OF CONTENTS

Contents Page No TABLE OF CONTENTS i ACKNOWLEDGMENTS v ABBREVIATIONS vii ENGLISH ABSTRACT ix ARABIC ABSTRACT xi LIST OF FIGURES xii LIST OF TABLES xiii 1. INTRODUCTION 1 1.1 Background information 1 1.2 Problem statement 2 1.3 Aim and Objectives 3 1. 4 Justifications 3 2. LITERATURE REVIEW 5 2.1 Neglected Tropical Diseases (NTDs) caused by protozoan 5 parasites 2.1.1 Contribution of Medicinal Plants to NTDs 6 2.1.2 Ethnopharmacology of Sudanese medicinal plants with special 7 emphasis on NTDs 2.2 Leishmaniasis 8 2.2.1 Leishmanial chemotherapy 12 2.2.2 Medicinal plants reported to exhibited antileishmanial activity 13

i 2.2.3 Alkaloids from medicinal plants reported to exhibited 27 antileishmanial activity 2.3 Plants investigated in the present study 88 2.3.1 Argemone mexicana L. (Papaveraceae) 88 2.3.1.1 The botanical description of A. mexicana L. 88 2.3.1.2 Ethnopharmacological importance of A. mexicana L. 50 2.3.1.3 Phytochemistry of A. mexicana L. 53 2.3.1.4 Biosynthesis of benzophenanthridine alkaloids 58 2.3.1.5 Pharmacological activity of protoberberine and protopine 66 alkaloids 2.3.2 Nauclea latifolia Smith. (Rubiaceae) 63 2.3.2.1 The botanical description of N. latifolia Smith. 63 2.3.2.2 Ethnopharmacological importance of N. latifolia Smith. 63 2.3.2.3 Phytochemistry of N. latifolia Smith. 66 2.3.2.4 Biosynthesis of monoterpene indole alkaloid Strictosamide 44 66 2.3.2.5 Pharmacological activity of β-carboline alkaloids 68 3. MATERIALS AND METHODS 07 3.1 General experimental conditions 76 3.1.1 Organic solvents, glassware, chemicals and reagents 76 3.1.2 Spectroscopic techniques 76 3.1.3 Chromatography 71 3.1.4 Detection method on TLC by spraying reagents 72 3.2 Plant Materials 72 3.2.1 Extraction and fractionation of plant materials 72

ii 3.3 Phytochemical screening 73 3.3.1 Isolation of compounds from Argemone mexicana L. 73 3.3.1.1 Isolation of compounds from petroleum ether fraction 73 3.3.1.2 Isolation of compounds from ethyl acetate fraction 74 3.3.2 Isolation of compounds from Nauclea latifolia Smith. 76 3.3.2.1 Isolation of compounds from petroleum ether fraction 76 3.3.2.2 Isolation of compounds from chloroform fraction 76 3.3.2.3 Isolation of compounds from ethyl acetate fraction 78 3.4 BIOLOGICAL ASSAYS 08 3.4.1 Materials 78 3.4.2 Antileishmanial assay 77 3.4.2.1 Maintenance of Parasites 79 3.4.2.1.1 Preparation of Biphasic medium (NNN medium) 79 3.4.2.1.2 Preparation of complete RPMI 1640 medium 79 3.4.2.1.3 Subculture of parasites 86 3.4.2.2 Preparation of sample 80 3.4.2.3 In vitro assay by colorimetric method 80 3.4.2.3.1 In vitro assay against extracellular promastigote 80 3.4.2.4 In vitro assay by macrophage method 81 3.4.2.4.1 Procedure for harvesting macrophages from the mice 81 3.4.2.4.2 In vitro assay against intracellular amastigotes 81

iii 3.4.3 Cytotoxicity assay 82 3.4.3.1 Cell-line 82 3.4.3.2 Method 82 4. RESULTS AND DISCUSSION 84 4.1 Secondary metabolites isolated from Argemone mexicana L. 84 4.1.1 Characterization of AM/1 as Sitost-4-en-3-one (β-Sitostenone) 84 113 4.1.2 Characterization of AM/2 as norchelerythrine 85 87 4.1.3 Characterization of AM/3 as berberine 10 87 4.2 Secondary metabolites isolated from Nauclea latifolia Smith. 71 4.2.1 Characterization of NL/1 as β-Sitosterol 114 71 4.2.2 Characterization of NL/2 as naucleficine 115 74 4.2.3 Characterization of NL/3 as strictosamide 44 78 4. 3Antileishmanial and cytotoxicity assays 162 4.4 Docking of the isolated compounds against leishmania enzymes 107 5. CONCLUSIONS AND RECOMMENDATIONS 110 5.1 Conclusions 11 0 5.2 Recommendations 11 2 6. REFERENCES 114

iv ACKNOWLEDGEMENTS

I would like to express my special appreciation and thanks to my supervisor Prof. Dr. Sami Ahmed Khalid. He has been a tremendous mentor for me. I would like to thank him for his precious and interesting suggestions, continuous support, patience, inspiration and immense knowledge. His guidance and encouragement helped me enormously in conducting my research and the writing up of this thesis.

I am deeply indebted to Prof. Dr. Atta ur Rahman of H.E.J, International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan, for his comments and valuable suggestions. I would like to extend my thanks to Prof. Dr. Mohammed Igbal Choudhary for his kind guidance and his enjoyable lectures in NMR spectroscopy and other topics. I would like to thank both of them for providing me the access to the laboratory and research facilities. Without their precious support it wouldn’t have been possible to conduct significant part of this research.

Sincere thanks and appreciation to Prof. Dr. Souad Abd Elaziz for her prayers, continuous encouragement and follow up and for taking over all my responsibilities at the University of Science and Technology during conducting my research in Pakistan. I am so grateful and obligated to the ex- dean of the Graduate College, University of Khartoum, Prof. Dr. Mohammed Ahmed Abu Alnour for his support and understanding of all circumstances and health conditions I went through during this research.

v My sincere thanks are due to Mr. Fouad Abd Aljalil, Faculty of Pharmacy, University of Khartoum, for his technical assistance in the preparation of the extracts.

I am deeply indebted to Dr. Farzana Navid for her kind support and being a caring and loving sister as well as a genuine friend. Special thanks to my fellow lab mates at H.E.J, ICCBS, for the stimulating discussions and the great fun we have had during our works. In particular, I am so grateful to the PhD fellow Mujeeb ur Rahman, Dr. Shabbir Husein, PhD fellow Ismaeel and Dr. Adighari. Thanks also to all technical staff of NMR, MS, HPLC laboratories and IT staff at ICCBS for their unlimited technical support during my stay in ICCBS.

I would like to extend my thanks to Dr. Sammer Yousuf, Miss. Samreen Khan and Miss. Nida Ghouri for performing the antileishmanial assay. I am also obliged to Dr. Omer Mohammed Elhaj for performing the cytotoxicity assays.

I do sincerely appreciate the support of Dr. Asaad Khalid and Dr. Mohammed Ahmed Mesaik for facilitating my mission at the ICCBS.

I would like to express my sincere thanks and gratitude to Prof. Dr. Moawia Mukhtar, and his lab. team, Institute of Endemic Diseases, University of Khartoum, for hosting me, providing me the access to the laboratory, and teaching me how to culture the leishmania parasite and macrophage cells.

Great thanks and appreciation to University of Khartoum for offering me the scholarship and the opportunity to achieve my post graduate studies.

vi ABBREVIATIONS

NTDs: Neglected Tropical Diseases CL: Cutaneous leishmaniasis MCL: Mucocutaneous leishmaniasis VL: Visceral leishmaniasis TDR: The special program for research and training in tropical diseases WHO: World health organization GDP: Gross domestic product HIV: Human immunodeficiency virus HAART: Highly active antiretroviral therapy DNDi: Drug for neglected diseases initiative DCM: Dichloromethane Pro: Promastigote Ams: Amastigote N. R.: Not reported DOPA: 3,4-dihydroxyphenylalanine TDC: Tryptophan decarboxylase G10H: Geraniol 10 hydroxylase SLS: Secologanin synthase STR: Strictosidine synthase SAP: Secreted aspartic protease MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (dye) DMEM: Dulbecco's Modified Eagle's Medium

vii MEM: Minimum Essential Medium eagle DMSO: Dimethyl sulfoxide EDTA: Ethylenediaminetetraacetic acid HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (buffer) RPMI 1640: Roswell Park Memorial Institute medium PBS: Phosphate buffered saline FBS: Fetal bovine serum FCS: Fetal calf serum NNN medium: Novy-MacNeal-Nicolle medium rpm: Revolutions per minute PI: Percentage inhibition

IC50: Inhibition concentration SD: Standard deviation

viii ABSTRACT

Introduction: Leishmaniasis a group of clinical diseases affecting millions of the world populations in 88 countries. Accordingly, this disease is considered as one of the Neglected Tropical Diseases (NTDs) besides malaria, sleeping sickness, mycetoma and other fifteen NTDs. Chemotherapy for leishmaniasis is still deficient and there is an urgent need to discover novel antileishmanial agents, hence most of the currently available drugs have serious limitations such as long-term administration, unaffordable cost, toxicity, and developing resistance by these parasites.

The objectives of this study is to isolate and elucidate the chemical structures of new anti-leishmanial secondary metabolites against both the promastigotes and amastigotes stages with special reference to alkaloids occurring in Argemone mexicana L. and Nauclea latifolia Smith, which are commonly growing plants in Sudan.

Methods: The extraction of plant materials was followed by bioactivity- guided fractionation using the promastigotes of L. donovani, L. major and L. tropica of the crude ethanolic extracts of A. mexicana and N. latifolia and their respective organic fractions as well as the water residue by colorimetric method. Isolation of pure compounds was achieved by high-performance liquid chromatography (HPLC). The isolated compounds were subjected to one- and two-dimensional Nuclear Magnetic Resonance (NMR) with special emphasis on 1D-NMR (1H, 13C, DEPT), 2D-NMR (COSY, HSQC, HMBC, NOESY) as well as UV, and mass spectrometry.

Results: Bioactivity-guided fractionation revealed a remarkable difference in susceptibility of the three leishmania species. The crude ethanolic extract of A. mexicana exhibited the most prominent activity against the promastigotes of L. donovani (IC50 11.39 μg/mL) followed by the water residue (23.93 μg/mL) which is indicative of the hydrophilicity of the bioactive compounds.

ix The crude ethanolic extract and water residue of Nauclea latifolia exhibited weak activity against the promastigotes of L. donovani with IC50 of 46.86 and 30.45 μg/mL, respectively.

Among the purified isolated alkaloids of Nauclea latifolia, strictosamide inhibited the proamastigotes of both L. major and L. tropica with IC50 of 77.67±1.11 and 80.40±3.00 μg/mL, respectively. The structurally related β- carboline alkaloid, naucleficine, however, showed no inhibition. Similarly, the isoquinoline alkaloid, norchelerythrine, isolated from A. mexicana, exhibited no leishmanicidal activity against L. major and L. tropica.

Attempting computational chemistry by docking of the isolated compounds against 14 leishmanicidal targets revealed that strictosamide has a relatively strong binding to certain protein targets such as phosphodiesterase B1 (LmajPDEB1) (PDB: 2R8Q) and Uridine 5'-monophosphate synthase (LdUMPS) (PDB: 3QW4) among all the isolated alkaloids.

Conclusion: The aerial part of Argemone mexicana yielded two benzophenanthridine alkaloids, norchelerythrine and berberine besides the tetracyclic terpenes, sitost-4-en-3-one (β-sitostenone. The extract of the root bark of Nauclea latifolia yielded the two β-carboline alkaloids, naucleficine and strictosamide besides the ubiquitous β-sitosterol.

Further research is required to reveal the possible mechanism of action of the isolated compounds.

x خالصة البحث

المقدمة: الليشمانيا هي مجموعة امراض سريرية تهدد ماليين من سكان العالم في حوالي 88 قطر. لذلك يعتبر هذا المرض من االمراض االستوائية المهملة بجانب المالريا، مرض النوم، المايسوتوما وغيرها. االدوية المستخدمة لعالج الليشمانيا لها عدة اثار جانبية كفترة العالج الطويلة، غالء االدوية ، السمية وكذلك مقاومتها من قبل الطفيل، مما يدعو للحاجة الماسة الكتشاف عقار جديد.

الهدف من هذه الدراسة عزل وتحديد البنية الكيميائية لمضادات ليشمانيا جديدة ضد الطور السوطي الخرجي والطور الخلوي الداخلي للطفيل. مع التركيز بشكل خاص علي القلويدات الموجودة في نباتي االرجيمون والكرمدودة التي تنمو في السودان.

الطريقة: استخالص اجزاء النبات متبوعة بفحص النشاط الحيوي للمستخلصات ضد الطور الخارجي السوطي لالنواع الثالثة من الليشمانيا للمستخلصات االيثانولية للخام النباتي لالرجيمون والكرمدودة واجزاء كل منها في االيثر البترولي ، الكلوروفورم، خالت االيثيل والماء بالطريقة اللونية. استخدمت تقنيتي كروماتوغرافيا العمود وكروماتوغرافيا الطبقة الرقيقة التحضيرية عالية االداء في عزل المركبات النشطة حيويا. تم توضيح هيكل المركبات المعزولة باستخدام طرق طيفية حديثة مختلفة: الرنين المغناطيسي، األشعة فوق البنفسجية ومطياف الكتلة.

النتائج: دلت نتائج فحص النشاط الحيوي علي فروقات ملحوظة في استجابة االنواع الثالثة من الليشمانيا ضد المستخلصات. اظهر المستخلص االيثانولي الخام والمائي لنبات االرجيمون النشاط االبرز ضد الطورالسوطي الخارجي للشمانيا donovani وقد كان التركيز المثبط )%56( يعادل 11.37 ميكروجرام/مل و23.73 ميكروجرام/مل علي التوالي. وهذا يدل علي ان المركبات النشطة بيولوجيا تركزت في المحلول المائي. كما اظهر المستخلص االيثانولي الخام والمائي لنبات الكرمدودة نشاطا ضعيفا ضد الطور السوطي الخارجي للشمانيا donovani وكانت التراكيز المثبطة ) %56( 86.86 و 36.85 ميكروجرام علي التوالي.

من بين القلويدات المعزولة نقية من الكرمدودة، strictosamide وقد قام بتثبيط الطور السوطي الخارجي لكل من اللشمانيا major وtropica وكانت قيم التراكيز المثبطة 77.67 و 86.86 ميكروجرام/مل، على التوالى. غير ان القلويد بيتا- كاربولين ، naucleficine، لم يظهر أى نشاط مضاد للشمانيا. وبالمثل فان قلويد آيسوكينولين، norchelerythrine المعزول من نبات االرجيمون، لم يظهر أى نشاط ضد الليشمانيا major و tropic.

أظهرت محاولة االلتحام الجزيئى للمركبات المعزولة ضد أربعة عشر من انزيمات الليشمانيا المستهدفة عن وجود عالقة ربط قوية ل strictosamide تجاه phosphodiesterase B و Uridine 5'-monophosphate synthase من بين كل القلويدات المعزولة.

الخالصة: تم بنجاح عزل اربعة قلويدات واثنان من التربينات رباعية الحلقة وقد تم تحديد البنية التركيبية باستخدام طرق طيفية حديثة. المركبات المعزولة من نبات االرجيمون كانت قلويدات ايسوكينولية، berberine,norchelerythrine باالضافة الي التربين رباعي الحلقة β-sitostenone . بينما مستخلص قلف الجذور لنبات الكرمدودة اعطي اثنان من القلويدات بيتاكاربولين، naucleficine و strictosamide باالضافة الي التربين رباعي الحلقة β-sitosterol.

المزيد من البحث لتجربة المركبات المعزولة داخل الخلية لمعرفة ميكانيكية عملها.

xi LIST OF FIGURES

Title of figure Page No Figure 1. Life cycle of leishmania parasite 16

Figure 2. Chemical structures of reported antileishmanial alkaloids 86

Figure 3. The main isoquinoline alkaloids of Argemone mexicana 58

Figure 4. Biosynthesis of (+)-Reticuline 92, a precursor of several 56 isoquinoline alkaloids

Figure 5. Formation of 9,10- or 10,11- substituted intermediates from 57 (+)-Reticuline 92 in the biosynthesis of protoberberine alkaloids

Figure 6. Biosynthesis of the benzophenanthridine alkaloids 57 Chelidonine 96 and Sanguinarine 88 from the common precursor (+)- Reticuline 92

Figure 7. Biosynthesis pathway of monoterpene indole alkaloid 67 Strictosamide 44

Figure 8. Chemical structures of indole alkaloids from Nauclea sp that 67 exhibited many biological activities

Figure 9. Selected COSY and HMBC Correlations of naucleficine 115 76

Figure 10. Selected HMBC Correlations of strictosamide 44 160

Figure 11. Interactions of β-Sitosterol and LmajHSP90. 109

Figure 12. Interactions of β-Sitosterol and LmajPDEB1. 109

xii LIST OF TABLES

Title of table Page No Table 1. Plants reported as antileishmanial 18

Table 2. Reported antileishmanial alkaloids 28

Table 3.1HNMR and 13CNMR spectral data for compound AM/1113 86

Table 4.1HNMR and 13CNMR spectral data for compound AM/2 85 88

Table 5.1HNMR and 13CNMR spectral data for compound AM/3 10 70

Table 6. 1HNMR and 13CNMR spectral data for compound NL/1 114 73

Table 7.1HNMR and 13CNMR spectral data for compound NL/2 115 77

Table 8. 1HNMR and 13CNMR spectral data for compound NL/3 44 161

Table 9. IC50 of the two plants extracts/fractions against the 163 promastigotes of L. donovani, L.major and L.tropica compared with the IC50 of the standard drugs Amphotericin B and Pentamidine by in vitro method (colorimetric)

Table 10. IC50 of the extracts/fractions against the amastigotes of L. 166 major and L. tropica compared with the IC50 of the standard drug, sodium stibogloconate (Pentostam®) (13.47±0.17 μg/mL) by macrophages method.

Table 11. IC50 of the pure compounds isolated from the two plants 167 against Leishmania major and Leishmania tropica compared with the IC50 of the standard drugs Amphotericin B (0.29±0.05 μg/mL) and Pentamidine (5.09±0.09 μg/mL) by in vitro method (colorimetric)

Table 12. Docking Binding free energies of the top ranked compounds 168 with leishmania enzymes

xiii 1. INTRODUCTION

1.1 Background information Protozoan parasites are among the most common chronic infections that occur primarily in rural and poor urban areas of tropical and subtropical regions around the world. They are responsible for a large number of severe and widespread diseases including malaria, leishmaniasis, Chagas’ disease (American trypanosomiasis), and sleeping sickness (African trypanosomiasis). These diseases belong to the group of Neglected Tropical Diseases (NTDs), since they are strongly linked with poverty and there is a lack of commercial markets for potential drugs. These diseases affect individuals throughout their lives, causing a high degree of morbidity and physical disability and, in certain cases, gross disfigurement. Patients can face social stigmatization and abuse as a result of contracting these diseases. The disability and the poverty associated with these diseases constitute large burdens on the health and economic development of low-income and middle-income countries in Africa, Asia, and the Americas. Overall global prevalence is approximately 550 million cases, and close to 2.7 billion people living in endemic areas are at risk of contracting any of these diseases. In total, there are about 280 million new cases each year causing important health and socio-economic problems where these diseases are endemic. Chemotherapy remains one of the key measures used to control the intolerable burden of protozoan parasitic and other tropical diseases, but most of the available drugs are no longer effective due to drug resistance. Moreover, some of those that are still effective suffer from problems associated with toxicity, compliance and high cost, resulting in an urgent need for new drugs (Osorio et al., 2008).

1.2 Problem statement Leishmaniasis a group of clinical diseases suffered by millions around the world, and affecting 88 countries in Africa, Asia, Europe and America, 72 of which are developing countries and 13 are among the least developed. The spectrum of the diseases divided into three major syndromes: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL). Annual incidences estimated at 1.5 million cases of the cutaneous forms (CL and MCL) and 500,000 cases of the visceral form (VL), resulting in approximately 51,000 deaths per year. Overall prevalence is 12 million people and the population at risk is 350 million. However, this estimated global burden of disease is believed to be inaccurate partly due to the passive case detection data used to estimate the disease prevalence in many endemic countries. Apparently, for each symptomatic case, there are estimated to be 10 asymptomatic infections. The total burden of Disability Adjusted Life Years (DALY) is 2.09 million, with 840,000 for women and 1.25 million for men. The Special Program for Research and Training in Tropical Diseases (TDR) has classified leishmaniasis as a group of emerging or uncontrolled diseases (Osorio et al., 2008).

Leishmaniasis is produced by at least 20 species of the protozoan Leishmania. Chemotherapy for leishmaniasis is still deficient. Most of the drugs have one or more limitations such as long-term administration, the route of administration is non orally, unaffordable cost and toxicity or even worse, inefficacy due to development of resistance in the parasite. Although these drugs constitute the main antileishmanial chemotherapy and have been used for over 50 years, information about their chemistry or precise mode of action and the identity of the biologically active components are uncertain (Osorio et

2 al., 2008). Therefore, there is an urgent need for new medicine , which are safe, efficient, effective, cheap, easy to administer, and for new lead compounds with novel mechanisms of action.

1.3 Aim and objectives The main aim of the present work is to investigate the anti-leishmanial activity of the secondary metabolites of two plants grown in Sudan, namely the aerial parts of Argemone mexicana L. and the root bark of Nauclea latifolia Smith. The specific objectives are: 1. To subject the plant extracts to bioactivity- guided fractionation to isolate the bioactive leishmanicidal compounds with special interest on alkaloids. 2. To elucidate the structures of the isolated compounds using modern spectroscopic methods, 3. To assess the anti-leishmanial activity against both the promastigotes and amastigotes.

1.4 Justifications The search for new and effective agents against leishmania and other diseases has led to increase interest in existing information about the remedies of these diseases from natural sources principally the plants. Efforts have been devoted and directed by many researchers to the isolation and characterization of the biological active principle of the plants. Natural products literature provides a growing research on plant derived antileishmanial agents and several natural products so far have been discovered with excellent activity against leishmania parasites. Alkaloids are the most potent therapeutic compounds of natural origin, they are an important class of secondary metabolites that occur in plants and also in certain higher animals and marine invertebrates (Okwu and Uchenna, 2009; Mishra et al., 2011). Alkaloids containing plants and their biosynthesized alkaloids have a remarkable potential to provide pharmaceutical and biological agents contributing to the development of future antiparasitic drugs (Osorio et al., 2008). It has been reported that alkaloids exhibiting significant anti-leishmanial activities (Mishra et al., 2011). Khalid (2012) reviewed the bioactivity of some isolated antiparasitic compounds from Sudanese medicinal plants against NTDs and still several compounds waiting to be discovered.

2. LITERATURE REVIEW

2.1 Neglected Tropical Diseases (NTDs) caused by protozoan parasites NTDs are a group of chronic disabling infections affecting more than a billion people worldwide, mainly in Africa and mostly those living in remote rural areas, urban slums or conflict zones. People suffering from NTDs are predominantly afflicted by poverty and they constitute an unattractive market to private-sector research and development (R&D) investment. These diseases not only affect our health but also represent a vicious cycle of socioeconomic events which reinforce and feedback on each other, leading towards inescapable poverty of a sizable number of the population (Khalid, 2012).

The global threat posed by NTDs compelled the World Health Organization (WHO) to release its first report on the situation five years ago (WHO, 2010). The number of NTDs enumerated in this report reached 17, including three soil-transmitted helminthiases. Although malaria is not mentioned among the NTDs in this report, possibly due to a resurgence at the global level of increased funding by various international bodies (e.g. Roll Back Malaria Initiative, RBM) and philanthropic organizations (e.g. The Bill & Melinda Gates Foundation) during the last decade, malaria still remains one of the NTDs; this is mainly due to the fact that the death toll from this disease in Africa constitutes over 90% of the total number of global death and due to its direct/indirect economic impact which results in an estimated US$ 12 billion annual income loss in sub-Saharan Africa, which translates to a 1.3% annual loss in gross domestic product (GDP) in this malaria endemic African region (Berger et al., 2010). Recent publications still include malaria together with

5 other NTDs such as leishmaniasis, trypanosomiasis, schistosomiasis, onchocerciasis, lymphatic filariasis and dengue fever (Chatelaine and Loset, 2011). Each year, there are between 300 and 500 million clinical cases of malaria; estimates of the number who die from the disease range between 1.0 and 2.5 million annually. A disproportionate number of deaths from malaria occur among the poor, and about half of those who die are children and pregnant women. Currently, there is no vaccine for malaria, and the parasite has already developed resistance against almost all the currently available antimalarials. Unfortunately, new reports indicate the emergence of resistance against artemisinin derivatives as well (Khalid, 2012).

2.1.1 Contribution of medicinal plants to NTDs The fascination of natural products, especially from plants with known medicinal properties, goes back to ancient times. The utilization of plants for and as medicines is old as civilization. Different traditional medicinal systems have been for a long time, making use of plants as effective medicines to treat many harmful diseases (Jyothirmayi and Prasad, 2012). At the beginning of the 19th century the discovery of pure compounds as active constituents from plants were described, and the art of exploiting natural products has become part of the molecular sciences (Kayser et al., 2002). Impressively within the last 25 years a large fraction of drugs recently approved have their origin in nature. Even more all approved anti-tumor drugs since the 1940s, of which more than 50% are of natural origin. However, many drugs used against infectious diseases are natural products or derivatives. Many reports estimated that approximately 80% of the population in developing countries still relies on traditional medicine for their primarily health care (Karou et al., 2011). About one quarter of the present prescription drugs dispensed by community

6 pharmacies in the United States contain at least one active principle originally derived from plant materials (Agyare et al., 2006). A milestone in the history of a remedy used successfully since ancient days against a protozoan infection would be the natural alkaloid quinine from Cinchona succiruba (Rubiaceae) and its subsequent development as drug which is still used successfully and increasingly against malaria today. Discovering untapped natural sources of novel antiprotozoal compounds from nature remains a major challenge and a source of novelty in the area of combinatorial chemistry and genomics (Schmidt et al, 2012a; Kayser et al., 2002). There are more than 2,70,000 higher plants existing in this planet, but so far less than 10% of recorded flora has been explored phytochemically as well as clinical evaluation for various biological activities (Reddy, 2010).

2.1.2 Ethnopharmacology of Sudanese medicinal plants with special emphasis on NTDs Sudan as an African tropical country is confronted with many parasitic and infectious diseases prevailing on this continent. Similar to other developing countries, traditional medical practices play an important role in Sudan. Herbal drugs are of major importance in Sudanese folk medicine. This was documented during comprehensive ethnobotanical investigations (El Kamali and Khalid, 1996; El Ghazali et al., 1994, 1997; El Kamali and El Khalifa, 1997, 1999; El Tahir et al., 1998, 1999a, 1999b; Ali et al., 2002; Khalid et al., 2012; Khalid, 2012).

A number of systematic attempts have been made to verify the claimed antiparasitic uses of Sudanese plants and to detect and/or isolate their bioactive agents (Khalid et al., 1986, 1989, 1998; El Kamali and El Khalifa, 1997; El

Tahir et al., 1998, 1999a,b). Among other natural products, the limonoid 2,6- dihydroxyfissinolide (Khalid et al., 1998) from Khaya senegalensis and the tetranortriterpenoid gedunin from Azadirachta indica (Khalid et al., 1989) were found to exhibit good antimalarial activity. The bioactivity of some isolated bioactive compounds from Sudanese medicinal plants against NTDs has been reviewed (Khalid, 2012). Inhibitory effects of Sudanese plants on HIV-1 replication and HIV-1 protease were also investigated (Hussein et al., 1999; Ali et al., 2002), as well as their declared antibacterial activities (Elegami et al., 2001). Ali et al., 2002 verified the ethnopharmacological uses of some selected Sudanese plants commonly employed to treat malaria and similar tropical diseases. The ethnobotanical information thus served as basis for the selection of the plants studied. They investigated the activities of diverse plant extracts against Plasmodium falciparum, Trypanosoma spp. and Leishmania donovani, their inhibitory activity towards HIV-1 reverse transcriptase and p56lck tyrosine kinase was also assessed.

2.2 Leishmaniasis Leishmaniasis represents a set of severe human diseases. These diseases are endemic in Africa, America, Indian sub-continent and in sub-tropical southwest Asia as well as the Mediterranean. Leishmania species are obligatory intracellular protozoan parasites exist in macrophages of their mammalian hosts. The extracellular stage, the promastigote, is transmitted into subcutaneous tissue in the human host (or animal) during the bite of an infected sandfly vector belong to the genus Phlebbotomus (Old World) and Lutzomyia (New World), both subfamily Phlebotominae, family Psychodidae. The promastigote is phagocytosed by a mononuclear phagocyte, after which it

8 converts into the obligatory intracellular form, the amastigote (Schmidt et al., 2012a).

The leishmania promastigotes are transmitted by sandfly to vertebrate hosts e.g. canines, marsupials, edentates and rodents. Once inside the blood stream of reservoirs for the disease, promastigotes are phagocytosed by the mononuclear phagocytic cells and are transformed to amastigotes that multiply by means of binary fission. On lyse of host cell, the free parasites spread to new cells and tissues of different organs including the spleen, liver and bone marrow. Amastigotes in the blood as well as in the monocytes are ingested during a blood meal by female sandfly. Once ingested, the amastigotes migrate to the midgut of the sandfly and transform into the promastigotes. After a period of four to five days, promastigotes move forward to the oesophagus reach to salivary glands of the sandfly. Infected sandfly during the second blood meal regurgitates the infectious promastigotes from its pharynx into the blood stream of the host vertebrates and the life cycle is repeated (Mishra et al., 2011).

Figure 1. Life cycle of leishmania parasite

Leishmania parasites were independently described by William Leishman and Charles Donovan in 1903 (Ross, 1903), but were previously observed by David D. Cuningham in 1885 and Peter Borovsky in 1989. The genus leishmania was proposed by James Wright in 1903 (Monzote, 2009).

There are about 20 known species of leishmania that have been associated with various disease forms in man. The severity of the disease ranges from the disfiguring cutaneous type in which nodules form at the site of infection, leaving scars upon healing, to the lethal visceral leishmaniasis. Some forms of the disease are anthroponotic (transmitted between humans), while others are zoonotic (involving an animal reservoir). Leishmaniasis is classified into three groups: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL) on the basis of their clinical symptoms. There

10 are two general forms, visceral leishmaniasis (VL) caused by two species of leishmania, Leishmania donovani and Leishmania infantum (chagasi) and tegumentary leishmaniasis (TL), as cutaneous leishmaniasis (CL) and disfiguring mucocutaneous leishmaniasis (MCL). TL caused by several dermatropic species of leishmania eg.; L. major, L. tropica, L. aethiopica, L. braziliensis, L. panamensis, L. amazonensis and L. mexicana. Leishmaniasis affects around 10 million people worldwide, with an annual incidence of approximately two million new cases, 350 million are living at the risk to be infected, and causes approximately 50,000 death cases annually. VL causes 500,000 new cases annually globally, 90% of the new cases occurring in just five countries, India, Bangladesh, Brazil, Nepal and Sudan. Tegumentary forms of the disease affects 1,500,000 people. Although not a killing disease as is VL, disfigurement, disability and social and psychological stigmas are all severe consequences of TL (Schmidt et al., 2012a; Osorio et al., 2008).

The leishmanial infection development depends on the parasite properties, such as infectivity, pathogenicity and virulence, beside the heterogeneity of the host regulatory response resulting in different clinical manifestations. There are a rising number of reports of leishmania/human immunodeficiency virus (HIV) co-infections across the world. leishmania/HIV co-infections has been globally controlled in Southern Europe since 1997 by highly active anti retroviral therapy (HAART), but it appears to be an increasing problem in other countries such as Ethiopia, Sudan, Brazil or India where both infections are becoming more and more prevalent. The situation is particularly alarming in southern Europe, where 50-75% of adult VL cases are HIV positive and among the 45 million people infected by HIV worldwide, an estimated one- third lives in the zones of endemic leishmania infections. To date, the greatest

11 prevalence of leishmania/HIV co-infections has been in the Mediterranean basin. Among more than 2.000 cases notified to the WHO, 90% of them belong to Spain, Italy, France and Portugal (Mishra et al., 2011).

2.2.1 Leishmanial chemotherapy The drugs recommended for the treatment of leishmaniasis are pentavalent antimonials (SbV compounds: sodium stibogloconate, Pentostam® and meglumine antimoniate, Glucantime®), these drugs since 1950s are problematic due to toxicity/side effects, and teratogenicity, as well as increasing resistance. Pentamidine, Pentacarinat® is used as alternative drug in cases resistant to antimonials, but severe side effects arising in the required long term treatment are reported (Schmidt et al., 2012a).

Amphotericin B (AmB), the antifungal polyene antibiotic is used as a second- line antileishmanial drug in developing countries and as a first-line drug in the industrialized countries. Miltefosine, Impavido®, the oral antineoplastic agent, an alkylphosphocholine, is available on the market for the treatment of leishmaniasis since 2002 (Schmidt et al., 2012a). In a combination with SbV compounds, interferon-γ can be administrated especially in immunosuppressed patients; it stimulates the phagocytic activity of macrophages in order to kill amastigotes that reside in them. However this treatment is cost intensive and due to lack of clinical trials it remains limited today (Schmidt et al., 2012a).

The triazole, the antimycotic posaconazole, currently under investigation by the Drug for Neglected Diseases initiative (DNDi) against Chagas disease, was found to be active against Leishmania amasonensis and Leishmania donovani in rodent models. Oxaboroles and nitroimidazole are compounds that investigated further by the DNDi showed in vivo efficacy and are currently in pharmacokinetic trials (Schmidt et al., 2012a).

Tafenoquine, the antimalarial compound was reported to be highly active in vitro and in a rodent model against leishmania. The orally administrated antimalarial, artemisinin was effectively reduced the parasite burden in BALB/c model of VL (Schmidt et al., 2012a). Although the causative agents of leishmaniasis have been known and studied since 1903 (Ross, 1903), to date an effective cure for the disease does not exist.

2.2.2 Medicinal plants reported to exhibited antileishmanial activity A bewildering array of secondary metabolites has been reported to exhibit remarkable antileishmanial activity. Table 1 compiles those plants reported to have antileishmanial activity.

Table 1. Plants reported as antileishmanial Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Acanthaceae N. R. In vitro (Pro.) L. donovani Carvalho and Ferreira, Acanthus illicifolius (Leaves) 2001 Andrographis paniculata (Roots) N. R. N. R. L. infantum Schmidt et al., 2012a Agavaceae Ethanol N. R. L. amazonensis Rocha et al., 2005 Yucca filamentosa L. (c) Anacardiaceae N. R. In vitro (axenicAms.) L. donovani Schmidt et al., 2012a Campanosperma panamense (N. R.) Annonaceae N. R. In vitro & in vivo L. amazonensis Rocha et al., 2005 Annona haematantha Miq (N. R.) (N. R.) Annona glauca Thonn (Seeds) DCM In vitro (Pro.) L. braziliensis. Carvalho and Ferreira, L. donovani. 2001 L. amazonensis Schmidt et al., 2012a Annona muricata L (Pericarp) Ethyl acetate N. R. L. braziliensis Rocha et al., 2005 L. panamensis Annona muricata (Seeds) N. R. In vitro (Pro.&Ams.) L. chagasi Schmidt et al., 2012a Annona senegalensis (Seeds) DCM N. R. L. donovani Schmidt et al., 2012a L. major Annona senegalensis (Seeds) Methanol & hexane In vitro (Pro.). L. major Schmidt et al., 2012a Annona aff. Spraguei Saff (Seeds) Chloroform N. R. L. braziliensis Rocha et al., 2005 L. panamensis L.infantum Annona squamosa (Leaves) N. R. In vitro (Pro.&Ams.) L. chagasi Schmidt et al., 2012a Duguetia furfuracea (Stem bark) N. R. In vitro (Pro.) L. braziliensis Schmidt et al., 2012a Friesodielsia obovata (N. R.) N. R. In vitro (axenicAms.) L. donovani Schmidt et al., 2012a Guatteria amplifolia (N. R.) N. R. N. R. L. panamensis Schmidt et al., 2012a L. mexicana Guatteria foliosa Benth (Stem bark) Alkaloid fraction N. R. L. amazonensis Rocha et al., 2005 L. braziliensis L. donovani Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Annonaceae Alkaloid fraction. N. R. L. amazonensis Rocha et al., 2005 Cardiopetalum calophyllum Schldl L. braziliensis (Leaves + Stem) L. donovani Duguetia spixiana Mart (Stem bark) Guatteria schoburgkiana Mart. (Leaves + stem bark) Oxandra espintana (Spruce) Baillon (Leaves + Stem bark)

Xylopia aromatica (Lam.) Mart (Leaves + Stem bark) Polyalthia longifolia var. pendula (N. R.) N. R In vitro & in vivo L. donovani Schmidt et al., 2012b (Ams.) Polyalthia macropoda king (Stem bark) N. R. In vitro (Pro.) L. donovani Carvalho and Ferreira, 2001 Rollinia emarginata Schdl. (Stem bark) DCM fraction of In vitro (Pro.) L. amazonensis Schmidt et al., 2012a methanol extract L. braziliensis L. donovaniz Unonopsis buchtienii R. E. Fries (N. R.) N. R. In vitro (Pro.) L. donovani Rocha et al., 2005 L. major Carvalho and Ferreira, 2001 Apiaceae N. R. In vitro (Pro.) L. major Schmidt et al., 2012a Ferula szowitsiana (N. R.) Apocynaceae N. R. In vitro (axenic Ams.) L. donovani Schmidt et al., 2012a Aspidosperma vargasii (Bark) Alstonia scholaris R.Br. (Stem) Ethanol N. R. L. donovani Rocha et al., 2005 Holarrhena curtisii King & Gamble Ethanol N. R. L. donovani Rocha et al., 2005 (Leaves) Mandevilla antennacea K. Schum. Ethanol N. R. L. amazonensis Rocha et al., 2005 (Leaves + stem) L. braziliensis Pentalinon andrieuxii (N. R.) N. R. N. R. Leishmania sp. Schmidt et al., 2012b Peschiera australis Miers. (Stem) Chloroform fraction In vitro (Pro.) & L. amazonensis Delorenzi et al., 2001 of Ethanol extract in vivo (Ams.) Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Apocynaceae Ethanol In vitro & in vivo L. amazonensis Rocha et al., 2005 Peschiera van. heurkii (Muell. Arg.) L. (Ams.) L. braziliensis Carvalho and Ferreira, Alloorge (Leaves + stem bark) 2001 Picralima nitida Th. &H. Dur. (Seeds) Chloroform N. R. L. donovani Rocha et al., 2005 Tabernaemontana obliqua (Miers) Leen Methanol N. R. L. amazonensis Rocha et al., 2005 wenb (Leaves) Araliaceae Ethanol In vitro (Pro.&Ams.) L. amazonensis Rocha et al., 2005 Hedera helix L. (Leaves) L. infantum Carvalho and Ferreira, L. tropica 2001 Cussonia zimmermannii (Root bark) Petroleum ether In vitro (axenic Ams.) L. donovani Schmidt et al., 2012a Oreopanax species (Leaves) Ethanol N. R. L. braziliensis Rocha et al., 2005 Asclepiadaceae Methanol N. R. L. major Rocha et al., 2005 Periploca graeca L. (twig) Gongronema latifolia Benth (Leaves) Methanol N. R. L. donovani Rocha et al.,2005 Asparagaceae N. R. In vitro (Pro.) L. major Schmidt et al., 2012a Asparagus africanus (Roots) Asteraceae Ethanol N. R. L. amazonensis Rocha et al., 2005 Acanthospermum hispidum DC. L. braziliensis (Entire plant) Achyrocline flaccida (Weinm.) DC. Ethanol N. R. L. braziliensis Rocha et al., 2005 (Entire plant) Ageratina pentlandiana (DC.) K. & R. Ethanol N. R. L. braziliensis Rocha et al., 2005 (Leaves) L. amazonensis Ageratum conyzoides (N. R.) N. R. N. R. L. donovani Schmidt et al., 2012a Artemisia herba-alba Asso. (c) Aqueus In vitro (N. R.) L. tropica Rocha et al., 2005 Baccharis retusa (N. R.) N. R. In vitro (Pro.) L. braziliensis Schmidt et al., 2012a L. amazonensis In vitro (Ams.) L. major L. chagasi Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Asteraceae Ethyl acetate N. R. L. braziliensis Rocha et al., 2005 Baccharis salicifolia (R. & P.) Pers. (Leaves) Calea uniflora (N. R.) N. R. In vitro (Pro.) L. major Schmidt et al., 2012a Chersodoma jodopappa (Sch. Bip.) Ethanol N. R. L. amazonensis Rocha et al., 2005 Cabrera (Leaves + Stem) L. braziliensis L. donovani Chromolaena hirsute (N. R.) N. R. In vitro (Pro.) L. amazonensis Schmidt et al., 2012a Cnicothamnus lorentzii Griseb Ethanol N. R. L. amazonensis. Rocha et al., 2005 (Leaves + Stem) L. braziliensis L. donovani Echinacea purpurea Moench (Entire Saponin N. R. Leishmania sp. Rocha et al., 2005 plant) Eupatorium perfoliatum (N. R.) N. R. In vitro (axenic Ams.) L. donovani Schmidt et al., 2012a

Inula montana L. (Aerial parts) Methanol N. R. L. infantum Rocha et al., 2005 Jasonia glutinosa DC. (Aerial parts) Acetone In vitro (Pro.) L. donovani Rocha et al., 2005 Schmidt et al., 2012a Lychnophora markgravii (N. R.) N. R. In vitro (Ams.) L. amazonensis Schmidt et al., 2012a Munnozia fournetii H. Robinson Ethanol N. R. L. amazonensis Rocha et al., 2005 (Leaves + Stem) L. braziliensis L. donovani Neurolaena lobata Ethanol N. R. L. mexicana Berger et al., 2001 R. Br. (Leaves) L. braziliensis Ophryosporus piquerioides (DC.) Benth Ethanol N. R. L. amazonensis Rocha et al., 2005 (Entire plant) L. braziliensis Perezia multiflora Less. (H. & B.) Less L. donovani (Leaves) Pterocaulon alopecuroideum (Lam.) DC. (Entire plant) Senecio clivicolus Wedd.(Leaves +Stem) Stevia yaconensis Hieron. (Entire plant)

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Asteraceae Chloroform In vitro (Ams.) L. aethiopica Carvalho and Ferreira, Vernonia amygdalina Delile (N. R.) 2001 Vernonia brachycalyx (Roots) N. R. In vitro (Pro.) L. major Schmidt et al., 2012a Carvalho and Ferreira, 2001 Vernonia squamulosa Hook. & Arn. Ethanol N. R. L. amazonensis Rocha et al., 2005 (Stem) L. braziliensis Werneria nubigena H.B. K. L. donovani (Leaves + Stem) Xanthium catharticum L. (Roots + Stem) Berberidaceae Alkaloid frction N. R. L. amazonensis Rocha et al., 2005 Berberis boliviana Lechl. (Bark + Stem) L. braziliensis Berberis bumeliaefolia Schum (Bark) L. donovani Berberis cf. laurina Epl. (Stem) Betulaceae N. R. In vitro (Pro.) L. major Schmidt et al., 2012b Betula species (N. R.) Berberis aff. Paucidentata Rusby Alkaloid frction. N. R. L. amazonensis Rocha et al., 2005 (Stem bark) L. braziliensis Bignoniaceae N. R. In vitro & in vivo L. amazonensis Rocha et al., 2005 Jacaranda copaia D. Don (Leaves) (Ams.) Tabebuia avellanedae (N. R.) N. R. In vitro (Pro.) L. braziliensis Schmidt et al., 2012a Kigelia pinnata DC. (Root bark) N. R. N. R. L. major Rocha et al., 2005 Bombacaceae Methanol N. R. L. panamensis Rocha et al., 2005 Huberodendron patinoi Cuatrec (Bark) Burseraceae N. R. N. R. L. donovani Schmidt et al., 2012b Boswellia serrata (N. R.) Protium amplum Cuatrec (Fruits) DCM N. R. L. amazonensis Rocha et al., 2005 L. braziliensis L. infantum Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Buxaceae N. R. In vitro (N. R.) L. major Schmidt et al., 2012a Sarcococca hookeriana (Whole air-dried plant) Calophyllaceae N. R. In vitro (Pro.& Ams.) L. amazonensis Schmidt et al., 2012a Calophyllum brasiliense (N. R.) Kielmeyera albopunctata (N. R.) N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012a Cannabaceae N. R. In vitro (Pro.). L. donovani Schmidt et al., 2012a Cannabis sativa (N. R.) Caparraceae N. R. N. R. L. major Rocha et al., 2005 Capparis spinosa L. (Branches) Chloranthaceae N. R. In vitro (axenic Ams.) L. amazonensis. Schmidt et al., 2012b Hedyosmum angustifolium (N. R.) L. infantum Celastraceae N. R. N. R. L. donovani Schmidt et al., 2012b Austroplenckia populnea (N. R.) Maytenus senegalensis (Lam.) Exell DCM In vitro (Pro.). L. major El Tahir et al., 1998 (Stem bark) Salacia madagascariensis (N. R.) N. R. N. R. L. donovani Schmidt et al., 2012b Cistaceae N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012b Cistus creticus (N. R.) Clusiaceae N. R. In vitro (Ams.) L. infantum Schmidt et al., 2012a Garcinia Livingstonei (N. R.) Garcinia lucida (Stem bark) DCM/ methanol In vitro (axenic Ams.) L. donovani Schmidt et al., 2012a (1 :1) Marila laxiflora Rusby (Leaves) DCM N. R. L. amazonensis Rocha et al., 2005 L. braziliensis Combretaceae N. R. In vitro (Pro.) L. amazonensis Schmidt et al., 2012b Combretum leprosum (N. R.) Crassulaceae N. R. In vitro (Pro.) L. tropica Schmidt et al., 2012b Aeonium lindleyi (N. R.) L. braziliensis Bryophyllum pinnatum Kurz (Leaves) Aqueus N. R. L. amazonensis Rocha et al., 2005 Kallanchoe pinnata (Kp) Bryophyllum Aqueus In vitro (Pro.) L. amazonensis Da Silva et al., 1995 pinnatum Kurz (synoname) (Leaves) Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Cupressaceae N. R.. In vitro (Pro.) L. donovani Schmidt et al., 2012b Juniperus procera (N. R.) Dilleniaceae Chloroform N. R. L. amazonensis Rocha et al., 2005 Doliocarpus dentatus Kubitzki (Stem) Ebenaceae N. R. In vitro (Pro.) & L. donovani Schmidt et al., 2012a Diospyros Montana (Bark) in vivo (Ams.) Hazra et al., 1995

Euphorbiaceae Alcohol (N. R) N. R. L. donovani Carvalho and Ferreira, Anthostema senegalense A. Juss 2001 (N. R.) Celaenodendron mexicanum (N.R.) N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012b Pera benensis Rusby Ethanol N. R. L. amazonensis Rocha et al., 2005 (Root bark + stem bark) L. braziliensis L. donovani Ricinus communis V. A. Moshkin N. R. N. R. L. major Rocha et al., 2005 (Branches) Illiciaceae N. R. In vitro (axenic Ams.) L. donovani Schmidt et al., 2012a Illicium floridanum (N. R.) Fabaceae N. R. In vitro (Pro.&Ams.) L. braziliensis Schmidt et al., 2012a Amburana cearensis (N. R.) L. donovani L. amazonensis Astragalus bicuspis (N. R.) N. Rp. In vitro (Pro.) L. major Schmidt et al., 2012b Caesalpinia echinata (N. R.) N. R. In vitro/ams. L. amazonensis Schmidt et al., 2012b Canavalia brasiliensis (Seeds) N. R. In vitro (Pro.) & L. amazonensis Barral-Netto et al., 1996 in vivo (Ams.) Crotalaria barbata R. Gach Ethanol N. R. L. donovani Rocha et al., 2005 (Entire plant) Desmodium gangeticum L. (Leaves) Methanol N. R. L. donovani Rocha et al., 2005 Glycyrrhiza sp. (N. R.) N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012a Lonchocarpus sp. (N. R.) N. R. In vitro (Pro.) L. braziliensis Schmidt et al., 2012a L. amazonensis L. donovani

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Fabaceae N. R. In vitro (Pro.) L. mexicana Schmidt et al., 2012a Lonchocarpus xuul (N. R.) Machaerium multiflorum (N. R.) N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012a Periandra mediterranea Taub. (Roots) Saponin fraction N. R. L. donovani Rocha et al., 2005 Prosopis glandulosa var. (Leaves) N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012a Psorothamnus polydenius (N. R.) N. R. In vitro (axenic Ams.) L. donovani Schmidt et al., 2012a Spartium junceum L. (Branches) N. R. N. R. L. major Rocha et al., 2005 Gentianaceae Ethanol In vivo (Ams.) L. donovani Rocha et al., 2005 Swertia chirata Buch. Ham.Ex Wall. Carvalho and Ferreira, (Entire plant) 2001 Geraniaceae Ethanol Invitro (Ams.) L. donovani Rocha et al., 2005 Pelargonium sidoides DC. (c) Schmidt et al., 2012a Lamiaceae N. R. In vitro (Pro.) L. major Schmidt et al., 2012a Perovskia abrotanoides (N. R.) Phlomis brunneogaleata (N. R.) N. R. N. R. L. donovani Schmidt et al., 2012a Ocimum sanctum (N. R.) N. R. In vitro (Pro.). L. major Schmidt et al., 2012a Lauraceae Ethyl acetate N. R. L. amazonensis Rocha et al., 2005 Aniba canelilla H. B. K. (Stem) L. braziliensis Aniba species (Stem) Ethanol N. R. L. amazonensis Rocha et al., 2005 L. braziliensis Ocotea duckei (N. R.) N. R. In vitro (Pro.) L. chagasi Schmidt et al., 2012a L. infantum Ocotea lancifolia (N. R.) Alkaloid crude In vitro (Pro.) L. amazonensis Schmidt et al., 2012a extract L. braziliensis L. donovani Liliaceae (c) In vivo (Ams.) L. major Rocha et al., 2005 Allium sativum L. (N. R.) Asparagus africanus Lam. (N. R.) N. R. In vitro (Pro.) L. major Carvalho and Ferreira, 2001 Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Liliaceae Methanol N. R. Leishmania sp. Carvalho and Ferreira, Dracaena arborea Hort. Angl. 2001 (Seed pulp) Dracaena. mannii Baker(Seed pulp) Leguminosae DCM In vitro (N. R.) L. amazonensis Bravo B. et al., 1999 Amburana cearensis A.C Smith. L braziliensis (Stem bark) L.donovani

Centrolobium sclerophyllum (Wood) Chloroform In vitro (Pro.) L. amazonensis Araujo et al., 1998 Maliaceae DCM N. R. L. amazonensis Rocha et al., 2005 Guarea polymera Little (Leaves) L. braziliensis Malpighiaceae N. R. In vitro (Ams.) L. amazonensis Schmidt et al., 2012b Lophanthera lactescens (N. R.) Malvaceae N. R. N. R. L. major Rocha et al., 2005 Malva nicaeensi All. (Branches) Melastomaceae Ethanol N. R. L. donovani Rocha et al., 2005 Tibouchina semidecandra Cogn. (Aerial parts) Meliaceae Methanol In vitro (Pro.) L. major El Tahir et al., 1998 Azadirachta indica A. Juss. (Stem bark) Guarea rhophalocarpa (N. R.) N. R. In vitro (Pro.&Ams.) L. donovani Schmidt et al., 2012a Khaya senegalensis A. Juss (N. R.) Alcohol (N. R) N. R. L. donovani Rocha et al., 2005 Khaya senegalensis (N. R.) N. R. In vitro (Pro.) L. major Schmidt et al., 2012b Menispermaceae Alkaloid fraction N. R. L. amazonensis Rocha et al., 2005 Abuta pahni Mart. (Stem) L. braziliensis Abuta rufescens Aublet (Bark) Alkaloid fraction N. R. L. amazonensis Rocha et al., 2005 Anomospermum bolivianum K ruk. & Alkaloid fraction N. R. L. amazonensis Rocha et al., 2005 Mold (Bark) L. braziliensis Stephania dinklagei (Aerial parts) N. R. In vitro (Pro.&Ams.) L. donovani Chan-Bacab and Peña- Rodríguez, 2001 Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Menispermaceae Ethanol N. R. L. donovani Rocha et al., 2005 Tinospora cordifolia (Wild) Hook.f. & Thoms (Stem) Moraceae Aqueus N. R. L. donovani Rocha et al., 2005 Dorstenia multiradiata Engl. (Leaves) Myristicaceae DCM N. R. L. amazonensis Rocha et al., 2005 Otoba novogranatensis Moldenke L.braziliensis (Leaves) L.infantum Otoba novogranatensis Moldenke Methanol N. R. L. amazonensis Rocha et al., 2005 (Leaves) L. braziliensis L. infantum Otoba parvifolia (mgf.) A. H. Gentry DCM N. R. L. amazonensis Rocha et al., 2005 (Bark) L. braziliensis Virola pavonis (Leaves) N. R. In vitro (Pro.&Ams.) L. donovani Barata et al., 2000 Virola surinamensis (Leaves) Myrsinaceae Ethanol N. R. L. braziliensis Rocha et al., 2005 Myrsine pellucida Spreng (Stem bark) Nepenthaceae N. R. In vitro (Pro.) & L. amazonensis Schmidt et al., 2012a Nepenthes thorelii (N. R.) in vivo (Ams.) L. donovani L. braziliensis L. mexicana. L.venezuelensis Olacaceae N. R. In vitro (N. R.) L. major Schmidt et al., 2012a Minquartia guianensis (Stem bark) Oleaceae N. R. In vitro & in vivo (N. L. donovani Rocha et al., 2005 Nyctanthes arbortristis L. (N. R.) R.) Carvalho and Ferreira 2001 Orobanchaceae N. R. N. R. L. donovani Schmidt et al., 2012a Melampyrum arvense (N. R.) Oxalidaceae N. R. In vitro (Pro.) L. amazonensis Schmidt et al., 2012a Oxalis erythrorhiza (N. R.) L. donovani Papaveraceae Alkaloid fraction. N. R. L. amazonensis Rocha et al., 2005 Bocconia integrifolia H & B L. braziliensis (Leaves + Stem bark) L. donovani

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Papaveraceae Alkaloid fraction N. R. L. amazonensis Rocha et al., 2005 Bocconia pearcei Hutch. (Leaves) L. braziliensis L. donovani Phrymaceae N. R. In vitro (axenic Ams.) L. donovani Schmidt et al., 2012a Mimulus bigelovii (N. R.) Phytolaccaceae Butanol N. R. L. enriettii Rocha et al., 2005 Phytolacca dodecandra L’Herit (Fruits) Piperaceae Ethanol N. R. L. amazonensis Rocha et al., 2005 Peperomia galioides H. B. & K. (Entire plant) Peperomia galioides H. B. & K Ethanol N. R. L. braziliensis Rocha et al., 2005 (Entire plant) Petroleum ether L. chagasi L. donovani Peperomia galioides H. B. & K. (N. R.) N. R. In vitro (Pro.) L. braziliensis Chan-Bacab and Peña- L. amazonensis Rodríguez, 2001 L. donovani Piper aduncum L. Petroleum ether In vitro (Pro.&Ams.) L. amazonensis Rocha et al., 2005 Schmidt et al., 2012a Piper elongatum (N. R.) N. R. In vitro (Pro.) L. braziliensis Schmidt et al., 2012a L. infantum L. tropica Piper nigrum (Fruits) N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012a L. amazonensis Piper rusbyi C. DC. (Entire plant) Ethyl acetate In vitro (Pro.) L. amazonensis Rocha et al., 2005 L. braziliensis L. donovani Schmidt et al., 2012a

In vivo (Ams.) L. amazonensis Piper sanguineispicum (Leaves) N. R. In vitro (axenic Ams.) L. amazonensis Schmidt et al., 2012a Plagiochilaceae N. R. In vitro (Ams.) L. amazonensis Schmidt et al., 2012b Plagiochila disticha (N. R.) Pleosporaceae N. R. In vitro (Ams.) L. amazonensis Schmidt et al., 2012a Cochliobolus sp. (N. R.)

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Ranunculaceae N. R. In vitro (Pro.) L. braziliensis Schmidt et al., 2012a Consolida oliveriana (N. R.) L. peruviana Thalictrum flavum (Roots) N. R. N. R. L. major Schmidt et al., 2012a Rubiaceae N. R. N. R. L. donovani Schmidt et al., 2012a Carapichea ipecacuanha (Roots) Faramea guianensis (Aublet.) Bremek Aqueus In vitro & in vivo L. amazonensis Rocha et al., 2005 (Leaves) (Ams.) Carvalho and Ferreira., 2001 Hintonia latiflora (N. R.) N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012a Nauclea latifolia (Root bark) Chloroform In vitro (axenic Ams.) L. donovani Schmidt et al., 2012a Rutaceae Ethyl acetate N. R. L. amazonensis Rocha et al., 2005 Dictyoloma peruvianum Planch Alkaloid fraction L. braziliensis (Stem bark) Haplophyllum bucharicum (N. R.) N. R. In vitro (Pro.&Ams.) L. infantum Schmidt et al., 2012a Helietta apiculata (Bark) N. R. In vitro (Pro.) & L. amazonensis Schmidt et al., 2012a in vivo (Ams.) L. braziliensis L. infantum Galipea longiflora Kr. Alkaloid fraction In vitro (Pro.) & L. amazonensis Carvalho and Ferreira., (Root bark + Stem bark + Leaves) in vivo (Ams.) L. braziliensis 2001 L. donovani L. venezuelensis Raputia heptaphylla Pittier (Leaves) N. R. In vitro (Pro.) L. panamensis Schmidt et al., 2012a Swinglea glutinosa Merr. (Bark) DCM N. R. L. amazonensis Rocha et al., 2005 L. braziliensis L. infantum Thamnosma rhodesica (N. R.) N. R. In vitro (Pro.) L. major Schmidt et al., 2012a Sapindaceae Ethanol N. R. L. amazonensis Rocha et al., 2005 Nicotiana glauca Grahm. L. braziliensis (Leaves + stem) L. donovani Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

Table 1. Plants reported as antileishmanial (Continued…) Botanical source Extract/Fraction Bioactivity/life stage Leishmania spp References Sapindaceae Ethanol N. R. L. amazonensis Rocha et al., 2005 Serjania tenuifolia Radlk (Leaves +stem) L. braziliensis Solanum actaeabotrys Rusby (Leaves) Ethanol N. R. L. braziliensis Rocha et al., 2005 L. donovani Scrophulariaceae DCM N. R. L. amazonensis Rocha et al., 2005 Conobea scoparioides (Cham.&Schltd.) L. braziliensis Benth (Leaves) Picrorhiza kurroa Royle, ex Benth Ethanol In vivo (Ams.) L. donovani Carvalho and Ferreira, (Roots + rhizome) 2001 Scrophularia scorodonia L. (Leaves) Methanol N. R. L. infantum Rocha et al., 2005 Solanaceae Ethanol In vitro (Pro.) L. amazonensis Rocha et al., 2005 Saracha punctata Ruiz & Pav. (Leaves) L. braziliensis Carvalho and Ferreira, L. donovani 2001 Solanum actaeabotrys Rusby (Leaves) Ethanol N. R. L. amazonensis Rocha et al., 2005 Solanum luteum Mill (Branches) N. R. N. R. L. major Rocha et al., 2005 Sterculiaceae Chloroform N. R. L. donovani Rocha et al., 2005 Cola attiensis Aubrev. & Pellegr. (Seeds) Ulmaceae Chloroform In vitro (Pro.) & L. amazonensis Rocha et al., 2005 Ampelocera edentula Kuhlm (Stem bark) in vivo (Ams.) L. venezuelensis Carvalho and Ferreira 2001 Verbenaceae Ethanol N. R. L. donovani Rocha et al., 2005 Nyctanthes arbortristis L. (Aerial parts) Vitex heterophylla Miq. (Leaves) Ethanol N. R. L. donovani Rocha et al., 2005 Winteraceae N. R. In vitro (Pro.) L. amazonensis Schmidt et al., 2012b Drimys species (N. R.) L. braziliensis L. chagasi Zingiberaceae N. R. In vitro (Pro.) L. donovani Schmidt et al., 2012b Aframonum sceptrum (N. R.) Zygophyllaceae (Nitrariaceae) N. R. In vitro (N. R.) L. donovani Schmidt et al., 2012a Larrea tridentata (N. R.) Peganum harmala (Seeds) N. R. In vitro (Pro.) & L. donovani Schmidt et al., 2012a in vivo (Ams.) Key: c: Data incomplete derived from an abstract; Pro.: promastigote; Ams.: amastigotes; DCM: dicloromethane; N. R.: not reported

2.2.3 Alkaloids from medicinal plants reported to exhibited antileishmanial activity Alkaloids reported to have antileishmanial activity from medicinal plants including diverse chemical structures, such as: quinolines, isoquinolines, indoles, diterpenoids, steroidal and amide alkaloids. Table 2 compiles the names, the antileishmanial activity of these alkaloids against various leishmania species, and their toxicity. The exact chemical structures of these alkaloids are presented in Figure 2.

Table 2. Reported antileishmanial alkaloids

QUINOLINE ALKALOIDS Compounds/botanical source Bioactivity/life Leishmania sp. Toxicity References stage γ-Fagarine 1 In vitro (Pro.) & L.amazonensis N. R. Schmidt et al., 2012b Helietta apiculata (Rutaceae) in vivo (Ams.) L. braziliensis L. infantum N-methyl-8-methoxyflindersine 2 In vitro (Pro.) L. panamensis N. R. Schmidt et al, 2012b Raputia heptaphylla (Rutaceae) Chimanine A 3 In vitro (Pro.) & L. braziliensis N. R. Carvalho and Ferreira, in vivo (Ams.) L. amazonensis 2001 Chimanine B 4 L. venezuelensis In vitro (Pro.) L.braziliensis Chimanine D 5 L. amazonensis Calipea longiflora (Rutaceae) In vivo (Ams.) L. venezuelensis Rocha et al., 2005 L. donovani Key: Pro.: promastigote; Ams.: amastigotes; N. R.: not reported