MASARYK UNIVERSITY FACULTY OF SCIENCE DEPT . OF BOTANY AND ZOOLOG Y BIOLOGICAL LY ACTIVE COMPOUNDS WIT H POTENTIAL ANTIPARASI TIC EFFECT AND THEIR IMPACT ON THE COURSE OF SELECTED PARASITOSES Ph.D. Dissertation Radka Pecková Supervisor: MVDr. Ivona Foitová, Ph.D . Brno 2018 Bibliographic Entry Author Mgr. Radka Pecková Faculty of Science, Masaryk University Department of Botany and Zoology Biological ly active compounds with potential Title of Thesis: antiparasitic effect and their impact on the processes of selected parasitoses Degree programme: Biology Field of Study: Parasitology Supervisor: MVDr. Ivona Foitová, Ph.D. Academic Year: 2017/2018 Number of Pages: 139 Keywords: Giardia intestinalis ; Cryptosporidium ; Anti - protozoal activity; Plant extracts; Drug of Choice; Natural Antiparasitics; Parasites; Archidendron fagifolium ; Diospyros sumatrana ; Piper betle ; Shorea sumatrana Bibliografický záznam Autor: Mgr. Radka Pecková Přírodovědecká fakulta, Masarykova univerzita Ústav botaniky a zoologie Biologicky aktivní látky s potencionálním Název práce: antiparazitárním účinkem a jejich působení na průběh vybraných parazitóz Studijní program: Biologie Studijní obor: Parazitologie Vedoucí práce: MVDr. Ivona Foitová, Ph.D. Akademický rok: 2017/2018 Počet stran: 139 Klíčová slova: Giardia intestinalis ; Cryptosporidium ; Antiprotozoární aktivita; Rostlinné extrakty; Alternativní léčiva; Přírodní antiparazitika; Paraziti; Archidendron fagifolium ; Diospyros sumatrana ; Piper betle ; Shorea sumatrana ABSTRAK T ABSTRACT This thesis deals with the study of the influence of extracts of selected plants from Indonesia on parasites Giardia intestinalis (Lambl) Alexeieff, 1914 and Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Jesudoss Chelladural, Prantlová & McEvoy, 2016 . Tested plants were selected based on behavioural data and the ability to reduce the intensity of parasitic infection in Sumatran orangutans. Subsequently, they were extracted in three types of solvent s: water, methanol and methanol - tetrahydrofuran (1:1). Testing was performed using both the in vitro and in vivo models. In vitro experiments on G. intestinalis , during which four plant extracts were tested, the antiparasitic activity was reported in two of them, namely Archidendron fagifolium and Piper betle . Piper betle was also tested in vivo on Mongolian gerbils , where this activity was confirmed. The effect of extracts on C. proliferans was investigated using an in vivo model (southern multimammate mouse ( Mastomys coucha ) ) . Although Diospyros sumatrana extracts slightly decreased the number of shed oocyst s , evaluation of histological sections did not confirm the decrease in the intensity of parasitisation of the gastric tissue. The thesis also contains two methodological studies; the first concerns the comparison of the in vitro excystation protocols of Cryptosporidium parvum Tyzzer, 1912 oocyst, while the second one deals w ith the study of the course of experimental giardiasis in the gerbils. Both of these studies can be used for the future planning of experiments to test the antiparasitic activity of plant extracts. ABSTRAK T ABSTRAKT Předložená práce se zabývá studiem vlivu extraktů vybraných rostlin z Indonésie na parazity Giardia intestinalis (Lambl) Alexeieff, 1914 a Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Jesudoss Chelladural, Prantlová & McEvoy, 2016 . Testované rostliny byly vybrány na základě behaviorálních dat a schopnosti snížit intenzitu parazitární infekce u sumaterských orangutanů. Následně byly extrahovány ve třech druzích rozpouštěd el: ve vodě, metanolu a metanol - tetrahydrofuranu (1:1). Te stování bylo provedeno s použitím in vitro i in vivo modelů. Při in vitro pokusech na G. intestinalis , kdy byly testovány čtyři rostliny, byla prokázána antiparazitární aktivita u dvou z nich, a to u Archidendron fagifolium a Piper betle . Piper betle byl t estován také na pískomilech, kde byla tato aktivita potvrzena. Vliv extraktů na C. proliferans byl zkoumán v in vivo modelu (krysa malá ( Mastomys coucha ) . Extrakty z rostliny Diospyros sumatrana mírně snižovaly počty vylučovaných oocyst, vyhodnocení histologických preparátů však nepotvrdilo snížení intenzity parazitární infekce v žaludeční tkáni. Práce dále obsahuje dvě metodologické studie; první se týká porovnání excystačních protokolů u oocyst Cryptosporidium parvum Tyzzer, 1912, druhá se zabývá studiem průběhu experimentální giardiózy u pískomilů. Obě tyto studie mohou být použity pro budoucí plánování experimentů zabývajících se testováním antiparazitární aktivity rostlinných extraktů. © Radka Pecková , Masaryk University, 2018 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS This thesis could be created thanks to the kind support of many people I would like to acknowledge here. First, I would like to express my very great appreciation to my supervisor, MVDr. Ivona Foitová, Ph.D., for her patient guidance, advices, enthusiastic encouragement and endless optimism during the whole time of my studies, as well as for the financial support. I would like to offer my special thanks to the Laboratory of Veterinary and Medical Protistology of the Institute of Parasitology, Biology Centre CAS in České Budějovice, namely Prof. Ing. Martin Kváč, Ph.D., RNDr. Bohumil Sak, Ph.D . and RNDr. Dana Květoňová. For the opportunity to work in this laboratory, the initiation into the experimental methods, inspiring advices and a pleasant collaboration. I would like to thank Assoc. Prof. RNDr. Milan Gelnar, CSc. for giving me the opportun ity to work in Parasitology Research Group and RNDr. Andrea Bardůnek Valigurová, Ph.D. for giving me initial training in some laboratory methods and useful advices on this thesis. Collaboration and advices concerning the use of plant extracts provided by M gr. Karel Doležal, DSc. were greatly appreciated. I wish to acknowledge the help provided by the Laboratory of Electron Microscopy, Biology Centre CAS in České Budějovice particularly their help with obtaining the data from scan electron microscopy. I am a lso grateful to all my colleagues who created a pleasant workplace environment and were always willing to help. My special thanks are extended to Mgr. Markéta Fikejzová for the drawings presented in this thesis. Last but not least, I would like to thank my family and friends for their support and encouragement throughout my studies. This thesis was implemented in the Parasitology Research group at the Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno. The studies were financiall y supported by the UMI – Saving of Pongidae Foundation project “Parasites and Natural Antiparasitics in the Orang - utan”, by the Czech Academy of Sciences, Grant No. P505/11/1163 and by Scholarship program to support creative activity - research collaborati on, No. PřF_04_27.10.2006. CONTENTS CONTENTS 1 INTRODUCTION ................................ ................................ ................................ ..... 11 2 AIMS OF THE THESIS ................................ ................................ ............................ 12 3 LITERA TURE OVERVIEW ................................ ................................ ..................... 13 3.1 Biologically active compounds ................................ ................................ ........... 13 3.1.1 History of herbal drugs ................................ ................................ ................ 14 3.1.2 Self - medicative behaviour in animals ................................ .......................... 15 3.1.3 Sources of secondary metabolites ................................ ................................ 17 3.1.4 Biologically active compounds from plants as a treatment of parasito ses ..... 20 3.2 Plant extracts ................................ ................................ ................................ ...... 27 3.2.1 Plant extract preparation ................................ ................................ .............. 27 3.2.2 Tested plants ................................ ................................ ............................... 28 3.3 Studied parasites ................................ ................................ ................................ 29 3.3.1 Giardia intestinalis ................................ ................................ ...................... 30 3.3.2 Cryptosporidium spp. ................................ ................................ .................. 34 4 MATERIALS AND METHODS ................................ ................................ ............... 41 4.1 Plant extracts ................................ ................................ ................................ ...... 41 4.1.1 Plant collection ................................ ................................ ............................ 41 4.1.2 Plant extract preparation ................................ ................................ .............. 41 4.2 Giardia intestinalis ................................ ................................ ............................. 42 4.2.1 In vitro experiments ................................ ................................ .................... 42 4.2.2 In vivo experiments ................................ ................................ ..................... 44 4. 3 Cryptosporidium spp. ................................ ................................ ......................... 49 4.3.1 In