Ethnopharmacology
of medicinal plants traditionally used in the Greater Mpigi region, Uganda
vorgelegt von M.Sc.
Fabien Schultz
ORCID: 0000-0003-1904-2430
an der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften – Dr. rer. nat. –
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Lorenz Adrian Gutachterin: Prof. Dr. Vera Meyer Gutachter: Prof. Dr. Juri Rappsilber Gutachter: Prof. Dr. Leif-Alexander Garbe Gutachter: Prof. Dr. Michael Heinrich
Tag der wissenschaftlichen Aussprache: 11. Juni 2021
Berlin 2021
Ethnopharmacology [constitutes] a respectful marriage between modern science and ancient wisdom with much to be gained in both directions.
Dr. Graham Jones (Heinrich and Jäger, 2015)
I Relevance: This PhD thesis provides an ethnopharmacological assessment of 16 medicinal plant species used by traditional healers for the treatment of various infectious and inflammatory diseases in the Greater Mpigi region of Uganda. It includes the first detailed report on selected medicinal plant use in the local traditional medicine system. The ethnobotanical data obtained supports the conservation of local traditional knowledge and will enable future drug discovery research. As conventional drug therapies are becoming increasingly limited and inefficacious, some of the greatest threats to global health today are addressed in this work, including antibiotic resistance, malaria, cancer, inflammation, and related symptoms such as pain and fever.
Aims of the PhD thesis: The overarching objectives were 1) the documentation of the medicinal use of (often understudied) plant species; 2) the development of a bibliographic assessment tool for selection of medicinal plants for lab studies; 3) the creation of a plant extract library;
4) the pharmacological in vitro assessment of these species regarding their respective traditional uses; 5) the contribution to early drug discovery stages and thereby the provision of a scientific basis for future drug lead identification endeavors; and 6) the integration of aspects of community work while fostering a bidirectional communication with indigenous communities.
Methods: The results of diverse ethnopharmacological field research methods and pharmacological lab assays are reported in this dissertation, including a) an ethnobotanical survey using structured interviews with 39 traditional healers from 29 villages; b) the novel Degrees of Publication (DoP) method; c) antibacterial screenings against a panel of clinical isolates of multidrug-resistant human pathogens; d) antivirulence experiments, e.g., monitoring of quorum sensing inhibition and direct protein output (į-toxin) in Staphylococcus aureus;
e) antiinflammatory investigations (e.g., cyclooxygenase-2 inhibition); f) antimalarial studies against chloroquine-resistant Plasmodium falciparum K1; g) cytotoxicity counterscreens (e.g., against human MRC-5SV2 lung fibroblasts and HaCaT keratinocytes); h) a genotoxicity assessment model with simulation of human liver metabolism; and i) a method for transferring research results back to traditional healers in rural indigenous communities.
Results: The results of the survey indicated a high level of local traditional use, as the 16 plant species are frequently used to treat a total of 75 different medical disorders. Many of these use reports were documented for the first time. The development and application of the DoP method allowed for assessment of what was already known about a species, while also estimating the quality of the evidence. In total, 634 peer-reviewed publications covering the period of 1960-2019 were reviewed. Six of the 16 species were then classified as being highly understudied, and three species as being understudied, which provided further justification for the subsequent lab investigations. The results of the pharmacological experiments generally showed a high correlation between the pharmacological activity and the respective traditional use. In the following, some examples of the strongest bioactive extracts are provided for a) growth inhibition activity: extracts of Zanthoxylum chalybeum and Harungana madagascariensis stem bark against S. aureus (MIC: 16 and 32 ȝg/mL) and Enterococcus faecium (MIC: 32 ȝg/mL); b) quorum sensing inhibition activity against four S. aureus
II calycinum subsp. angustifolium leaves (IC50: 1–64 ȝg/mL); c) selective COX-2 inhibition activity: an extract of Leucas calostachys leaves (IC50: 0.66 ȝg/mL); and d) antiplasmodial activity against P. falciparum K1: extracts of W. ugandensis stem bark and L. calostachys leaves (IC50: 0.5 and 5.7 ȝg/mL). The transfer of lab results back to the traditional healers culminated in a video article showing a two-day workshop.
Conclusions: The conservation of traditional knowledge continues to be vital for future generations, especially when facing the need for novel and more effective drugs, which have often been discovered from natural products in the past. The newly introduced DoP method is a useful tool for selecting traditionally used species for future lab studies, like costly pharmacological experiments and time-consuming isolation procedures. The results of the pharmacological studies provided scientific support for the potential therapeutic effects of the medicinal plants used in the Greater Mpigi region.
III Relevanz: Diese Doktorarbeit beschäftigt sich mit der ethnopharmakologischen Untersuchung von 16 Medizinpflanzen, die von traditionellen Heilerinnen und Heilern zur Behandlung von diversen Infektions- und Entzündungskrankheiten in der Großregion Mpigi in Uganda genutzt werden. Sie beinhaltet erstmalig einen detaillierten Bericht zur selektiven Medizinpflanzennutzung im örtlichen traditionellen Gesundheitssystem. Die erhobenen ethnobotanischen Daten tragen zum Erhalt des lokalen traditionellen Wissens bei und ermöglichen zukünftige Medikamentenwirkstoffentdeckungen und -entwicklungen. Da herkömmliche medikamentöse Therapien zunehmend in ihrer Anwendung beschränkt und wirkungslos sind, werden in dieser Arbeit einige der größten Bedrohungen für die globale Gesundheit thematisiert, darunter Antibiotikaresistenzen, Malaria, Krebs und Entzündungen sowie damit in Zusammenhang stehende Symptome wie Schmerzen und Fieber.
Ziele der Doktorarbeit: Die übergeordneten Ziele sind 1) die Dokumentation der medizinischen Anwendung von (oftmals bislang wenig erforschten) Pflanzenspezies; 2) die Entwicklung einer bibliographischen Bewertungsmethode für die Auswahl von Medizinpflanzen für Laborstudien; 3) der Aufbau einer Pflanzenextrakt-Datenbank; 4) die pharmakologische in vitro Untersuchung dieser Spezies bezüglich ihrer traditionellen Anwendungen; 5) die Mitwirkung an frühen Wirkstofffindungs- und -entwicklungsphasen durch die Bereitstellung der wissenschaftlichen Basis für die zukünftige Identifikation von Arzneimittel-Leitstrukturen;
und 6) die Integration von Aspekten der lokalen Community-Arbeit in die Forschung mit dem Ziel der Förderung einer bidirektionalen Kommunikation mit indigenen Gemeinschaften.
Methoden: In dieser Dissertation werden Resultate von diversen ethnopharmakologischen Feldforschungsmethoden und pharmakologischen Labor-Assays präsentiert, unter anderem von a) einer ethnobotanischen Umfrage bei 39 traditionellen Heilerinnen und Heilern aus 29 Dörfern mittels strukturierten Interviews; b) der neuartigen „Degrees of Publication“- Methode (DoP-Methode); c) antibakteriellen Screenings gegen ein Panel bestehend aus klinischen Isolaten multiresistenter Humanpathogene; d) Antivirulenz-Experimenten, z.B. der Überwachung der Quorum Sensing Inhibition und der direkten Protein-Sekretion (į-Toxin) bei Staphylococcus aureus; e) antiinflammatorischen Untersuchungen (z.B. der Cyclooxygenase- 2-Inhibition); f) Antimalaria-Studien gegen den chloroquin-resistenten Plasmodium falciparum K1-Stamm; g) Cytotoxizitäts-Counterscreenings (z.B. gegen menschliche MRC-5SV2-Lungenfibroblasten und HaCaT-Keratinozyten); h) einem Gentoxizitäts- Beurteilungs-Modell mit Simulierung des menschlichen Lebermetabolismus; und von i) einer Methode zum Transfer von Forschungsergebnissen zurück zu den traditionellen Heilerinnen und Heilern in ländlichen indigenen Gemeinschaften.
Ergebnisse: Die lokalen Umfrageresultate zeigten, dass die 16 Pflanzenarten häufig bei der traditionellen Behandlung von insgesamt 75 Erkrankungen angewendet werden. Viele dieser Anwendungsberichte wurden erstmalig dokumentiert. Die Entwicklung und Anwendung der DoP-Methode ermöglichte eine Bewertung darüber, inwiefern eine Spezies bereits erforscht wurde. Zudem wurde die Qualität der wissenschaftlichen Evidenz eingeschätzt. Insgesamt wurden 634 von Experten begutachtete wissenschaftliche Publikationen aus einem Zeitraum von 1960 bis 2019 geprüft. Sechs der 16 Spezies wurden dabei als hochgradig untererforscht und drei Spezies als untererforscht eingestuft, was die darauffolgenden Laboruntersuchungen
IV Allgemeinen eine hohe Korrelation zwischen der pharmakologischen Aktivität und der jeweiligen traditionellen Anwendung. Im Folgenden sind einige Beispiele für die in den Versuchen am stärksten wirkenden Extrakte aufgeführt: a) Wachstumshemmungsaktivität:
Extrakte aus Zanthoxylum chalybeum- und Harungana madagascariensis-Stammrinde gegen S. aureus (MIC: 16 und 32 ȝg/mL) und Enterococcus faecium (MIC: 32 ȝg/mL); b) Quorum Sensing-Hemmaktivität gegen vier S. aureus-Accessory-Gene-Regulator-Allele: Extrakte aus Solanum aculeastrum-Wurzelrinde und Sesamum calycinum subsp. angustifolium-Blättern (IC50: 1–64 ȝg/mL); c) selektive COX-2-Hemmaktivität: ein Extrakt aus Leucas calostachys- Blättern (IC50: 0,66 ȝg/mL); und d) antiplasmodiale Aktivität gegen P. falciparum K1: Extrakte aus W. ugandensis-Stammrinde und L. calostachys-Blättern (IC50: 0,5 und 5,7 ȝg/mL). Die Rückführung der Laborergebnisse zu den traditionellen Heilerinnen und Heilern resultierte in einem Videoartikel, der einen zweitägigen Workshop veranschaulicht.
Fazit: Die Konservierung des traditionellen Wissens der lokalen Heilerinnen und Heiler ist fortwährend unerlässlich für zukünftige Generationen. Dieses ist vor allem bezogen auf die Nachfrage nach neuen, effektiveren Medikamenten der Fall, die in der Vergangenheit bereits häufig in Naturstoffen entdeckt und daraus entwickelt wurden. Die neu vorgestellte DoP-Methode erwies sich als ein leistungsfähiges Instrument für die Auswahl von traditionell genutzten Spezies für zukünftige Laborstudien, wie z.B. kostenintensive pharmakologische Experimente und zeitaufwendige Naturstoffisolationen. Die Ergebnisse der pharmakologischen Studien unterstützen die potenziellen therapeutischen Wirkungen der in der Großregion Mpigi genutzten Medizinpflanzen.
V
Abstract I
Zusammenfassung III
Acknowledgements VI
Abbreviations VII
Thesis structure IIX
Introduction 1
Publication I:
"Ethnobotanical study of selected medicinal plants traditionally used in the rural
Greater Mpigi region of Uganda" 13
Publication II:
"A bibliographic assessment using the Degrees of Publication method: Medicinal plants from the rural Greater Mpigi region (Uganda)" 32 Publication III:
"Targeting ESKAPE pathogens with anti-infective medicinal plants from the
Greater Mpigi region in Uganda" 51
Publication IV:
"Antiinflammatory medicinal plants from the Ugandan Greater Mpigi region act
as potent inhibitors in the COX-2 / PGH2 pathway" 86
Manuscript V:
"Pharmacological assessment of the antiprotozoal activity, cytotoxicity and genotoxicity of medicinal plants used in the treatment of malaria in the Greater
Mpigi Region in Uganda" 126
Manuscript VI – Video article with accompanying short written article:
"Transferring ethnopharmacological results back to traditional healers in rural
indigenous communities – The Ugandan Greater Mpigi region example" 169
Discussion and conclusions 181
Awards, achievements, plenary talks, conference and poster presentations,
honorary positions, and press features linked to the PhD studies 193
References 200
VI My eternal gratitude to my supervisor, supporter, mentor, and friend Professor Leif-Alexander Garbe, who always put his trust in me, let me independently define my own research approach, and unconditionally facilitated the opportunity for me to follow my dreams, while turning my passion into an occupation. Thank you, Leif.
Mein größter Dank gebührt meiner Familie, insbesondere meiner Mutter, die immer an mich glaubte. Durch ihre unerschöpfliche Liebe wurde ich zu der Person, die ich heute bin.
My heartfelt thanks to the traditional healers in the Greater Mpigi region and neighboring regions who provided the ethnobotanical information that formed the foundation of this thesis.
I would also like to thank my supervisors, Professor Vera Meyer and Professor Juri Rappsilber, for their valuable guidance and for being available as reviewers for my dissertation. Thanks to my mentors, Professor Michael Heinrich and Professor Luc Pieters, for providing important feedback on my studies, and for always being available.
Thanks to Professor Cassandra Quave, who has been an outstanding, supportive, and inspiring Fulbright host and mentor to me. Dr. Quave has always been nothing but kind, while pushing me towards greater productivity with her professionalism and expertise.
Thanks to my Ugandan colleague Godwin Anywar and our student assistants for the fruitful collaboration.
Thanks to Fulbright Germany, the BMBF, the DFG, and the DAAD for funding my research.
Special thanks to the HSNB for letting me conduct research on rather out-of-the-ordinary topics, and to Mrs. Gaschler, who has been the best university third-party-funded project coordinator I could ever imagine.
Thanks to my buddy Logan Penniket for the (most likely tiresome) proofreading of most of our publications.
And lastly, but most importantly, my deepest gratitude to my beautiful wife and best friend, Inken, who always has my back and supports me unconditionally. Thank you for putting all your efforts into the various fieldwork stays in Uganda, which we always experienced together.
I am beyond lucky and so grateful to have had you always by my side, for better or for worse, for more than 12 years now. And it makes me even happier that our partnership will now be crowned by the birth of our wonderful daughter. I love you, Inken.
VII
AA: arachidonic acid
AChE: acetylcholinesterase
ACTs: artemisinin combination therapies
2-AF: 2-aminofluorene
agr: accessory gene regulator AMR: antimicrobial resistance
ATCC: American Type Culture Collection BHI: brain heart infusion
BUTHGA: Buyijja Traditional Healers Association CAMHB: cation-adjusted Mueller Hinton Broth CBD: Convention on Biological Diversity CC50: 50% cytotoxic concentration
CDC: Centers for Disease Control and Prevention CFU: colony forming units
CHA: chlorogenic acid
COPE: Committee on Publication Ethics
COX: cyclooxygenase
COX-1: cyclooxygenase-1
COX-2: cyclooxygenase-2
DHA: docosahexaenoic acid
DMSO: dimethyl sulfoxide
DoP: Degrees of Publication
DPPH: 1,1-diphenyl-2-picrylhydrazyl radical
DSMZ: German Collection of Microorganisms and Cell Cultures ELISA: enzyme-linked immunosorbent assay
EPA: eicosapentaenoic acid ESI: electrospray ionization
ESKAPE: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species
FC: frequency of citation
GEO: Emory University Herbarium
GI: growth inhibition
GMR: Greater Mpigi region
12(S)-HpETE: 12-S-hydroxyeicosatetraenoic acid 15(S)-HpETE: 15-S-hydroxyeicosatetraenoic acid IC50: half maximal inhibitory concentration
IL-1: Interleukin-1
IL-4: Interleukin-4
LC-FTMS: liquid chromatography-Fourier transform mass spectrometry LDH: lactate dehydrogenase
LOX: lipoxygenase
12/15-LOX: 12/15-lipoxygenase
LXA4: lipoxin A4
m.a.s.l.: meters above sea level
MHB: Mueller Hinton Broth
MI: mutagenicity index
MIC: minimum inhibitory concentration
VIII MRSA: methicillin-resistant Staphylococcus aureus
MS: mass spectrometry
MUH: Makerere University Herbarium
NADP: ß-nicotinamide adenine dinucleotide phosphate disodium salt 2-NF: 2 nitrofluorene
NGO: non-governmental organization NSAIDs: nonsteroidal antiinflammatory drugs
OD: optical density
PG: prostaglandin
PGD2: prostaglandin D2
PGE2: prostaglandin E2
PGF2Į: prostaglandin F2Į
PGG2: prostaglandin G2
PGH2: prostaglandin H2
PGI2: prostaglandin I2
QSI: quorum sensing inhibition RFCs: relative frequencies of citation
RvE3: resolvin E3
SDS: sodium dodecyl sulfate
SERNEC: SouthEast Regional Network of Expertise and Collections
SI: selectivity index
sox. Soxhlet extraction
sox. succ.: successive Soxhlet extraction.
TI: therapeutic index
TNF: tumor necrosis factor TNFĮ: tumor necrosis factor alpha TSA: tryptic soy agar
TSB: tryptic soy broth TPC: total phenolic content
TX: thromboxane
WHO: World Health Organization
YFP: yellow fluorescent protein
IX This doctoral thesis encompasses six combined manuscripts, which in the following sections are referred to by their corresponding Roman numerals (I-VI). The dissertation is therefore presented in cumulative form. All manuscripts were drafted with first authorship and subsequently submitted to international peer-reviewed journals. Each manuscript, including a prefaced statement regarding my personal contribution, information about its publication, and a subsequent supplementary material document (where applicable), represents a separate chapter of the thesis. Four of these manuscripts have recently been published and are inserted herein as postprint versions. The remaining two manuscripts are currently undergoing peer- review processes and are included as preprint versions. The last manuscript is presented in an innovative new format: a 26-minute video article, which has been submitted to the Video Journal of Education and Pedagogy. An access link to the video is provided in this thesis, using an online hosting platform, along with a brief written manuscript, which was also part of the submission to the journal. In addition, the print version of this thesis includes an enclosed USB flash drive containing the video article file.
The six manuscripts are listed below:
Publication I:
"Ethnobotanical study of selected medicinal plants traditionally used in the rural Greater Mpigi region of Uganda"
Schultz, F.; Anywar, G.; Wack, B.; Quave, C.L.; Garbe, L.-A.
published in the Journal of Ethnopharmacology (publisher: Elsevier) Volume 256, 28 June 2020, https://doi.org/10.1016/j.jep.2020.112742
Permission by Elsevier to reprint the material at no charge in my thesis was obtained, and the reproduction of the article is confined to the purpose for which permission was given.
Publication II:
"A bibliographic assessment using the Degrees of Publication method: Medicinal plants from the rural Greater Mpigi region (Uganda)"
Schultz, F.; Anywar, G.; Quave, C.L.; Garbe, L.-A.
published in Evidence-Based Complementary and Alternative Medicine (publisher: Wiley/Hindawi)
6661565, 2021, https://doi.org/10.1155/2021/6661565
This is an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0).
X
"Targeting ESKAPE pathogens with anti-infective medicinal plants from the Greater Mpigi region in Uganda"
Schultz, F.; Anywar, G.; Tang, H.; Chassange, F.; Lyles, J.T.; Garbe, L.-A.; Quave, C.L.
published in Scientific Reports (publisher: Nature Research)
volume 10, Article number: 11935, 2020, https://doi.org/10.1038/s41598-020-67572-8 This is an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0).
Publication IV:
"Antiinflammatory medicinal plants from the Ugandan Greater Mpigi region act as potent inhibitors in the COX-2 / PGH2 pathway"
Schultz, F.; Osuji, F. O.; Wack, B.; Anywar, G.; Garbe, L.-A.
published in Plants (publisher: MDPI)
10(2), 351, 2021; https://doi.org/10.3390/plants10020351
This is an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0).
Manuscript V:
"Pharmacological assessment of the antiprotozoal activity, cytotoxicity and genotoxicity of medicinal plants used in the treatment of malaria in the Greater Mpigi Region in Uganda"
Schultz, F.; Osuji, O. F.; Nguyen, A.; Anywar, G.; Scheel, J. R.; Caljon, G.; Pieters, L.;
Garbe, L.-A.
submitted to Frontiers in Pharmacology on March 9, 2021 (in review, publisher: Frontiers Research Foundation)
If accepted, this will be an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0).
Manuscript VI – Video article and accompanying written short article:
"Transferring ethnopharmacological results back to traditional healers in rural indigenous communities – The Ugandan Greater Mpigi region example"
Schultz, F.; Dworak-Schultz, I.; Olengo, A.; Anywar, G.; Garbe, L.-A.
submitted to the Video Journal of Education and Pedagogy on February 18, 2021 (in review, publisher: Brill)
If accepted, this will be an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0).
1 Ethnopharmacology
The science of ethnopharmacology seeks to investigate the medicinal use of natural materials, such as plants, macrofungi, microorganisms, animals, and minerals, by humans, while applying pharmacological, anthropological, and socio-cultural research methods. Various other branches of science may also be involved in this highly interdisciplinary field (Heinrich, 2014;
Heinrich and Jäger, 2015). For example, some ethnopharmacologists study how indigenous peoples use plants for the treatment of diseases and healing, and pharmacologically investigate the recorded and collected species in a laboratory or clinical setting regarding their potential medicinal effects. Here, the initial fieldwork stages of research are often closely linked to the scientific discipline of ethnobotany, the study of relationships between plants, culture, and humans (Alexiades and Sheldon, 1996; Martin, 2004; Balick and Cox, 2020). Ethnobotanical information, such as the identification of plants used and the traditional methods of harvesting, preparation, and administration, are often vital for the successful replication and assessment of pharmacological effects in in vitro and in vivo models (Harrison et al., 2015; Balick and Cox, 2020). The results can be the starting point for the discovery of novel drug candidates and botanical formulations for therapies, as well as evidence-based safety assessments (Mukherjee et al., 2010; Heinrich and Jäger, 2015; Balick and Cox, 2020). In today's drug discovery led by the modern Western pharmaceutical industry, the key role of traditional medicine is evident, as up to 50% of all FDA-approved drugs on the market are currently derived directly or indirectly from natural sources (Veeresham, 2012; Atanasov et al., 2015; Li et al., 2019). The most famous example of an ethnopharmacology-driven success story with worldwide significance for human health was the discovery and isolation of artemisinin (Qinghaosu) from Artemisia annua (sweet wormwood), a Chinese medicinal herb called Qinghao (Qinghaosu Research Group, 1977; Tu, 2004; Tu, 2011). The research leading to this discovery was conducted by Dr. Youyou Tu and her team, who found a diethyl ether extract of the plant's leaves that strongly inhibited malaria (Czechowski et al., 2020). Initially, Youyou Tu had obtained the ethnobotanical information on the traditional use against malaria fevers from an ancient Chinese herbal book compiled by Ge Hong in China's Eastern Jin Dynasty around 317–420 A.D. (Kong and Tan, 2015; Czechowski et al., 2020). Today, A. annua remains the sole global source of the drug, and derived artemisinin combination therapies (ACTs) are considered first-line drugs to combat malaria, saving millions of lives, especially among
2 2020; Ma et al., 2020). In honor of her contribution to the discovery of artemisinin, the Chinese ethnopharmacologist Youyou Tu was awarded the Nobel Prize in Physiology or Medicine in 2015 (Croft and Ward, 2015; Efferth et al., 2015). Other well-known examples of outstanding ethnopharmacological research in the past have been the discovery of 1) paclitaxel (Taxol), first isolated from the Pacific yew (Taxus brevifolia) and widely used in the chemotherapy of various types of cancers today; 2) morphine, an analgesic isolated from the opium poppy (Papaver somniferum); and 3) quinine, derived from the bark of Cinchona spp., which was originally used medicinally by the indigenous peoples of the Andes and most likely first used for the treatment of malaria by a European in the 1630s (Klockgether-Radke, 2002; Heinrich et al., 2015; Permin et al., 2016; Walker and Nesbitt, 2019). An example of a traditionally used botanical drug that provides the basis for modern phytomedicines is the herb St. John's wort (Hypericum perforatum), which is prescribed globally to treat mild and moderate cases of depression (Heinrich et al., 2015; Volz, 2020; Moragrega and Ríos, 2021).
In my personal interpretation of the science of ethnopharmacology, research activities involve a) field studies (such as ethnobotanical studies in local communities, surveys, interviews, first-time documentation of medicinal use, ritual use, or religious aspects); b) the pharmacological assessment of species regarding their respective traditional uses in a laboratory setting ("bioactivity studies"); and c) the discovery of pharmacologically active natural products via pharmacognostic approaches, e.g. bioassay-guided fractionation and isolation of bioactive secondary metabolites (Schultz et al., 2021b). However, these activities may be further expanded to include community work because I believe that ethnopharmacologists have a responsibility for the respective indigenous communities they collaborate with and should therefore also act as their advocates.
Ugandan traditional medicine and its significance
My field research was conducted in the tropical country Uganda, which is located on the northern shores of Lake Victoria, bordered by Rwanda and Tanzania to the south, the Democratic Republic of Congo to the west, South Sudan to the north, and Kenya to the east.
Uganda is characterized by a very rich biological diversity due to its unique bio-geographical location (Kalema, 2005). Seven of Africa's 18 phytogeographical regions occur in Uganda (Davenport and Matthews, 1995), making it the most phytogeographically diverse country on the African continent (White, 1983). In total, there are more than 5,000 species of higher plants present in the indigenous Ugandan flora (Hamilton et al., 2016). From an anthropological
3 (Brockerhoff and Hewett, 2000).
Humans around the world have used plants as medicine since time immemorial, and traditional medicine continues to be of highest importance for all human beings (Yuan et al., 2016; Bussmann et al., 2018; Kigen et al., 2019). Africa is more reliant on traditional medicinal healthcare than any other continent, as up to 90% of its population seeks traditional treatment provided by healers (WHO, 2013; Ekor, 2014). According to the World Health Organization (WHO, 2016), Uganda has one physician for every 10,752 patients, which is the lowest proportion of healthcare professionals with modern medical training per capita in East Africa (a ratio of modern physicians to inhabitants of 1:1,000 is considered an indicator of a good health system by the WHO). At the same time, Ugandan traditional healers provide significant basic medical treatment that has been passed down by previous generations for centuries (estimated healer to patient ratio in Uganda: 1:100) (Green, 1997; Nyamukuru et al., 2017;
Schultz et al., 2020b). It is important to mention that, in most cases, traditional information on the use of local herbal drugs has never been pharmacologically investigated or even documented. These traditional practices and applications of medicinal plants in Uganda significantly vary between different ethnic groups, cultures, and even neighboring villages. It is an unfortunate fact that much of this traditional knowledge has already been lost due to economic drift to the cities (rural-urban migration, Western influence, and deforestation) (Bussmann et al., 2018). Thus, the work on the documentation of medicinal applications of understudied plant species that is presented in this PhD thesis further contributes to the preservation of traditional knowledge, which will be vital for future generations.
The Greater Mpigi region
My field research site in West-Central Uganda covered an area of 715 km2 in the Mpigi, Butambala, and Gomba districts, which I refer to in the following as the "Greater Mpigi region". The area of the research site was calculated by drawing a perimeter line around the locations of 29 villages and 39 traditional healers (study participants). At this field site, the local vegetation is described as a tropical, moist evergreen forest / savanna mosaic (Barbour, 1987; Howard, 1991). The Greater Mpigi region, including geographical, anthropological, and socio-economic data, is further described in "Publication I" as part of this PhD thesis (Schultz et al., 2020b). There is a high dependency on local traditional healers and medicinal plants for covering primary healthcare needs in this remote area. Nevertheless, reports on medicinal plant use and the documentation of traditional methods of preparation and administration in the
4 2014; Kibuuka and Anywar, 2015; Nyamukuru et al., 2017).
PhD research
In my PhD studies, I investigated 16 selected Ugandan medicinal plants that had been identified in preliminary studies during my previous field research in Uganda. The approach applied in this dissertation is highly interdisciplinary and based on ethnopharmacological, ethnobotanical, ethnomedicinal, pharmacological, and anthropological best-practice methods.
For example, procedures for plant collection, species identification, and the assignment of scientific names followed the current standards for conducting and reporting ethnopharmacological field studies at all times (Alexiades and Sheldon, 1996; Martin, 2004;
Heinrich et al., 2018; Weckerle et al., 2018).
The overarching objectives of this thesis were a) the first-time documentation of the medicinal use of (in many cases understudied) plant species; b) the development of a bibliographic assessment tool for selection of medicinal plants for lab studies; c) the creation of a plant extract library; d) the pharmacological in vitro assessment of the 16 species regarding their respective traditional uses; e) the contribution to early drug discovery stages and thereby the provision of a scientific basis for future drug lead identification endeavors; and f) the integration of aspects of community work while fostering a bidirectional communication with indigenous communities. More detailed objectives for the individual studies summarized in this thesis are specified in each of the six manuscripts.
The research structure, including individual study stages and the resulting publications/manuscripts, is illustrated in Figure 1. Each of the six presented manuscripts contains a thorough introduction, leading to a more profound understanding of the individual study objectives and their background. Therefore, the various research topics addressed in this thesis are only introduced briefly in the following paragraphs and subsequently presented in detail in the manuscripts' introductions (including the corresponding literature reviews).
The first stage of field research was the collection of plant material, guided by traditional healers. This work included the preparation of herbarium voucher specimens for taxonomic identification or confirmation, which was carried out subsequently at the Makerere University herbarium. The collected plant material was processed (e.g., bark was cut into small pieces) and dried in the shade. An ethnobotanical and anthropological survey among 39 traditional healers followed, resulting in "Publication I" (Schultz et al., 2020b).
Figure 1: Overview of the structure of my PhD research, including individual study stages and 5
the resulting publications/manuscripts. GMR: Greater Mpigi region; ESKAPE: Enterococcus
6
Pseudomonas aeruginosa and Enterobacter species; agr: accessory gene regulator; MRSA:
methicillin-resistant S. aureus; LC-FTMS: liquid chromatography-Fourier transform mass spectrometry; COX-1: cyclooxygenase-1; COX-2: cyclooxygenase-2; 12/15-LOX: 12/15- lipoxygenase; TPC: total phenolic content
In addition, the survey assessed the healers' general knowledge of Western medicine, including their understanding of infectious diseases, the concept of microbial pathogens, and cancer. Another topic addressed was the identification of the traditional healers' needs and future expectations regarding our collaboration.
Next, I planned to develop an urgently needed novel tool for literature assessment in ethnopharmacological research that would determine which species merit the costly lab studies, e.g., pharmacological assays and subsequent isolation of active natural product compounds.
This novel method should therefore assess the degree to which a species has already been studied in the past and, additionally, allow to estimate the quality of the journals in which the studies have been published. This development work led to the introduction of the "Degrees of Publication" (DoP) method in a standalone publication ("Publication II"), which also included a thorough literature review for the 16 selected medicinal plant species from the Greater Mpigi region (Schultz et al., 2021a).
The plant samples collected during fieldwork on multiple research stays between 2013 and 2017 were regularly taken to the lab, where a total of 86 different extracts were produced.
The extraction procedures are further described in "Publications III and IV" (Schultz et al., 2020a; Schultz et al., 2021c) and in "Manuscript V." One objective of this PhD research was the creation of a unique extract library of medicinally used species, which I intend to continuously expand over the course of my academic career. Once this library had been established, diverse pharmacological investigations, screening the extract library, were performed in accordance with the use reports given by the traditional healers (in vitro bioactivity assessment).
In the context of the ethnobotanical survey in the Greater Mpigi region, the 16 medicinal plant species were found to be critical to anti-infective traditional medicine practices (in particular, wound infections, skin infections, and symptoms associated with bacterial infections). Therefore, one study involved screening for growth inhibitory activity against a panel of life-threatening ESKAPE pathogens. In addition, the extract library was screened for antivirulence activity against quorum sensing processes in methicillin-resistant Staphylococcus aureus (MRSA), which represents a promising alternative therapeutic, yet non-antibiotic, strategy. The study also involved a library counterscreen for cytotoxicity against human
7 protein output assessment (į-toxin), and the elucidation of putative matches of compounds present in the best-performing extracts via LC-FTMS. In order to realistically assess the results of this study for future advances in drug discovery, multidrug-resistant clinical isolates were used in the experiments. Antimicrobial resistance (AMR) is considered "one of the biggest threats to global health, food security, and development today," potentially affecting anyone of any age around the globe (WHO, 2018). For example, in 2017, MRSA alone was associated with 19,832 deaths and 119,247 severe blood-stream infections in the United States (Kourtis AP, 2019). It is estimated that, by 2050, more than 10 million people will die annually due to AMR and associated multidrug-resistant bacterial pathogens worldwide. This figure is even higher than the current number of annual cancer-related fatalities (O’Neill, 2016). Bacteria can develop and accumulate adaptations that enable them to survive the drug exposure originally designed to eradicate them. AMR develops through the long-term accumulation of these adaptations (Lomazzi et al., 2019), accelerated by the broad deployment of antibiotics (Llor and Bjerrum, 2014). The worldwide consumption of antibiotics increased by 65% between 2000 and 2015, substantially driven by low- and middle-income countries, where the use of antibiotic drugs doubled (Klein et al., 2018). Factors leading to the overuse and misuse of antibiotics in humans, animals, and plants included complacency about the consequences, a fear of bacteria, a lack of knowledge, and the passing of responsibility between physicians, patients, pharmacies, and the pharmaceutical industry (Vazquez-Lago et al., 2012). As a consequence, current antibiotic drug therapies are becoming increasingly inefficient and limited. This is where traditionally used Ugandan plants and the development of novel therapeutic strategies come into play. Part of my research therefore sought to tackle the global health threat posed by AMR in an initial drug discovery study, investigating crude extracts of species medicinally used by Ugandan traditional healers for the treatment of bacterial infections. This study subsequently resulted in "Publication II" (Schultz et al., 2020a).
In a second "bioactivity" study, the 16 medicinal plant species were investigated via in vitro assessment of inhibition of proinflammatory enzymes such as cyclooxygenases (COX) in the human PGH2 pathway. The ethnopharmacological justification for this study was the fact that the Ugandan traditional healers had been cited to use the plants in the treatment of inflammation and related disorders, including pain, redness, heat, fever, wounds, general infections, and even types of cancer (Schultz et al., 2020b). Inflammation is regarded as one of the most important human host defense mechanisms, as it represents the immune system's
8 George et al., 2014). Diverse medical disorders and their pathogeneses are implicated with (over- or chronic) inflammation, potentially leading to tissue damage, failure of vital organs and death (Michaëlsson et al., 1995; Stuhlmüller et al., 2000; George et al., 2014). Mediators of inflammation are involved in various biochemical signaling pathways. A key role is attributed to the COX-2 and COX-1 pathways that describe the biosynthesis of prostaglandin signaling molecules via the precursor prostaglandin H2 (PGH2) (Williams et al., 1999; Ricciotti and FitzGerald, 2011). However, the main human cyclooxygenase isoforms, COX-2 and COX-1, do not both play a major role in inflammatory response. COX-1, which is constitutively present in human cells, catalyzes the biosynthesis of signaling molecules involved in normal, hemostatic functions, e.g., macrophage differentiation, cytoprotection of gastric mucosa, renal blood flow, hemostasis, and regulation of cells (Kurumbail et al., 2001;
Botting, 2006). By contrast, the isoform COX-2 is underexpressed in cells under normal conditions and is rapidly upregulated to elevated levels during inflammation (Botting, 2006;
Cao et al., 2010). The resulting prostanoids include potent proinflammatory mediators that contribute to or induce medical disorders such as pain, swelling, and fever, and are even implicated with allergies, arthritis, stroke, types of cancer, asthma, and Alzheimer's disease (Konturek et al., 2005; Young et al., 2008; Saba et al., 2009; Cao et al., 2010; Ricciotti and FitzGerald, 2011; Schneider and Pozzi, 2011; Bitto et al., 2017; AlFadly et al., 2019; Hashemi Goradel et al., 2019; KuĨbicki and BroĪyna, 2020; Sheng et al., 2020). Commercial painkillers of the group of nonsteroidal antiinflammatory drugs (NSAIDs) are sold and administered globally on a large scale. Examples of NSAIDs are Paracetamol, Aspirin, and ibuprofen, which share the capacity for COX inhibition, thus causing a reduction of pain, fever, and inflammation in the patient. However, these NSAIDs exhibit low selectivity to COX-2, thereby generating various side effects due to unwanted COX-1 inhibition (Pirlamarla and Bond, 2016; Ho et al., 2018; Wongrakpanich et al., 2018; Varrassi et al., 2020). Therefore, both human recombinant isoforms, COX-2 and COX-1, were included in the pharmacological experiments of this study to make the assessment of the selectivity of the strongest COX-2 inhibitors among the plant extracts in the library possible. There is a vital need for discovery of novel antiinflammatory drug leads, and medicinal plants and ethnopharmacological approaches have regained momentum for the treatment of inflammatory disorders (Ghasemian et al., 2016; Shaikh et al., 2016). In addition to screening for potential in vitro antiinflammatory effects of extracts, the study also screened for 12/15-lipoxygenase (12/15-LOX) inhibition activity in the
9 activity against multidrug-resistant strains of Listeria innocua, Listeria monocytogenes, Escherichia coli K12, and S. aureus MRSA, applying a resazurin-based bioassay. In order to help rule out a potential mechanism of action for the COX-2/1 and 15-LOX inhibition activity due to an increased presence of polyphenols and free radical scavengers in the complex extracts, an in vitro antioxidant activity screening model and the determination of total phenolic contents (TPCs) of individual extracts were also included in the experimental design of this study. The results were reported in "Publication IV" (Schultz et al., 2021c).
The next study to investigate the pharmacological effects of the 16 medicinal plants claimed by the traditional healers focused on their antimalarial properties and safety in terms of cytotoxicity and genotoxicity. During the ethnobotanical survey in the Greater Mpigi region, the plant species were additionally found to be frequently used in the treatment of malaria, fever, and related disorders (Schultz et al., 2020b). Although the prevalence of malaria infection and incidence of related clinical treatment have significantly decreased in sub- Saharan Africa in recent years (Bhatt et al., 2015; Snow et al., 2017), malaria remains one of the most severe public health problems in the world (CDC, 2021). In 2020, 5% of the world's malaria cases were reported in Uganda, while the whole continent of Africa accounted for 94%
of the cases (215 million) and 384,000 deaths (WHO, 2020). Human malaria is transmitted through the bites of female Anopheles mosquitoes that are infected with protozoan parasites of the genus Plasmodium (Kotepui et al., 2020). Here, the greatest malaria threat globally is posed by the species Plasmodium falciparum, which is also responsible for 94% of the malaria cases in Africa (WHO, 2020; CDC, 2021). Children below five years of age continue to be the most vulnerable patients, accounting for 274,000 of the malaria deaths worldwide in 2019 (WHO, 2020). The objectives of this study were, therefore, a) the pharmacological investigation of the 16 species regarding their traditional use in treatment of malaria and related fevers by identifying promising candidates from the extract library via the application of a hemozoin formation inhibition screening model. Active hits were subsequently followed up through the assessment of their antiprotozoal effects against chloroquine-resistant Plasmodium falciparum K1. A cytotoxicity counterscreen against human MRC-5SV2 lung fibroblasts was conducted, and selectivity indices were calculated. In addition to the traditional use of the plants against malaria, traditional healers at another of our study sites near the village Nakawuka in Wakiso District indicated that one of the plants, Plectranthus hadiensis, is frequently used as a ritual plant to "prepare young women and teenagers for marriage" and to boost their fertility. Details
10 these Ugandan communities and according to anecdotal reports, health providers observed a high incidence of undiagnosed, rapidly growing large breast masses in young female patients (not necessarily breast cancer). At the study site, breast cancer detection and treatment are scarce due to inefficiencies in the healthcare system and are often unaffordable for the rural Ugandan population (Foerster et al., 2019; Nakaganda et al., 2021). During puberty and shortly thereafter, the development of mammary glands in lifecycle windows represents a window of susceptibility for breast cancer in teenagers and young women due to DNA damage caused by mutagens/carcinogens during the increased proliferation of cells (Davis and Lin, 2011; Macias and Hinck, 2012; Martinson et al., 2013; Natarajan et al., 2020). The frequent ritual use of P. hadiensis in the Nakawuka area could potentially cause a first incident of cell growth perturbation. In the long term, this could facilitate more incidents, leading to tumor growth.
Few studies report plants that are suspected of promoting the formation of breast tumors through growth stimulation by phytoestrogens or mutagenesis (Stopper et al., 2005; Bilal et al., 2014). Therefore, in the study, the potential genotoxic effects derived from the 16 medicinal plants, including P. hadiensis, were preliminarily investigated for the first time (objective b)).
This was accomplished by a genotoxicity assessment of the extract library using a Salmonella reverse mutation bioassay, both without and with metabolic bioactivation after pre-treatment of extracts with human S9 liver fraction (simulation of human liver metabolism). A manuscript, resulting from this study, is currently under review with the journal Frontiers in Pharmacology.
The research was conducted in accordance with the international, national, and institutional recommendations considering the Convention on Biodiversity and the Nagoya Protocol (at the beginning of the project, no active implementation was in place based on the information obtained from our Ugandan collaborators). Collaboration agreements between the universities were signed and are available upon request. Export permits for transfer of plant samples were obtained from the Ugandan Ministry of Agriculture, Animal Industry and Fisheries / Plant Health and Inspection Services. Researchers and academic institutions involved in no way sought to benefit financially from the traditional knowledge shared. Written informed consent was obtained from all study participants. All results from the lab work on plants collected and the survey data was transferred back to traditional healers and local participants. The sixth manuscript therefore addresses an often-neglected issue in ethnopharmacological research and is of particular importance for this PhD thesis. In the past, the intellectual property rights of indigenous peoples have often not been recognized. The
11 international agreements for financial benefit sharing and a nation's sovereignty over its biodiversity (Alexiades and Sheldon, 1996; Heinrich and Jäger, 2015; Heinrich et al., 2018;
Balick and Cox, 2020). However, non-financial benefits, such as the transfer of knowledge in both directions, are poorly defined in these agreements. Unfortunately, ethnopharmacologists still rarely return to the local communities once a study has been completed and published (Maregesi et al., 2007; Schultz et al., 2020b). As a consequence, the extraction of ethnomedicinal information from indigenous communities and subsequent collection of samples under the traditional healer's guidance often marks the end point of a one-sided collaboration. However, the research continues, as samples and data are analyzed, interpreted, and published, potentially leading to unique, significant discoveries, and the results of these studies would certainly be of high interest to the local study participants. This issue has been addressed in the past, e.g., in the book Giving Back - Research and Reciprocity in Indigenous Settings (Herman et al., 2018), and remains a major problem in the field of ethnopharmacology today. During the ethnobotanical survey ("Publication I"), one question was added to the questionnaires, asking the traditional healers about their motivation to collaborate with me, a Western scientist, and with my Ugandan colleague from Makerere University. Moreover, we specifically asked them what they expected from us researchers in this study. Their responses were quite interesting. Although the traditional healers live in rather low socio-economic circumstances, only 5% stated that they would like to benefit financially from the collaboration.
In fact, the three most frequent responses were, in descending order, that they would like 1) to receive scientific support through information on whether the investigated plants actually possessed the claimed medicinal properties; 2) to get feedback on the actual findings of the pharmacological lab studies that followed the fieldwork; and 3) to continue and improve our collaboration (Schultz et al., 2020b). These findings indicate that there is a vital need for feedback and a strong interest in continued collaboration after the study is completed. The transfer of research results back to local study participants usually fosters an equal co-partnership and might even empower them locally (Cordell, 1995; Unander et al., 1995;
Vandebroek et al., 2011). I believe that ethnopharmacologists have the important responsibility to ensure that their collaboration and communication with local study participants is bidirectional. This can be accomplished, for example, by sharing information, knowledge, and scientific results, thereby generating a benefit for both the local informant and the scientist. In this sixth study, a transfer of research results back to the traditional healers who initially
12 The description of this method, providing an example for transferring fieldwork and lab results back to indigenous communities, is presented as a 26-minute video article in this PhD thesis.
The video article uses the same structure as a written scientific article (introduction, methods, results and discussion, conclusions). It is currently under review with the Video Journal of Education and Pedagogy, along with a brief accompanying written manuscript. Once accepted, it will be freely available to the traditional healers and the public via online video platforms.
13 Publication I:
"Ethnobotanical study of selected medicinal plants traditionally used in the rural Greater Mpigi region of Uganda"
Pages: 14-31 Personal contribution
In the following, my personal contribution to the presented study and manuscript is briefly described: I designed the overall strategy of the study and the questionnaires for the ethnobotanical survey. I conducted part of the fieldwork. I contributed to the processing of the survey data and the collection of plant material for future lab analysis. Moreover, I interpreted the majority of the data and wrote most parts of the manuscript. A more detailed author- contribution statement is given in the published article.
Information on publication
This study was published in the Journal of Ethnopharmacology in June 2020 and is available at https://www.sciencedirect.com/science/article/abs/pii/S0378874119332313. Permission by Elsevier to reprint the material at no charge in my thesis was obtained, and reproduction of the article is confined to the purpose for which permission was given.
Schultz, F.; Anywar, G.; Wack, B.; Quave, C.L.; Garbe, L.-A.: Ethnobotanical study of selected medicinal plants traditionally used in the rural Greater Mpigi region of Uganda. Journal of Ethnopharmacology, Volume 256, June 28, 2020
https://doi.org/10.1016/j.jep.2020.112742 Graphical abstract
Contents lists available atScienceDirect
Journal of Ethnopharmacology
journal homepage:www.elsevier.com/locate/jethpharm
Ethnobotanical study of selected medicinal plants traditionally used in the rural Greater Mpigi region of Uganda
Fabien Schultza,c,d,∗, Godwin Anywarb, Barbara Wackc, Cassandra Leah Quaved,f, Leif-Alexander Garbea,c,e
aInstitute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Gustav-Meyer-Allee 25, Berlin, 13355, Germany
bDepartment of Plant Sciences, Microbiology and Biotechnology, Makerere University, P.O Box 7062, Kampala, Uganda
cDepartment of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Brodaer Str. 2, Neubrandenburg, 17033, Germany
dDepartment of Dermatology, Emory University School of Medicine, 615 Michael St., Atlanta, 30322, Georgia, USA
eZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Seestraße 7A, Neubrandenburg, 17033, Germany
fCenter for Study of Human Health, Emory University College of Arts and Sciences, 615 Michael St., Atlanta, 30322, Georgia, USA
A B S T R A C T
Ethnopharmacological relevance: This study provides the first report on selected traditional medicinal plant use, including parts used and methods of preparation, in the Greater Mpigi region of Uganda. This data supports the conservation of local traditional ecological knowledge and will facilitate future drug discovery research.
Aim of the study: Our study aimed to conserve culturally and scientifically-valuable medical knowledge of 16 plant species traditionally used in the Greater Mpigi region in Uganda, namelyAlbizia coriaria,Cassine buchananii,Combretum molle,Erythrina abyssinica,Ficus saussureana,Harungana madagascariensis,Leucas calostachys, Microgramma lycopodioides,Morella kandtiana, Plectranthus hadiensis,Securidaca longipedunculata,Sesamum calycinum subsp. angustifolium, Solanum aculeastrum, Toddalia asiatica,Warburgia ugandensisandZanthoxylum chalybeum. An additional objective of the study was an ethnological investigation of the socio-cultural background and medical understanding of diseases treated by traditional healers in the study area.
Materials and methods: A pilot survey in the study area revealed that 16 plant species were frequently used in treatment of a variety of medical disorders. In order to obtain more complete information, we conducted a broader ethnobotanical survey using structured interviews with 39 traditional healers from 29 villages, speci- fically asking about the traditional uses of these 16 medicinal species.
Results: Results of the survey confirmed a high level of traditional use of these species in the Greater Mpigi region. In addition, various other traditional uses and methods of preparation were recorded, most of them for the first time. In total, 75 different medical disorders treated with the plants were documented.
Conclusions: Conservation of traditional knowledge for future generations is vital, as loss has already been recorded due to multiple causes. The need for novel and more effective drugs derived from natural products is more important than ever, making future studies on herbal remedies both justified and urgently required. The traditional healers surveyed in this project also have expectations of the research – they would like to be updated about any resulting studies into the pharmacological efficacy of medicinal plants so that the research findings can inform their confidence in each herbal remedy.
1. Introduction
Plants have been used traditionally as a source of medicine and natural remedies throughout history by humans across the globe and medicinal plant use is still the predominant form of healthcare services in East and Central Africa (Bussmann et al., 2018;Kigen et al., 2019). In Uganda, four out of five people primarily seek care from traditional healers and previous studies report that there is at least one traditional healer per village (Abbo, 2011;THETA, 2001). Subsequently, Uganda has been reported to have many more indigenous traditional healers than Western-trained doctors. The traditional healer-to-population ratio in Uganda is 1:200 compared to 1:20,000 for Western-trained physi- cians (Abbo, 2011;King, 2002;Tuck and Green, 1997), thus resulting in 100 times as many traditional healers as Western-trained physicians.
Especially in rural areas, these Western-trained physicians are absent, although the WHO recommends a ratio of at least 1 physician to 1000 people (WHO, 2016). On the other hand, traditional healers meet the healthcare needs of most Ugandans in a culturally appropriate manner.
Uganda has a very rich biological diversity deriving from its unique bio-geographical location despite its small size (Kalema and Bukenya- Ziraba, 2005). The East African country boasts seven of Africa's 18 phytogeographical regions (Davenport and Matthews, 1995). This tally is higher than that of any other African country (White, 1983). Its biodiversity includes more than 5000 species of higher plants in the indigenous flora (Hamilton et al., 2016).
Roughly 86% of Uganda's population are predominantly farmers who rely on subsistence agriculture and live in rural areas (Turyahabwe et al., 2013). These farmers are generally poor and 40% live on less than
https://doi.org/10.1016/j.jep.2020.112742
Received 16 August 2019; Received in revised form 2 March 2020; Accepted 3 March 2020
∗Corresponding author. Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Gustav-Meyer-Allee 25, Berlin, 13355, Germany.
E-mail address:Fabien.Schultz@mailbox.tu-berlin.de(F. Schultz).
Available online 26 March 2020
0378-8741/ © 2020 Elsevier B.V. All rights reserved.
T
a US dollar per day, which is below the global poverty line (Turyahabwe et al., 2013). Previous studies amongst the traditional medicine practitioners in our study region indicate they generally have very low levels of literacy and schooling (Adia et al., 2014;Nyamukuru et al., 2017).
Practices of African traditional medicine and application of medic- inal plants vary between different cultures, geographic and climatic regions, and even between neighboring villages. In most parts of Africa, including Uganda, traditional knowledge of healing using plants is transferred orally from one generation to the other and is often never documented (Adia et al., 2014; Nyamukuru et al., 2017). Un- fortunately, much of this traditional knowledge on the medicinal ap- plication of plants has already been lost due to deforestation, economic drift to the cities (rural-urban migration), or Western influence in general (Bussmann et al., 2018). Thus, this first documentation of tra- ditional knowledge concerning the use of selected medicinal plants in the Greater Mpigi region is vital for the conservation of traditional knowledge for future generations.
The aims of this study were two-fold: 1) To document the traditional use of selected medicinal plant species from tropical Uganda, specifi- cally in the Greater Mpigi region; and 2) To undertake an ethnological assessment of the socio-cultural background and medical understanding of diseases treated by traditional healers in the study area.
2. Materials and methods 2.1. Study area
The study was conducted with traditional healers in the Greater Mpigi region of central Uganda–a tropical country located on the northern shores of Lake Victoria, bordered by Kenya to the east, South Sudan to the north, the Democratic Republic of Congo to the west, and Rwanda and Tanzania in the south. Apart from the high biodiversity, Uganda is also characterized by its diversity in terms of ethnic groups (> 50) and languages (Brockerhoff and Hewett, 2000).
The Greater Mpigi region consists of Mpigi, Butambala and Gomba districts. Butambala and Gomba were originally counties within Mpigi district, but were elevated to district status in 2010 (Mpigi-Local- Government, 2019;UBOS, 2012). Mpigi District is located 0.2274° N, 32.3249° E, whereas Butambala is located at 0° 8′ 16 N and 32° 13′ 5 E and Gomba is 0° 12′ 0 N and 31° 45′ 0 E. The population of Mpigi, Butambala and Gomba districts is 273,900, 105,400 and 169,000 re- spectively, according to the Uganda Bureau of Statistics (UBOS, 2018).
The study area lies between 1182–1341 m above sea level and receives heavy rainfall in the range of 1513 mm per annum. The main ethnic group here are the Buganda and the main language spoken is Luganda (UBOS, 2012). The local vegetation is characterized as a tropical, moist evergreen forest/savanna mosaic (Barbour et al., 1987;Howard, 1991).
A total of 39 traditional healers from 29 different villages were in- terviewed. The locations of the villages are shown inFig. 1. The peri- meter in which the 29 villages are located adds up to an estimated total study area of 715 km2. People living in this remote area are highly dependent on medicinal plants and local traditional healers to cover their primary healthcare needs.
Most of the traditional healers visited and interviewed belonged to the Buyijja Traditional Healers Association (BUTHGA), which is af- filiated with an international NGO called PROMETRA that has a branch in Uganda (www.prometraug.com). The PROMETRA Uganda head- quarters is located within the study area in Buwama-Buyija, 67 km from Kampala along Masaka road. The institution is situated on more than 100 acres of forested land. Through the platform created by PROME- TRA, traditional healers meet once a week to share ideas, remedies and learn from each other during workshops.
2.2. Collection of ethnopharmacological data
During preceding pilot study field research expeditions in 2015 and 2016, 16 different medicinal plant species were collected, identified and the traditional use was recorded through three informants, ac- companied by a thorough literature review. All of these 16 species were verbally reported to be highly used medicinally in the rural Greater Mpigi region. In 2018, ethnobotanical interviews were conducted in the study area with 39 informants. The three traditional healers from the pilot study were not among these 39 informants, but all informants were local practicing traditional healers in their respective home vil- lages. The ethnopharmacological survey questionnaires were specifi- cally designed to collect in-depth data on each species (use, parts used, methods of preparation) because for many of these species, this would be the first report of their medicinal use in this particular region of Uganda.
Our research group specializes in ethnopharmacological fieldwork in Africa and subsequent evaluation of traditional use through phar- macological bioassays. Therefore, the questionnaires were designed to:
a) collect specific information on the traditional use in treatment of those diseases where ourin vitromodel expertise lies, namely ma- laria, bacterial infections, antiinflammatory disorders and cancer.
This strategy will guide towards further selection and prioritization of medicinal plants for future pharmacological studies through the ethnobotanical approach;
b) gather data on the totality of ethnopharmacological uses of the studied plants within the study area;
c) assess the general knowledge regarding Western Medicine of the traditional healers in the study area, including understanding of infectious diseases, the concept of microbial pathogens, and cancer.
Listing known infectious diseases contributed to the identification of the culturally most-important diseases;
d) identify the traditional healers' needs and future expectations of our research endeavor and collaboration.
The methodological standards of the survey and the questionnaires were evaluated prior to the field research according to established re- commendations (Heinrich et al., 2009;Weckerle et al., 2018). Before undertaking the survey, written prior informed consent was obtained from all traditional healers participating in the study after explaining to them the study aims and what would be involved in obtaining the data.
The questionnaires were in the English and Luganda language (Appendix A and B Supplementary data). Interviews were conducted in the Luganda language by GA.
2.3. Collection of plant material and identification of specimens
Linking local plant names with collected plant samples is one of the major challenges of ethnopharmacological field research (Bennett and Balick, 2014;Rivera et al., 2014). The 16 selected plant specimens were collected with representative morphological features under guidance of the traditional healers. The collection was conducted following the standard collection procedures (Martin, 2004).
The methodology for plant identification and assignment of scien- tific names was adapted fromWeckerle et al. (2018)in terms of col- lecting specimens for herbarium vouchers, linking plant names given during interviews to plants collected for herbarium voucher prepara- tion, as well as exhaustive collection of plant material and application of visual aids for identification (KEW database). Scientific names were cross-checked withhttp://www.theplantlist.orgon August 11th,2019 and family assignments follow The Angiosperm Phylogeny Group IV guidance (The Angiosperm Phylogeny, 2016). Voucher specimens of all species collected were deposited at Makerere University Herbarium in
2
