UNIVERSITY OF HELSINKI Viikki Tropical Resources Institute VITRI TROPICAL FORESTRY REPORTS 50 Paavola, M. 2012. The impact of village development funds on community welfare in the Lao People’s Kallio, M.H. 2013. Factors influencing farmers’ tree planting and management activity in four case Enass Yousif Abdelkarim SALIH Ethnobotany, phytochemistry and antimicrobial activity of Combretum, Terminalia and Anogeissus species (Combretaceae) growing naturally in Sudan UNIVERSITY OF HELSINKI Viikki Tropical Resources Institute VITRI TROPICAL FORESTRY REPORTS ______________________________________________________________________ – TROPICAL FORESTRY REPORTS contains (mainly in English) doctoral dissertations, original research reports, seminar proceedings and research project reviews connected with Finnish-supported international development cooperation in the field of forestry. _____________________________________________________________________________ Publisher Viikki Tropical Resources Institute (VITRI) P.O. Box 27, FI-00014 University of Helsinki, Finland (address for exchange, sale and inquiries) _____________________________________________________________________________ Editor Markku Kanninen Telephone +358-9-02941 58133 E-mail markku.kanninen@helsinki.fi Website https://www.helsinki.fi/en/researchgroups/viikki-tropical-resources-institute ___________________________________________________________________________ Cover Design Lesley Quagraine ______________________________________________________________________________ Suggested reference abbreviation: Univ. Helsinki Tropic. Forest. Rep. – Ethnobotany, phytochemistry and antimicrobial activity of Combretum, Terminalia and Anogeissus species (Combretaceae) growing naturally in Sudan Enass Yousif Abdelkarim SALIH Academic dissertation for the degree of Doctor of Science (Dr. Sc.) in Agricultural and Forestry under the Doctoral School of Biological, Environmental and Food Sciences, Doctoral Programme in Interdisciplinary Environmental Sciences (DENVI) To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public discussion in Auditorium 235 at the University of Helsinki Info Centre Korona, on Friday 27th September 2019, at 12 o'clock noon. Helsinki 2019 Main Supervisor: Pia Fyhrquist, PhD Division of Pharmaceutical Biosciences Faculty of Pharmacy University of Helsinki Finland Co-supervisors: Professor (Em.) Olavi Luukkanen Viikki Tropical Resources Institute (VITRI) Department of Forest Sciences, University of Helsinki Finland Professor Riitta Julkunen-Tiitto Department of Environmental and Biological Sciences Faculty of Science and Forestry University of Eastern Finland, Joensuu Finland Docent Anna-Maija Lampi Department of Applied Chemistry and Microbiology Faculty of Agriculture and Forestry University of Helsinki, Finland Reviewers: Professor Gabriele König Institute for Pharmaceutical Biology University of Bonn Germany Adjunct Professor Maarit Karonen Laboratory of Organic Chemistry and Chemical Biology Department of Chemistry University of Turku Finland Opponent: Professor Jacobus Nicolaas Eloff The Phytomedicine Programme Department of Paraclinical Sciences Faculty of Veterinary Sciences University of Pretoria South Africa Custos: Professor Markku Kanninen Director, Viikki Tropical Resources Institute (VITRI) Department of Forest Sciences, University of Helsinki Finland ISBN 978-951-51-5420-0 (paperback) ISBN 978-951-51-5421-7 (PDF) ISSN 0786-8170 Unigrafia Oy, Helsinki 2019 2 Abstract A variety of tree species, belonging to the genera Combretum, Anogeissus and Terminalia (Combretaceae) are well known for their uses in African traditional medicine. Many of these species are used in applications for the treatment of infectious diseases and wounds. In this study, twelve species belonging to the genera Anogeissus, Combretum and Terminalia were studied for their traditional medicinal uses in the Blue Nile and Kordofan regions in Sudan. Of these species, Anogeissus leiocarpus, Terminalia brownii and Terminalia laxiflora were selected for in-depth studies on their antibacterial and antimycobacterial effects and phytochemical constituents. The main objectives of this research were (1) to perform ethnobotanical and ethnopharmacological documentation of the traditional uses of the medicinal plants found in the study areas in Sudan, with special emphasize to the species belonging to the genera of Terminalia, Combretum and Anogeissus, (2) to study the antibacterial and antifungal effects of extracts of various polarities, obtained from A. leiocarpus, T. brownii and T. laxiflora (3) to elucidate the chemical structures in extracts with promising antimicrobial activity and (4) to isolate fractions with antibacterial activity using preparative TLC and column chromatography. Three expeditions were made to the Blue Nile and Kordofan regions in south-eastern and south-western Sudan in 2006, 2012 and 2014. The expeditions were performed during the dry and rainy seasons in order to make the species identification of the plants easier. Ethnopharmacological and ethnobotanical information was collected from seven villages. Traditional healers, village elders and other local people in the villages, who belonged to different ethnic groups, were interviewed on their uses of selected medicinal plants, with special emphasis on the plant family Combretaceae. The results of the interviews indicated that three plants could be selected for in-depth studies on their antimicrobial effects and phytoconstituents. Thus, root, stem bark, stem wood and leaf material from A. leiocarpus, T. brownii and T. laxiflora were dried and extracted using conventional extraction techniques, such as Soxhlet methanol and cold methanol extraction as well as sequential and liquidliquid extraction techniques. Moreover, macerations and decoctions in water were prepared according to the ways used by traditional healers. A total of 125 extracts and 11 pure compounds, found to be present in some of the antimicrobial extracts, were subjected to antimicrobial screening, using agar disk diffusion, agar well diffusion, agar dilution method and microplate methods. The microorganisms used for the screenings included the potentially human pathogenic Gram-positive bacteria, Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, Micrococcus luteus ATCC 4698 and Mycobacterium smegmatis ATCC 14468 and the Gram-negative bacteria, Pseudomonas aeruginosa ATCC 27853. In addition the plant pathogenic fungi, Aspergillus niger ATCC 9763, Aspergillus flavus ATCC 9763, Nattrassia mangiferae ATCC 96293 and Fusarium verticilloides (syn. F. moniliformis) ATCC 24378 were used. Various phytochemical extraction and preparative chromatography isolation techniques were used, such as reversed and normal phase thin layer chromatography (TLC) and column chromatography (Sephadex LH-20). The mass spectrometric techniques, including GC-MS, UHPLC/QTOF-MS and LC-MSn Tandem Mass Spectrometry were employed in order to characterize the obtained molecular ions, such as [M]+ and [M-H]-of the various chemical constituents in the antimicrobial extracts. In study I, the ethnopharmacological and ethnobotanical data of the uses of Terminalia brownii, T. laxiflora and Anogeissus leiocarpus in traditional medicine in the villages in Sudan, demonstrated that infectious diseases with diarrhea or cough as symptoms, such as dysentery and TB, as well as wound inflammation and stomach problems are treated with various parts of these plants. Some of these ethnopharmacological uses could be justified by the in vitro antibacterial results of extracts of these plants, demonstrated in this study. Thus, extracts obtained from various parts of Terminalia 3 brownii, T. laxiflora and Anogeissus leiocarpus, showed good activity against the growth of Staphylococcus aureus, S. epidermidis, Micrococcus luteus and Pseudomonas aeruginosa. The most active extracts gave MIC values of 39 μg/ml. The chemical profiling of the most active extracts, such as the methanolic Soxhlet extract of the root of Terminalia brownii, the ethyl acetate extract of the root of T. laxiflora and the stem bark and root extracts of Anogeissus leiocarpus, showed the presence of a high variety of chemical classes, including ellagitannins, gallotannins, condensed tannins, flavonoids and stilbenes. All species were rich in hydrolysable tannins, and especially in ellagitannins. The ellagitannins were detected based on their characteristic UVO absorption maxima spectra, often showing three distinct absorption peaks, and methyl-(S)flavogallonate [M-H]- at m/z 483.0795 and its isomer showing an [M-H]- at m/z 483.0811 were found. In a methanolic Soxhlet extract of Terminalia brownii roots, methyl-(S)-flavogallonate [MH]- at m/z: 483.0795 and its isomer [M-H]- at m/z: 483.0811, were characterized for the first time. In an ethyl acetate extract of Terminalia laxiflora roots, corilagin and its isomer sanguiin H-4, [MH]- at m/z: 633.0750 and 633.0743, respectively, and punicalagin [M-H]- at m/z: 1083.1541, were characterized. The ETs mentioned have not been reported before in T. laxiflora. In a methanolic Soxhlet extract of the root of Anogeissus leiocarpus, we identified for the first time, the stilbenes pinosylvin and methyl pinosylvin. Moreover, five flavonoids were found in the roots or A. leiocarpus. These flavonoids were, aromadendrin syn. dihydrokaempferol [M-H]- 288,0000, ampelopsin syn. dihydromyricetin, [M-H]- 319,1417, taxifolin syn. dihydroquercetin [M-H]303,0365, methyltaxifolin syn. (+)-dihydroisorhamnetin and it is isomer ([M-H]- 317,0678 and 317,0676, respectively). Of these flavonoids, ampelopsin was also found in the stem bark of A. leiocarpus in this study. The mentioned flavonoids have not been reported before in A. leiocarpus. In study II, hydrophilic and lipophilic extracts were extracted from the stem bark and stem wood of Terminalia brownii, using sequential extraction and solvent partition. The hydrophilic ethyl acetate and aqueous extracts demonstrated good growth inhibitory results against the plant pathogenic fungi, Aspergillus niger, Aspergillus flavus, Nattrassia mangiferae and Fusarium verticilloides, with diameters of inhibition zones ranging from 17 – 20 mm and MIC values of 250 – 500 μg/ml. The most antifungal ethyl acetate extracts of the stem bark and stem wood of Terminalia brownii were analyzed for their chemical constituents, using LC-MSn Tandem Mass Spectrometry. This analysis revealed the presence of two stilbenes, trans- and cis-resveratrol-3-Oβ-galloyl-glucoside. These stilbenes have not been detected before in these species, nor in the genus Terminalia. Likewise, the flavonoids, quercetin-7-β-O-di-glucoside, quercetin-7-O-galloylglucoside, naringenin-4′-methoxy-7-pyranoside and 5,6-dihydroxy-3′,4′,7-tri-methoxy flavone were detected in the stem wood of T. brownii. Moreover, gallagic acid dilactone syn. terminalin ([M-H]- at m/z 601) and corilagin and its derivative ([M-H]- at m/z 633) as well as arjunglucoside I ([M-H]- at m/z 725) and methyl-(S)-flavogallonate ([M-H]- at m/z 483; Appendices, Figs. 50, 51, 52) were characterized in the ethyl acetate extract of the stem wood of T. brownii. In study III, seventy-seven extracts of various polarities from the roots, stem bark, stem wood, fruits and leaves of Terminalia laxiflora and T. brownii, were tested for their growth inhibitory effects against Mycobacterium smegmatis ATCC 14468, a model bacterium for tuberculosis. The purpose of this study was also to find the most active part of a methanol root extract of T. brownii, using Sephadex LH-20 fractionation. Moreover, reversed phase preparative thin layer chromatography was used to separate antimycobacterial fractions of an ethyl acetate extract of the roots of T. laxiflora. The lowest MIC value among the extracts was observed for an acetone extract of the root of Terminalia laxiflora (625 μg/ml). In general, large zones of inhibition were observed especially for the polar extracts of the roots of both Terminalia species. Good growth inhibitory effects were obtained with methanol Soxhlet extracts, ethyl acetate extracts and decoctions and macerations of the roots (IZ, 21 –26 mm). The MIC values of the mentioned extracts were 4 moderate, however, ranging from 1250 –5000 μg/ml. In general, the MIC values obtained with polar extracts of T. laxiflora were lower than those resulting from the corresponding extracts in T. brownii. Interestingly, Sephadex LH-20 purification of a methanol Soxhlet extract of T. brownii resulted in a strong increase in the growth inhibitory activity for both resulting fractions. Thus, the acetone wash, which contained a higher proportion of ellagitannins, and especially methyl-(S)flavogallonate and its isomer, gave a MIC value of 62.5 μg/ml. The ethanol wash contained a smaller number of ellagitannins than the acetone wash, but in this fraction, the isomer of methyl(S)-flavogallonate was present in a higher concentration. In addition, a number of ellagic acid derivatives and pure ellagic acid, which were absent from the acetone part, were present in the ethanol fraction. Compared to the crude methanol extract (MIC 5000 μg/ml), also the ethanol wash exhibited increased growth inhibitory activity (MIC 125 μg/ml). The good antimycobacterial effect of the ethanol fraction was thus suggested to be due to a favourable combination of ellagic acid and its derivatives and the ellagitannins in this fraction. In contrast to the Sephadex LH-20 purification, preparative thin layer chromatography of an ethyl acetate extract of the root of T. laxiflora led to a smaller increase in growth inhibitory activity compared to the crude extract (MIC 500 μg/ml for the TLC fraction, Fr5, versus MIC 2500 μg/ml for the crude extract). Compared to the crude ethyl acetate extract of T. laxiflora, the TLC fraction Fr5 contained a higher concentration of corilagin, but less punicalagin. Thus, the growth inhibitory effects of pure commercial corilagin was tested and was found to be quite modest (MIC 1000 μg/ml). Therefore, the improved growth inhibitory activity of Fr5, when compared to the crude ethyl acetate extract could not be due to an increase in the concentration of corilagin alone, but is suggested to be due to synergistic effects of this ET together with other ETs and ellagic acid derivatives in this fraction. In study III it could be demonstrated that in addition to the polar extracts, also hexane extracts of the stem wood and barks of T. brownii and T. laxiflora showed good growth inhibitory effects against M. smegmatis (IZ, 19.5 –21 mm). Thus, the phytochemical composition of the hexane stem bark extracts of both species was investigated using GC/MS analysis. Altogether 30 compounds, including triterpenoids, steroids, long-chain fatty acids and fatty alcohols were characterized. The long-chain fatty acids, 1,18- octadec-9-ene-dioate 18:1 (C18H32O4, tR in GC/MS 8.30 min, MW. 456), tetracosanoic acid 24:0 (C24H48O2, tR in GC/MS 9.23 min, MW. 368.6), hexacosanoic acid 26:0 (C26H52O2, tR in GC/MS 10.45 min, MW. 396.7), and octacosanoic acid 28:0 (C28H56O2, tR in GC/MS 11.94, MW. 424.7) were characterized for the first time in T. laxiflora and T. brownii stem bark. Of these fatty acids, octadec-9-ene-dioate (18:1) was present in high concentration in the hexane extracts of the stem bark of both species of Terminalia, and based on the to date knowledge on the good antimycobacterial effects of some unsaturated fatty acids, this fatty acid could be responsible for a part of the antimycobacterial effects shown by these extracts in our study. In addition, this study revealed the occurrence of the fatty alcohols, octacosanol (C 28H58O, tR in GC/MS 11.18 min, MW. 410.7), triacontanol (C30H62O, tR in GC/MS 12.87, MW. 438.81) and dotriacontanol (C32H66O, tR in GC/MS 14.97, MW. 438.81). These fatty alcohols have not previously been reported in T. laxiflora and T. brownii. We found that triacontanol gave a moderate MIC of 250 μg/ml against M. smegmatis. Thus, the contribution of this fatty alcohol to the antimycobacterial effects of the hexane extracts of T. laxiflora and T. brownii is suggested to be small. Besides the steroid compounds, β-sitosterol (C29H50O, tR in GC/MS 13.50, MW. 486), stigmast-4-en-3-one (sitostenone, C29H48O, tR in GC/MS 14.84, MW. 412) and 5α-stigmastan-3,6dione (C29H48O2, tR in GC/MS 16.96, MW. 428) were characterized. Of these compounds, 5αstigmastan-3,6-dione has not been found in the genus Terminalia previously. We found that βsitosterol gave a MIC of 500 μg/ml against M. smegmatis. However, β-sitosterol has given good 5 growth inhibitory effects against M. tuberculosis in a previous investigation (Saludes et al. 2003). In addition, five triterpenes, β-amyrine (C30H50O, tR in GC/MS 14.14, MW. 498), friedelin (C30H50O, tR in GC/MS 16.20, MW. 426), betulinic acid (C30H48O3, tR in GC/MS 16.32, MW. 600) and two oleanane-type triterpenoids (tR in GC/MS 17.56 and 18.41, MW. 570) were identified in the stem bark of T. laxiflora and T. brownii in this study. Of the mentioned triterpenes, friedelin was found in the highest concentration in both species of Terminalia. Previous authors have found that friedelin is growth inhibitory against Mycobacterium bovis (MIC 4.9 μg/ml). However, we found that friedelin is not very active against M. smegmatis (MIC 250 μg/ml). In conclusion, we have found that the species of Terminalia laxiflora, T. brownii, and Anogeissus leiocarpus possess good antibacterial, antifungal and antimycobacterial activity. These results support their uses in African traditional medicine. Extracts of the investigated plants could moreover be used in traditional agriculture for the protection of sensitive crop plants against fungal contamination. This study also shows that the investigated plants contain a high variety of both nonpolar and polar compounds, which could have further uses as antibiotic scaffolds and adjuvants. Author’s current address: Enass Yousif Abdelkarim Salih Viikki Tropical Resources Institute (VITRI), University of Helsinki, Finland & Faculty of Pharmacy, Division of Pharmaceutical Biosciences University of Helsinki, Finland Email: enass.salih@helsinki.fi Author’s permanent address: Enass Yousif Abdelkarim Salih Department of Forest Products and Industries Faculty of Forestry, University of Khartoum, PO BOX 13314, Shambat, Khartoum-North, Sudan E-mail addresses: enass7@yahoo.com eyabdelkareem@uofk.edu 6 PREFACE This dissertation comprises multidisciplinary research work and was performed at the Division of Pharmaceutical Biosciences, Faculty of Pharmacy, Department of Food and Environmental Sciences, and the Viikki Tropical Resources Institute (VITRI), Department of Forest Sciences at the University of Helsinki, Finland. In addition, some parts of the current research were carried out at the Department of Environmental and Biological Sciences, Faculty of Science and Forestry, University of Eastern Finland, Joensuu and in the Department of Forest Products and Industries, Faculty of Forestry, University of Khartoum, Sudan. One of the main reasons for my motivation to consider this research work was that the study provided fundamental knowledge on alternative aspects for the uses of forest products in Sudan (and other countries in Africa) and, in particular, on the medicinal values of some species of the family Combretaceae as anti-infectives, based on their uses in Sudanese traditional medicine for curing a wide range of infectious diseases. The value of these plants as phytomedicines, especially for the people who live in the vulnerable rural areas of Sudan, was thus elucidated. Secondly, this research added new findings to previous studies that had focused on local ethnopharmacological knowledge as a base for reverse drug discovery and for consideration of plant species that could be considered for further investigations. After many years of hard work and attention that are now bearing fruit, I want to thank my first supervisor, Dr. Pia Fyhrquist, for her contribution with her time and ideas as well as for her help with a project grant (Ekhaga Foundation, Stockholm, Sweden). During this thesis work, she has had an impact for improving my knowledge in the field of pharmaceutical biosciences and built my capacity to support my future career. Thank you, Pia, also for sharing with me an enjoyable and beneficial time during various scientific international conferences and during practical work at the University of Eastern Finland, Joensuu. I am blessed to have had you as supervisor. Thank you for your continuous support to deal with the challenges of doing this research in Finland. I am highly indebted to Professor Olavi Luukkanen for his kind help. Without his moral support this work could not be materialized, and without his coordination of the crucial stages that I faced in 2017, I would not have been able to complete my doctoral research work. Thank you, Prof. Olavi for appreciating my research and astutely encouraging me to improve the weak areas in manuscript drafts. I am grateful to Professor Markku Kanninen for his willingness to be my official responsible professor, who in the distant 2012 saw my research proposal and believed enough in what he read, to accept me as one of his doctoral students, thus becoming my major professor. Many thanks also go to Professor Heikki Vuorela, who facilitated my stay at the Division of Pharmaceutical Biosciences, for performing and completing the practical laboratory work for this thesis. I am grateful to Professor Riitta Julkunen-Tiitto, who treated me with love and patience and from whom I learned the basics of UPLC-QTOF techniques. Her guiding and sense of humor, epitomized by “no niin”) were often needed in stressful situations. I would like to thank Professor Marketta Sipi, who gave me the chance to be part of the International Doctoral Programme in Bioproducts Technology, PaPSaT and to be able to discuss and enrich my work by attending many courses related to my research work at Aalto University. 7 Many thanks are due to Dr. Anna-Maija Lampi at the Department of Food and Environmental Sciences, for her amazing sense of respect; she always gave me a hand to deal with the GC/MS. Thanks for providing me the support when I most needed it. This work is also dedicated to the memory of Professor Raimo Hiltunen (1944-2014), who will remain unforgettable in my thoughts, due to his effective support, mentorship and guidance, especially when he introduced me to Pia Fyhrquist and asked her to supervise my thesis work at the Faculty of Pharmacy. I am also thankful to all the co-authors who helped me with the experimental work and in the writing process of the various publications. I would also like to dedicate this work to Dr. Hiba Abdelrhaman Ali (1966-2016), who taught me the principles of phytochemical analysis. Her outstanding lectures in the field of phytochemical analysis inspired me. Without these lectures, I might not ever had pursued this challenging area of phytochemical analysis, overlooked by so many. Also I would like to thank the deceased Dr. Huda Sharwii who passed away from us in 2011. I am grateful for her efforts, to facilitate and encourage me to be able to accompany my husband, Dr. Mustafa Fahmi, during his research work in Finland and to start my own research work for my doctoral thesis. I am most grateful to the traditional healers and the assistant plant collectors in the Ed-Damazin and Kordofan regions in Sudan, who so kindly gave their time and expertise to complete the primary ethnobotanical survey and provided the basic information for this research work. Special thanks go to Amo Abdella, Mr. Balla Alfadel, Mr. Bashir Abaker Adam, Hateel Al gasim. Similarly, I am grateful to Dr. Abdalla Gaafar, Dr. Abdelazim Mirghani Ibrahim and Mr. Musa Alsafori at the Forest National Cooperation, and to Dr. Abdallah Mirghani Altaybe, Dr. Abdelazim Yassin Abdelgadir, Dr. Zaynab Abdelhamid, Dr. Haytham Hashim Gibreel and Dr. Ashraf Mohamed Ahmed Abd Alla at the Faculty of Forestry, University of Khartoum, and, finally, to Dr. El Sheikh Abd Alla Al Sheikh at Soba Forest Research Center, Khartoum, Sudan. My thanks also go to the family members of Mr. Balla Alfadel and Mr. Bashir Abaker Adam, with whom I spent nice moments during the fieldwork and plant collections. I would like to thank all other colleagues in Sudan, who provided great help, at the University of Khartoum, the International Research Center, as well as at the Forest National Corporation at Khartoum, Kordofan and Ed-Damazin, and at the Forestry Research Center, Soba. My thanks go to all friends in my home country Sudan and in Finland, who gave me confidence and enlightening words of encouragements. I also express my gratitude to the reviewers of this thesis, Professor Gabriele König at the University of Bonn, Germany, and Docent Maarit Karonen, University of Turku, Finland, for their valuable suggestions, and to Prof. Kobus Eloff at University of Pretoria for accepting the invitation to act as the official opponent. Special thanks goes to our skilful and helpful lab-technicians, Tarja Hiltunen and Krista Virtanen. I am very grateful to Senior Software Engineer, Tomas Wilkman for modifying Excel to be suitable for our antimicrobial data analysis. I cannot adequately express how thankful I am to my former and present colleagues at Department of Forest Sciences, VITRI, and Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry and to my colleagues at the Division of Pharmaceutical Biosciences, 8 Faculty of Pharmacy, University of Helsinki. Also many thanks goes to my colleagues at Department of Environmental and Biological Sciences, Faculty of Science and Forestry, University of Eastern Finland, Joensuu. I would like to express my thanks to Tanja Malo, HR at the University of Helsinki and Mervi Leppäkorpi for advice on various issues related to my residence in Finland in 2017. This research would not have been possible to complete in Finland without the financial support of more than three hundred donors, who made my staying in Finland possible through a Mesenaatti crowdfunding campaign initiated by Prof. Olavi Luukkanen. Hereby I also express my deepest appreciation to the practical help offered by Jasmin Etelämäki, Mikko Telttari and Pauliina Seppälä. I would also like to express my gratitude to the University of Khartoum and the Ministry of Higher Education, Sudan, for financial support during the first stage of this research work. I gratefully acknowledge the financial support from Ella and Georg Ehrnrooth Foundation, Mikko Kaloinen Foundation, the Faculty of Agriculture and Forestry, the University of Helsinki, Tiina and Antti Herlin Foundation, and Ekhaga Foundation, Stockholm. I am also thankful for the travel grants I have received for participating in various international conferences and courses granted by FinCEAL Plus, PaPSaT and University of Helsinki. Most importantly, I would like to dedicate this work to the soul of my Father, Yousif Abdelkarim Salih and to my loving Mom, Amna Mohamed Al Faki Ahmed who visited Finland two times to support and help me taking care of my kids during my doctoral research work. I am sure that her special prayers have pushed my work to a successful end. You are extremely amazing and I am so proud of being your daughter. My brothers and sisters, Mohamed, Omima, Nasir, Amel, Mutaz, Eman, Musab and Esra, thanks for all moments we have had as sisters and brothers and for supportive discussions to keep on following my dreams, even though I have been away from you all - I dedicate this thesis to YOU. Finally, I want to thank my husband, Dr. Mustafa Fahmi, for support, encouragement and patience. You have been in my thoughts during this doctoral thesis study, giving me strength. I feel that we both learned a lot about the happiness of life, about commitment and dedication of our life to important causes. Thanks for keeping things going and for always showing how proud you are of me. Many thanks also go to my lovely kids, Tala, Ahmed and Sama, for their tolerance, for giving me extra strength, for letting me organize my limited time and for motivating me to push this work through, even if I have taken too much of their time in order to achieve and reach this stage. Helsinki, June 2019 Enass Yousif Abdelkarim SALIH 9 LIST OF ORIGINAL PUBLICATIONS (CONTRIBUTIONS) This study is based on the following original publications, referred to in the text by their roman numbers. I. Salih, E.Y.A., Kanninen, M., Sipi, M., Luukkanen, O., Hiltunen, R., Vuorela, H., JulkunenTiitto, R. & Fyhrquist, P. 2017a. Tannins, flavonoids and stilbenes in extracts of African savanna woodland trees Terminalia brownii, Terminalia laxiflora and Anogeissus leiocarpus showing promising antibacterial potential. South African Journal of Botany 108: 370 –386. II. Salih, E.Y.A., Fyhrquist, P., Abdalla, A.M.A., Abdelgadir, A.Y., Kanninen, M., Sipi, M., Luukkanen, O., Fahmi, M.K.M., Elamin, M.H. & Ali, H.A. 2017b. LC-MS/MS Tandem Mass Spectrometry for Analysis of Phenolic Compounds and Pentacyclic Triterpenes in Antifungal Extracts of Terminalia brownii (Fresen). Antibiotics 6(4): 2 –18. III. Salih, E.Y.A., Julkunen-Tiitto, R., Lampi, A.-M., Kanninen, M., Luukkanen, O., Sipi, M., Lehtonen, M., Vuorela, H. & Fyhrquist, P. 2018. Terminalia laxiflora and Terminalia brownii contain a broad spectrum of antimycobacterial compounds including ellagitannins, ellagic acid derivatives, triterpenes, fatty acids and fatty alcohols. Journal of Ethnopharmacology 227: 82 –96. In the studies I, II and III, Enass Salih introduced the research idea, performed collections of plant material and ethnobotanical and ethnopharmacological data, as well as antimicrobial testing and phytochemical research. Salih was solely responsible for the field work in Sudan. In study I, Salih presents some of the ethnobotanical results from the field work in Sudan. In the studies I and III, Salih performed antibacterial and antifungal testing under the supervision of P. Fyhrquist. Moreover, in the studies I and III, Salih collaborated with R. Julkunen-Tiitto to analyze and to interpret the phytochemical composition of extracts and fractions of the studied plants as well as the molecular masses of compounds in extracts showing good antimicrobial activities, using HPLC-UV/DAD and UHPLC/Q-TOF MS. Besides, in the study III, Salih collaborated with A.-M. Lampi to analyze the lipophilic compounds using GC/MS. In the study II, Salih and H. Ali collaborated on the interpretation and analysis of the chemical constituents of Terminalia brownii, using Tandem mass spectroscopy (LC/MSn). In the study II, Salih collaborated with M. Elamin, to perform antifungal testing. P. Fyhrquist, O. Luukkanen and A.-M. Lampi have given a major contribution for the drafting of all manuscripts. R. Julkunen-Tiitto, M. Kanninen, O. Luukkanen, A.M. Abdalla, H. Vuorela, M. Sipi, M. K. Fahmi, A.Y. Abdelgadir and M. Lehtonen have made additional contributions to the manuscripts. 10 LIST OF MAIN ACRONYMS AND ABBREVIATIONS AAMPS; African Association for Medicinal Plants Standards ATCC; American type culture collection AU; Absorbance units BSTFA; N,O-bis (trimethylsilyl) trifluoroacetamide, with formula CF3C[=NSi(CH3)3]OSi(CH3)3 CITES; Convention on International Trade in Endangered Species CLSI; Clinical Laboratory Standards Institute DBH; Diameter at breast height DHHDP; dehydrohexahydroxydiphenic acid DPPH; 2,2-Diphenyl-1-picrylhydrazyl, chemical formula C18H12N5O6 ESBL; Extended-spectrum beta-lactamase ET; Ellagitannin FAD; Food and Drug Administration FNC; Forest National Cooperation GC/MS; Gas chromatography coupled to mass spectrometry GIFTS; Global Initiative for Traditional Systems of Health HHDP; hexahydroxydiphenic acid HPLC-DAD; High performance liquid chromatography with diode-array detection HTs: Hydrolyzable tannins IC; Inhibitory concentration IFNγ; Interferon gamma IZ; Diameter of inhibition zone in mm Köppen-Geiger climate classification (AW); Tropical wet and dry or savannah climate Köppen-Geiger climate classification (BSh); B (dry) and (S) steppe (h) hot Köppen-Geiger climate classification (BWh); B (dry) and (W) warm (h) hot LCFA; long-chain fatty acids (more or equal to ≥ C16) M+; Molecular ion MBC; minimum bactericidal activity m-DOG; valoneoyl MDR; Multi drug-resistant MFC; Minimum fungicidal activity m-GOD; sanguisorboyl m-GOG; isodehydrodigalloyl [M-H]-; Negative ion resulting from negative ion chemical ionization in mass spectrometry MIC; Minimum inhibitory concentration MRSA; Methicillin-resistant Staphylococcus aureus m/z; Mass-to-charge ratio NK cells; Natural killer cells “a type of cytotoxic lymphocyte critical to the innate immune system” NHTP; nonahydroxytriphenoyl OADC; Oleic Albumin Dextrose Catalase PAHs; poly-aromatic hydrocarbons PBPs; Penicillin-binding proteins ppm; parts per million mass accuracy Rt; retention time Rf; retardation factor SAR; Structure-activity relationship SCFA; short-chain fatty acids (near to C8) SEC; Size exclusion chromatography STRC; Scientific, technical and research commission of the organization of African unity 11 TB; Tuberculosis T cell or T lymphocyte; is a type of lymphocyte that plays a central role in cell-mediated immunity TLC; Thin layer chromatography TMCS; Trimethylchlorosilane with molecular formula (CH3)3SiCl TMS; Trimethylsilyl TMP; Traditional medicinal practitioner UDP; Uridine diphosphate glucose UHPLC/QTOF-MS; Ultra-high performance liquid chromatography coupled to quadrupole time of flight mass spectrometry UPEC; Uropathogenic E. coli VRE; Vancomycin-resistant Enterococci UVOmax.; Ultraviolet absorption spectrum WCMC; World conservation monitoring centre WHO; World health organization XDR; Extended drug-resistant 12 1. INTRODUCTION 1.1. Documentation of African medicinal plants In Africa, considered to be the cradle of mankind, plants have been used as medicines since prehistoric times (Edwards et al. 2012, Mahamoodally et al. 2013, Buckley et al. 2014, GuribFakim 2006). Ebers Papyrus is among the oldest African medical texts, and was written in hieratic Egyptian circa 1550 BC (Malterud 2017, Sreeramu 2004, Petrovska 2012). This extensive text on a papyrus scroll includes 800 prescriptions, based on 700 plant species as the main ingredients (Allen 2005, Sreeramu 2004). In addition, the ancient Egyptian civilization produced a number of other medical texts, such as Cahun Gynaecological Papyrus from ca. 1800 BC and Edwin Smith Papyrus from 1600 BC (Vargas 2012, Smith 2011). In the medieval Islamic age, the Arabic culture contributed with their own Materia medica on African medicinal plants, the The Book of Healing (Kitab Al-Shifa), which was written by Ibn Sina (Latin; Avicenna) ca. 980-1037 AC. The Book of Healing has been acknowledged as one of the forty most significant medicinal texts worldwide (Rahman and Kabir 2017, Bennison 2014, Fyhrquist 2007). In addition to this book, Avicenna also wrote The Canon of Medicine (El-Qanun fi al-Ṭibb). These books are still used in Africa, both in primary healthcare and amongst traditional healers (Moosavi 2009, Buranova 2015). In addition to these historical texts, a number of modern books have also been written on African traditional medicine. Examples of these are the Handbook of Medicinal Plants, which documents the medicinal uses of over 1000 medicinal plants in Africa (Iwu 1993) and The African Pharmacopoeia volume 1, which describes African traditional medicine and 96 plants and drugs used in many African countries. The later book have been published by Scientific, Technical and Research Commission of the Organization of African Unity (STRC) in 1985 (Hostettmann et al. 2000). The most recent book, The African Herbal Pharmacopoeia, describes the botanical, commercial and phytochemical information of 50 plant species that are regarded as the most important medicinal plants in Africa (African Association for Medicinal Plants Standards, AAMPS 2010 and Brendler et al. 2010). Despite of these books, there is a still a large number of African plant species, especially south of the Sahara, which have not been studied comprehensively, nor for their traditional uses or biological activities and pharmacologically active compounds (Gurib-Fakim 2006). This is due to the ethnopharmacological knowledge existing predominantly as verbal information, as well as to the vast number of plant species in Africa with an estimated 40,000 – 45,000 species growing on the continent (Gurib-Fakim 2006). Moreover, many of these species are endemic, with their occurrence restricted to certain areas in Africa (Iwu 1993). To date, only 5000 species are known to be used as medicinal plants in Africa (Hostettman 1998, Gurib-Fakim 2006, Mahomoodally 2013). The accelerating loss of various forest habitats in Africa due to anthropogenic activity and the erosion of traditional medicinal knowledge warrants for a thorough documentation on medicinal plants before this information is lost (Gurib-Fakim and Mahomoodally 2013). 17 1.2. The significance of plants in African traditional medicine and in medicinal plant trade In Africa, depending on the country, up to 70 – 90 % of the population relies primarily on traditional medicines with plants as the main ingredients for their primary healthcare (GIFTS 1997, Mahomoodally 2013, Benzie and Wachtel-Galor, 2011). The high dependence on traditional medicine has been suggested to be due both to cultural and socio-economic reasons, as well as to the plant-based remedies being readily available and affordable to the people (GIFTS 1997, Mahomoodally 2013). This can be exemplified by Tanzania, where 60 % of the people consult a traditional healer (TMP, traditional medical practitioner) as their first point of contact in case of illness (Fyhrquist 2007). The importance of traditional healers in many African countries is also reflected by their integration and promotion in the official health care systems (Mahomoodally 2013). In addition to the rural regions, even in urban regions in Africa people commonly prefer and rely on traditional medicine (Gurib-Fakim 2006, GIFTS 1997, Gurib-Fakim and Mahomoodally, 2013). The national and regional trade of medicinal plants in Africa is increasing, stimulated by rural unemployment, rapid urbanization and population growth and thus an ever increasing demand for herbal remedies in the big cities (Kayombo et al. 2013, GIFTS 1997). This trade is affecting especially those plant species which are popular medicinal plants and therefore at risk for overexploitation, and thus also risking the direct incomes of traditional healers. In addition to this trade, an international trade also occurs, focused on a limited number of species. When it comes to various species of trees, their logging and their export for extraction of pharmaceuticals, both inside and outside Africa, is competing with their uses by TMPs for traditional medicine. In order to benefit the local people, such as the traditional medicinal practitioners and their communities, the collections of medicinal plants must be controlled and restricted and resource replacement costs should be payed to the local people. The sustainable use of medicinal plants in Africa is of importance even globally to preserve valuable genetic material for future research on chemical lead compounds (GIFTS 1997). 1.3. African plants as sources of (new) bioactive compounds and extracts Natural products and structures derived from natural products continue to play a significant role in drug discovery and development (Newman and Cragg 2016). Molecular structures from natural products show a high diversity in terms of the number of chiral centers in the molecules as well as the combination of heteroatoms, thus increasing the chances of finding lead molecules with promising biological activities (Reayi and Arya 2005). However, although higher plants are known to contain a variety of antimicrobial compounds, none of the 71 antimicrobial agents derived from natural products which were developed between 1981 and 2014 were based on molecular structures from botanical sources (Newman and Cragg 2016). African plants are sources for a high diversity of secondary compounds, many of which might be potential leads for the development of new antibiotics and other medicines. Thus, it is likely that new remedies will result from extracts and compounds of these plants. The high diversity in secondary compound chemistry in African plants is reflected in the high biodiversity of species, especially in various forest habitats, such as rain forests and savanna woodlands (Mahomoodally 2013). Secondly, it has been suggested that plants in tropical and subtropical environments, such as in Africa, would be forced to produce and accumulate a high diversity and content of secondary 18 compounds throughout the year, as defense compounds against UV radiation, pathogenic microorganisms, insects and herbivores, as well as for attraction of pollinators and as signal compounds (allelochemicals) (Mahomoodally 2013, Gurib-Fakim and Mahomoodally 2013). Thus, African plants would perhaps contain a higher quantity and quality of secondary compounds than plants growing in non-tropical regions (Abegaz et al. 2004, Mahomoodally 2013). However, in spite of this large diversity and potential, only a small number of drugs originating from African plants have been commercialized globally, and none of these drugs are antimicrobials (http://www.aamps.org). 1.4. Approaches to select plant material as sources for biologically active compounds When selecting plant materials as sources for new phytomedicines (extracts) and/or new active compounds, ethnopharmacological information is often of crucial importance. It has been estimated that 74 % of the pharmacologically active, plant-derived compounds have been found with the guidance of ethnobotanical information (Sheldon et al. 1997, Atanasov et al. 2015). Thus, plants which are used for the treatment of infectious diseases and their symptoms in traditional medicine, are likely to contain antimicrobial compounds. The chance of finding biologically active extracts and compounds in plants is also increased by using a phylogenetic approach. In this approach, the plant species, which are related to each other, such as species belonging to the same genus or family, are chosen for the biological screenings, since it can be expected that they contain chemically related compounds (Dounias 2005, Vuorela et al. 2004, Gullo 2013). 1.5. The threat of multi-resistant bacterial and fungal infections warrants for the search of new antimicrobials New antibiotic resistant bacterial strains are continuously emerging, such as multidrug-resistant and extensively-drug resistant Mycobacterium tuberculosis (MDR-TB and XDR-TB), penicillinresistant Streptococcus pneumoniae (PRSP), methicillin-resistant Staphylococcus aureus (MRSA), pan-drug-resistant Acinetobacter and Pseudomonas (PDR), as well as vancomycin-resistant enterococci (VRE) (Ventola 2015). Tuberculosis (TB) is affecting millions of people in Africa, and due to HIV the proportion of antibiotic resistant strains is especially high, compared to other continents (WHO 2010). Currently, the number of candidates in the drug pipeline for the development of new anti-TB and other antibacterial drugs is insufficient (Marrakchi et al. 2014, Zumla et al. 2013). Natural compounds derived from higher plants could inhibit the growth of bacteria with different mechanisms of action compared to conventional antibiotics (González Lamothe et al. 2009, Cowan 1999). Thus, the risks of development of resistance could be minimized. Recently, there has been a revival in the interest to develop new antibacterial agents from natural sources (Dashti et al. 2014, Santhosh and Suriyanarayanan 2014, Shilpi et al. 2015). To date, there are only four classes of antifungal agents available to treat human fungal infections. These include polyenes, azoles, echinocandins, and pyrimidine analogues, all of them originating from fungal sources (Carmona and Limper 2017). At the same time, globally, 1.3 million deaths result from systemic fungal infections (Drummond and Brown 2011). Serious systemic fungal diseases are often associated to other diseases and infections and are results of immunosuppression due to HIV infection, cancer and aging (Faini et al. 2015, Xiao et al. 2014). Cryptococcal meningitis, resulting from infection with Cryptococcus neoformans and systemic candidiasis, 19 mostly resulting from Candida albicans and Candida glabrata infections are among the most common systemic fungal diseases. Moreover, people with chronic respiratory illnesses such as cavitary tuberculosis and cystic fibrosis are at risk to develop systemic Aspergillus infection (Denning et al. 2011). Extracts and compounds from medicinal plants used to treat fungal infections in traditional medicine could increasingly be used as sources/scaffolds for new antifungal compounds (Hostettmann and Marston 1994, Aqil et al. 2010). Fungal infections in plants, due to Aspergillus, Fusarium and Nattrassia species, amongst others, affect the production of staple crop plants, such as maize, potato and rice, and both pre- and postharvest damages are common worldwide (Salih et al. 2017b). Synthetic fungicides such as Vinclozolin and Ziram, which are harmful to the environment and health, are still in use in some African and Asian countries (Chippaux et al. 1996, Archer and van Wyk 2015, Melvin et al. 2018), although they were prohibited by UNEP/FAO in 2004 (Ioakeimidis, United Nation, 2004). Moreover, phytopathogenic fungi have developed resistance to many synthetic fungicides (Hahn 2014). Plant-derived extracts and compounds, such as volatile and other compounds with antifungal properties, could serve as new, natural fungicides (Milano and Donnarumma 2017, Stenberg et al. 2015, Pereira et al. 2012). 1.6. Aims of the study The aim of this study was to A) perform ethnobotanical documentation of the medicinal uses of taxonomically selected species of the genera Terminalia, Combretum and Anogeissus in Sudan and to B) study the antibacterial and antifungal effects of extracts of the plant specimen collected in Sudan, and to C) study the phytochemical composition of and molecular structures of compounds in extracts and fractions showing promising antimicrobial activities, and D) finally, to isolate fractions and compounds with good antimicrobial activity. Terminalia laxiflora, Terminalia brownii and Anogeissus leiocarpus were selected on the basis of the ethnopharmacological study, as well as based on previous literature showing that especially their polyphenol chemistry and the biological activities of these polyphenols have not been studied sufficiently. Moreover, extracts of some other species of the studied genera have shown promising antimicrobial effects and are known to produce a high variety of (new) antimicrobial compounds (Eloff et al. 2008). The goals of this study have been achieved as follows: 1) The plant material used for this study was collected during three expeditions to Blue Nile and Kordofan regions in Sudan, each of them lasted more than one month in 2006, 2012 and 2014. During these expeditions, a number of interviews were performed on the uses of the collected plants in traditional medicine. 2) Crude methanol extracts and extracts of various polarities, obtained using sequential extraction as well as decoctions and macerations, of which the two last mentioned preparations are common in Sudanese traditional medicine, were studied for antibacterial and antifungal effects using human pathogenic and phytopathogenic strains (I, II and III). 20 3) In an attempt to find fractions and compounds with antibacterial and antifungal activity, preparative TLC (RP-18) and column chromatography (Sephadex LH-20) were used (I and III). 4) Extracts and fractions showing especially promising growth inhibitory activities were chosen for phytochemical investigations, using GC-MS, HPLC-DAD, UHPLC/QTOF-MS and LCMS/MS tandem mass spectrometry as the main methods for the identification of the molecular structures of the compounds. Special emphasis was put in the identification of ellagitannins in the studied species, since this group of hydrolysable tannins are especially common in the investigated plants but yet not sufficiently studied. Moreover, some flavonoids, triterpenoids, stilbenes and fatty acids were examined further, since they have not earlier been reported to occur in the studied species. (I, II and III). 21 Sudanese medicinal plants and their antimicrobial activity and phytochemistry Ethnobotanical and ethnopharmacological documentation of Sudanese medicinal plants. Evaluation of antimicrobial activity of extracts and compounds from the ethnobotanically selected species of Terminalia, Combretum and Anogeissus against bacteria and fungi causing infectious diseases. Identification of antimicrobial compounds from the active extracts of the studied spp.. Study (I) Human pathogenic bacteria Antibacterial effect of Terminalia and Anogeissus extracts. Model bacteria: Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis (MIC 39 μg/ml). UHPLC/QTOF-MS for structure elucidation of polyphenols. Study (II) Filamentous fungi Study (III) Tuberculosis (TB) T. brownii extracts affecting the storage and growth of crops in Africa. Extracts, fractions and compounds of T. brownii and T. laxiflora tested against M. smegmatis. Extracts tested against plant pathogenic fungi: Aspergillus niger, Aspergillus flavus, Nattrassia mangifera and Fusarium moniliforme. Sephadex purification of a root extract of T. brownii resulted in significantly increased activity (MIC 62.5 of an acetone fraction vs. MIC 5000 μg/ml for the crude root extract). Tandem mass spectrometry for structure elucidations of stilbenes, flavonoids, triterpenes and polyphenols. UHPLC/QTOF-MS and GC/MS for structure elucidation of polyphenols and non-polar compounds Figure 1. General framework of this study. The studies have focused on issues illustrated by the above flowchart. Images source: Software CS, ChemDraw professional, Featuring SciFinder, https://www.dreamstime.com/ and https://www.vectorstck.com/ and personal lab work images. 22 1. LITERATURE REVIEW 1.2. The plant family Combretaceae The pantropical plant family Combretaceae consists of an estimated 600 species and twenty genera (Tan et al. 2002), including Anogeissus, Combretum, Conocarpus, Guiera, Laguncularia, Lumnitzera, Pteleopsis and Terminalia, all of which are found in Africa. The genera Combretum and Terminalia are the largest ones, with a total number of 250 - 370 and 200 - 250 species, respectively (Masoko and Eloff 2006; Wickens 1973, Mbwambo et al. 2011, The Plant List, 2010). In the African mainland, 140 species of Combretum and 30 species of Terminalia have been distinguished (Maroyi 2012, Mosango 2013). In addition, the total number of officially accepted species within the genus Anogeissus is nine (The plant List 2010) and only one species, Anogeissus leiocarpus (syn. leiocarpa), is native to Africa (Jansen and Cardon 2005). 2.1.1. Botanical description and geographical occurrence of Terminalia, Anogeissus and Combretum species used in this study 2.1.1.1. Terminalia laxiflora Engl., English name: Yellow pod Terminalia, Sudanese Arabic name: Adrot or Subag Terminalia laxiflora is a deciduous tree, reaching heights of 15 – 22 m (Hassan et al. 2013). The crown and branches are spreading widely around the stem, with some of the branches growing in a low position around the trunk. The bark is deeply fissured, blackish to dark or brownish grey and inner bark is thick and fibrous with a pleasant scent. Twigs are brownish or dark grey, purplish to brown and glabrous when sprouting. The leaves are simple and arranged spirally and the leaf blade is elliptic-obovate. The white and scented flowers are arranged in axillary spikes, up to 12 cm long, and flowering from February to March in Sudan (Fig. 2, Mosango 2013). The fruits are winged nuts (samaras) with two dark yellow papery, mostly semi-lanceolate wings. Fruiting occurs twice a year from May to June and August to November (Table 2). The morphology of the fruits and the characteristic, fissured bark are the main features to distinguish T. laxiflora from the closely related T. brownii (Salih, field observations). T. laxiflora is widely distributed in the Sahel region and grows in a belt (the Sahel belt) from Senegal and Gambia in West Africa to Sudan, Ethiopia and Uganda in East Africa (Fig. 4A). It is extremely drought resistant and can occur in semi-arid and arid regions, such as in shrubland and bushland areas of the Kordofan and Blue Nile regions, with an annual rainfall of 701 and 684 mm, respectively (https://en.climate-data.org), (Gorashi 2001, FAO 2010, Munyua and Mbiru 2011, Hassan et al. 2013). T. laxiflora also occurs in deciduous woodland and bushy grassland, and in Ethiopia it occurs in areas with an annual rainfall of 1400 – 2000 mm. In addition, T. laxiflora is common in gallery forests along rivers, and it can occur on both well and poorly drained soils such as loam, sand, clay and Gardud (an Arabic Sudanese name) soils (Mosango 2013). Gardud means sand and clay soil (Elgubshawi 1995). 23 2.1.1.2. Terminalia brownii Fresen, English name: Red pod Terminalia, Sudanese Arabic name: Alshaf, Adrot or Subag Terminalia brownii is a tree, reaching heights of 13 –25 m. The crown and branches are spreading widely around the stem. The leaves are simple and arranged spirally with elliptic-obovate blades. The bark is blackish and longitudinally fissured. The flowers are white to cream colored and arranged in axillary spikes, scented and glabrous. The flowering period occurs from April to June. The fruits are samaras (winged nuts), with two purplish red wings and mostly semi-ovate in shape (Fig. 3). The fruiting period occurs in October to November (Mosango, 2013). T. brownii is widely distributed in the African Horn, in East and Central African countries, such as Cameroon, Central African Republic, Chad, Djibouti, Eritrea, Ethiopia, Kenya, Nigeria, Somalia, Sudan, Tanzania, Uganda and Zaire (Fig. 4B), with a mean annual rainfall ranging from 500 –1300 mm (Mosango 2013). T. brownii is drought resistant and occurs in semi-arid and arid regions, in shrubland and bushland vegetation in Kordofan and Blue Nile states. Moreover, T. brownii is also common in deciduous woodland, wooded grassland and gallery forests, and is commonly growing on various types of soils, such as rocky, sand, loam and Gardud soils (Wickens 1973, Bosch and Gurib-Fakim 2006). Both mentioned species of Terminalia are dependent on bushfires for their germination (Zida 2007, Dayamba 2010). According to Hassan et al. (2013), the germination frequency of T. laxiflora seeds is very low, and thus this species is vulnerable. 24 B A C E D Figure 2. (A) Terminalia laxiflora Engl. & Diels.; (B) bark is deeply fissured, showing an anastomosing pattern; (C) leaves; (D) fruits are yellow; (E) inflorescence. Photos: E. Y. A. Salih and H. H. Gibreel 2006. A B C D Figure 3. (A) Terminalia brownii Fresen; (B) bark is longitudinally fissured, and is not forming an anastomosing pattern as in T. laxiflora. (C) fruits are dark brown; (D) leaves and inflorescence. Photo: E. Y. A. Salih and H. H. Gibreel 2006. 25 (A) (B) Desert Bush-grass savanna (Semi desert) Rainforest Savannah woodland Coastal Forest Figure 4. Geographical occurrence of Terminalia laxiflora (A) and T. brownii (B) in Africa. Source: African Plant Database. 26 2.1.1.3. Anogeissus leiocarpus (DC.) Guill. & Perr. syn. A. leiocarpa, A. schimperi, Terminalia leiocarpa, Conocarpus leiocarpus, Conocarpus schimperi, English names: Chew-stick, African birch. Sudanese Arabic name: Al-Sahab A. leiocarpus is an evergreen or occasionally, deciduous tree with a slightly grooved, strait stem which can reach diameters of up to 1 m at breast height. It reaches heights of 15 –18 m in Sudan and Senegal, and even 30 m in moister woodlands in Nigeria. The crown is variable with uneven branches, branching from low positions on the stem and the branches are drooping. The bark is grey to dark brown and relatively soft compared to Terminalia and Combretum species, typically scaly, fibrous, and flaking off in rectangular patches and producing a gum exudate. The leaves are arranged alternately to nearly oppositely and lack stipules. They are simple and entire with ovateelliptical blades, covered densely with silky hairs when young. Flowers are greenish yellow and fragrant. The fruit is a semi-globose (rounded) samara with a dark brown to yellowish brown color and with a short beak (Fig. 5) (Jansen and Cardon 2005). In Sudan, A. leiocarpus flowers from September to November and fruiting occurs between August and January (Table 2). A. leicarpus is a drought and salt resistant tree and it is highly competitive, producing allelochemicals, killing species of grass. Its area of occurrence extends from Nigeria and Senegal in West Africa to Sudan and Ethiopia in East Africa (Fig. 7A). In Sudan, A. leiocarpus is distributed widely in the west, east and south (El-Amin 1990). A. leiocarpus occurs in various habitats, ranging from deserts and dry savannas to wooded bushlands and moist woodlands, such as gallery forests with a mean annual rainfall ranging from 200 –1900 mm. A. leiocarpus is very sensitive to fire. However, after the bushfire season, it has the ability to produce new coppice from the surface root (Dayamba 2010, Andary et al. 2005). It can grow on a wide variety of soil types, including Vertisols (clay soils) (Sacande and Sanogo 2007, Burkill 1985). 2.1.1.4. Combretum hartmannianum Schweinf., syn. Poivrea hartmanniana Schweinf., English name: not registered; Sudanese Arabic name: Al-Habeel C. hartmannianum is a deciduous tree, reaching heights up to 25 m with a diameter of 70 –80 cm at breast height (DBH), and thus, it is amongst the tallest and thickest of the African Combretum species (El-Mahi 1990, El-Amin 1990, Herrmann et al. 2003). The crown and branches are spreading widely and the characteristic shape can be distinguished from a long distance. The leaves are arranged alternatively and sometimes opposite on glabrous branches. They are simple, ovate and petiolate with characteristic, extremely long tips, which are nearly as long as the leaf blade itself (Fig. 6). The bark is whitish grey, smoothly fissured. The flowers are small and greenish white and arranged in small axillary spikes on pubescent branches. The flowering occurs in January to February. The fruits are samaras with a greenish color and with four wings. Fruiting occurs in April to May (Fern et al. 2016). C. hartmannianum has a limited occurrence in northeastern Africa, where it grows in Sudan, SouthSudan, Ethiopia and Eritrea (Fig. 7B). It typically grows in wooded grasslands and in CombretumTerminalia woodlands, both on alluvial and on loamy soils (Ken Fern 2014, Walter and Gillett 1998 UNEP-WCMC). Increasing human activity and expansion of agricultural practices have contributed to its decline, and to date, C. hartmannianum is on the IUCN red list of threatened species (UNEP-WCMC, Walter and Gillett 1998). In common with many other of the Combretum species growing in savanna biomes in East Africa, Combretum hartmanninum is relatively fire resistant, and its fruits need regular bushfires to release their seeds in order to germinate (Dayamba 2010) 27 B A D C Figure 5. (A) Anogeissus leiocarpus (DC.) Guill. & Perr. (B) scaly bark; (C) leaves and flowers; (D) leaves. Photos: E. Y. A. Salih and H. H. Gibreel 2006. A C B D E Figure 6. (A) Combretum hartmannianum Schweinf.. (B) Bark rough and finely fissured; (C) samara fruits; (D) leaves; (E) flowers. Photos: E. Y. A. Salih and H. H. Gibreel 2006. 28 (A) (B) Desert Rainforest Bush-grass savanna (Semi-desert) Savannah woodland Coastal Forest Figure 7. Occurrence of Anogeissus leiocarpus (A) and Combretum hartmannianum (B) in Africa. Source: African Plant Database. 29 2.2. Commercial uses and traditional uses of African species of Terminalia, Combretum and Anogeissus 2.2.1. Uses in medicinal plant trade and in traditional medicine and for other traditional applications in Africa 2.2.1.1. Trade African Terminalia and Combretum species are traded and exported within various African countries and to Europe, USA and Asia for many medicinal purposes. For instance, the bark of Terminalia sericea, one of the 50 most important medicinal plant species in Africa (Nair et al. 2012), is exported from Mozambique by MEDIMOC, a government enterprise, with an estimated total amount of 24-25 tons/ year, and the main buyers are German companies (GIFTS 1997, UNIDO 1994). Besides, from Tanzania, the root bark of T. sericea is exported to Indena, a privately owned pharmaceutical company in Italy, where sericoside, isolated from T. sericea, is used to produce anti-aging and anti-wrinkle skin cosmetic creams (http://www.indena.com). Likewise, the bark of Anogeissus leiocarpus has contributed to reduce poverty among the local people of Koro village in Burkina Faso, who collect the bark from the wild, to be used by cosmetic and pharmaceutical companies in France for producing various cosmetic products (Andary et al. 2005). For example, the company of Givaudan France Fragrances SAS, produces a cosmetic agent from bark extracts of A. leiocarpus, which is commercially known as ELLAGI-C (https://www.givaudan.com). This agent improves the condition of the skin and has been approved and certified by Ecocert (Certification body for sustainable developments) (www.cosmosstandard-rm.org). Besides, in Mali, local communities produce traditionally dyed “Bogolan cloth”, using the tanninrich plant extracts of the stem bark of Terminalia macroptera and the decoction of the leaves of A. leiocarpus. For each 20,000 m2 of the dyed cloth 6000 kg of leaves from A. leiocarpus is required (Blanchart et al. 2010, Limaye et al 2012, Andary et al. 2005). In many western and central African countries, such as Ivory Coast, Mali, Burkina Faso, Senegal, and Niger, Bogolan cloth are sold mainly by women on local market. However, in late 2004, the Bogolan industry started to flourish, and many African companies started to invest in Bogolan craft, including Groupe Bogolan Kasobane in Mali and ADT (Aissa Dione Tissus) in Sengal, while the demand increased in Europe, Japan, USA and Canada (Lemmens et al. 2012, Andary et al. 2005, https://www.contemporaryafrican-art.com/african-textiles.html). In West African countries where A. leiocarpus is absent, the leaves of Combretum glutinosum is used in the Bogolan industry, and the Bogolan fabric workshops are mainly run by women and are considered culturally important (Marquet 2005, Jansen and Cardon 2005, UNESCO 2016). In Sudan, the stem wood and roots of Terminalia laxiflora and Terminalia brownii are sold in the domestic market as fuelwood and for cosmetic purposes as “burning wood for smoke and incense or Dokhan” for the softening, lightening and enhancing of the skin appearance (Fig. 29). 30 2.2.1.2. Patents resulting from African medicinal plants African plants are sources for a number of effective therapeutic drugs and cosmetic products, which either are extracted directly from the plants or synthesized by mimicking the molecules found in the source plants (Table 1). Catharanthus roseus (Apocynaceae), which is endemic to Madagascar, but also introduced to Ghana, Nigeria and Congo, was originally found to be effective for the treatment of diabetes, but later some effective anti-leukemia alkaloids were discovered from this plant. C. roseus has been commercialized, and drugs from this plant have been developed by many companies worldwide, such as Sigma Aldrich (USA), Eli Lilly (US) and AdooQ BioScience (in Canada and USA) (Stoianoff 2004). The two most important dimeric alkaloids patented from Catharanthus roseus for use against leukemia and Hodgkin's lymphoma are Vinblastine (C46H58N4O9) and Vincristine (C46H56O10), which generate sales of US $ 100 –180 million/year (Stoianoff 2004, Nejat et al. 2015). Among Combretum and Terminalia species, some of them originally from Africa, there are a number of patents for cosmetical products and drugs. Combretum caffrum (Combretaceae), a native species to South Africa, was discovered by the American scientist, G.R. Pettit, as a source of stilbene compounds, the combretastatins (Pettit et al. 1987, 1988, 1989 and 1995). These combretastatins inhibit the formation of new blood vessels in cancer tumours (Pettit et al. 1995). To date, the pharmaceutical companies, of Oxigene (Nottinghamshire, England) and Bristol-Myers Squibb (New York, USA) develop vascular disrupting agents (VDA) from the phosphate salt of combretastatin A-4, which are used for the treatment of solid tumours (Michael 2000, Malik 2017, Zhou et al. 2018). In addition to C. caffrum, also Combretum kraussii is a source of anti-tumour combretastatins, and thus an Italian research group has patented combretastatin derivatives from this plant. In the West-African species, Combretum micranthum, rare piperidine-flavan-alkaloids have been found, which have been patented for their uses for the treatment of diabetes. In addition, extracts from Terminalia cattappa (Combretaceae) were patented due to their contents of antiwrinkle compounds that were found to cosmetically enhance the skin (Renimel et al. 2002). Likewise, Terminalia sericea, which is an endemic tree to Southern and East Africa, was patented due to the triterpene compound, sericoside (syn. Arjunglucoside I, C36H58O11), which is exclusively traded by the Italian pharmaceutical company, “Indena” (Shackleton et al. 2011). The new pipelines of antimalarial compounds, the artemisinin, which were discovered by the Chinese Nobel Prize winner, Professor Tu Youyou, originated from various species of Artemisia (Asteraceae). Of these species, A. judaica grows nauturally in North Africa and the Middle East and is used in the Libyan traditional medicine to treat diabetes. Moreover, the most important source plant for artemisinin, A. annua, was used traditionally for thousands of years in China to treat malaria and a wide range of infectious diseases (Ellman et al. 2010, Alkangar et al. 2015). Crude extracts of A. annua and other Artemisia spp., contain artemisinin, a sesquiterpene lactone (C15H22O5), which has been patented and commercialized globally by Phytopharm for the treatment of chloroquine resistant malaria (United Kingdom) (Barnett 2006, MC Gown 2006, Connolly and Constenla 2009). Last century, A. annua was introduced in many African countries, which had an epidemic malaria situation, such as Kenya, Madagascar, Uganda, Sudan and Tanzania. This occurred especially after the discovery of the active core of artemisinin in 1972 by Prof. Tu (a nobel prize winner in 2015) and after the official approval of artemisinin for malaria treatments in 1986 (Callaway et al. 2015). Species belonging to the genus Cassia (Fabaceae) are native to East, Northeast, Southern, and West-Central Africa. These species produce laxative agents, the Sennosides A and B (C42H38O20) and Danthron C14H8O4 in their leaves. 31 Pygeum africanum syn. Prunus africana (Rosaceae) has a wide occurrence in Africa and is known from the central, eastern and southern parts of Africa, including Madagascar and some other islands near the African continent. Its extracts are used for the treatment of urinary tract infections and prostatic disorders in African traditional medicine (Dawson et al. 2000, Neuwinger 2000, Stewart 2003, Komakech et al. 2017). The extracts of P. africana mainly contain the fatty compounds of n-docosanol, lauric acid, myristic acid, β-sitostenone and sitosterol. The African traditional application of P. africana has been patented by Debat in 1974. Subsequently, in the late 1990s, the global market of P. africana stem bark was estimated to be US $ 220 million/3225 metric tons (Chupezi and Ndoye 2006, Bodeker et al. 2014). In 1995, France, Germany and Spain imported 3190 tons of naturally growing Prunus africana from African countries such as Cameroon, Madagascar, Kenya, Zaire and Uganda (FAO 1997). Since the invention of an anti-prostatic extract from P. africana, several companies, such as Madaus (Germany and Spain), Prosynthese (France) and Indena (Italy) began to produce an alternative phytotherapeutic agent-based on P. africana extract (Bodeker et al. 2014). Recently, P. africana has been classified by the Convention on International Trade in Endangered Species (CITES) to belong to one of the most endangered tree species in Africa (Keehn et al. 2016, Stewart 2003). Pausinystalia johimbe (Rubiacaeae) or Yohimbe is a native tree to western and central Africa, and extracts of its stem bark are used as aphrodisiacs, as mild hallucinogens, and for the treatment of impotency in West African traditional medicine. The stem bark contains indole alkaloids, and of these alkaloids, yohimbine (C21H26N2O3) was patented for the treatment of erectile dysfunction (Omar 2000). Moreover, yohimbine was found to be useful in veterinary medicine as a reverse anaesthesia to promote the wake up process after animal surgery (FDA 2013). In many African countries, the exportation of the stem bark from naturally growing P. johimbe is increasing annually. For example, in 1998, Cameroon alone exported the total amount of 286 ton stem bark to France, Holland, Germany, Luxembourg and Belgium (GIFTS 1997), and the total price of this export was estimated at US $ 600.000 for that year (Clark and Sunderland 2004). Physostigma venenosum, the Calabar bean (Fabaceaeae), is a perennial climber, reaching 10 –15 m in height. This poisonous climber is native to Sierra Leone, Zimbabwe, Mozambique, Nigeria, Gabon, Zambia and Congo. The plant is a source to the alkaloid, physostigmine (C15H21N3O2), used for treatment of glaucoma and as an agent for reversing the influence of drugs and toxins causing the anti-cholinergic syndrome (reduction in cholinergic activity in the central nervous system). Until the last century, the total export of the fruits from wild populations of P. venenosum from African countries, such as Ivory Coast and Nigeria, to Germany was estimated to be 70 – 80 ton/year (GIFTS 1997). In 2002, the global market for the sales of plant-based drugs was valued at US $ 30.7 billion, with a yearly increase of 6.3 % (Nirmal et al. 2013). In general, more than 5.000 compounds, originating from medicinal plants, have been patented at global trade offices, with a total cost of US $ 150 million (Randeep 2009). Of these compounds, more than 122 are classified and approved as therapeutic drugs (Atanasov et al. 2015, Taylor 2000, Farnsworth et al. 1985). In addition, more than fifty compounds have been registered as active drugs synthesized from molecular models occurring in the source plants (Gurib-Fakim 2006). North African countries and sub-Saharan Africa have been reported to produce 29 plant-derived agents (Boer 2008). 32 Table 1. Patents resulting from African medicinal plants belonging to the plant family Combretaceae. Plant species name Terminalia Terminalia chebula Patent No., inventors and assignee, year of patent Native or introduced to African countries Description of patent Chinese patent CN 107929411; inventors: Nagong, Bilige; Assignee: Peoples Republic of China, 2018 Introduced and cultivated in African countries, such as Ivory Coast, Congo and Tanzania Ointment for treating grasserie (virus disease of silkworms). Terminalia chebula Indian patent IN 201641031883 and WO 2018051363; inventors: Polachira,Suja Kizhiyedath, Vidya, Anu Geetha, Jayalekha, Reshmi Nair Rajagopal;Assignee: HLL Lifecare Limited,India, 2018 Pharmaceutical gels against candidiasis. Terminalia bellerica Chinese patent CN 107823486; inventors: Rong, Guifu; Assignee: Peoples Republic of China; 2018 Introduced to mainland Africa, Ivory Coast, Uganda and Tanzania Oral tablet for preventing chronic sore throat Terminalia sericea Japanese patent 2016210755; Inventors: Ueda, Koji; Assignee: Narisu Cosmetic Co., Ltd., Japan, 2016 Native to South Africa, Kenya, Tanzania and Congo Essential oils as cosmetic liquid and for prevention of grey hairs. Terminalia sericea Italian patent 5166139; inventors: Ezio Bombardelli, Gianfranco Patri Roberto Pozzi; Assignee: Indena, 1992 and Italian patent 6475536; Inventors: Ezio Bombardelli, Paolo Morazzoni, Aldo Cristoni, Roberto Seghizzi, Assignee: Indena, 2002 Glycosidic triterpene (saponin) identified from the root and stem bark part as anti-inflammatory agents. Combination of saponin, triterpene aglycone and phospholipids in pharmaceutical and cosmetic applications for treatment of the skin. Terminalia sericea Japanese patent 3908245 and JP 2006045157; inventors: Nakaoji, Koichi; Tonokaito, Hiroshi; Sakai, Kaoru; Hamada, Kazuhiko; Assignee: Pias Corp, Japan 2006 and 2007 Oily composition from the stem bark to stimulate hair growth and preventing Alopecia. Terminalia sericea Italian patents WO 9817293 and US 6267996; inventors: Bombardelli, Ezio; Cristoni, Aldo ; Morazzoni, Paolo; Seghizzi, Roberto 1998 and 2001. Assignee: Indena, Italy Lipophilic extracts containing anti-inflammatory saponins for anti-acne use. Terminalia catappa Japanese patent 2005053864; inventors: Aniya, Yoko; Inafuku, Morio; Nakamoto, Katsuo. Assignee: Ryukyu Bio-Resource Development Co., Ltd., Japan; Nakazen Yakusho Nojo Y. K. 2005 Introduced to West and East African costal area Food additives from the stem, rhizome, trunk, leaf and flower to treat diabetes and to reduce cholesterol. Anogeissus leiocarpus United kingdom GB 2543822 and WO 2017071822; inventor: Bell, Michael David, Sitaram, Aneshkumar, Dineshchandra, Wibawa, Judata Iskandar, Darma, Tomlinson, Paul James. Assignee: The Boots Company PLC, UK, 2017 Native from east to west Africa in the sahel belt Lipophilic extracts for skin care. Anogeissus leiocarpus Japanese patent JP 2016183137; inventors: Fujita, Takashi; Assignee: Ritsumeikan University, Japan; 2016 Hair growth enhancer agent. Anogeissus leiocarpus Chinese patent CN 103254210; inventors: Su, Liuhua; Wan, Dongmei; Assignee: Method for preparing castalagin of high purification level. Anogeissus 33 Nanjing Zelang Agriculture development Co., Ltd., Peop. Rep. China; 2013 Anogeissus leiocarpus United states patent WO 2012050745, CA 2812111, Inventors; Daly, Susan M.; Declercq, Lieve; Assignee: ELC managment LLC, USA 2012 Skin care composition. Anogeissus leiocarpus Australian patent WO 2010111745; Inventors: Wolf, Florian. Assignee: Jurlique international Pty Ltd, Australia 2010 Method for delaying skin aging and for enhancing the uptake of vitamin C. Combretum micranthum United states patent US 8642769, US 20130143921 and WO 2011140066. inventors: Simon, James E.; Wu, Qingli; Welch, Cara; Assignee: USA; 2014, 2013 and 2011 Native in West African countries such as Benin, Senegal and the Gambia Piperidine-flavan alkaloid as anti-diabetic agent. Combretum caffrum Chinese patent CN 102838461; inventors: Liu, Dongfeng; Assignee: Nanjing sorun Herbal Technology DevelopmentCo., Ltd., Peop. Rep. China 2012 Native to Southern Africa, also found in East Africa in Tanzania Method to produce a high yield of combretastatin. Combretum cafrum United states patent US 4996237; inventors: Pettit, George R.; Singh, Sheo B; Assignee: Arizona Board of Regents, USA 1991 Combretum kraussii Italian patent WO 9405682; inventors: Pelizzoni, Francesca; Colombo, Roberto; D'Incalci, Maurizio; Verotta, Luisella; Assignee: Italy 1994 Combretum Identification of the combretastatin A- series. Combretastatin A-4P capsules, tablets, and powder for treatment of leukemia and human colon cancer. Now in clinical trials. Native to East and Southern Africa 34 Combretastatin derivatives as anti-tumor agent. 2.2.1.3. Traditional medicine Terminalia species In many southern, western and eastern African countries as well as in the Sahel region of Africa (a semi-arid region forming a belt from eastern to western Africa and situated between Sahara in the north and the Sudanian savanna in the south), Terminalia spp. are used to treat traditionally diagnosed infectious diseases. In Guinea, Terminalia macroptera and T. albida are used against diarrhoea, dysentery and dermatophytic fungi (Traoré e t al. 2015, Silva et al. 1997). Likewise, in Tanzania and Mozambique, T. kaiserana. T. sambesiaca, T. spinosa, T. stenostachya and T. sericea are used for a variety of infections, including tuberculosis (Moura et al. 2018, Fyhrquist et al. 2014, Fyhrquist et al. 2004). In Nigeria, Kenya, Congo, Eritrea and Ethiopia, Terminalia laxiflora and T. brownii are used against symptoms related to microbial infections, such as diarrhea, fever as well as for bacterial vaginosis and sexually transmitted diseases (Abraham et al. 2016, Machumi et al. 2013, Muraina et al 2010, Mbwambo et al. 2007). Combretum species In the Sahel region and in many East African countries, Combretum spp. are used customarily against fever, diarrhea, cough and other symptoms of infectious disease. For instance, in Guinea, Combretum micranthum, which is among the 50 most important African medicinal plants (http://www.aamps.org) is used against tuberculosis (Welch 2010), while, C. glutinosum is used against dermatophytic fungi and bacterial infections (Traoré et al. 2015). In Mozambique, the decoction of various parts of C. zeyheri and C. hereroense are used against abdominal ulcers, diarrhea, cough, Chlamydia symptoms and wounds (Moura et al. 2018). In Cameroon, Togo, Tanzania, Niger and Ethiopia, C. apiculatum, C. collinum, C. constrictum, C. fragrans, C. molle, C. padoides, C. psidioides, and C. obovatum are likewise used against traditionally diagnosed infectious diseases, such as bacterial and fungal infections (Manzo et al. 2017, Wondmnew and Teshome 2016, Vroumsia et al. 2015, Batawila et al. 2005, Fyhrquist et al. 2004, Fyhrquist et al. 2002). Anogeissus leiocarpus In Burkina Faso, Nigeria, Niger, Ivory coast and Ghana, decoctions of various parts of Anogeissus leiocarpus are used against abdominal pain, ulcers, skin infectious diseases, fungal infection, for bacterial vaginosis and for wound healing (Anyanwu and Okoye 2017, Muraina et al. 2010, Andary et al. 2005). 2.2.1.4. Brief insight into the uses of Terminalia and Combretum spp. and Anogeissus leiocarpus as medicinal plants in Sudan There are no records on the yearly consumption of Terminalia, Anogeissus and Combretum spp. as medicinal plants in Sudan. At the local level, herbalists collect various parts of medicinal plants from the forests in the vicinity of their homes and sell them in the local market places as remedies to cure a wide range of the diseases (Fig. 8). In Sudan, the various parts of T. laxiflora, T. brownii, A. leiocarpus and C. hartmannianum are used against eye infection, skin inflammation, stomach, diarrhea, venereal infections, dental caries, fever and related symptoms and as anti-dandruff agents 35 (Ali et al. 2017, Mohieldin et al. 2017, Salih et al. 2017a, Mariod et al. 2014, Osman et al 2014, Albagouri et al 2014, Ali et al. 2009, Eldeen and Van Staden 2007, El Ghazali et al. 1997 and 2003). Plant beverages Health Food Food Food Insects for food (beekeeping) Plants for primary health care (hospitals, health care centers) Traditional Drugs Forest Food and medicine for animal Plant derived herbicides and insect repellents Drug Modern Drugs (synthetic and modified molecules based on plant-derived compounds) Forest for cosmetics Forest-Based Products Environmental conservation”Desertification” Timber, construction wood Drugs from higher plants (Drug molecules obtained directly from plant) Forest-based agriculture For grazing Pulp and paper Fuelwood Figure 8. Patterns of woody plant utilization modified from FAO 1999 and Riewpaiboon 2003, in Singh, A. (Ed.) 2011. 2.3. Antimicrobial extracts and phytochemicals in African Terminalia, Combretum and Anogeissus species 2.3.1. Extracts Phytochemical research and antimicrobial screenings of African species of the genera Terminalia, Combretum and Anogeissus (Combretaceae) has rapidly increased during the last decades (Cock 2015, Mann et al. 2008). Extracts and pure compounds from a number of species have been found to give promising antimicrobial activities (Kuete et al. 2010). African Combretum and Terminalia species as well as Anogeissus leiocarpus have been found to contain a large variety of secondary compounds, including ellagitannins, ellagic acid derivatives, pentacyclic triterpenes, stilbenes (including the combretastatins), phenanthrenes, fatty acids and oils, flavonoids and the rare piperidine-flavan alkaloids, and some of them have been demonstrated to possess promising antimicrobial effects (Kim et al. 2018, Makhafola et al. 2017, Jaroch et al. 2016, Cao et al. 2010, Katerere et al. 2003, Welch 2010). Thus, among the mentioned chemical groups, promising new antimicrobial lead molecules might be found. Several in vitro studies have validated the traditional uses of decoctions and macerations of Terminalia, Anogeissus and Combretum spp. against infectious microorganisms. Most of these in 36 vitro studies have validated crude extracts and fractions, whereas a smaller number of studies also included investigations on the phytochemical profiles and even on pure compounds in extracts showing promising activity. For example, the stem bark of Terminalia superba, which is used in traditional medicine as a maceration against bacterial infections, was found to be growth inhibitory against a wide range of bacteria and fungi, including Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Shigella dysenteriae, Pseudomonas aeruginosa, Salmonella typhi, Citrobacter freundii, Candida albicans, Candida glabrata, Microsporum audouinii, Trichophyton rubrum, Mycobacterium smegmatis and Mycobacterium tuberculosis with MIC values ranging between 19.53 – 78.12 μg/ml (Kuete et al. 2010). Extracts of the stem bark of T. sericea were found to be active against a multidrug-resistant strain of M. tuberculosis (MIC < 100 μg/ml) (Green et al. 2010). Likewise, Mann et al. (2008) found that root extracts of Anogeissus leiocarpus and Terminalia avicennioides were active against the filamentous fungi Trichophyton rubrum, Aspergillus niger, Penicillium spp., Aspergillus fumigatus and Microsporum audouinii, with MIC values ranging between 0.03 – 0.04 μg/ml. Moreover, the crude methanol extracts of the macerated leaves from Combretum adenogonium and the stem bark of Anogeissus leiocarpus were found to be relatively active against Escherichia coli, Staphylococcus aureus and Micrococcus luteus, with MIC values ranging from 1.25 – 10 mg/ml (Nounagon et al. 2017). Examples of some investigations which also resulted in the phytochemical profiling of active extracts are presented below. Terminalia chebula is not native to Africa, but is common as an introduced species in many African countries (Jansen and Cardon 2005). In vitro studies showed that ethanol and acetone extracts of its fruits exhibited significant growth inhibitory activity against fungi and bacteria, and the active extracts were found to be rich in ellagitannins (Joshi and Aneja 2009). Moreover, T. chebula was found to suppress the growth of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) (Lipińska et al. 2014, Sato et al. 1997). Methanol and water extracts of Terminalia mollis and Combretum adenogonium were found to give moderate activity against bacterial and fungal growth, with MIC values ranging from 500 –1000 μg/ml (Maregesi et al. 2008). Decoctions of the root of Terminalia macroptera are used in Guinean traditional medicine against gastric ulcers and infectious diseases. Accordingly, when tested in vitro against Helicobacter pylori, this decoction gave a MIC value of ˃200 μg/ml (Silva et al. 2012). Moreover, Terminalia macroptera root extracts have shown good antimicrobial activity against Shigella dysentheriae, and four ellagitannins were identified in the active extracts (Silva et al. 1997). In addition, extracts of the leaves of T. macroptera were found to inhibit the growth of Neisseria gonorrheae (Silva et al. 2002). Also, the stem bark of T. macroptera was found to give good growth inhibitory activity against bacteria and fungi, and the active extracts contained triterpenes that were found to give very low MIC values (2.5 – 5 μg/ml) (Conrad et al. 1998). All these studies justify the uses of various species of Terminalia and Combretum and Anogeissus leiocarpus for the treatment of topical and internal bacterial and fungal infections in African traditional medicine. 37 2.3.2. Phenolic compounds The term "Polyphenol" should be used to define plant secondary metabolites derived exclusively from shikimate-derived phenylpropanoid and/or the polyketide pathway (s), featuring more than one phenolic ring and being devoid of any nitrogen-based functional group in their most basic structural expression (Quideau et al. 2011). 2.3.2.1 Hydrolysable tannins Hydrolysable tannins (HTs) are polyphenolic, water-soluble compounds that precipitate proteins and other macromolecules in aqueous solutions (they bind to proteins at their phenolic hydroxyl groups, mostly with hydrogen bonds), and their occurrence is restricted to dicotyledonous plants (Okuda et al. 2011, Salminen and Karonen 2011). Hydrolysable tannins are classified into simple gallic acid derivatives (five or less galloyl groups), gallotannins (six or more galloyl groups) and ellagitannins (Fig. 9). HTs are thought to be defense compounds against oxidative stress, herbivores and microorganisms (Quideau 2009, Tanaka 2009, Barbehenn et al. 2009). Ellagitannins and gallotannins, both of which comprise significant structural diversity, are two subgroups of HTs (Salminen and Karonen 2011, Constabel et al. 2014). Ellagitannins Chemistry Ellagitannins (ETs) are a structurally diverse group, and their molecular structures are distinct from all other types of tannin groups (Fig. 9) (Quideau 2009, Salminen and Karonen 2011, Tanaka 2009). To date, over 500 ellagitannins are known from several plant families, the plant family Combretaceae being the fifth richest in these molecules (Lamy et al. 2016, Okuda and Ito 2011, Yoshida et al. 2010). ETs are medium to high molecular weight, water-soluble and hydrolysable polyphenols. Currently, the biosynthesis of ellagitannins is not fully known (Salminen and Karonen 2011). However, the general view implies that ETs are produced from pentagalloyl glucose or from HHDP esters (dehydro-HHDP ETs) by stepwise oxidation and polymerization reactions involving the intramolecular C-C oxidative coupling of spatially closely located galloyl residues to form a hexahydroxydiphenoyl ester unit (HHDP) as well as coupling to a sugar or polyol unit (Fig. 9, 10 , and 11) (Okuda et al. 2009, Yoshida et al. 2010). The HHDP groups can be further oxidized to DHHDP groups. Although the majority of the ellagitannins contain β-D-glucose as their core unit, ellagitannins containing a polyol, caffeoyl glucose and even hydroxytriterpenes as core units also have been identified (Tanaka 2009, Nishimura et al. 1984 and 1986, Jiang et al. 2001, Chen et al. 1999). Ellagitannins are classified into six subgroups according to their subunits (continued Fig. 10): hexahydroxydiphenoyl (HHDP) ester (Fig. 10 A), dehydro-hexahydroxydiphenoyl (DHHDP) ester (Fig. 10 B) and their modifications (Fig. 10 C), nonahydroxytriphenoyl (NHTP) ester (Fig. 10 D), flavonoellagitannins (Fig. 10 E) and their oligomers. The type of bond between the monomers is also important for ETs classification (Fig. 10 F and G) (Salminen and Karonen 2011, Moilanen et al., 2013). For example, the simple ellagitannin, tellimagrandin II, is derived from pentagalloyl glucose and thus contains β-D-glucose as its core unit. Tellimagrandin II is a component in many ellagitannins (Tanaka 2009). Acid hydrolysis of ETs yields sugar, gallic acid and hexahydroxydiphenic acid 38 (HHDP) for HHDP containing ellagitannins (Fig. 11 and12) (Smeriglio et al. 2017, Quideau 2009). The hexahydroxydiphenic acid lactonizes spontaneously to ellagic acid, and thus free ellagic acid is often found in extracts of ellagitannin-rich plants, such as Terminalia and Combretum species (Pfundstein et al. 2010, Uzuner and Acar 2012). However, for ETs in which the HHDP group has been further oxidized, the dehydro-HHDP ETs, ellagic acid is not produced upon hydrolysis (Salminen and Karonen 2011). According to their degree of polymerization and the nature of the oxidative linkages, ellagitannins can be categorized into monomeric ellagitannins (punicalagin, tellimagrandin I and II, chebulinic acid, chebulagic acid, terflavin A and B, sanguiin H-4 and corilagin), all found in Terminalia spp. and oligomeric ellagitannins including dimeric ellagitannins (rhoipteleanin A, cornusiin and sanguiin H-6), macrocyclic dimers and trimers (oenothein B and A) and higher oligomers (pentamers, such as melastoflorins A-D). Moreover, oligomeric ellagitannins contain either C-C oxidative bonds, such as in C-glycosidic ellagitannins with an open chain glucose core (castalagin, casuarinin, casuariin, punicacortein C and D in Terminalia spp. and Combretum spp.) or C-O-C oxidative bonds (gemin A) (Okuda and Ito 2011, Yoshida et al. 2010, Diop et al. 2019). Corilagin forms the primary part of many ellagitannins and dehydroellagitannins, such as in geraniin and chebulagic acid (Okuda and Ito 2011). A special subclass of ellagitannins are the complex ellagitannins (flavano-ellagitannins), which are composed of a C-glycosidic ellagitannin combined with a flavan-3-ol (acutissimin A in Quercus, Castanea and Anogeissus acuminata and the dimeric anogeissusin A and B and anogeissinin in Anogeissus spp. (Continued Fig. 46) (Yoshida et al. 2010). Moreover, the position of the HHDP units on the sugar core lead to either (S) - or (R)-configurations of the ellagitannin molecule (Fig. 13B). The dehydroellagitannins containing a dehydrohexahydroxydiphenoyl (DHHDP) group are an especially stable form of tannins, such as chebulinic and chebulagic acids in Terminalia chebula (Yoshida 2010). Of the ET categories, the C-glycosidic ETs represent an especially interesting and perhaps unique class of polyphenols (Quideau 2009). 39 Tannins Proanthocyanidins (Condensed tannin CTs) Hydrolyzable tannins Flavan-3-ol based Pentagallyol glucose based (Gallic acid as a base or HHDP or DHHDP unit) depside bond depside bond HHDP Phloroglucinol In red brown algae (C-C) or (C-O-C) bond (HHDP) Ellagitannins (ETs) Simple gallic acid dervatives, contained five or less galloyl group Phlorotannins gallotannins (GTs), contained six or more galloyl group Catechin Epicatechin Gallic acid ester of flavonoids DHHDP Modified- NHTP Flavono-ETs Flavono-ETs oligmers DHHDP HTs found in kingdom plantae including green algae Figure 9. Classification of tannins and their subgroups found in the plant kingdom (Quideau 2009 and Quideau et al. 2011). ETs, ellagitannins; NHTP, nonahydroxytriphenoyl; DHHDP, dehydro hexahydroxydiphenoyl; HHDP, hexahydroxydiphenoyl. 40 carbon-carbon (C-C) linkage between the monomers of the C-glycosidic ETs (A) HHDP ester Punicalagin (B) Dehydro-HHDP ester geraniin (C) Modified dehydro-HHDP chebulagic acid m-DOG An ether of (C-O-C) linkage between the monomers of ETs (D) NHTP ester castalagin m-GOG An ether of (C-O-C) linkage between the monomers of ETs (+)-catechin unit (E) A flavano-ellagitannin Anogeissinin A (F) A dimeric ellagitannin with two bonds between the monomers Oenothein B (G) A dimeric ellagitannin with one bond between the monomers Gemin A Figure 10. Chemical classification of ellagitannin subgroups. NHTP, nonahydroxytriphenoyl; DHHDP, dehydro-hexahydroxydiphenoyl; HHDP, hexahydroxydiphenoyl subunits of some oligomerized ellagitannins. Modified from Salminen and Karonen 2011. 41 UDP-glucose + Gallic acid Simple galloylglucoses (simple gallic acid derivatives) Mono GG DiGG TriGG TetraGG dehydro ETs PentGG monomeric HHDP ester gallotannins C-glucosidic ETs m-GOG oligomeric ETs m-GOD oligomeric ETs m-DOG oligomeric ETs Macrocyclic m-DOG oligomeric ETs Continued figure 10. Biosynthetic pathway of ellagitannin subclasses. The biosynthetic pathway of the dehydro ETs starts either from PentaGG or/and HHDP ester. Modified from Engström et al. 2016. ½ O2 (B) Ellagitannins H2O Oxidation -2 H (A) 1,2,3,4,6 pentagalloylglucose Hydrolysis e.g. HCL C-glucosidic oligomeric ETs Tellimagrandin II Lactonization n Ellagic acid -2H2O Hexahydroxydiphenic acid (HHDP) Figure 11. (A), Biosynthesis of HHDP ellagitannins and (B), their hydrolysis to hexahydroxydipenic acid which lactonizes to ellagic acid. (Grundhöfer et al. 2001, Yahia 2017). 42 (A) unit of gallic acid + HHDP (B) galloyl Unit (C) hexahydroxydiphenic acid (HHDP) Oxidation + galloyl group ½ O2 H2O -2 H Oxidation (D) gallagyl (E) valoneoyl unit (m-DOG). An ether of (C– (F) dehydrohexahydroxydiphenic acid O–C) linkage between the monomers of in ETs (DHHDP) (G) chebuloyl (modified dehydroEts) Figure 12. Basic molecular units (structural groups) in ellagitannins. (A), gallic acid oxidating to (B), a galloyl unit and (C), hexahydroxydiphenic acid (HHDP) resulting from the oxidative coupling of two galloyl units. (D), gallagyl functional group. (E), valoneoyl group (m-DOG). (F), dehydro-hexahydroxydiphenic acid (DHHDP) these unit polymerize around a core molecule of sugar (glucose, arabinose, xylose) to form ellagitannins. (Yoshida et al. 2010). 43 ETs in plant defense and their in vitro antimicrobial effects In the plant, ETs occur in both vegetative parts, such as the roots, stem bark, heartwood and the leaves, and in reproductive parts such as fruits and seeds (Koponen et al. 2007, Lee et al. 2013, Constabel et al. 2014). Moreover, ETs are compounds of constant structure and can be found at any season and mode of growth of the plant that is, ETs can always be found in the plants producing those (Okuda and Ito 2011). Lately, it has been observed for ellagitannins, as for other tannins that their synthesis is precisely regulated, and it is restricted to certain cells or tissues in the plant (Constabel et al. 2014). Thus, various organs of a plant may differ from each other to their ellagitannin composition (Salminen and Karonen 2011). It has been suggested that there is a tradeoff between the biosynthesis of ellagitannins and other secondary metabolites, such as flavonoids and condensed tannins, so that cells/tissues which produce a high quantity and quality of ellagitannins do not contain high levels of flavonoids at the same time (Salminen and Karonen 2011). Moreover, young tissues often contain high levels of ellagitannins and mature tissues higher levels of condensed tannins (Salminen and Karonen 2011). Ellagitannins usually occur in high concentrations, like other tannins, and have often been thought to be the major components of the plant constitutive chemical defense (Salminen and Karonen 2011). However, there is some evidence that plants use ellagitannins also for their inducible defense against fungal pathogens. This has been exemplified by the foliar application of a pentameric ellagitannin to strawberry leaves, which resulted in induced resistance against Colletotrichum fragariae (Mamaní et al. 2012). Ellagitannins have been thought to be the biologically active components in many traditional medicinal preparations used for infectious diseases, such as decoctions and macerations (Quideau 2009, Okuda and Ito 2011). Thus, a number of ellagitannins have been investigated in vitro for their antimicrobial properties in order to validate ethno-medicinal preparations scientifically. However, the ellagitannin composition of a decoction does not necessarily reflect that of its plant ingredients, since many ellagitannins undergo hydrolysis during the preparation of the decoction (Okuda and Ito 2011). On the other hand, ellagitannins produced from hydrolysation have been found to give noticeable health effects (Das et al. 1985). Amongst the earliest investigations on in vitro antimicrobial effects of pure ellagitannins was performed by Kolodziej et al. (1999), who reported weak antibacterial effects of ellagitannins, with MIC values of 1000 – 2000 μg/ml against bacteria, but better growth inhibition against fungi. Among one of the most noticeable in vitro antimicrobial effects of ellagitannins is their ability to produce synergistic effects together with known antimicrobial agents (Hatano et al. 2005, Okuda et al. 2009). For example, tellimagrandin I and corilagin were found to decrease the MIC of oxacillin against MRSA (Shimizu et al. 2001). Moreover, ETs from Davidia involucrata were found to inhibit the growth of vancomycin resistant Enterococci (VRE) and methicillin-resistant S. aureus (MRSA), with MIC values varying between 16 – 64 μg/ml (Shimozu et al. 2017). In general, the good antimicrobial activity of ellagitannins is thought to be due to their unique chemical stability and their antioxidative potential (Yoshida et al. 2018, D'Urso et al. 2018, Afshari et al.2016, Lin et al. 1990, Tanaka et al. 1991). The distinctive chemical structures of the (S) (2,3,4,6- and 1,6- HHDP in the 4C1 glycosidic part in ETs) and (R) (3,6- HHDP in the 1C4 glycosidic part in ETs) configurations of the ETs (Fig. 13 B), especially in the alkyl gallate part (Fig. 13 A), make the ETS unique and stable compounds (Quideau 2009). Nonetheless, some oligomers of ETs and ETs with low molecular weights are not stable even in moderate condition (Quideau 2009). However, the antimicrobial mechanisms of action of tannins are still not well-understood (Constabel et al. 2014). Some in vitro structure-activity relationship (SAR) investigations indicate 44 that the antifungal potential of ETs is dependent on the presence of a hexahydroxydiphenic acid (HHDP) moiety, which especially affects the growth of Cryptococcus neoformans (Latte and Kolodziej 2000). Likewise, dehydrohexahydroxydiphenic acid (DHHDP) groups might also determine the antimicrobial potential of ETs (Shiota et al. 2004). According to Shimozu et al. (2017), the presence of galloyl groups make ETs more active against bacteria, whereas they found that ETs containing HHDP and DHHDP groups were less active. Moreover, it has been found that a high number of pyrogallol groups in polyphenols, including some ETs, make them more antibacterial (Marín et al. 2015). Antimicrobial mechanisms of actions of ellagitannins include inactivation of extracellular enzymes, substrate deprivation as well as the disintegration of the bacterial outer membrane and interference with membrane proteins, leading to disruption of the cellular integrity and/or transport (Marín et al. 2015, Constabel et al. 2014). Moreover, it has been found that smaller phenolics, which do not precipitate protein, were as effective as larger polymers, thus suggesting another antimicrobial mechanism of action than protein binding for these compounds (Scalbert 1991). A number of in vitro antimicrobial studies have been made both on ellagitannins, and ellagitannincontaining extracts from species of Terminalia. This genus is known to be a rich source of ellagitannins (Pfundstein et al. 2010) and some of them with promising antimicrobial activities (Silva and Serrano 2015). Tellimagrandin I, punicalin, punicalagin, terflavin, chebulic, chebulagic and chebulinic acid have been identified in Terminalia horrida, T. chebula, T. bellerica and T. calamansai (Pfundstein et al. 2010, Afshari et al. 2016, Lin et al.1990, Tanaka et al 1991, Chen and Li 2006). Moreover, it has been found that a high proportion of Terminalia species contain punicalin and punicalagin, both of which were originally found in pomegranate (Punica granatum) (Lin et al. 1999, Yoshida et al. 2010). Punicalagin derived from the extracts of Terminalia mollis and T. brachystemma revealed growth inhibitory activity against Candida spp., with MIC values from 6.25 – 50 μg/ml (Liu et al. 2009). Punicalagin (α and β), terchebulin and terflavin A were also identified in antibacterial extracts of the root of T. macroptera, which showed good growth inhibitory effects against Neisseria gonorrheae and Shigella dysentheriae (Silva et al. 2000, Silva et al. 2002, Silva et al. 2012, Silva et al. 1997). Muddathir et al. (2013) reported on the occurrence of flavogallonic acid dilactone and terchebulin in the stem wood of Terminalia laxiflora, and these ellagitannins were thought to be responsible for the good antimicrobial effects of this plant against Propionibacterium acnes. Geraniin, which was originally found in Geranium species, has also been found in Terminalia chebula (Lin et al. 1990) and gave good growth inhibitory activity especially against the gram-positive S. aureus and B. subtilis (MIC 25 –100 μg/ml) (Adesina et al. 2000). Besides, geraniin gave moderate antifungal activity against Candida albicans with a MIC value of 1990 μg/ml (Gohar et al. 2003). Punicalagin and punicalin have shown antifungal properties against C. albicans, Cryptococcus neoformans and Aspergillus fumigatus (Marín et al. 2015). Shiota et al. (2004) found that corilagin and tellimagrandin I, both found in various species of Terminalia, suppress the activity of the β-lactam ring degrading enzyme, β-lactamase of methicillin-resistance S. aureus with 55 and 75 %, respectively, compared to an untreated control. A number of ellagitannins, some of which have been found to possess antimicrobial properties, have been found in the genera Combretum and Anogeissus. Some of these ETs are common with those found in Terminalia spp. Terchebulin and flavogallonic acid were found in Combretum hartmannianum and showed MIC values of 500 and 1000 μg/ml, respectively, against the anaerobic, Gram-negative bacterium, Porphyromonas gingivalis (Mohieldin et al. 2017). Combreglutinin and punicalagin were characterized from Combretum glutinosum and C. molle, and punicalagin, isolated from the stem bark of C. molle, was found to possess good antimycobacterial activity, with a MIC value of 600 μg/ml (Jossang et al. 1994, Asres et al. 2001). 45 Grandinin, castalagin (1-epi-vescalagin), castalin (neovescalin), anogeissusin A and B and anogeissinin where characterized in Anogeissus acuminata, whereas only castalagin has been characterized in A. leiocarpus (Fig. 13) (Shuaibu et al. 2008, Lin et al. 1991). Dimeric ellagitannins, such as anogeissusin A and B, have not been found in the genera Combretum and Terminalia (Continued Fig. 46) (Yoshida et al. 2010). Thus, Anogeissus spp. could be sources for ETs with special biological properties. Although, extracts of the roots, stem and leaf of A. leiocarpus have shown remarkable antibacterial and antifungal activities, it was not shown whether the ETs in these extracts could be responsible for these activities (Adigun et al. 2000, Chaabi et al. 2008, Elsiddig et al. 2015, Singh et al. 2016). Moreover, interestingly, a methanol extract of the stem bark of A. leiocarpus, which contains high concentrations of castalagin, was found to quench quorum-sensing in Pseudomonas aeruginosa (Ouedraogo and Kiendrebeogo 2016). Since Adonizio (2008) had earlier demonstrated that castalagin inhibits quorum sensing, it was thought that this ET could stand for part of the anti-quorum sensing effect observed for the A. leiocarpus extract. Metabolism of ETs Tannins have earlier been defined as “toxic molecules that damage the liver” (Robinson and Graessle 1943). However, from the end of the last century (Silanikove et al. 1996a and Silanikove et al. 1996b) and till to date (Hu et al. 2017), this definition has been changed due to the large daily intake of various kinds of foods, such as grapes, blueberry, tea, chocolate, pomegranate and coffee, which are renowned for their rich content of tannins (Al-Rawahi et al. 2013, Ackar et al. 2013). Despite of the good antimicrobial activities that many ellagitannins have shown in vitro, it has been suggested that their in vivo antimicrobial effects would result mainly from their colonic metabolites, rather than from the original compounds, which are poorly absorbed (Manach et al. 2004, Rechner et al. 2002, Cerda et al. 2005). These bioactive metabolites behave as hormone analogs, which empower the immune system against harmful microorganisms (Lipińska et al. 2014). Ellagitannins, ellagic acid and its derivatives are converted to ellagic acid-dimethyl ether glucuronides and different forms of 6H-dibenzo(b,d) pyran-6-one derivatives, such as urolithin and its derivatives (urolithin A, B and C) by the human gut microbial flora in the large intestine (Espín et al. 2013, Fig. 14). The urolithins and their derivatives (Fig. 14) are transferred from the large intestine via the blood circulation system to tissues as well as to the urinary bladder (Piwowarski et al. 2016, Cheng et al. 2017). The ratio of the urolithin production depends on the molecular class of the ellagitannins as well as on the species of bacteria in the microbial gut flora, and thus has been found to vary inter-individually (Saha et al. 2016, Piwowarski et al. 2016). Thus, a high intake of ellagitannins does not necessarily result in a high yield of urolithin metabolites (Cerda et al. 2005, Saha et al. 2016). It has been suggested that the urolithins exert their beneficial health effects mainly through their high antioxidative capacity, and this might imply also for their antimicrobial effects (Okuda and Ito 2011). However, in an earlier ex vivo investigation it was found that, after several days of ETs consumption, a small amount of intact ETs, such as punicalin and corilagin, could be found in the feces and in the blood (Cerdá et al. 2003, Ito 2011, Espín et al. 2013). Thus, these undegraded ETs could play a more significant role than was earlier assumed as antimicrobial agents in vivo. 46 R = H, gallic acid R = CH3, Methyl gallate R = -CH2-CH3, Ethyl gallate R = -CH2-CH2-CH3, Propyl gallate R = -CH2-CH2-CH2-CH3, Butyl gallate R = -CH2-CH2-CH2-CH2-CH3, Pentyl gallate R = - CH2-(CH2)6-CH3, Octyl gallate (A) (B, I) (B, II) C-glycosidic ETs with carbon-carbon (C-C) linkage between the monomers Figure 13. (A), Chemical structure of alkyl esters (gallates) derivatives. (B), (S) and (R) configuration in the ellagitannin of the (I) revised chemical structure and (II) previously proposed structure of Castalagin (R1= H; R2 = OH) and Vescalagin (R1= OH; R2 = H). HHDP in dark-blue color (Edwards et al. 2012 and Matsuo et al. 2014) 47 acid or alkaline hydrolysis ETs HHDP & DHHDP e.g. HCL Alkaline base & colonic microbiota COMT Figure 14. Conversion of ellagitannins to urolithins by the human gut due to hydrolysis and the colonicmicrobial flora. Urolithin M-5 and its derivatives are 6H-dibenzo [b.d] pyran-6-one based. COMT is the enzyme catechol-O-methyltransferase, produced in the human gut, which methylates catechol polyphenols. Source Larrosa et al. 2006, Gonzalez-Barrio et al. 2011, Selma et al. 2017. 48 Ellagic acid and derivatives Chemistry Ellagic acid is a phenolic compound based on the gallic acid molecule and found in the form of free ellagic acid, as methylated and glycosylated derivatives of ellagic acid, and as a chemical constituent of the ellagitannins (De et al. 2018). As shown in Fig. 15, in the strawberry, ellagic acid has been proposed to be produced de novo mainly through the phenylpropanoid and shikimate pathways from gallic acid and glucose, via galloylglucose and di-galloylglucose (Schulenburg et al. 2016, Ossipov et al. 2003). Besides, ellagic acid can also be produced from acid and alkaline hydrolysis of ellagitannins (Fig. 11 and 14). Ellagic acid derivatives are known from a number of Terminalia and Combretum spp., such as Terminalia mantaly, T. brownii, T. laxiflora, Combretum dolichopetalum and C. leprosum. From these species3-O-methyl ellagic acid,3,3′,4-tri-O-methylellagic acid,3,3´-di-O-methylellagic acid4-O-β-D-glucopyranoside, 3-O-methylellagic acid-4´-O-α-L-rhamnopyranoside, 3,3´,4-tri-Omethylellagic acid-4´-O-β-D-glucopyranoside, and 3,3´-di-O-methylellagic acid have been found (Facundo et al. 2005, Uzor et al. 2015, Tchuente Tchuenmogne et al. 2017, Salih et al. 2017a). In vitro and in vivo Antimicrobial effects Ellagic acids and their methylated and glycosylated derivatives have shown antimicrobial effects against Staphylococcus aureus, Pseudomonas aeruginosa (MIC 125 – 250 μg/ml) and Helicobacter pylori (MIC 5 – 30 μg/ml) (Salih et al. 2017a, De et al. 2018). Methyl ellagic acid, isolated from Terminalia macroptera, was found to possess growth inhibition against Helicobacter pylori (MIC 200 μg/ml) (Silva et al. 2012). Moreover, the ellagic acid derivatives, 3,3′-di-Omethylellagic acid 4′-O-α-rhamnopyranoside and 3-O-methyl ellagic acid, isolated from Terminalia mantaly, demonstrate anti-Candida activity against C. parapsilosis, C. albicans and C. krusei with the MIC value ranging from 9-5000 μg/ml (Tchuente Tchuenmogne et al. 2017). In addition, the urolithins, resulting from the conversion of ellagic acid and its derivatives in the human gut, were found to possess antimicrobial activity against a wide range of microorganisms (Wang et al. 2018). In silico investigations have demonstrated that ellagic acid and its derivatives, such as pteleoellagic acid, have a high affinity for the binding site of enzymes responsible for the cell wall biogenesis of the tuberculosis bacterium (Cozza et al. 2006, Shilpi et al. 2015). This could be due to the position of the functional groups, such as hydroxyl groups in these ellagic acid derivatives (Cozza et al. 2006, Joshi et al. 2018). 49 Figure 15. Biosynthesis of ellagic acid in Fragaria vesca (Schulenburg et al. 2016). 50 Simple gallic acid derivatives and gallotannins Chemistry Gallotannins and simple gallic acid derivatives are hydrolyzed to sugar and gallic acid (Smeriglio et al. 2017). Simple gallic acid derivatives are built from one to five galloyl moieties (Continued fig. 10, Fig. 12 and 15) esterified with a sugar residue (usually D-glucose) through depsidic bonds (linkages) (Fig. 16). Gallotannins are made up of six or more galloyl groups (Niemetz and Gross 1999, Salminen and Karonen 2011). The galloylation process can be partial or lead to the galloylation of all hydroxyl groups in the glucose core molecule. Initially monogalloyl-glucose is formed and additional galloylations lead to di-, tri-, tetra-, penta-, hexa- and octagalloylglucoses. Pentagalloylglucose is one of the most common gallic acid derivatives in plants and is usually presented as the gallotannin prototype molecule (Smeriglio et al. 2017). Gallotannins containing other polyols than glucose, such as xylose, fructose and glucitol, shikimic acid, hamamelose and quercitol, are not very common (Smeriglio et al. 2017). Gallotannins have a broad range of structural variation, and the oxidation of their galloyl groups to hexahydroxydiphenoyl acid leads to the production of ellagitannins, as already mentioned previously. For example, the oxidation of pentagalloyl glucose leads to the formation of the ellagitannin, tellimagrandin II (Grundhöfer et al. 2001, Fig.11 A). Gallotannins in plant defense and their in vitro antimicrobial effects Gallotannins are common in woody plants and they can be found in the roots, stem bark, heartwood, leaves, fruits and seeds (Constabel et al. 2014). However, gallotannins are much rarer in plants than ellagitannins (Salminen and Karonen 2011). The in vitro antimicrobial activities of gallotannins (as of other hydrolysable tannins) suggest that they might play a role in plant defense against phytopathogenic bacteria and fungi (Constabel et al. 2014). Traditional medicinal plant remedies and preparations, used against a wide range of infectious diseases are often rich in tannins, and thus also gallotannins (Salih et al. 2017a, Kolodziej et al. 1999, Liu et al. 2011). Thus, the antimicrobial activities of these preparations has often been attributed to gallotannins and other polyphenols (Kolodziej et al. 1999, Liu et al. 2011, PuupponenPimiä et al. 2005). Indeed, recent investigations show that some gallotannins exhibit potent in vitro antibacterial and antifungal activities. Pentagalloylglucose gave promising antifungal and antibacterial activity with low MIC values of 39 μg/ml and 130 μg/ml against Candida glabrata and Staphylococcus aureus, respectively (Torres-León et al. 2017). Many gallotannins which have been identified and/or isolated from species belonging to the family Combretaceae have shown significant antibacterial and antifungal activity. Tri-, tetra- and pentagalloylglucose identified in Terminalia muelleri (Fahmy et al. 2016) exhibited significant activity against methicillin-resistant Staphylococcus aureus and against ceftazidime- and imipenem-sensitive Pseudomonas aeruginosa, with MIC values ranging from 48.8 – 97.6 μg/ml (Kassi et al. 2014). Similarly, 1,2,6-tri-O-galloyol-β-D-glucopyranose, isolated from T. chebula, showed more than 50% inhibition of the biofilm formation activity in multidrug-resistant, uropathogenic E. coli (UPEC) (Bag and Chattopadhyay 2017). Engels et al. (2011) demonstrated that GTs are more effective growth inhibitors of Gram-positive than Gram-negative bacteria. This might be related to the lipopolysaccharide layer of the outer membrane of the Gram-negative bacteria, which could be important for the resistance to gallotannins. Moreover, the antimicrobial activity of gallotannins is related to their ability to bind 51 iron (Cho et al. 2010). Gallotannins have been suggested to be structural analogues to siderophores, small molecular structures in bacteria which sequester iron, and thus gallotannins might compete with these siderophores for bacterial iron sequestering (Engels et al. 2011). Also, it has been found that bacteria lacking siderophores are more susceptible to the growth inhibition of gallotannins and that bacteria growing in iron-supplemented medium are less sensitive to gallotannins (Engels et al. 2011). The antimicrobial effects of gallotannins has also been suggested to be related to the inactivation of membrane-bound proteins (Engels et al. 2011). SAR studies on the antimicrobial mechanism of action of the gallotannin molecule indicate that the number of the galloyl groups in gallotannins correlates significantly with their antimicrobial activity (Vlietinck et al. 1998). For example, it was found that the octa- and hepta-galloylglucose compounds were more active compared to gallotannins composed of one to three galloyl groups (Vlietinck et al. 1998, Engels et al. 2011). Figure 16. Depside bond in gallotannin (hexagalloyglucose). 2.3.2.2. Flavonoids Chemistry Flavonoids have been found to occur in all parts of the plant (Panche et al. 2016). Vegetables, fruits, cocoa, wine and grains are rich in dietary flavonoids. Flavonoids are also found in the bark, roots, stems and flowers (fruits) of medicinal plants (Calado et al. 2015, Panche et al. 2016). The concentration of flavonoids is dependent both on biotic and abiotic factors, such as herbivory, microbial colonization (phytoalexins and others), allelopathy, light, temperature, rainfall and soil type (Bouzoubaã et al. 2016, Majuakim et al. 2014, Panche et al. 2016, Andreote et al. 2014). Flavonoids are phenolic, low-molecular weight and water-soluble compounds with the basic skeleton of C6-C3-C6 (Fig. 17). To date, about 6000 structures of flavonoids are known from various plants (Panche et al. 2016). Flavonoids are classified into six main subgroups and further subgroups, depending on to which carbon atom on the C-ring the B-ring is attached (Fig. 17). Flavonoid subgroups are listed as follows: flavans, flavan alkaloids, flavones, anthocyanins (= flavanolols), leucoanthocyanidin, flavanonones (= flavanone-3-OH), flavonols, flavanol (flavan-3ol), flavanonols (= dihydroflavonols), flavanones (=dihydroflavones), isoflavonoids (= 52 isoflavones), isoflavanone (isoflavan-4-one), dihydrochalcone and chalcones. neoflavonoids, aurones, flavolignans, In isoflavones, the B-ring is linked to position 3 in the C-ring, in neoflavonoids the B-ring is linked to position 4 and in the other subgroups the B-ring is linked to position 2 of the C-ring (Panche et al. 2016). Flavones, such as apigenin and luteolin are common in leaves, flowers and fruits as glucosides. In flavones, a hydroxyl group at position 5 in the A-ring is common. Moreover, for flavones, hydroxylation at position 7 in the A-ring and at 3`and 4`in the B-ring are also common. Flavonols contain a ketone group and are building blocks of anthocyanins. Moreover, flavonols contain a hydroxyl group in position 3 of the C-ring, which can be glycosylated. Kaempferol, quercetin, myricetin and fisetin belong to the flavonols. Flavanones have a saturated C-ring and are thus known to have good free radical scavenging properties. Flavanones are common in Citrus peels and juice. Isoflavonoids, such as genistein and daidzein, are predominantly found in leguminous plants and some of them have even been reported to be present in microbes (Matthies et al. 2008). Isoflavonoids are precursors of phytoalexins, which are important for plant-microbe interactions (Aoki et al. 2000, Dixon and Ferreira 2002). Flavanonols are 3-hydroxylated derivatives of flavanones and the double bond between carbon 2 and 3 in the C-ring, which is present in other flavonoids, is absent from the flavanonols. Anthocyanins are color pigments usually produced in the outer layers of organs, such as in fruit peels, flower petals and barks and their colors are pHdependent as well as dependent on methylations and acylations of the hydroxyl-groups at the Aand B-rings. Pelargonidin, delphinidin and malvidin are examples of anthocyanidins which are found both in herbaceous and in woody plants. In chalcones, the C-ring is absent, and thus they are open-chain flavonoids. Cardamonin, isoliquiritigenin, arbutin, phloretin and phloridzin are chalcones (Panche et al. 2016, Lima et al. 2012, Garcez et al. 2006). 53 Basic structure of flavonoids Flavan Flavone Flavanone Hesperidin Rutin, Apigenin Naringenin Luteolin Pinocembrin Sakuranetin Isoflavone Genistein Daizein Glycitein Flavonol Quercetin Kaempferol Flavanol (Flavan-3-ol) Catechin Proanthocyanidin Epicatechin Isoflavanone Eriotrichin B Ficustikounone Neotenone Isosakuranetin Flavanonone Chalcone (Flavanone-3-OH) Hesperetin Eriocitrin Eriodictyol Narirutin Cardamonin Isoliquiritigenin Anthocyanidin Aurone Dihydrochalcone Flavan-3,4-diol (leucoanthocyanidin) & Anthocyanin Trilobatin Neohesperidine -DHC Leucoanthocyanidin Pelargonidin Malvidin Delphinidin Rosinidin Hispidol Flavolignan Neoflavonoids Piperidine flavan alkaloid Silymarin Isohydnocarpin Arylcoumarin Piperidine Neoflavene flavan Dihydroaryl- alkaloid coumarins Aurone base Apigenin in T. arjuna, T. brownii Genistein in T. arjuna Catechin in T. bellerica Flavanonone base Cardamon in C. apiculatum Isoliquiritigen in T. fagifolia Naringenin in C. albiflorum, T. brownii; Pinocembrin in C. apiculatum; Sakuranetin in T. fagifolia Flavan-3,4-diol base (leucoanthocyanidin) Isohydnocarpin Dihchalcone base Kaempferol in C. hartmannianum,C. erythophyllum and C. apiculatum Isosakuranetin in T. fagifolia T. arjuna pelargonidin, Neoflavonoid base Piperidine-flavan alkaloid in C. micranthum Figure 17. Classification of flavonoids. T. Terminalia; C. Combretum. The basic skeletons of various classes of flavonoids are indicated with different colors: A, blue color: flavan base; B, pink color: flavonol base; C, green color:flavanone base; D, purple color: isoflavonebase; E, red color: flavone base; F, light turquoize color: flavanone base; G, brown color: chalcone base; H, gray color: flavan-3,4-diol (leucoanthocyanidin) base; I, yellow color: diydrochalcone base; J, light blue color: anthocyanidins base; H, orange color: aurone base; I, light purplecolor: flavanonone base; J, dark turquoise color: neoflavonoid base; K, black color: flavolignans. Source: Kshatriya et al. 2018, Martins et al. 2015, Arries et al. 2016. 54 Flavonoids in plant defense and their in vitro antimicrobial effects Flavonoids and other low molecular weight secondary compounds, play a major role in plant communication (plant-plant, plant-herbivore and plant-pollinator interactions) and for plant defense (plant-microbe relations) (Samanta et al. 2011). Plants use flavonoids both for their constitutive (phytoanticipins) and induced defense (phytoalexins) against phytopathogenic fungi and bacteria (Ahuja et al. 2012). Accordingly, flavonoids have been found to possess potent in vitro antimicrobial effects and several authors have reported good antibacterial and antifungal activity both against plant and human pathogenic microorganisms, including MRSA (Iinuma et al. 1994). Flavonoids have been found to significantly affect microbial growth due to their unique chemical structures and their functional groups (Wu et al. 2013). In general, flavonoids have been thought to exert their antimicrobial activity via interactions with extracellular soluble proteins and via their attachment to the bacterial cell walls and membranes, leading to the disruption of the membrane integrity (Cowan 1999, Tsuchiya et al. 1996, Babii et al. 2018). The large number of flavonoid subgroups, and thus molecular structure diversity has been found to give a number of mechanisms of action on microbial growth inhibition. For example, structure activity relationship (SAR) studies have revealed that the chalcones inhibit the metabolism of the ATP, flavones inhibit or weaken the integrity and thus the function of the cell membrane, and flavonols inhibit the synthesis of the DNA in bacteria via interacting with DNA gyrase (Swanson 2016, Cushnie and Lamb 2005, Wu et al. 2013). For DNA gyrase inhibitory activity it was found that hydroxyl group substitutions at the position C-5 in the A-ring and C-4` in the B-ring, as well as methoxyl group substitutions at positions C-3 and C-8 in the A-ring are important for good activity (Wu et al. 2013). Thus, flavonoids with optimal functional group substitutions might be used for the development of highly active and less toxic antimicrobial agents (Wu et al. 2013, Cushnie and Lamb 2005). Moreover, various glycosides of flavonoids have been found to possess good growth inhibitory activity against pathogenic microorganisms, although, sometimes, the aglycone part of flavonoids have been found to be more antimicrobial than their glycosides (Gaitan 1989, Kanwal et al. 2009, Xiao 2014). For example, quercetin and its glycosides, identified in the extracts of the stem bark of Terminalia paniculata and T. tomentosa, were found to give good growth inhibition against Neisseria gonorrheae, Haemophilus ducreyi and Candida albicans, and this result could justify the uses of traditional preparations of these plants, as macerations or decoctions, for the treatment of gonorrhea and fungal infections (Ganjayi et al. 2017, Gahlaut et al. 2013, Jadhav et al. 2015). Flavonoids, and especially C-glycosidic flavonoids from Terminalia catappa demonstrated good anti-Candida activity (Terças et al. 2017). In general, it has been found that O-glycosylation in flavonoids leads to a reduction in their antimicrobial effects (Xiao 2014). A number of flavonoids have been characterized from the genus Combretum (Roy et al. 2014). For example, dillenetin, isorhamnetin, quercetin, 3-O-methyl quercetin, apigenin, kaempferol and rhamnocitrin were found from the flowers of Combretum lanceolatum and the leaves of Combretum erythrophyllum and showed growth inhibitory effects against Vibrio cholerae and Enterococcus faecalis with MIC values ranging between 25 – 50 μg/ml (Araujo et al. 2013, Martini et al. 2004). Moreover, flavonoid-rich extracts of the leaves of Combretum edwadsii and C. kraussii, showed promising effects against the growth of E. coli, S. aureus and Klebsiella pneumoniae (Chukwujekwu and van Staden 2016). A number of flavonoids were found in extracts of Anogeissus leiocarpus, showing good antifungal effects against the eumycetoma fungus, Madurella mycetomatis (Eltayeb et al. 2016, Arbab 2014). Among the flavonoids in these extracts, 55 chalcone, isorhamnetin, quercetin-7-O-β-rhamnoside, quercetin 3-methoxy-7-O-β– glucopyranoside, kaempferol-7-O-β-glucopyranoside, quercetin-7-O-β-glucopyranoside, were characterized. 2.3.2.3. Stilbenes, and phenanthrenes Chemistry Stilbenes and phenanthrenes are secondary metabolites originating from the phenylpropanoid pathway (Zhang et al. 2015, Iriti and Faoro 2011, Austin and Noel 2003). Stilbenes are colorless and hydrophobic compounds (Likhtenshtein 2009). They are polyphenols based on the 1,2-diphenylethene (C6-C2-C6) nucleus (Fig 18) (Shen et al. 2009, Vogt 2010). Furthermore, stilbenes are classified into monomeric and oligomeric stilbenes (Shen et al. 2009). Monomeric stilbenes occur in 23 plant families, including the Combretaceae family, from which a series of tubulin polymerization inhibitors, the combretastatins, and resveratrol and its glycosides are known (Pettit et al. 1987, 1988, 1989, 1995, Shen et al. 2009, Joseph et al. 2007; Fig. 20). Oligomeric stilbenes result from the coupling of monomers and are further classified into a variety of groups. According to the most recent classification, there are oligomers based on I) resveratrol, II) isorhapontigenin, III) piceatanol, IV) oxyresveratrol (OH)4, resveratrol (OH)3 and miscellaneous oligomers (Shen et al. 2009). The two stilbenes of methyl E-viniferin and E-viniferin were found in extracts of Anogeissus leiocarpus, showing good antifungal effects against the eumycetoma fungus, Madurella mycetomatis (Eltayeb et al. 2016, Arbab 2014). Phenanthrenes (PAHs) are a rather uncommon group of aromatic secondary compounds (Kovács et al. 2008). They occur in Orchidaceae, Combretaceae, Dioscoreaceae and Betulaceae, in which a large number of differently substituted phenanthrenes have been characterized (Kovács et al. 2008). Besides, phenanthrenes also occur in Hepaticae (liverworts). Phenanthrenes are produced from the oxidative coupling of the aromatic rings of stilbene precursors (Kovács et al. 2008, Fig 19). Moreover, there are some phenanthrenes that most likely originate from diterpenoid precursors (Kovács et al. 2008). Phenanthrenes in plants are classified into three major groups, the monophenanthrenes, the diphenanthrenes and the triphenanthrenes (Xue et al. 2006, Kovács et al. 2008). Most phenanthrenes, about 210 compounds, are of the monomeric form. About one hundred of these monomeric phenanthrenes are only hydroxyl- or methoxy-substituted, and these compounds are either 9,10-dihydro or -dehydro derivatives. In the plant family Combretaceae, phenanthrenes are mainly found in Combretum species, such as C. apiculatum, C. caffrum, C. psidioides, C. hereroense and C. molle (Kovács et al. 2008, Malan and Swinny 1993). In addition, phenanthrenes are known in Terminalia catappa (Owolabi et al. 2013). 56 Figure 18. The 1,2-diphenylethene (C6-C2-C6) nucleus unit in stilbenes. Figure 19. Production of phenanthrenes from the oxidative coupling of the aromatic rings of cis (Z)- and trans (E)-stilbenes (Kwasniewski et al. 2003). Stilbenes and phenanthrenes in plant defense, their antimicrobial effects and in vivo metabolites Various hydroxylated stilbene derivatives have been found to function as phytoalexins in the plant (Rühmann et al. 2013, Khan et al. 2017). Pinosylvin, a naturally occurring stilbenoid in the leaves and wood of Pinus species, has been found to increase the resistance of heartwood to wood decay (Fu et al. 2018). The antifungal and antibacterial mechanisms of action of resveratrol and stilbenes are still not very well understood. However, there are some studies suggesting that resveratrol inhibits fungal cell respiration (Caruso et al. 2011, Hart 1981). It has also been suggested that hydroxystilbenes and pterostilbene cause membrane damage in fungi (Pan et al. 2008, Pezet et al. 1995, Pezet et al. 1990). Species of the genus Combretum are known to contain a diverse array of stilbenes, especially in their heartwood and stem bark, but also in their leaves and fruits. The stilbenes, combretastatin-A-4 and its derivatives, which were discovered by Pettit et al. (1987) from the stem wood of Combretum caffrum, have attracted profound interest due to their potential in vitro and in vivo anti-cancer effects (Pettit et al. 1988, Pettit al. 1995). In addition, combretastatin 57 A-4 and its derivatives as well as combretastatin B5, combretastatin A1 and its derivatives, the later identified in Combretum woodii and C. erythrophyllum, have shown good antibacterial or antifungal activity (Man et al. 2018, Eloff et al. 2005, Ma et al. 2016, Jaroch et al. 2016, Schwikkard et al, 2000). Combretastatin A-4 and its derivatives were identified as strong fungicides against the soil-borne pathogen, Rhizoctonia solani (EC50 46μM) and Fusarium oxysporum (inhibition zones of 53 mm at concentrations of 50 μg/ml) (Ma et al. 2016). Moreover, many authors reported antimicrobial activity of stilbene-rich extracts from species of Combretum and Terminalia, especially against human pathogenic microorganisms. For example, Welch (2010) identified resveratrol from antimicrobial extracts of Combretum micranthum. Moreover, Banfi et al. (2014) demonstrated considerable antimicrobial activities of stilbene-rich crude extracts of Combretum micranthum, with MIC values of 625 and 125 μg/ml, respectively, against methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile, respectively. Phenanthrenes from a number of medicinal plants have shown a diverse spectrum of antimicrobial activity and strong potency against methicillin-resistant Staphylococcus aureus (MRSA) as well as against extended spectrum beta-lactamase (ESBL) producing E. coli, P. aeruginosa and B. subtilis (Tóth et al. 2016, Yoshikawa et al. 2014). Mushi et al. (2015), reported moderate antibacterial activity of 2,3,8-trihydroxy-4,6-dimethoxyphenanthrene and 2,3,8-trihydroxy-4,6-dimethoxy9,10-dihydrophenanthrene against Pseudomonas aeruginosa (MIC 160 μg/ml). Both phenanthrenes were isolated from the root of Combretum adenogonium. Despite of a number of in vitro results on the antimicrobial effects of phenanthrenes, the antimicrobial mechanisms of action of compounds of this chemical class are still ambiguous (Iwatsuki et al. 2010). Phenanthrenes are highly lipophilic and thus their bioavailability after ingestion is significant, and high levels of phenanthrenes have especially been detected in adipose tissue (Abdel-Shafy and Mansour 2016). A mixed-function oxidase enzyme system is responsible for the metabolism of phenanthrenes, involving epoxidation and further conjugations with glutathione, leading to the production of dihydrodiols, which have been found in the plasma, urine and liver (Schober et al. 2010, Abdel-Shafy and Mansour 2016). The metabolism of stilbenes includes glucuronization, hydroxylation and methylation (Fu et al. 2018). In vivo research has shown that resveratrol metabolizes rapidly to glucuronide and sulfate derivatives (Muzzio et al. 2012). 58 Combretastatin-A4, identified from Combretum caffrum R=H R = OCH3 Phenanthrenes, found in Combretum caffrum Combretastatin-A4 phosphate (Fosbretabulin). a drug designed from combretastatin R = CH3, R1 = R2 = H R = R1 = CH3, R2 = H R = H, R1 = R2 = CH3 R = R2 = H, R1 = CH3 Dihydrophenanthrenes, found in Combretum caffrum,C. psidioides, C. molle and C. apiculatum Figure 20. Stilbenes and phenanthrenes found in Combretum spp. and the phosphate derivative of combretastatin A4, CA4P, (Pettit et al. 1988, Pettit al. 1995 and Pettit et al. 1987) 2.3.2.4. Lignans Chemistry Lignans (C6 – C3)2 are phytoestrogens or phytohormones based on a 2,3-dibenzylbutane skeleton (Fig 21 A) which is produced via oxidative and cyclization processes from phenylpropanoid units, via p-coumaric acid and coniferyl alcohol as transitional precursors (Suzuki and Umezava 2007). Lignans and norlignans are found in large quantities in the heartwood part of soft and hardwood species and in many herbaceous plants, including food and crop plants (Suzuki and Umezawa 2007). Lignans have estrogenic and anti-estrogenic activities, and they are classified according to their chemical structure into eight subclasses, including furans (lariciresinol), furofurans (pinoresinol), aryltetralins, arylnaphthalenes, dibenzylbutyrolactones (matairesinol), bibenzylbutyrolactols, dibenzylbutanes (secoisolariciresinol) and dibenzocyclooctadienes (Zhu et al. 2017, Linder et al. 2016). 59 Antimicrobial effects and in vivo metabolites In the family Combretaceae, the lignan terminaloside and its derivatives have been reported in Terminalia citrina and T. tropophylla (Muhit et al. 2016, Cao et al. 2010), while trachelogenin and dibenzylbutyrolactone were reported in Combretum fruticosum (Fig 21 B) (Moura et al. 2018). Moreover, in Anogeissus acuminata, the occurrence of new lignans, anolignan A and B as well as secoisolariciresinol was reported (Fig 21 C and D) (Rimando et al. 1994). Lignans have showed good antiviral (Rimando et al. 1994), antibacterial (Raghavendra et al. 2017, Yamauchi et al. 2016, Yuan et al. 2007) and antifungal activity (Nishiwaki et al. 2017, Raghavendra et al. 2017, Yamauchi et al. 2016). However, their mode of action remains unclear (Satake et al. 2013). Lignans are converted to enterodiol and/or enterolactone metabolites by the gut microflora in the human large intestine and these metabolites are commonly known as enterolignans or mammalian lignans (Imran et al. 2015, Landete 2012). Many of the lignan metabolites have been found in the blood and urine (Buck et al. 2010). Mammalian lignans were found to have lipid peroxidation activity and antioxidant activity (Kitts et al. 1999, Landete 2012). However, although a number of studies have been performed on their biological effects (Thompson et al. 1996, Liu et al. 2017), the antimicrobial activity of lignan metabolites has not been well studied. (B) Trachelogenin in Combretum (A) Dibenzylbutane skeleton based structure for lignans molecules (C6-C3)2 fruticosum (C) Anolignan A in Anogeissus acuminata (D) Secoisolariciresinol in Anogeissus acuminata Figure 21. Basic skeleton of lignans and lignans identified from Anogeissus and Combretum spp. (Zhu et al. 2017, Linder et al. 2016, Rimando et al. 1994). 60 2.3.3. Terpenes and saponins Chemistry Terpenes or terpenoids are hydrocarbon molecules derived mainly from isopentenyl diphosphate and dimethyl allyl diphosphate to form the isoprene unit (C5H8) through the mevalonate pathway (Fig. 22). According to the number of the isoprene units, terpenes are classified into ten types, the main groups being mono-, di- and sesquiterpenes (Kumari et al. 2018, Pazouki and Niinemetz 2016). More than 60,000 terpenes are known from the plant kingdom (Pazouki and Niinemets 2016). Terpenes are classified as the main compounds in essential oils (Roberts and Stevens 1962). Derivatives of squalene result in steroid compounds (Vriet et al. 2013) which are also classified as non-polar metabolites in the plant. Saponins contain of an aglycone part called a sapogenin, which is usually a triterpene or a steroid connected with a sugar. The foaming of saponins is caused by a combination of a hydrophobic sapogenin and a hydrophilic sugar molecule (Bone and Mills 2013). Antimicrobial effects Pentacyclic triterpenes from African Combretum and Terminalia spp. have shown good in vitro antibacterial and antifungal effects. For example, in combinations, arjunolic acid and asiatic acid, both isolated from the leaves of Combretum nelsonii (Syn. C. kraussii), showed growth inhibitory effect against filamentous fungi and Candida spp. (Masoko et al. 2006). Betulinic acid, ursolic acid, terminolic acid, arjungenin and quadranoside II from Combretum racemosum exhibited good activity against Staphylococcus aureus, Escherichia coli and Enterococcus faecalis with an average MIC value of 128 μg/ml (Gossan et al. 2016). Likewise, oleanolic acid derivatives from Terminalia superba showed good growth inhibition against Salmonella typhi and Bacillus subtilis in terms of large inhibition zones (IZ) (Tabopda et al. 2009). Moreover, oleanolic acid, derived from extracts of Terminalia mollis and Terminalia brachystemma, revealed activity against Candida spp., with MIC values from 6.25 – 50 μg/ml (Liu et al. 2009). Arjunglucoside, sericoside, arjunetin and chebuloside are saponins known from many African species of Terminalia and Combretum. These saponins have been found to give promising growth inhibitory effects against fungi and bacteria (Wang et al. 2010, Li et al. 2009, Dawé et al. 2017). Saponins in methanolic extracts of the leaves of Combretum albidum were found to significantly contribute to the growth inhibition of Pseudomonas aeruginosa (Sahu et al. 2014). Antimicrobial extracts from Terminalia macroptera contain a wide range of saponins and triterpenes, such as 23galloylarjunolic acid-28-O-β-D-glucopyranosyl ester, terminolic acid, arjunic acid, arjungenin, arjunglucoside 1, sericic acid and sericoside (Conrad et al. 1998). In general, triterpenes from many medicinal plants have shown antibacterial (Monte et al. 2014) and antifungal effects (Lee et al. 2017), with MIC values ranging from 42.5 – 500 μg/ml against Staphylococcus and and 70 – 250 μg/ml against Candida strains (Avato et al. 2006, Tsuzuki et al. 2007). In a study on the antibacterial effects of a number of saponins and their aglycones, it was established that the aglycone unit was more active than the saponin itself, and thus the sugar part of the saponins was considered not to be important for their antibacterial effects (Avato et al. 2006). The opposite has been found for the antifungal effects of some saponins versus their aglycones, so that some saponins have been found to be more antifungal than their aglycones (Sajjadi et al. 2016). 61 or or Isopentenyl diphosphate (isopentenyl pyrophosphate) or or (C5H8) Isoprene unite Dimethyl allyl diphosphate Via the mevalonate pathway via the acetate mevalonate pathway Fatty acids Terpenes As (C5H8)3 Sesquiterpene (C5H8)2 Docosanoic acid Octadecanoic acid Tetracosanoic acid Monoterpene (steric acid) C18H36O2 (behenic acid) C22H44O2 (Lignoceric acid ) C24H48O2 (C5H8)4 Diterpene (C5H8)6 Triterpene Figure 22. Biosynthesis of the isoprene unit, which is the basic unit involved in the synthesis of the various classes of terpenes and fatty acids (Kumari et al. 2018, Pazouki and Niinemetz 2016). 62 (C5H8)8 Tetraterpene (C5H8) ˃8 Polyterpene 2.3.4. Fatty acids Chemistry In plants, fatty acids are synthesized de novo primarily in the plastids, and they are used for the synthesis of plastid and other cell membranes (Rawsthorne 2002). In seeds, they are also used for the synthesis of storage oils. Fatty acids are carboxylic acids produced from isoprene units, via the acetate-mevalonate pathway (Fig. 22), starting from acetyl-CoA via malonyl-CoA. Fatty acids have either a short hydrocarbon chain (near to C8, SCFA, short-chain fatty acids) or a long hydrocarbon chain (more or equal to ≥ C16, LCFA, long-chain fatty acids) (Rawsthorne 2002, Ulbright et al. 1982, McGaw et al. 2002). Fatty acids of plant origin contain a large percentage of unsaturated fatty acids based on C16 or C18 (McGaw et al. 2002). Moreover, fatty acids with cis double bonds occur widely in nature, whereas trans double bonds are rare (McGaw et al. 2002). In plants, fatty acids occur mainly in the bound form as fats and lipids, esterified to glycerol, and are important constituents of cell membranes, chloroplasts and mitochondria (Jeffery and Baxter 1983, McGaw et al. 2002). Fatty acids are also produced from enzymatic degradation of the lipids (Olbrich and Müller 1999). In the liver, fatty acids are metabolized to triglycerides, phospholipids, hormones and ketone bodies, of which the first mentioned are important components of cell membranes (Chiang 2014). Moreover, the fatty acids are important producers of ATP in animal cells (Panov et al. 2014). Antimicrobial effects Since the 1970s, it has been well known that fatty acids are antibacterial and antifungal (Anyanwu et al. 2018, Veličkovska et al. 2018, Abe et al. 1969, Kabara et al. 1972). During bioassay-directed fractionation of plant extracts it has occasionally been observed that fatty acids, at least partly, were responsible for the antimicrobial effects of the crude extract (Cerdeiras et al. 2000, McGaw et al. 2002, Yff et al. 2002). A number of fatty acids with antibacterial activity have been isolated from a variety of plant species (Dubey et al. 2014, Mohadjerani et al. 2014, Hilmarsson et al. 2006, McGaw et al. 2002, Cedeiras et al. 2000). Some of these fatty acids, such as 11-O-(6'-O-acetyl-β-D-glucopyranosyl)-stearic acid inhibited the growth of S. aureus with a MIC value of 9 μg/ml (Cedeiras et al. 2000). Likewise, linoleic and oleic acid were found to inhibit the growth of Bacillus subtilis, Bacillus cereus and S. aureus, but showed no effect against Gram-negative bacteria (Dilika et al. 2000). In the genus Terminalia, mainly Asian species, and especially the seeds of Terminalia chebula, have been investigated for their fatty acid composition (Janporn et al. 2015, Onial et al. 2014). T. chebula seed oil was found to contain palmitic (= Hexadecanoic acid), stearic (= Octadecanoic acid), arachidic (= eicosanoic acid) and behenic acids (= Docosanoic acid), whereas the linoleic (= 9,12-Octadecadienoic acid) and oleic acids (= 9-Octadecenoic acid) were found to represent the major fatty acids in the kernel of the fruits (Onial et al 2014). Combretum micranthum is one of the few African species that has been investigated for its fatty acids, and palmitic, oleic and linoleic acids were found in this species (Welch 2010, Tahiri et al. 2012). The occurrence of similar fatty acids, such as oleic, linoleic, hexadecanoic (palmitic acid) and octadecatrienoic acids in the leaves of Terminalia coriacea, T. ferdinandiana and Combretum micranthum, as well as in the fruits of T. catappa, might explain the oral, fumigation and ointment applications of extracts from these species as folkloric medicines for treatment of infectious diseases, urticarial rash and skin infections (Akter et al. 2018, Patel et al. 2017, Ladele et al. 2016, Bony et al. 2014, Welch 2010, Daulatabad and Ankalgi 1983). 63 Fatty acids have been shown to function as important intracellular mediators or extracellular signals in plant defense and interspecies communication (McGaw et al. 2002). Fatty acids have a high affinity to proteins and through this mechanism they could interfere with the function of microbial enzymes such as efflux proteins as well as enzymes needed for the synthesis of the bacterial cell envelope (wall and membrane) (McGaw et al. 2002, Gurr and James 1980a and B, luo et al. 2011). In Mycobacterium, fatty acids have been found to inhibit the synthesis of mycolic acid, an important constituent of the cell wall (Rugutt and Rugutt 2012). However, the antimicrobial mechanisms of actions in detail of many plant-derived non-polar and polar lipid compounds are still poorly clarified, although a considerable number of authors agree that fatty acids are involved in cell wall damage (Desbois and Smith 2010). 64 3. MATERIAL AND METHODS 3.1. Field work (Studies I and III) Ethnobotanical and botanical field work, comprising plant collections and interviews, was performed in the Blue Nile and Kordofan regions of Sudan. These study areas were chosen for the ethnobotanical surveys since relatively little research has been performed on the uses of medicinal plants and other useful plants in these regions. Earlier, El Ghazali et al. (1987, 1997 and 2003), and Musa et al. (2011), have performed some ethnopharmacological investigations in these areas, but there are limited data on the uses of plants belonging to the plant family Combretaceae, and medicinal plants in general in these regions. Similarly to many other regions in Africa, most of the information on the uses of medicinal plants in these regions is oral, and thus the documentation of this information is important. 3.1.1. Geographical area and vegetation types of the study areas Two geographical sites in south-eastern and south-western Sudan, the Blue Nile and the Kordofan regions, were selected as research areas, based on common occurrence of the medicinal plant species belonging to the family Combretaceae (Fig. 23). The vegetation in the study areas ranges from semi-arid desert to high rainfall (wet) savanna forest (Fig. 23). The following vegetation types and climatic conditions can be found in the study areas: 1) The savanna zone found in the Blue Nile and Kordofan regions is characterized by a wet-dry tropical climate (AW), with a long dry and cold season in December- February, during which the temperatures lie between +20°C and +25°C, and a wet season in June-October with heavy rains and warmer temperatures (Köppen-Geiger climate classification). The average annual precipitation of the savanna zone is between 15 – 25 inches (381 – 635 mm/year), and the temperature ranges from +20°C to +30°C (Köppen-Geiger climate classification). 2) The southern parts of Sudan, including the Blue Nile and Kordofan regions, is covered by warm semi-deserts with a warm, semi-arid climate (BSh), which is characterized by an annual average rainfall of 250 to 500 mm and an average annual temperature of +20°C (Köppen-Geiger climate classification). 3) The largest area of Sudan, including the middle and northern parts, is characterized by a warm desert climate (BWh) with an average annual rainfall of 0–250 mm, and temperatures ranging from +21 to + 32° C. The northern parts of the Kordofan and Blue Nile reginos belong to this desert zone. The river Nile and the mean annual rainfall are the main elements shaping the vegetation types in Sudan (Mahmoud et al. 1996). In the far north, the country is covered by desert plants. Further south, with increasing rainfall, from the semi-desert to low rainfall savanna and in regions nearer to the Nile, these deserts gradually change from Acacia shrub deserts to Acacia bush land with short grass and finally, in moister places, to Acacia tall grass forests (Mahmoud et al. 1996). The Acacia forests contain a number of herbal and woody plant species belonging to the families Balanitaceae (Balanites aegyptiaca), Asclepiadaceae (Calotropis procera), Capparaceae (Capparis decidua), Rhamnaceae (Ziziphus spinachristi), Combretaceae (Anogeissus leiocarpus, Terminalia brownii, Terminalia laxiflora, Combretum hartmannianum) and Tamaricaceae (Tamarix aphylla) (Mahmoud et al. 1996; Chileshe 2011, Gafaar 2011). In the southern part of Sudan (Fig. 23), the dominant vegetation types are savanna bushlands, savanna woodlands, swamps and grasslands. The vegetation in the savanna bushlands and woodlands is characterized by the plant families Combretaceae, Rubiaceae, Anacardiaceae, Bombacaceae, Sterculiaceae, Ochnaceae, Loganiaceae, Fabaceae, Burseraceae and Tiliaceae, whereas the semi-arid grasslands are characterized by species such as Capparis decidua, Ziziphus spina-christi, Balanites aegyptiaca and Calotropis procera (Mahmoud et al 1996, Chileshe 2011, Gafaar 2011). Besides the natural vegetation types also various vegetation types modified by agricultural practices, such as 65 traditional rain fed agriculture, mechanized rain fed agriculture and irrigated agriculture, are common in Sudan (Robinson et al. 2011, Mahgoub 2014). 3.1.2. Ethnobotanical (reconnaissance) survey and collection of the plant material 3.1.2.1. Plant collection methods and voucher samples Voucher specimens and plant bulk material as well as ethnobotanical information (the later mentioned explained in paragraph 4.1.), were collected during three expeditions to the Blue Nile and Kordofan regions in Sudan in May-June 2006, in June-August 2012 and in February-March 2014. The plant specimens were collected from healthy individuals, which were mostly trees and some shrubs. The species identification was performed according to the fruit, leaf and stem bark morphology (for Terminalia, Combretum and Anogeissus spp., the fruit and stem bark characteristics were mainly used). The diameter of the collected trees at breast height varied from 40 – 70 cm and their height ranged from 12 – 25 m (Table 2). During the plant collections, apart from two botanists from University of Khartoum and Forest Research Centre, also traditional healers were involved to confirm the identity of the medicinal plants discussed in the interviews (Fig. 24 and 25). Collected plants were photographed for further documentation and species identification was performed by the writer of this thesis book as well as Dr. Gibreel H. Hashim, from the Faculty of Forestry, University of Khartoum, Sudan and Dr. El Sheikh Abd Alla Al Sheikh, from Forest Research Centre at Soba, Khartoum, Sudan. Voucher specimens were deposited at the Faculty of Science and Faculty of Forestry at the University of Khartoum, and at the Commission for Biotechnology and Genetic Engineering, National Centre for Research, Ministry of Science and Technology, Khartoum, Sudan. More than 83 species of trees and shrubs were recorded for the interviews. Of these, 14 species belonged to the plant family Combretaceae (Table 2) and 69 to other plant families. These species were listed to be investigated for their traditional medicinal uses as well as for their other possible traditional applications, since very little research on these aspects have been performed in the chosen study areas. Besides, attention was put on the various multiple local uses of the plant species, in order to estimate their total ethnobotanical values. The plant species were selected due to their common uses in traditional medicine, with focus on those used for infectious diseases and wound infections. Moreover, the species were chosen as related to the scarcity of written documents on their medicinal uses, despite of their long history of uses in Sudanese traditional medicine (Machumi et al. 2013, Muddathir and Mitsunaga 2013, Muddathir et al. 2013, Mosango 2013, Opiyo et al. 2011, Mbwambo et al. 2007, Morgan et al. 2018, Mohieldin et al. 2017). The family Combretaceae is known to contain a number of secondary compounds with a variety of biological activities, and among them antimicrobial effects, but the species which were selected for this investigation had not been studied thoroughly in this aspect. For example, in 1987, Pettit et al. identified and isolated anticancer molecules belonging to the chemical class of the stilbenes (for example, the combretastatin) from the stem bark of the South African species, Combretum caffrum. Therefore, compounds with similar structures or their derivatives could be expected found in other, closely related species. The collections of the plant material for further phytochemical and antimicrobial analysis focused especially on the family Combretaceae (Terminalia laxiflora, Terminalia brownii, Anogeissus leiocarpus, Combretum hartmannianum), but also some species of Rubiaceae (Xeromphis nilotica and Gardenia lutea) were collected. 66 Warm desert climate (BWh) Warm semi-desert climate (BSh) Tropical savanna climate (AW) Blue Nile region (A) Kordofan region (B) Figure 23. The location of Sudan in Africa and the ethnobotanical study areas, the Blue Nile state (A) and Kordofan (B) regions. Source: Peel et al. 2007. 3.1.2.2. Interviews Altogether seven villages were visited for the ethnopharmacological interviews. Four of them, were situated in the Blue Nile region and three in the Kordofan region. In addition, some marketplaces in the mentioned villages and in Khartoum were visited in order to perform some additional interviews. The ethnobotanical work, including interviews and the collections of plant material, was carried out according to the established ethical guidelines (International Society of Ethnobiology. 2006) and according to general guidelines for ethnobotanical work (Towns et al. 2014, Cotton 1996, Martin 1995, Martin 2010). In each village, traditional healers and other villagers, such as household members including house wives and elder family members, were interviewed with focus on their medicinal (and other) uses of the plant species of Combretaceae and some other plant families shown to them. Seven healers and eighty other informants, belonging to different ethnic groups, were interviewed in Arabic and other local languages (various dialects). The informants were either Muslims (Baggara, Rizeigat, Ta’isha, Misseriya, Hawazma, Borgo, Fallata, Hausa, Bornu, and Masalit tribes) or Christians (a minority among the Nuba tribe). Of these tribes, the Baggara, Fallata, Kababish and Nuba live in the Kordofan region and the Mapan and Angassana tribes in the Blue Nile region. Moreover, the informants were either of Afro-Arab or African ethnicity. In the regions included in this study, 41 dialects are spoken (out of 400 total dialects in Sudan) (Nelson 1982, CIA 2007). Of these dialects, eleven are spoken in South Kordofan (Kome, Tegali, Kajakja, Ngakom, Kinderma, Dar El Kabira, Tira, Dagig, Luman, and Kamdang), whereas thirty dialects are spoken in the Blue Nile state (Shuru, Bake, Undu, Mayu, Fadashi, Maiak, Mufwa, Ragreig, Bogon, Mugo-Mborkoina, Mughaja, Mugaja, Jemhwa, Gombe, Kukur, Kulang, Kelo, Beilla, Buwahg, Dewiya, Kukwaya, Disoha, Dakunza, Sai, Dekoka, Gombo, Modea, Beni, Sheko and Chali). Semi-structured interviews 67 Questionnaires with pre-determined questions were used for the ethnobotanical surveys (Appendix 1). The questionnaire consisted of a part with more general questions regarding the profession, education and the age of informants, as well as of questions regarding who possessed ethnobotanical knowledge in the village. Informants were also asked how they had learnt to use plants, e.g. whether their knowledge had been inherited. After these more specific questions were made on the uses of the selected plants for medicinal and other purposes. For medicinal plants, their frequency of use and modes of uses were clarified. Focus was put on symptoms which could be related to infectious diseases such as cough, fever, chest pain, soar throat, wound infections, skin rashes and diarrhea. Moreover, the informants were specifically asked whether plants were used for dysentery, respiratory infections and tuberculosis (Appendix 1). In addition, the uses of the plants against protozoan diseases was also documented. These so called “neglected diseases” (WHO 2012, Karl et al. 2015, CDC 2017, Hotez and Damania 2018), included malaria, bilharzia (Schistosomiasis), sleeping sickness (Trypanosomiasis) and leishmaniasis (also known as kala-azar or black fever in Sudan), which are all very common in the rural parts of Sudan (Appendix1). Open-ended and institutional interviews Open-ended interviews were used in order to encourage the informants into longer informal and formal discussions (cf. Mustalahti and Nathan 2009, Jamshed 2014). During these interviews, for most of the time, the Sudanese Arabic language and the local language “dialects” were used (Fig. 26 B). The openended questions were dependent on the semi-structured questions and allowed us to extend and broaden the specific questions. As shown in the questionnaire (Appendix 1), more data, evidence and information regarding the plants studied was generated when using the open-ended questions. These open-ended questions were additional ones attached next to the main questions in the questionnaire in order to specify the answers to the main questions. Institutional interviews Researchers and administrators, belonging to the organization of the Forestry and Agricultural sectors, were also interviewed (Appendix 1). The questions were divided into scientific, political, environmental, and economic ones and included issues that directly or indirectly affected the study areas and influenced the management (including regeneration), conservation, and use of the plant species now studied. 68 Figure 24. Collection and identification of medicinal plants in the Blue Nile region (Al-Azaza Reservation Forest). Photos by the writer of this thesis book 2012. Figure 25. Air drying, in the shade of various parts of the collected medicinal plants. Photos by the writer of this thesis book 2012. Figure 26. An interview in El Nour (El Azaza) village at Hamag civil administration in Blue Nile region. Interview with traditional healers in the village and in the forest. Photos by the writer of this thesis book 2012. 69 3.2. Phytochemistry 3.2.1. Sample preparation (Studies I, II and III) The collected plant material was dried in the shade at ambient temperature (Fig. 25). Roots and stems were debarked manually using slashers, axes, knives and a cutter to separate the stem bark from the stem wood and the root bark from the root (Fig. 27 A and B). Excluding the leaves and fruit, the other parts of the plants were chipped to small chips before sending them for hammer mill mesh grinding (Fig. 27 C and D). This final mesh grinding resulted in finely grinded plant powders. The plant material was investigated at the plant Quarantine Services at the Khartoum International Airport. A Phytosanitary Certificate No. 0005784, was issued for the plant sample to be transported for further laboratory analysis at University of Helsinki, Finland (Appendix 2). B) A) C) D) Figure 27. (A and B), Debarking and chipping the stem bark, stem wood, rootbark and root of the selected medicinal plants; (C and D) grinding the various plant parts using a hammer mill to obtain fine grained powder. Photos by the writer of this thesis book 2012. 70 3.2.2. Extraction 3.2.2.1. Crude extracts Macerations and decoctions (Studies I and III) Macerations and decoctions were made according to the traditional preparations of the plants, since this kind of preparation is common in Sudan according to our ethnopharmacological data. For the decoctions, 20 g plant material was soaked with 300 -500 ml water and was brought to the boil for 5 minutes. For the macerations, the same amount of plant material in relation to water was used, but the plant material was left overnight in the lukewarm water and a magnetic stirrer was used to facilitate the extraction. The extracts were then centrifuged at 2000 – 3000 rpm for 10 – 20 min (Eppendorf AG centrifuge 5810 R, Germany), wherafter they were filtered using Schleicher & Schuell filter paper (‡ 150 mm, Germany) and vacuum filtration technique (Ceramic Porcelain Vacuum Suction Filter KitTM 500 ml Büchner Funnel Glass). The filtered extracts were frozen at -20qC before lyophilization. Hot and cold methanol extraction (Studies I and III) The Soxhlet distillation extraction method was used with methanol as solvent. For this extraction technique, a heating mantle and a Pyrex®Soxhlet extractor (Sigma-Aldrich) were used. A Borosil boiling flask with a capacity of 1000 ml was filled with 800 –1000 ml methanol and this flask was connected to the distillation apparatus containing of a condenser, siphon arm and a cellulose thimble with the sample to be extracted, placed in the extraction chamber. A total of 20 – 100 g of the plant sample was weighed in the thimble which was placed into the Soxhlet extraction chamber, after which 500 – 1000 ml methanol was added to the boiling flask. The methanol was boiled for 4 – 5 h resulting in methanol fume which condensated in the distillation apparatus and flowed through the dry plant material after which the liquid with the extract dropped back into the boiling flask. The resulting extracts were then evaporated to dryness using a rotary evaporator (Heidolph VV2000) connected to a water bath not exceeding + 40°C. Cold methanol extractions were made using 20 g plant powder and 300 – 500 ml methanol. Extraction was performed overnight using a magnetic stirrer and a magnetic rod. The extract was filtered and evaporated in the same way as the other crude extracts. All extracts were freeze-dried in a lyophilizer (HETO LyoPro 3000 Freeze Dryer, Denmark) for 1– 3 days. The dried extracts were dissolved in methanol to obtain stock solutions of 50 mg/ml for primary antimicrobial screenings, using the agar diffusion method. 3.2.2.2. Sequential extraction and liquid-liquid partition (Studies I, II and III) Sequential extraction and liquid-liquid partition were performed in order to separate compounds of various polarities from each other (Fig. 28). 100 – 200 g of milled plant material was used for the extractions. Depending on the part of the plant to be extracted, different quantities of extraction solvents were used. For example, more solvent was needed for the extractions of the stem wood and leaves, whereas the smallest volumes of the extraction solvents were needed for the root, fruit, and stem bark. Volumes of the extraction solvents were adjusted according to the density and unique anatomical character of the plant material (stem wood, leaves, bark and roots), such as the size and number vessel elements, the porosity and fibrosity (Azwanida 2015). The sequential extraction was initiated by using the most non-polar solvent, n-hexane or petroleum ether. About 1000 –1600 ml of petroleum ether or hexane was used to extract non-polar compounds, such as 71 long-chain fatty acids, waxes, fatty alcohols, sterols, steroids and non-glyosidic terpenes, as well as some less polar triterpenes and their glycosides (saponins). The n-hexane/petroleum ether extraction was followed by chloroform or dichloromethane extraction of the marc. These solvents were used in volumes ranging from 1000 – 1400 ml. Acetone would have been a preferred solvent to use for all the extractions, but due to the limited availability of some of the plant samples, acetone was used only for some samples. For these samples, acetone was used since Eloff (1998) had demonstrated that acetone has the capacity to dissolve a large number of hydrophilic and lipophilic compounds in Combretum spp.. When acetone extraction was used, 800 – 1000 ml of this solvent was used, following the chloroform/dichloromethane extractions. For each extraction solvent, the extracted plant material was stirred continuously overnight for 24 hours using a RCT basic digital magnetic stirrer and a magnetic stirring bar (TRIKA® 25 – 40 mm), at no heating. The final step of the extraction procedure, following the sequential extraction, was liquid-liquid partition (Fig. 28). For liquid-liquid partition, freshly prepared 80% methanol in water and 100% ethyl acetate were used. An equal volume of these two immiscible solvents were added to a Nalgene® FEP separation funnel to create two layers resulting from the denser solvent of water remaining in the bottom of the separation funnel and the ethyl acetate layer forming an upper layer. This separation process allows the hydrophilic compounds to be extracted in the aqueous part (mainly glycosidic, phenolic compounds and many tannins), whereas the more non-polar glycosidic, aglycones of phenolic compounds and many ellagitannins dissolve in the ethyl acetate layer. The liquid partition (or solvent partition) layers were dried at +40º C, using a rotary evaporator (Heidolp VV2000), whereafter they were freeze-dried in a lyophilizer (HETO lyoPro 3000 Freeze Dryer, Denmark) for 1–5 days. All extracts resulting from the sequential extraction were dried in a fume cabinet at room temperature or by using rotary evaporator before subjecting them to freeze-drying. The weights and percentages of the yields of the obtained extracts were calculated, and each extract was assigned with a code or an abbreviation for further and easier use. For the major and primary antimicrobial screening, 50 mg/ml, stock solutions were prepared by dissolving the lyophilized extracts either in methanol, 2.5% v/v dimethylsulfoxide (DMSO, Sigma-Aldrich, France) or hexane. 72 Plant material (100-200 g) Hexane or Petroleum ether Petroleum ether or hexane extracts Marc Dichloromethane or Chloroform CH2Cl2 or CHCl3 Ex. Marc Acetone 80 % Methanol Ex. Acetone extracts MeOH extract Marc Liquid/liquid separation EtOAc layer Ex. Aqueous layer Ex. Figure 28. Schematic diagram of the sequential extraction and liquid/liquid partition. 100-200 g, of plant materials were used. Dichloromethane, CH2Cl2, refers to Dichloromethane extracts; chloroform extracts, CHCl3, refers to chloroform extracts; ethyl acetate (EtOAc); marc, refers to the extraction residue; Ex., refers to extracts. 73 3.2.3. Column and thin layer chromatography 3.2.3.1. Sephadex LH-20 (Study I and III) Size exclusion chromatography (SEC) (also called gel filtration chromatography) with the dextran-based polymer, Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) as the stationary phase, was used in order to separate the high molecular weight compounds (mainly ellagitannins and other tannins) from lower molecular weight compounds (flavonoids, lower molecular ellagitannins and other phenolic compounds) in a root extract of Terminalia brownii. Acetone and 80 % ethanol were used as eluents. Sephadex LH-20 seperation was performed in centrifuge tubes (Eppendorf, 50 ml volume) as follows. 10 ml of 80 % (v/v) ethanol was used to dissolve 200 mg of a methanolic Soxhlet extract of the roots of T. brownii. Subsequently, the resulting extract was centrifuged and the supernatant was added to a 50 ml centrifuge tube which contained 2.5 g of Sephadex LH-20. Then the centrifuge tube was mixed carefully by shaking and centrifuged for 3 min at 3000 rpm (SIGMA 3-18K Centrifuge), after which the supernatant (the 80 % ethanol extract) was discarded and collected in and Erlenmeyer flask for analysis. The adding of 80 % ethanol (8 –10 ml at a time) was repeated until the color of the upper phase changed from yellow to colorless or transparent (absorbance near to zero) at the wavelength of 280 nm. The fractions resulting from the 80 % ethanol wash, i.e. the “non-tannin or low molecular component fractions” were combined and concentrated to dryness under a rotary evaporator. Following the ethanol wash, 70% acetone (v/v) was used (2 u 15 ml) to release the high molecular weight tannins the ET, enriched fraction or tannin wash) from the pores of the Sephadex LH-20 phase. The acetone wash fraction was also evaporated to dryness using a rotary evaporator. Liquid nitrogen treatment was used for two days to dry the Sephadex LH-20 fractions. For agar diffusion antimycobacterial screenings and microplate assays, 1000 μg/ml from the ethanol wash and acetone wash, were dissolved in one ml methanol (stock solution of 1000 μg/ml), and stock solutions of 3 mg/ml were used for the antibacterial microplate assays. The Sephadex LH-20 fractions, at 2 mg/ml (injection volume 10 μl), were analyzed with HPLC-DAD for total compound profiling of the fractions, especially focusing on phenolic compounds (hydrolysable tannins and flavonoids). The UV absorption maxima and retention times of the HPLC peaks of the ethanol and acetone fractions were compared to the chemical profile of the crude methanolic Soxhlet extract. 3.2.3.2. Preparative RP-18 thin layer chromatography (TLC) (Study III) Preparative thin-layer chromatography using reversed phase thin-layer chromatography on glass-backed and aluminum-backed silica plates (TLC plates, 20 u 20 cm, coated with RP-18F 254, Merck, Darmstadt, Germany) were used to collect ellagitannin-enriched fractions of a methanolic root extract of Terminalia laxiflora. Corilagin, ellagic acid and gallic acid were used as reference compounds. The thin layer chromatography was performed as follows. 5 – 10 microliters of the crude extracts (50 mg/ml in MeOH) were applied to the thin layer plates using micro-capillary pipettes (1 – 5μl, P 4518-5X, AccuppetteTM Pipets/ DADE and 5 – 10μl, DURAN®, ring Caps, Code No. 9600210, Germany). For the separations on RP-18 reversed phase silica gel, a mobile phase containing methanol: water: orthophosphoric acid (50:50:1) was used. The eluted plates were photographed using a Camag Video documentation System (Camaq Reprostar 3 TLC Visualizer). The retardation factors (Rf values) of the fractions and the pure compounds were calculated and the Rf values of the compounds were compared to reference compounds, such as corilagin, ellagic acid and gallic acid. All compounds which had fluorescent and/or quenching properties at the wavelengths of 366 nm and 254 nm respectively, were detected and marked on the 74 plates using a soft pencil. The spots, representing pure compounds or fractions, were removed from altogether 30 glass plates and placed in centrifuge tubes. 5 ml of methanol was then added to the tubes before placing them in a vacuum rotation centrifuge (Mini Spin®plus, Eppendorf, Germany) at 5000 rpm for 10 minutes in order to separate the compounds and fractions from the silica. The supernatants were transferred into clean test tubes, whereafter the methanol was evaporated using an Eppendorf tube evaporator (Concentrator Plus, Eppendorf). Stock solutions at concentrations of 1000 μg/ml, where then prepared from the dried fractions and compounds to be used for antimicrobial test and phytochemical analysis (HPLC-DAD). 3.2.3.3. DPPH-TLC qualitative method for measuring antioxidative activity (Study III) A qualitative TLC bioautography method (Wang et al. 2012) were used to identify compounds with antioxidative properties in the extracts of Combretum hartmannianum, Anogeissus leiocarpus, Terminalia brownii and Terminalia laxiflora. Reversed phased, aluminum-backed TLC-plates (RP-18 F254s TLC plates, 20×20 cm, Merck, Darmstadt, Germany) and aluminum-backed normal phase TLC plates (Silica Gel 60 F254, 20×20 cm, MERK, Germany) was used. A volume of 1 μl of the extracts (50 mg/ml) were applied equidistantly on the TLC plates. For the RP-18 plates, methanol: water: orthophosphoric acid (50:50:1, v:v:v) was used as mobile phase and the plates were developed to a distance of 8 – 12cm. Chloroform: ethyl acetate: formic acid (50:40:1, v:v:v) was used for the normal phase silica plates. Both the undeveloped and the developed plates were investigated at 366 and 254 nm, using the Camaq Reprostar 3 TLC Visualizer documentation system, and the spots were marked with a soft pencil. The developed plates were then sprayed with a DPPH (0.2 % w/v in methanol) (2,2-Diphenyl-1picrylhydrazyl, Sigma-Aldrich, D9132-1G, Germany) reagent. After spraying, the plates were rapidly dried using a hair dryer under the fume hood, whereafter they were photographed in visible light. Compounds and fractions with antioxidative properties were visible as yellow spots against a purple background. Standard compounds known to be present in the extracts, such as corilagin, ellagic acid and gallic acid, were used as a reference compounds. For the identification of antioxidative fractions/compounds, the sprayed plates were compared to unsprayed replicates, and the retardation factors (Rf) of the antioxidative fractions/compounds were measured and compared to the reference compounds. The retardation factors were measured as the distance moved by the compound (from starting point to the center of the spot) divided by the distance moved by the solvent front. 3.2.3.4. Qualitative normal phase and reversed phase thin layer chromatography (Study II) Normal phase and reversed phase silica gel thin layer chromatography were used for qualitative investigations on the secondary compound contents of ethyl acetate extracts of the stem wood and bark of Terminalia brownii. For the normal phase TLC runs, aluminum backed Kieselgel 60 F254, (Merck, Darmstadt, Germany) was used, and toluene: ethyl acetate: formic acid (4:5:1, v:v:v) was used as an eluent. For the reversed phase runs, RP-18 F254s (Merck, Darmstadt, Germany) was used and methanol: water: acetic acid (6:2:2) was used as eluent. For all runs, 5 μl of the ethyl acetate extracts (5 mg/ml) was used. The development distances were 8 cm. Vanillin-H2SO4, was used for the detection of phenolic compounds, whereas Dragendorff reagent was used for the detection of alkaloids and Natural Products reagent for flavonoids. Plates sprayed with the reagent were compared to non-sprayed plates at 254 and 366 nm. The plates were documented using a Camaq Video documentation system. 75 3.3. Analytical chemistry 3.3.1. HPLC-DAD (Studies I and III) For rapid and accurate phytochemical analysis of phenolic compounds, a HPLC method explained in Taulavuori et al. (2013) was used to identify phenolic compounds such as hydrolyzable tannins and flavonoids in Terminalia brownii, T. laxiflora and Anogeissus leiocarpus. An HPLC-DAD apparatus coupled to Agilent Chemstation Software (Water Corp., Milford, USA) and to an auto-sampler was used. Moreover, the high performance liquid chromatography (HPLC) system consisted of a Waters 600 E pump, a reversed phase column (Hypersil Rp C18 column, length: 60 mm; ID: 2 mm) and a detector (991 PDA detector). All the extracts were dissolved in 50% methanol to a concentration of 2 mg/ml, of which 10μl was injected into the HPLC apparatus. 1.5% tetrahydrofuran and 0.25% orthophosphoric acid were used as solvent (A) and solvent (B) was 100 % MeOH. Gradient elution was performed and the flow rate was 2 ml/min. The chemical profiles of the various chromatograms were achieved at the wavelengths of 220, 270, 280, 320 and 360 nm. Agilent Chemstation software was used to obtain the UVλ absorption maxima spectra of the studied peaks at wavelengths ranging from 210 and 400 nm. The peaks of interest were compared to a database of natural compounds available in the computer and to the literature, such as Conrad et al. (2001) and Pfundstein et al. (2010). 3.3.2. UHPLC/QTOF-MS (Studies I and III) An ultra-high performance liquid chromatography method described in Taulavuori et al. (2013) was used to separate the phenolic constituents in the extracts. This UHPLC-apparatus was coupled to a time of flight mass spectrometer, which has a capacity of estimating molecular masses with the accuracy of four decimals. For the separations and mass determinations, UHPLC-DAD (Model 1200 Agilent Technologies)JETSTREAM/QTOFMS (Model 6340 Agilent Technologies) and a reversed phase column of C18 (2.1 u 60 mm, 1.7 μm, Agilent Technologies) were employed. Solvent (A) was 1.5% tetrahydrofuran and 0.25% acetic acid in ionized water. Solvent (B) was 100% methanol (HPLC grade). Gradient elution was used; starting from 0 to 1.5 min, B 0 %, from 1.5 to 3 min, 0 to 15% B, from 3 to 6 min, 10 to 30 % B, from 6 to 12 min, 30 to 50% B, from 12 to 20 min, 50 to 100% B, and from 20 to 22 min, 100 to 0% B. Negative ion mode [M-H]- and a mass range between 100 – 2000 m/z were used. The negative ion mode has been found to be more accurate for the mass elucidation of ellagitannins (Pfundstein et al. 2010) and the results of the analyses were compared to Pfundstein et al. (2010) and Conrad et al. (2001). The parts per million (ppm) or the mass accuracy was calculated according to Brenton and Godfrey (2010) and is shown in the formula below. The mass accuracy measures the accuracy of the mass spectrometric measurement of the molecular ions [M-H]- compared to the exact calculated molecular masses of the analyzed compounds. The mass accuracy is calculated as follows: PPM (ppm, parts per million mass error) mass accuracy = (M measured – M calculated) u 106/M calculated Where M measured stands for the measured mass in Q-TOF-MS and M calculated stands for the exact calculated mass according to the molecular formula of the compound. Since the negative mode of Q-TOF-MS was used, the monoisotopic mass of the hydrogen atom (1.0078) was subtracted from all the calculated masses. 76 3.3.3. Tandem mass spectrometry (LC-MSn) (Study II) An Agilent LC system (1100 series, Waldbronn, Germany) consisting of a binary Agilent 1200 series binary pump system and a diode-array (DAD) detector was used. Gradient elution was performed using acetonitrile and water containing formic acid (0.005 %) as solvent A and acetone and glacial acetic acid as solvent B. A flow rate of 0.5 ml/min was used. Chromatograms were scanned at 254, 320, 360 and 380nm. This LC was connected with electrospray ionization (ESI, Bruker Daltonics HCT Ultra, Bremen, Germany) and ion-trap mass spectrometer and operated in MSn scan mode using Collision Induced Dissociation (CID) energy, to achieve a series of smart fragment ions in m/z form. This system made it possible to construct the molecules with the aid of the molecular fragments resulting from their fragmentations. The negative mode condition [M-H]- was used. 3.3.4. GC/MS 3.3.4.1. Sample preparation for GC/MS analysis The GC/MS method reported by Münger et al. (2015), was used in order to identify fatty acids, sterols, triterpenes and their glycosides (saponins) and steroids present in n-hexane, petroleum ether, chloroform or dichloromethane extracts of the stem and roots of Terminalia laxiflora and T. brownii. The extracts were treated with silylation reagents, such as trimethylchlorosilane (TMCS, GC grade, Sigma-Aldrich, Buchs, Switzerland) and N,O-bis (trimethylsilyl) trifluoroacetamide, BSTFA, GC grade (Sigma-Aldrich, Buchs, Switzerland) in order to increase their degree of volatility. 10 mg of the extracts was dissolved in 2 ml dichloromethane (HPLC-grade), and 200μl of this extract was used for the silylations and dried using nitrogen gas (PIERCE Modle 18780 Reacti-VapTM). Silylation was performed using 100μl of the silylation reagent which consisted of 99% BSTFA, 1% TMCS and 100μl of pyridine). The sample was heat, treated in an oven, where the silylation was performed for 30 – 40 min at 60qC, whereafter the silylation reagent analytes, containing trimethyl silyl ethers (TMS), were evaporated to dryness using nitrogen gas (PIERCE Modle 18780 Reacti-VapTM). For further analysis, the silylated samples were dissolved in 200μl heptane (chromasolv® for HPLC, Germany) to be scanned using Agilent technology GC/MS. 3.3.4.2. GC/MS run conditions Gas chromatography coupled to mass spectrometry was performed using a GC/MS HP6890 (Agilent technologies) apparatus which consisted of an auto-sampler, a split/splitless injector and a fused silica RTXTM-5 capillary column (5% diphenyl and 95% dimethyl polysiloxane phase; 60 m, 0.32 mm, ID “inner diameter”, 0.1 mm film, Restek Corp., Bellefonte, PA, USA). For the gas chromatography, the oven temperature was +150qC for 1 min and then increased to a maximum of +275q C, with a temperature increase rate of +15qC/min. Finally, the temperature was set to +310qC at a rate 5.00qC/min and held at that temperature for 10 minutes. 2.0 μl of samples (in heptane) were injected to the GC at a temperature of +275q C. Helium was used as sheath gas at a pressure of 21.50 psi. For the mass spectrometry, electron impact ionization was used. The interface and ion source temperatures were +280qC and +230q C, respectively. The MS spectra were acquired using a mass range from 80 to 650 m/z. 77 3.3.5. Data analysis methods for the identification of the compounds The retention times (Rt), molecular ions (M+), main fragment ions (m/z value), percentage of the relative intensity of the peaks and the peak areas (%) were used for analysing compounds in the GC- and MSchromatograms. The AOCS lipid library (Wiley, NIST) and Scifinder libraries of reference compounds were used as references. The resulting data were compared to the data reported in the literature. 78 3.4. Antimicrobial assays 3.4.1. Bacterial and fungal strains ATCC and other standard strains of bacteria, molds and yeasts were obtained from the Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, Finland and National Research Center and University of Khartoum, Sudan. Four gram-positive and one gram-negative bacterial strains were used. Among the gram-positive bacteria, Mycobacterium smegmatis ATCC 14468, was used as a fast growing model strain for tuberculosis. Moreover, Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 25923 and Micrococcus luteus ATCC 4698 were used as other Grampositive model strains. Pseudomonas aeruginosa ATCC 27853, was used as model strains of Gramnegative bacteria. The filamentous fungi (molds), Aspergillus niger ATCC 9763, Nattrassia mangiferae ATCC 96293, Aspergillus flavus ATCC 9763 and Fusarium moniliforme ATCC 24378, were used as model plant pathogenic fungi, occasionally and increasingly also known to cause infections in immunodeprived humans. 3.4.2. Pure compounds used as reference compounds for the antimicrobial tests Standard compounds present in the extracts of the studied plants were used as reference compounds to compare their antimicrobial activities to those of the fractions and crude extracts in order to assess their possible contribution to the growth inhibitory activities of fractions and extracts obtained from the studied plants. Purchased pure compounds representing the main chemical classes present in the studied plants were used. These compounds included the ellagitannins punicalagin (Sigma-Aldrich, Germeny) and corilagin (Sigma-Aldrich, USA) and their monomer, ellagic acid (Sigma-Aldrich, England). Moreover, gallic acid (Sigma-Aldrich, China), which occurred in almost all more polar extracts, was used. Triacontanol (Sigma-Aldrich, Germany), sitosterol (Sigma-Aldrich, Germany) and stigmasterol (SigmaAldrich, Germany), were used as representatives of the fatty alcohol and sterols which were found in this study in Terminalia brownii and T. laxiflora. In addition, the fatty acids stearic acid (octadecanoic acid, Sigma-Aldrich, Germany) and behenic acid (docosanoic acid, Sigma-Aldrich, Germany), were used. Friedelin (Extrasynthese, France), was used since we have found this triterpene in Terminalia brownii. Quercetin (Merk, Darmstadt, Germany), apigenin (HPLC, Etrasynthese, France) and luteolin-7-glycoside (Sigma-Aldrich), were also used, since these flavonoids were found to be present in both studied species of Terminalia as well as in Anogeissus leiocarpus. 79 3.4.3. In vitro antimicrobial methods 3.4.3.1. Agar well and disk diffusion methods Bacteria (Study I) For the primary screenings, an agar diffusion method was used (Elegami et al. 2002, Fyhrquist et al. 2002 and 2014). For the agar disk diffusion method, sterile paper disks with a diameter of 12.7 mm were used (Schleicher and Schuell 2668). For the well diffusion method, wells with a diameter of 10 mm were bored in the agar. The test was initiated by inoculating the bacterial strains on nutrient agar slants for 24 hours at +37° C. Subsequently, a few colonies from this overnight bacterial culture were transferred to 2 ml of an isotonic sodium chloride solution. 1 ml from this suspension was pipetted into a disposable UV Cuvette (BrandTech®, USA), and the turbidity of the solution was measured at 625 nm using a UVVisible Spectrophotometer (Pharmacia LKB-Biochrom 4060). According to the turbidity measurement result, the bacterial suspension was further diluted with 0.9% (w/v) of isotonic sodium chloride to obtain an absorbance of 0.1 at 625 nm, which equals to approximately 1 × 108 CFU/ml (0.5 McFarland Standard). 200μl of this suspension was spread evenly in a petri dish (‡ = 14 cm, VWR Finland). These petri dishes contained 25 – 30 ml of Base agar (DifcoTM, VWR Finland) as bottom layer and a similar amount of Isosensitest agar (OXOID, Thermo Fisher Scientific) as a top layer. If paper disks were used, 4 u 50 μl of the extracts (50 mg/ml), and pure compounds and antibiotics (10 mg/ml), were applied to the disks, and the disks were allowed to dry well before placing them equidistantly on the inoculated petri dishes. Tetracycline hydrochloride (Sigma-Aldrich, St. Louis MO,USA, penicillin-G (Fluka, Germany), ampicillin (Sigma-Aldrich, St. Louis MO, USA) and gentamycin (Sigma-Aldrich, St. Louis MO, USA) were used as standard antibiotics. The same concentrations were used for the agar well diffusion method, with the difference that the whole sample volume could be added to the wells at the same time. The solvents used to dissolve the samples, such as methanol and hexane, were used as negative controls, and 200 μl of each solvent was pipetted into the agar wells or onto the filter paper disks. Prior to incubation, the petri dishes were kept in the cold room at +4° C for 1 – 2 hours, in order to facilitate the diffusion of the extracts and the compounds into the agar. The plates were then incubated at +37 °C for 24h. Each experiment had three to six replicates, and the experiments were performed at least two times. The diameters of the resulting inhibition zones (IZ) were measured using a caliper and a petri dish magnifier, and the results were calculated as the mean of three – six replicates ± standard error of mean (SEM). A Camaq video documentation system (Camaq Reprostar 3 TLC Visualizer) was used to photograph the petri dishes containing the inhibition zones. Mycobacterium smegmatis (Study III) An agar disk diffusion method described in Fyhrquist et al. (2014) and Salih et al. (2018), was used for the primary screenings of the crude extracts, chromatographic fractions (Sephadex LH-20 and RP-18 TLC) and pure compounds, as well as the reference antibiotic (rifampicin) against Mycobacterium smegmatis ATCC 14468. Prior to the test, M. smegmatis was grown on a Löwenstein-Jensen agar slant (Becton-Dickinson & Company, USA) for five days at +37 °C. After completed growth, one colony was transferred from the Löwenstein-Jensen medium to fresh Dubos broth (Difco) and the turbidity of this suspension was measured at 625 nm, using a UV-visible spectrophotometer (Pharmacia, LKB-Biochrom 4060). A625 of the suspension was then adjusted to 0.1, which equals to approximately 1 u 108 CFU/ml. 200 μL of this 80 inoculum was then pipetted onto each petri dish (‡ = 14 cm, VWR Finland). The petri dishes contained 25 – 30 ml of Base agar (Difco™) as bottom layer and 25 – 30 ml of Middlebrook 7H10 agar (Difco™) containing OADC supplement (Difco™) as a top layer. Sterile filter paper disks with a diameter of 12.7 mm (Schleicher and Schuell 2668) were saturated with 200μl of the crude extracts (50 mg/ml), Sephadex LH-20 fractions and RP-18 TLC (1000 μg/ml), and rifampicin (10 mg/ml) (Sigma-Aldrich). 200 μl of methanol and hexane were used as solvent controls and were found to possess no activity against the growth of M. smegmatis. Prior to incubation, the petri dishes were kept for 1 – 2 hours in a cold room at +4° C. The petri dishes were then incubated for five days at + 37qC. Each sample was tested in triplicates, and the growth inhibitory effects were measured as the mean of the diameters of these triplicates r SEM. Moreover, the growth inhibitory effects of the extracts and chromatographic fractions were compared to rifampicin using a formula for calculating the activity index that indicates the % of activity compared to rifampicin: AI (Activity index) = Inhibition zone of the plant extract/ Inhibition zone of rifampicin Filamentous fungi (Aspergillus, Fusarium, Nattrassia) (Study II) For the primary screenings of the growth inhibitory effects of extracts of various polarities of the stem bark and stem wood of Terminalia brownii against filamentous fungi, a slightly modified cup well agar diffusion method, based on Elegami et al. (2002), was used. All fungal strains were grown overnight at +35° C on Sabouraud dextrose agar petri-dishes. 100 ml of sterile, isotonic NaCl was used, in order to obtain a conidial suspension. For the agar diffusion test, the cell density of the conidial inoculum was adjusted to 1 × 108 CFU/ml. 200μL of this inoculum was mixed with 20 ml of molten Sabouraud dextrose agar, and poured in petri dishes (‡= 9 cm). The agar with the conidia was left to set, whereafter four wells (‡ = 10 mm) were bored in the agar. Each well was filled with 100μl of the extracts and amphotericin B (1 mg/ml in 50 % methanol). 100 μl of 50 % methanol was used as a negative control. The petri-dishes were left in the cold (+4qC) for 1 h, whereafter they were incubated at +35 °C for 24 hours. Five replicates were used for each experiment, and the results were calculated as the mean of four diameters r SEM. 3.4.3.3. Assays for measuring MIC, MBC and MFC Microdilution assays for MIC values (bacteria and M. smegmatis) (Studies I and III) A turbidimetric microdilution assay was used to measure the antibacterial and antimycobacterial growth inhibitory effects of crude extracts, solvent partition fractions and chromatographic fractions of the investigated plants. The method used for each of the three categories of micro-organisms was essentially the same, but differed when it came to the media used (Mueller-Hinton broth for bacteria, and Dubos broth for M. smegmatis) and the inoculum sizes. Basically for each method the guidelines of Clinical Laboratory Standards Institute (CLSI 2010 and CLSI 2013) were followed. In brief, the methods reported in Salih et al. (2017a, 2018) were used. For all microorganisms, two-fold serial dilutions of the extracts were made with concentrations ranging from 5000 – 78.12 μg/ml. For chromatographic fractions, pure compounds and antibiotics, two-fold dilution of concentrations ranging from 1000 μg/ml to 0.030 μg/ml were used. The dilutions were performed in Mueller-Hinton (bacteria) or Dubos broth (Mycobacterium smegmatis). For the microdilution tests, colonies of bacteria, grown overnight (at +37qC on Nutrient agar slants for bacteria) or for five days at + 37qC on Löwenstein-Jensen agar slants (Mycobacterium smegmatis), were transferred to 2 ml of Mueller81 Hinton or Dubos broth, respectively, in order to measure the turbidity at 625 nm using a spectrophotometer (UV-Visible Spectrophotometer, Pharmacia LKB-Biochrom 4060). The suspensions were diluted according to the guidelines of the Clinical and Laboratory Standards Institute (2012, 2013). For the bacteria an inoculum containing 1.0×106 CFU/ml was used, whereas for Mycobacterium smegmatis an inoculum of 2.5 × 105 CFU/ml was used. A volume of 100 μl of the microbial inoculi was added to the test wells (containing plant extracts, fractions, pure compounds or antibiotics) and the growth control wells of the microplates (96-Well™ ,400 μl/well, polystyrene, clear, flat bottomed microplate, Thermo Scientific™, Nunc, Nunclone, Denmark). Besides a volume of 100 μl of the diluted plant extracts, fractions and pure compounds, the solvents (negative control) and broth alone (in the growth control wells) were added to the 96-well plate. Thus, the total volume per well was 200 μl. Moreover, for each plant extract and fraction, sample controls were made, which consisted only of that extract/fraction and broth. These sample controls were made, since some of the plant extracts were found to give a high absorbance at 620 nm. Thus these absorbances were subtracted from the results obtained from the corresponding test wells, containing the extracts/fractions and bacteria/fungi. The microplates were incubated for 18 – 24 hours at +37°C for bacteria, respectively, whereas Mycobacterium smegmatis was incubated for four days at +37qC. After completed incubation, the turbidity at 620 nm was measured using a Victor 1420 microplate reader (Wallac, Finland). The turbidimetric data were analyzed using Excel software, and the growth inhibitions were calculated as the mean percentage of growth of test well triplicates, compared to the growth control. The minimum inhibitory concentration was taken as those concentrations of the tested samples which resulted in 90 % or more growth inhibition compared to the growth control. The following formulas were used to calculate the percentage of microbial growth and the percentage of growth inhibition: % bacterial growth = [(x̄ GT A620 – x̄ SC A620)/ x̄ GC A620) u 100] % inhibition of growth = 100 (% growth of the growth control) – [(GTA620 –SC A620)/GC A620) u 100] Where the GTA620 is the turbidity of the test well at 620 nm (containing plant samples, pure compounds or antibiotics and microbial cells), SCA620 is the sample control (consisting of the plant samples, the compounds or antibiotics alone, without microbial cells) and GC A620 is the turbidity of the growth control at 620 nm (containing only bacterial cells). x̄ is the average of the triplicates - quadruplicate. Agar diffusion assay for MIC values (bacteria) (Studies I and III) An agar diffusion method was used to determine the minimum inhibitory concentration for extracts that could not be tested with the microdilution method. The microplate method was found to be inappropriate to use for these extracts forming precipitation due to molecules present in the extracts which did not dissolve properly in the broth (non-polar compounds in hexane extracts or large molecular compounds such as tannins in the aqueous fractions) or due to color absorbing at wavelengths of 620 nm. The agar diffusion method, described in chapter 4.3.1. was used for these MIC assays. Two-fold serial dilutions of the extracts were made in methanol for the polar extracts and in n-hexane for the non-polar extracts in order to dissolve the extracts properly. Concentrations ranging from 5 mg/ml – 78.12 μg/ml and 1000 μg/ml to 0.030 μg/ml were used for the extracts and antibiotics respectively. 200 μl of each dilution of the extracts were pipetted onto Whatman filter paper discs (Ø = 12.7 mm, Schleicher and Schuell 2668) which were placed equidistantly on the inoculated petri dishes (∅ = 14 cm, VWR Finland). The approximate MIC value was determined as the mean of the diameters of the inhibition zones of triplicates 82 r SEM, and was taken as the smallest concentration of the extracts resulting in a visible inhibition zone (IZ) around the filter paper disc. Agar dilution assays for MIC value (filamentous fungi) (Study II) A method described by Guarro et al. (1997), was used to determine the minimum inhibitory concentration of the extracts of the stem bark and stem wood of Terminalia brownii and amphotericin B against Aspergillus, Fusarium and Nattrassia spp..100μL of the fungal suspension, adjusted with 0.9 % w/v NaCl to contain 1.0u106 CFU/ml, was mixed with 10 ml of molten Sabouraud dextrose agar and poured to petri dishes (Ø = 9 cm). 10 mL, containing 9.5 ml molten Sabouraud agar and 500 μl of the two-fold dilutions was poured as a top layer onto the petri-dishes. The dilutions of the stem bark and stem wood extracts of Terminalia brownii (500 – 31.25 μg/ml) and antibiotics (500 – 15.62 μg/ml) were made in 50 % methanol or hexane. For the negative controls, 9.5 ml molten Sabouraud agar was mixed with 500 μl of 50 % methanol or 500 μl hexane. The petri dishes were incubated at +35 qC for 24 h. The MIC was taken as those concentrations of extracts or amphotericin-B resulting in clear petri-dishes containing no visible growth of the fungi. The experiments were performed in triplicates. 83 4. RESULTS AND DISCUSSION 4.1. Results from the ethnobotanical and ethnopharmacological investigations in Sudan 4.1.1. General observations Several tribes belonging to various ethnic groups and with different religions are resident in both study areas (El Hassan et al. 1993, Casciarri 2009, Salim et al. 2011, Abdalla 2013, Daak et al. 2016, Human Right 2016). In the Blue Nile region, the people belong to the Afro-Arabian tribes Kenana, Rufaa, Fallata, Hausa and Bornu and the African tribes Masalit and Dinka. In the Kordofan region, the major AfroArabian tribes are Baggara “Rizeigat, Ta’isha”, Misseriya, Hawazma, Borgo, Fallata, Hausa and Bornu and the African tribes are Masalit and Nuba (El-hassan et al. 1993, Casciarri 2009, Salim et al. 2011, Abdalla 2013, Daak et al. 2016). The people in the Blue Nile and Kordofan regions are either nomadic herders, keeping animals such as cows, sheep, camels and goats, or settled farmers or craftspeople (Watch 1990, El-Harizi and Prato2007). The farmers grow various food crops such as groundnuts, sorghum, pearl millet, sesame, maize and vegetables such as arugula, lettuce, jute mallow, purslane, potato, onion, tomato, okra, eggplant, cucumber, watermelon and pumpkin (Karimi et al. 2012). Both among the nomads and the settled farmers people work as traditional medicinal practitioners (TMP). Moreover, also village elders, both women and men, possess knowledge on the traditional use of medicinal plants. However, after the current war erupted in South Kordofan in 2011, the communities started to lose their animals and their farmland (Elhassab and Elhassab 2017). Therefore, the people have shifted to work in gold mining or oil boring, in order to provide income for their families. Due to the increasing population, however, there is an increasing trend in using plants for medicinal purposes, and especially for the treatment of infectious diseases and their symptoms. Results from the ethnobotanical and ethnopharmacological interviews are presented in appendices Table 2. The scientific names of the listed plant species are updated with the latest officially accepted nomenclature (The Plant List 2010). Moreover, also the Sudanese Arabic names, plant parts used, methods of preparation, frequency and the preferred time of collections, are listed. As shown in appendices Table 2, the most important species for curing infectious diseases in local traditional medicinal applications (labeled with three asterisks), include Anogeisssus leiocarpus, Terminalia brownii and Guiera senegalensis, (moderate importance), and the plants of lowest importance are marked with an asterisk. Traditional uses for treating fungal diseases in animals, humans and crop plants are presented in appendices Table 4. 4.1.2. Traditional uses of Terminalia species Four Terminalia spp. occurred commonly in woodlands and dry savannas of the study areas, namely T. laxiflora Engl., T. brownii Fresen, T. macropetra Guill. & Perr. (Syn. Terminalia chevalieri Diels) and T. avicennioides Guill. & Perr. In four villages in the Blue Nile region and three villages in the Kordofan region, the traditional healers and villagers interviewed described the genus Terminalia and especially T. laxiflora and T. brownii as the most popular plant species for cosmetic and medicinal uses (appendices Table 2). There was a good consensus on the medicinal uses of these species, with most traditional healers reporting similar uses. However, it appeared that it is difficult both for the local people and the traditional healers to distinguish between T. laxiflora and T. brownii, especially before the fruits are ripening and before the trees have reached maturity. The stem bark morphology is species specific only in mature individuals, with T. brownii bark characterized by clear, longitudinal fissures, whereas in T. laxiflora the stem bark is deeply anastomosed and fissured (Figs. 2 and 3). 84 All parts of T. laxiflora and T. brownii are mostly administrated orally as decoctions or macerations to cure a wide range of infectious diseases such as dysentery, diarrhea, cough, abdominal pain, chest pain, fever, eye infections and respiratory infections. Both species are also used externally as ointments or poultices against rheumatism or to cure skin infections such as acne, for wound healing and skin ulcers and to treat itchy skin. In some cases the gum exudate from Terminalia spp. and Combretum hartmannianum (Combretaceae) and the gum arabic exudate from Acacia senegal and A. seyal (Mimosaceae), is mixed with extracts of T. laxiflora and T. brownii to stabilize and homogenize the liquid and as preserve it for the longest storage. Besides the medicinal uses of T. laxiflora and T. brownii, all interviewees mentioned that fumigations obtained by burning of the stem bark of these species were used for cosmetic and other well-being purposes among women (appendices, Table 2). This result is in accordance with earlier findings indicating that in both rural and urban regions of Sudan, most of the married women, including widows and divorced women, use the smoke fumigation called “Dokhan“ for various purposes (Mariod et al. 2014). “Dokhan” results from the burning of the stem wood and roots of Terminalia brownii or T. laxiflora and the stem wood of Acacia seyal. “Dokhan” is used for several different purposes: (1) Before delivery, “Dokhan” works as a natural massage against fatigue and to relieve contractions. (2) After the delivery, “Dokhan” is used to accelerate the healing process of the vagina as well as for the tightening of the vagina and for avoiding venereal inflammations and infections. (3) Before wedding, the elder women are daily preparing the brides for their wedding day by treating the brides with “Dokhan” for at least one month continuously, in order to obtain a bright and soft skin (Fig. 29) with a nice smell, as well as a long healthy hair. These customary applications are normally continued even after the wedding day by the Sudanese women. Moreover, the smoke produced from wood is used for making a natural skin scrub known as “Dilka”, for which the women are mixing sorghum porridge with the smoke produced from the burned wood and roots, of Terminalia laxiflora, T. brownii and Acacia spp. (Fig. 29). “Dilka” balls can be stored and kept at room temperature for many years without rotting, and just a small volume of water is added to moisten them before use. (C (A (C (B ) (D) (E) Figure 29. (A), Burning wood for Dokhan application; (B), Stem wood in a clay jar in a hole surrounded by a mat made from palm fibers, the wood is burned beneath a special blanket placed on a basket, the blanket is used to cover the body and retain the smoke produced from wood; (C), The skin is exposed to the smoke, resulting in a brown to dark yellow color; (D), Wood chips of Terminalia brownii and T. laxiflora used for perfume and insect repellent; (E), Sorghum balls “dilka” used for scented organic massage and as natural skin scrub especially after dokhan and during normal days, besides dilka used as facial organic cream. Photos by Enass Salih. 2014. 85 4.1.3. Combretum hartmannianum Altogether seven different species of Combretum were found to occur in both study areas. These species were C. hartmannianum, C. molle, C. nigricans, C. aculeatum (Syn. C. alternifolium; C. denhardtiorum; C. leuconili; C. ovale), C. glutinosum, C. lamprocarpum, and C. ghasalense. Results from the ethnobotanical interviews on the traditional uses of Combretum spp. in the villages in Kordofan and Blue Nile regions are listed in appendices Table 2. All species mentioned were used to treat infections and their symptoms. Our results also revealed that in Sudanese traditional medicine, C. hartmannianum is more extensively used than other the species of Combretum for the treatment of infectious diseases, due to its good availability in the area. Various parts of C. hartmannianum, C. molle, C. glutinosum, C. lamprocarpum and C. aculeatum, such as roots, leaves, the stem bark and stem wood, are prepared as macerations and decoctions and administered orally to cure abdominal pain, sore throat, dysentery and fever. In addition, the stem wood and roots of C. hartmannianum are utilized externally as fumigations to cure sexually transmitted diseases, fungal nail infections and against rheumatism and fatigue. Also, tonics from the leaves and stem wood, as well as pastes and ointments from these plant parts are used against itchy skin, acne, leprosy and for wound healing and against ulcer infections. Our results are in accordance with other investigations on the ethnopharmacological uses of C. hartmannianum (Zenebe et al. 2012, Issa et al. 2018). Furthermore, the traditional healers and other informants reported that in addition to the stem and roots of T. brownii and T. laxiflora, also the stem bark of Combretum hartmannianum is used for “Dokhan” resulting from the burning of the stem wood and root. “Dokhan” is used as an insect repellent, especially against the malaria transmitting mosquito (Anopheles arabiensis) and against lice and flies transmitting cholera (Vibrio cholera) and dysentery (Shigella dysenteriae) (Kereru et al. 2007, Ibrahim and Osman 2014). In Sudan we have observed of Terminalia laxilfora, T. brownii and Combretum hartmannianum that the price of one sack of the stem wood and root part “Dokhan” of Terminalia laxiflora, T. brownii and Combretum hartmannianum (approximating 50 – 40 kg, depending on the wood type and density), fluctuates according to the season. During the rainy season the prices are at their peak (equaling 800 – 900 € in 2017 compared to 250 € in 2012). This increase in price has resulted from a growing consumption of wood for fumigations and could also be due to recent fluctuations in the duration and severity of the rainy season as well as to the deterioration of the local economy and to an increase in the inflation rate (Elfaig et al. 2013, Stiftung 2015). Difficulties in collecting the species mentioned from the forest during the rainy season, may be another reason. 4.1.4. Anogeissus leiocarpus Although a total of eight species of Anogeissus are known, only one, Anogeissus leiocarpus (DC.) Guill. & Perr. occurs in Africa (Eloff 1999, Singh et al. 2016). We found that A. leiocarpus occurs abundantly in both the Blue Nile and Kordofan regions in Sudan, which is in agreement with other authors (Ibrahim and Osman 2014, Gibreel et al. 2013). According to our results, all the traditional healers and other villagers interviewed, used A. leiocarpus as a medicinal plant. Also, we found that this species was used in a similar way by most of the healers i.e. as decoctions and macerations of the stem bark, root and leaves against cough which could also be due to TB, bronchitis, fever, respiratory infections and gastric disorders (appendices Table 2). The doses of administration of decoctions or macerations differed according to the age and weight of the patients, but, in general, half of a small teacup (≈ 250 ml in size), was adminstrated. In addition, some healers informed us that the smoke of the stem and root of A. leiocarpus is used in traditional therapy against rheumatism (appendices Table 2). 86 4.2. Extraction yields from Terminalia brownii, T. laxiflora, Anogeissus leiocarpus and Combretum hartmannianum (Studies I, II and III) Based on our ethnopharmacological results, and according to literature searches, four species, namely Terminalia brownii, T. laxiflora, Combretum hartmannianum and Anogeissus leiocarpus, collected during our fieldwork expeditions in Sudan, were chosen for further investigations on their in vitro antimicrobial effects and phytochemical composition. Another criterion for choosing these species was that they are closely related to each other, all belonging to the family Combretaceae. Thus, similar compound classes, such as ellagitannins, were expected to be found from these species. A further criterion for choosing these plant species was that the phytochemistry of these species is relatively poorly studied. Although the active compounds were not isolated from the species during this research, the phytochemical composition of growth inhibitory (antibacterial and antifungal) extracts and fractions purified to contain a few compounds (Sephadex-LH 20 and TLC fractions) was investigated. A classical bioassay-guided fractionation procedure was used to find those extracts with the most prospective antimicrobial activities. In this fractionation procedure, the fractionation and bioassay testing continues until active fractions containing a few compounds and/or pure compounds with good activity are isolated (Cragg and Boyd 1996). The choice of extraction solvents is important in order to be able to extract target compounds with biological activities from the plant matrix (Eloff et al. 2007, Bergs et al. 2013). However, since the targets are often unknown, a conventional solid-liquid extraction (sequential extraction) process using a series of solvents, from non-polar to polar, is often used for natural compounds (Sarker et al. 2006, Bergs et al. 2013). In the early stages of bioactivity-guided isolation, solvents with broad extraction capacity are recommended to be used, such as acetone, methanol or ethanol (Eloff 1998). Eloff et al. (2007) reported that for species belonging to the family Combretaceae, an approach of using solvents with a broad extraction capacity in the beginning of the search for bioactive compounds is the best approach, and especially acetone was found to be a good extractant for Combretum and Terminalia spp. In our search for extracts of Terminalia laxiflora, T. brownii, and Anogeissus leiocarpus that would be rich in antimicrobial compounds, we have chosen to use methanol as a basic solvent, extracting a high variety of compounds. We were aware of the toxicity problems with methanol in the bioassays, but at a maximum percentage of 5 %, we found that methanol was not toxic to the bacterial and fungal strains used in our screenings. Also, for the agar diffusion methods, methanol was evaporated before the start of the assays. For extraction using methanol, both cold and hot methanol (Soxhlet) extractions were performed. In addition solid-liquid or sequential extraction was used routinely, using a series of solvents with increasing polarities, such as n-hexane or petroleum ether, chloroform or dichloromethane, acetone, ethyl acetate, methanol and water. Moreover, water was also chosen as a solvent for the powdered plant samples in accordance with the traditional uses as macerations and decoctions of the plant species studied. Since the total antimicrobial activity of a plant extract is dependent on its extraction yield, the extraction yields were calculated for each extraction. Eloff (2000) proposed that the total activity of an extract is equal to the extraction yield (yield from extraction of 1 g plant material) divided to the minimum inhibitory concentration of the same extract. 87 The yields resulting from the various kinds of extraction used in this thesis work are presented in Figs. 30 and 31. Yields resulting from single, solvent extractions are presented in Fig. 30. The by far highest yields of extraction resulted from hot methanol Soxhlet extraction (Fig. 30 A). We found that the extraction yields were species specific and origin specific however, and there were large variations in yields between the different species and even, within a species, between the different organs. From calculations based on the formula below, we found that the highest extraction yield of 81 % resulted from hot MeOH Soxhlet extraction of the leaves of T. brownii (Fig. 30 A). In addition, high yields of extraction were achieved when using hot methanol extractions of the bark of T. brownii (78%) followed by leaves of T. laxiflora (76%). Our result on the extraction yields for Terminalia laxiflora, T. brownii, Combretum hartmannianum and Anogeissus leiocarpus are in accordance with the finding of other authors, that alcohol solvents, such as methanol and ethanol, give good extraction yields for various species of Combretum and Terminalia (Danmalam et al. 2011, Zou et al. 2014, Mbouna et al. 2018, Anokwuru et al. 2018). Moreover, we found that fairly high extraction yields for all the plant species studied resulted from cold water (maceration) and hot water (decoction) extractions. Cold methanol extractions were not as effective as the hot ones in terms of yields (Fig. 30 B). However, caution must be taken when using hot methanol extraction since it can lead to the degradation of sensitive compounds. Our results are in agreement with those of Eloff (2007), showing that solvents with broad extraction capacity, such as methanol (or acetone), are the best choice for extraction of antimicrobials from Combretaceae. Maceration of the leaves, bark and roots of the species studied, resulted in good to moderate extraction yields (Fig. 30 B, D). The highest extraction yields for macerations were obtained for the root parts of Terminalia laxiflora, T. brownii and Combretum hartmannianum, with yields of 20, 19.3 and 14.8% respectively (Fig. 30 C). The highest yield for decoctions were obtained for the leaves of Terminalia brownii (14.4%), followed by the root bark of Anogeissus leiocarpus and T. brownii, both giving yields of 11.4% (Fig. 30 D). We observed that the high molecular compounds, such as ellagitannins and other phenolic constituents, which were present in methanol extracts, and believed to contribute to the antimicrobial effect of those extracts, were also present in water extracts (HPLC-DAD data). Adding to this, water extracts usually contain salt and sugar constituents, which could contribute to make the extracts better tasting for customary use (Weib and Alt 2017, Koh et al. 2009, Ogushi et al. 2005). The good extraction yields that we found for the water extracts are in agreement with Chang and Lin (2012) and Alzeer et al. (2014) and could justify the traditional medicinal uses of these species as hot and cold water extracts. When sequential extraction was used, it was found that a large proportion of the compounds in T. laxiflora and T. brownii were soluble in the aqueous fractions that is, fractions obtained from 80 % methanol in water with 8 – 39% extraction yields and in acetone fractions with an extraction yields ranging from 15 to 43 % (Fig. 31 C and E). Ethyl acetate and dichloromethane gave smaller extraction yields (3.5 to 12.4 % and 0.6 to11 %, respectively), and the n-hexane extracts gave by far the smallest extraction yields (0.7 to 8 %) (Fig. 31 B, D). However, for some species of Terminalia, such as T. catappa, hexane extraction was found to give high extraction yields for the fruits (Wright et al. 2016). In agreement with our results, methanol and acetone extracts have earlier given good extraction yields when using sequential extraction of some other species of Terminalia, such as for the extraction of compounds from the leaves of Terminalia prunioides, T. brachystemma, T. sericea, T. gazensis, T. mollis and T. sambesiaca (Masoko 2006, Eloff 1999). As shown in Fig. 31C, when the sequential extraction was used, for the various parts of Combretum hartmannianum, the highest extraction yield was obtained from the acetone extracts of the root part with a percentage 88 yield of 35 %, followed by the aqueous and ethyl acetate fractions with extraction yields ranging from 5.0 to 24 % and 4.5 and 11.5 %, respectively (Fig. 31 D and E). Hexane and dichloromethane (Fig. 31 A, B) showed the lowest extraction yields (0.9 to1.1 and 1.5 to 3.7 %, respectively. Accordingly, also for Anogeissus leiocarpus acetone extracts of the various parts displayed the highest yields (15 to 22 %) (Fig. 31 C), whereas hexane, dichloromethane and ethyl acetate showed the lowest ones (Fig. 31 A, B, D). In agreement with our results, other authors have found that for many other species of the genus Combretum, such as Combretum erythrophyllum and C. vendae, acetone gives a high extraction yield for the leaves (Suleiman et al. 2010, Eloff 1998). Likewise, the aqueous extracts of the leaves of Combretum paniculatum were found to give fairly good exatrction yields, with the total percentage of extraction of 13.5% (Stanley et al. 2014), whereas the methanol extracts from the root of Combretum dolichopetalum gave a yield of 12.05% (Uzor et al. 2015). The following equation was used: Percentage of extraction yield (%) = Dry weight of crude extracts / Dry weight of plant powder × 100 89 Percentage yield (% w/w) (B) Extracts resulting from cold methanol extraction 19,3 12,9 10,0 10 4,6 13,3 12,7 14,8 10,3 9,7 7,5 4,2 2,2 2,0 Anog. W.Me Anog. R.Me Anog. B.Me Anog. RB.Me hartman. B.Me hartman. W.Me brownii. L.Me hartman. R.Me brownii. B.Me 2,8 3,5 2,1 2,8 2,7 brownii. W.Me 8,0 7,2 Anog. L.Me 8,0 (D) Extracts resulting from decoctions (C) Extracts resulting from maceration 20,0 3,0 3,5 brownii. R.Me 10,8 6,7 5,3 brownii. RB.Me 20,5 3,9 4,5 Laxi. L.Me 20,6 3,8 5,2 Laxi. F.Me 24,6 9,6 Laxi. B.Me 28,8 12,0 10,0 8,0 6,0 4,0 2,0 0,0 Laxi. W.Me 29,6 50,7 47,1 43,5 41,1 15 5 81,1 Laxi. R.Me 34,0 25 20 78,2 76,9 Percentage yield (% w/w) (A) Hot methanolic soxhlet extracts 2,4 4,5 7,1 6,7 1,7 0 Percentage yield (% w/w) Percentage yield (% w/w) 90 80 70 60 50 40 30 20 10 0 16 14 12 10,8 10,0 10 8 6 4 2 11,4 5,3 6,0 6,8 14,4 12,8 9,8 11,4 10,7 6,9 6,8 2,8 2,3 3,5 7,4 5,2 0 Figure 30. Percentage yield of extraction using single solvent extraction for various plant parts of Terminalia brownii, T. laxiflora, Combretum hartmannianum and Anogeissus leiocapus (A) hot methanol extraction (Soxhlet), (B) cold methanol cold, (C) maceration (D) decoctions. Rb, root bark; R, root; W, stem wood; B, stem bark; F, fruit; L, leaf; Anog, Anogeissus leiocarpus; Hartman, C. hartmannianum; brownii, T. brownii; laxi, T. laxiflora; MeSox, methanol Soxhlet extract; Me, cold methanol; HH2O, hot water; H2O*, cold water. 90 8,3 6,2 5,9 1,1 1,7 2,4 1,0 1,3 0,8 1,1 0,7 0,9 1,0 1,0 2,2 1,5 0,7 (C) Extracts resulting from sequential extraction using acetone solvent 50 43,0 45 40 35,1 35 30 25,2 25,1 22,1 25 16,5 20 15,1 15,1 15 10 5 0 Percentage yield (% w/w) Percentage yield (% w/w) (A) Extracts resulting from sequential extraction using hexane solvent 9 8 7 6 5 4 3 2 1 0 Percentage yield (% w/w) (B) Extracts resulting from sequential extraction using dichloromethane solvent 12 11,2 10,0 10 8 4 2 6,4 4,9 6 2,7 2,9 2,8 1,2 0,8 3,4 2,5 4,6 3,7 1,5 1,2 1,9 0,6 0 Figure 31. Percentage of extraction yield resulting from sequential extraction using (A), hexane; (B), dichloromethane; (C), acetone; (D), ethylacetate; (E), 80% MeOH (aqueous layer) of the various part of of the studied species. brownii, Terminalia brownii; laxi, T. laxiflora; Hartman, Combretum hartmannianum; Anog, Anogeissus leiocarpus; hex, hexane extracts; acet, acetone extracts; Dic, dichloromethane extracts; EtOAc, ethyl acetate fraction; aqu, aquous fraction; Rb, root bark; R, root; W, stem wood; B, stem bark; L, leaf; F, fruit. 91 Percentage yield (% w/w) (D) Extracts resulting from sequential extraction using ethyl acetate solvent 14 12 10 8 6 4 2 0 12,4 7,2 Percentage yield (% w/w) (E) 45 40 35 30 25 20 15 10 5 0 7,4 7,9 11,5 9,3 5,5 5,3 6,8 9,5 7,9 7,6 4,5 4,4 3,5 5,4 6,4 Extracts resulting from sequential extraction of the aqueous layer (80% MeOH) 39,1 22,2 21,0 18,0 24,8 16,1 8,1 20,9 25,8 24,0 20,0 5,0 10,0 20,0 7,0 8,0 Contiuned figure 31. Percentage of extraction yield resulting from sequential extraction using (A), hexane; (B), dichloromethane; (C), acetone; (D), ethylacetate; (E), 80% MeOH (aqueous layer) of the various part of of the studied species. brownii, Terminalia brownii; laxi, T. laxiflora; Hartman, Combretum hartmannianum; Anog, Anogeissus leiocarpus; hex, hexane extracts; acet, acetone extracts; Dic, dichloromethane extracts; EtOAc, ethyl acetate fraction; aqu, aquous fraction; Rb, root bark; R, root; W, stem wood; B, stem bark; L, leaf; F, fruit. 92 4.3. Results from the antimicrobial screening 4.3.1 Antibacterial effects (Study I) In order to verify the traditional medicinal uses as anti-infectives of the species of Terminalia, Combretum and Anogeissus examined in this study, extracts of various polarities as well as chromatographic fractions and some pure compounds occurring in these plants were evaluated for their antibacterial effects using common human pathogenic bacteria. For the screenings, three Gram-positive and one Gram-negative bacterial strain were used. Of these strains, Staphylococcus aureus is known to cause bacteremia, osteomyelitis, endocarditis, pneumonia and skin infections and sometimes-even infections in the central nervous system (Liu et al. 2011). Moreover, food-poisoning caused by S. aureus is one of the most common food-borne diseases worldwide (Kadariya et al. 2014). Methicillin- and multridrug resistant strains of S. aureus are an increasing health threat globally (de Oliveira et al. 2011, Diep et al. 2008, Howden et al. 2011). The Gram-negative model bacterium used for our screenings, Pseudomonas aeruginosa, is an opportunistic pathogen causing a range of infections, including wound infections, intra-abdominal and urogenital sepsis and pneumonia in immune-compromised individuals and in children suffering from cystic fibrosis (Arancibia et al. 2002, Fujitani et al. 2011, Thaden et al. 2017). Pseudomonas aeruginosa has been found to have a high intrinsic resistance against antibiotics, and the incidence of strains resistant to many classes of antibiotics (XDR and MDR strains), including combination therapies using an aminoglycoside or a fluoroquinoline in combination with a β-lactam antibiotic, has been reported to increase (Poole et al. 2011, Page et al. 2009, Lister et al. 2009, Rasamiravaka et al. 2015, Ji et al. 2019). 4.3.1.1. Extracts Using agar diffusion, a total of 39 extracts of various polarities from the stem bark, stem wood, leaves, roots and root bark of T. laxiflora, T. brownii and A. leiocarpus were assayed for their antibacterial effects. The results of these screenings are presented in Figs. 32 to 34. Of these extracts, especially the ethyl acetate, aqueous, hot methanol (Soxhlet) and hot and cold water extracts gave large diameters of inhibition (Figs. 32 and 34). Micrococcus luteus was the most sensitive bacterium for the ethyl acetate extracts from both Terminalia species and A. leiocarpus, and large inhibition zones from 32-38 mm were recorded against this bacterium (Figs. 32 and 33). Ethyl acetate extracts from the species mentioned gave good activity also against the Gram-negative bacterium Pseudomonas aeruginosa, with diameters of inhibition zones ranging from 18 – 26 mm and MIC values from 39 – 312.50 μg/ml (Figs. 32 and 33). The best growth inhibitory effect by far, against P. aeruginosa, was obtained with a chloroform extract of the stem wood of T. laxiflora, showing an inhibition zone of 32 mm. However, this result did not correlate well with the high MIC of 2500 μg/ml for the chloroform extract of the stem wood of T. laxiflora against P. aeruginosa, which might be due to the low solubility of this extract in aqueous broth (appendices Table 3). The lowest MIC value of 39 μg/ml in our screenings was obtained with hot methanol (Soxhlet), acetone, ethyl acetate and hot water extracts (decoctions) of the roots of Terminalia laxiflora against all bacteria. Notably, the methanolic Soxhlet extracts of the roots of Terminalia laxiflora, T. brownii and Anogeissus leiocarpus, were more growth inhibitory than the tested antibiotics, gentamycin and tetracycline against P. aeruginosa, which gave a MIC of 65.5 and 125 μg/ml, respectively against this bacterium (Figs. 33 and 34). 93 In general, as shown in Fig. 33, the MIC values of the crude methanol Soxhlet extracts and especially the roots were low against all the bacterial strains studied. This could be related to the rhizosphere being a habitat rich in various bacterial strains and among them also pathogenic bacteria against which the roots are forced to produce various signal and defense compounds (Lareen et al. 2016). In the roots, and also in other plant parts, several compounds are known to act in concert (synergistically) and thus maximize their antimicrobial effects and reduce the possibilities for the development of antimicrobial resistance (Salih et al. 2017a, Sanhueza et al. 2017, Zeng and Lin 2013, Ghostaslou et al. 2018, Elisha et al. 2017). Thus, standardized extracts, especially from the roots of T. laxiflora, T. brownii and A. leiocarpus could be used for the treatment of antibiotic resistant bacterial infections. Our results for Terminalia brownii are in accordance with earlier investigators (Machumi et al. 2013, Opiyo et al. 2011) who have stated that especially the root extracts, but also stem bark and wood extracts of Terminalia spp. give good antibacterial effects. Moreover, in agreement with our results, especially polar to medium polar extracts of the roots of African Terminalia spp., have been found to be antibacterially active (Tshikalange et al. 2005, Moshi and Mbwambo 2005, Wright et al. 2016). Our results could justify the uses of hot water extracts (decoctions) of especially the roots of Terminalia brownii and T. laxiflora for the treatment of infectious diseases in Sudan and in other African countries, where these species of Terminalia are readily available. Moreover, since several Anogeissus spp., including A. leiocarpus, are used topically as decoctions for the treatment of wound and skin infections (Singh et al. 2016), our low MIC values, especially for the roots of this species against Pseudomononas aeruginosa and S. aureus could justify these uses. The bacteria mentioned are frequently found in wounds and on infected skin (lu et al. 2018, Tashiro et al. 2018, Shlyapnikov et al. 2018). 4.3.1.2. Pure compounds present in antibacterial plant extracts A number of commercial standard polyphenolic compounds (see paragraph 3.4.2), which we had identified in crude extracts and fractions of T. laxiflora, T. brownii and A. leiocarpus, giving promising antibacterial activities, were investigated for their growth inhibitory activities in order to assess their activity in relation to the crude extracts. The results are shown in Fig. 33. Punicalagin, found in the roots of T. laxiflora and ellagic acid, found in both T. laxiflora and T. brownii roots both showed an MIC value of 125 μg/ml against S. aureus. Our MIC for punicalagin against S. aureus is in accordance with earlier reports (Taguri et al. 2006, Burapadaja and Bunchoo 1995). However, when compared to the crude extracts with MIC values of 39 μg/ml against S. aureus our MIC values for punicalagin and ellagic acid are still quite high. Therefore, the good growth inhibitory effects of the crude root extracts might be due to some other unknown ellagitannins, since we have found that polar to medium polar root extracts of T. brownii and T. laxiflora are rich in a high variety of unknown ellagitannins and that this compound group occurs more abundantly than any other compound chemical classes in these extracts. In addition to punicalagin, we have found methyl-(S)-flavogallonate and its isomer in antibacterial root extracts of T. brownii. According to our literature search, to date, there are no investigations on the antibacterial effects of methyl-(S)-flavogallonate. However, castalagin, flavogallonic acid (Fig. 46 A and B) and flavogallonic acid dilactone (Fig. 49 D), of which the later two are structurally closely related to methyl-(S)-flavogallonate (Fig. 43 A), have been found to be effective inhibitors of the growth of various bacteria, including S. aureus (Taguri et al. 2004, Mohieldeen et al. 2017). Thus it could be predicted that also methyl-(S)-flavogallonate, which we have found in the roots of T. brownii, would be anti-staphylococcal. Moreover, we detected fairly high concentrations of corilagin in antibacterial root extracts of T. laxiflora. Corilagin has been found 94 to reduce the growth of S. aureus with a MIC of 250 μg/ml (Quideau 2009). In addition, corilagin has been shown to slightly decrease the production of penicillin binding protein 2` and thus to reduce the resistance level of MRSA to β-lactam antibiotics (Shiota et al. 2004). It has also been observed that the ellagitannins tellimigrandin I and oenothein B work synergistically with β-lactam antibiotics, such as oxacillin, and thus restore their effects against MRSA (Yoshida et al. 2018). Overall, the various unknown ellagitannins we found in T. laxiflora, T. brownii and A. leiocarpus should be explored for their precise molecular structures and for possible synergistic effects together with antibiotics. Regarding the safety of ellagitannins for use as antimicrobial medications, it has been observed that corilagin and tellimagrandin I, at concentrations ˃ 50 μg/ml, do not adversely affect the growth of some lactic acid bacterial strains that are regarded health promoting in the human intestinal system (Quideau 2009). In addition to their direct antibacterial properties, ellagitannins are immunomodulatory, and amongst other effects, they enhance the production of interferon gamma (IFNγ) by human NK and bovine cells (Asami et al. 2018, Yoshida et al. 2018, Wallet et al. 2017, Ince et al. 2017) . Thus, these findings together with our results support the traditional curative effects of ellagitannin-rich water extracts and decoctions of Terminalia spp. against infectious diseases. In accordance with our study, several researchers have demonstrated the antibacterial effects of ellagitannins and ellagic acid derivatives. For example, Silva et al. (2000), found that ellagitannins in Terminalia macroptera were antibacterial. Besides, Muddhathir et al. 2013, found that some ellagitannin-rich extracts of T. laxiflora gave an anti-acne activity. Moreover, in our search for antibacterial compounds in T. brownii and T. laxiflora, we found that gallic acid was less effective than the other tested compounds, punicalagin and ellagic acid, against S. aureus, giving a MIC value of 500 μg/ml against this bacterium (Fig. 33). This result is in accordance with Vu et al. (2017) who found that gallic acid was less active than other hydrolysable tannins against plant pathogenic bacteria. Differences between the MIC values of the crude extracts and the pure compounds were more pronounced for Pseudomonas aeruginosa, than for S. aureus, so that punicalagin gave a MIC of 500 μg/ml and ellagic acid a MIC of 250 μg/ml, compared to 39 μg/ml for the methanolic Soxhlet extracts of the roots of T. brownii, T. laxiflora and A. leiocarpus. From these results it is obvious that the compounds in the crude extracts of the species studied, Terminalia species and A. leiocarpus, may act synergistically to inhibit the growth of P. aeruginosa. Thus, one approach for the isolation of active compounds from these plant species could be to test combinations of some of the compounds known to be present in the extracts, such as ellagitannins, ellagic acid derivatives and flavonoids. Moreover, we have found a number of methylated and glycosylated ellagic acid derivatives, including di-methyl ellagic acid, di-methyl ellagic acid glucoside and di-methyl ellagic xyloside, in the antibacterial root extracts of Terminalia brownii and T. laxiflora and in antibacterial root and stem bark extracts of Anogeissus leiocarpus. According to previous research, ellagic acid derivatives are antibacterial. Dimitrova et al. (2017) and Ochoa-Pacheco et al. (2017), reported good antibacterial activity of pure 3,3′-di-O-methyl-ellagic acid-4-O-β-D-glucopyranoside against S. aureus and Pseudomonas aeruginosa. Mechanistic studies have revealed that ellagic acid derivatives inhibit the function of efflux pumps located in the bacterial cell membrane and thus would trap the antibiotics inside the bacterial cell (Rampioni et al. 2017, Dimitrova et al. 2017). Some condensed tannins (CTs) have been found to give good antibacterial effects. For example, epigallocatechin gallate gave a MIC value of 50 μg/ml against Staphylococcus epidermidis (Nishino et al. 1987, Dey et al. 2016). We found that an ethyl acetate extract of the stem bark of A. leiocarpus contained a fairly high concentration of epigallocatechin gallate, and thus this 95 condensed tannin might be responsible for a part of the good antibacterial effects of this extract against S. epidermidis (Fig. 33). New antimicrobial agents do not necessarily need to be single compounds. The approach to use standardized, complex plant extracts as antimicrobials instead of single compounds is gaining increasing interest (Kelber et al. 2017, Aiello et al. 2018, Kelber et al. 2018). Another recent trend is to use plant-derived compounds as adjuvants in combinations with conventional antimicrobials (Abreu et al. 2012). Many authors claim that using a single compound, such as an antibiotic alone, increases the risks for the development of multi-resistant bacteria (Alekshun et al. 2007). For example, resistance has evolved against β-lactam and glycopeptide antibiotics, such as vancomycin and colistin (Band and Weiss 2015, Wang et al. 2017, Munita and Arias 2016; Falagas et al. 2008; Demler and Mulcahy 2018). When combating bacterial infections with standardized plant extracts or with combination therapies using adjuvants and antibiotics, there are several compounds simultaneously acting on the bacteria, so that some of the compounds might act as efflux inhibitors and thus enhance the transport of antibacterial compounds into the bacterial cell (Wang et al. 2018). Therefore, also plant-based antimicrobial single compounds might stimulate bacteria to develop resistance. However, plantderived compounds might inhibit bacteria by different mechanisms of action compared to conventional antibiotics, which are usually derived from microbial sources (Eloff 1998). Thus, high MIC values for the pure compounds tested in our screenings do not necessarily tell that these compounds could not be good candidates for the development of new antimicrobials. As previously, mentioned, plant-derived compounds, such as ellagitannins, are known to exert their antimicrobial effects via enhancing the immune system, and these effects are not visible in in vitro antibacterial assays. Plant-derived compounds are also known to inhibit microbial signal molecules involved in the biofilm formation, often associated with bacterial infections (Elnaggar et al. 2016). In the human gut, metabolites from the acid hydrolysis of ellagitannins, such as gallic acid and hexahydroxydiphenic acid (HHDP), have been found to significantly reduce the growth of harmful bacteria, and the metabolites mentioned were found to damage the cell membrane of these bacteria, without affecting health beneficial lactic acid bacteria (Selma et al. 2014, Yahia 2017). Moreover, pure plant-derived compounds, such as various ellagitannins and other hydrolysable tannins, are known to enhance the effects of conventional antibiotics. For example, it has been found that corilagin reduces the MIC values for β-lactam antibiotics 100 – 2000-fold against various strains of MRSA (Shimizu et al. 2001), and tannic acid below MIC concentrations has been reported to reduce the MIC of oxacillin and cefdinir against S. aureus (Akiyama et al. 2001). In this perspective, our new species studied of Terminalia, and other species belonging to this genus as well, might be rich in still unknown new antimicrobial ellagitannins, which might have uses especially for topical bacterial infections caused by S. aureus and other bacteria. Moreover, owing to the fact that the bioavailability of ellagitannins is still poorly known (Ito 2011), there might be small-molecular ellagitannins which reach their microbial targets in the body without being modified, and thus the in vitro results on the antibacterial effects of these ellagitannins might come closer to the in vivo situation. Since we found that the root methanol Soxhlet extracts of Anogeissus leiocarpus are rich in flavonoids, it could be predicted that many of these compounds would contribute to the growth inhibitory activity of this extract. Of the flavonoids we detected in this extract, taxifolin, aromadendrin and ampelopsin have been reported to possess antibacterial effects especially against Staphylococcus aureus (Tajuddeen et al. 2014, Raj et al. 2017, Wu et al. 2018). In addition, we found the stilbene pinosylvine (syn. trans-3,5-dihydroxystilbene) in this same methanol root extract of A. leiocarpus, and this compound might also contribute to the antibacterial effects of this extract. 96 Accordingly, stilbenes and their derivatives have been reported to metabolize to urolithin A, which could inhibit the growth of harmful bacteria (Selma et al. 2009, Selma et al. 2017). Although we did not screen the phytochemical composition of the methanolic Soxhlet stem bark extract of A. leiocarpus, previous investigators have found castalagin and flavogallonic acid as major compounds in this part of A. leiocarpus (Shuaibu et al. 2008, Fig. 46 A and B). Moreover, castalagin is known to possess good growth inhibitory effects against S. aureus (Taguri et al. 2004), and that could explain our low MIC of 39 μg/ml for the stem bark methanol extract against this bacterial strain. 97 IZ in mm 50 (A) Terminalia laxiflora 32 40 26 26 23 21 2521 23 21 30 19 17 22 1818 20 19 18 20 10 0 (B) T. brownii 40 35 30 25 20 15 10 5 0 (C) Anogeissus leiocarpus IZ in mm IZ in mm 50 40 30 20 10 0 18 19 18 30 15 19 22 17 23 21 16 17 27 20 21 20 19 2018 15 22 19 23 21 39 252425 20 17 18 28 16 18 15 19 26 20 20 19 16 24 23 21 21 18 26 20 20 21 16 22 25 16 17 20 26 19 20 24 17 19 17 28 22 23 19 22 16 24 21 26 24 19 21 19 19 30 24 25 22 22 25 22 39 37 24 39 21 20 22 32 24 18 16 36 22 21 22 39 22 36 28 23 24 23 36 36 2325 22 212022 38 22 21 37 22 20 20 19 20 23 22 24 Figure 32. Agar diffusion results on the antibacterial activity of 39 extracts (50 mg/ml) of the stem bark, stem wood and root of (A) Terminalia laxiflora; (B) T. brownii and (c) Anogeissus leiocarpus. Results are expressed as the diameters of the inhibition zones of four replicates. IZ, diameter of inhibition zone in mm; laxi, Terminalia laxiflora; Brow, T. brownii; Anog, Anogeissus leiocarpus; B, stem bark; W, Stem wood; R, root; EtOAc, ethyl acetate fraction; MeSox, methanolic soxhlet extracts; Pt, petroleum ether extracts; CHCl3, chloroform extracts; aqu, aqueous fraction; acet, acetone extracts; HH2O, decoction. Study I (Salih et al. 2017a). 98 3000 MIC in μg/ml 2500 2500 2500 (A) Terminalia laxiflora 2000 1500 1250 1250 1250 1000 1250 625 312 500 625 625 625 156 312 0 3000 W.CHCl3 W.EtOAc MIC in μg/ml 312 625 312 W.MeSox 39 B.EtOAc 2500 2500 500 312 787878 39393939 39 B.MeSox R.HH2O R.MeSox 39 39 39 39 39 39 39 R.acet 39 39 39 125 39 R.EtOAc 125 Puni 2500 250 500 EA 250 GA (B) T. brownii 2000 1250 1500 1000 500 625625 312 0 39 78 B.EtOAc 1500 MIC in μg/ml W.aqu 156 78 78 312 39 B.aqu 1250 39 39 1250 625 625 312 78 B.MeSox 156 W.MeSox 39 78 R.HH2O 78 39 39 39 39 39 39 R.MeSox 312 39 R.acet 1250 1250 78 312 250 125 39 R.EtOAc EA 500 250 GA (C) Anogeissus leiocarpus 1000 625 500 156 312 312 312 156 312 312 156 0 W.EtOAc W.MeSox B.aqu 78 156156156 B.MeSox 312 39 78 39 39 39 39 39 39 R.HH2O R.MeSox 39 39 39 R.acet 500 500 156 39 39 78 R.EtOAc 125 Puni 125 EA 250 250 GA Figure 33. Minumum inhibitory concentration (in μg/ml) of extracts of the stem bark, stem wood and root of (A), Terminalia laxiflora; (B) T. brownii; (C), Anogeissus leiocarpus, and pure compounds identified in the study spp.. Result were obtained using agar diffusion method and expressed as diameter of inhibition zones in triplicates, also microdiltution method using 96-well microplate well was used. Puni, Punicalagin; EA, ellagic acid; GA, Gallic acid; aqu; aqueos extracts; MeSox, methanolic soxhlet; acet, acetone extracts; EtOAc, ethyl acetate extracts; CHCl3, Chloroform extracts. Study I (Salih et al. 2017a). 99 (A) 68 65 (B) 130 120 110 100 90 80 70 60 50 40 30 20 10 0 67 68 60 53 53 MIC in μg/ml IZ in mm 70 64 58 50 45 33 40 31 125 28 30 20 0,061 0,488 0,976 M. luteus 10 0 M. luteus S. aureus Ampicillin Tetracycline S. epidermidis P. aeruginosa 1,95 0,031 0,488 S. aureus 0,061 0,976 62,5 0,976 S. epidermidis P. aeruginosa Gentamycin Penicillin Figure 34. (A) Diameter of inhibition zones (in mm) and (B) minimum inhibitory concentration (in μg/ml) of the reference antibiotic compounds against the studied bacterial strains. Results were obtained using an agar diffusion method and are expressed as the diameters of the inhibition zones of triplicates r SEM and the MIC values were obtained using a microdilution method. Study I (Salih et al. 2017a). 100 4.3.2. Antifungal effects against filamentous fungi (Study II) A total of eight extracts from the stem bark and stem wood of Terminalia brownii, obtained from sequential extraction, were investigated for their growth inhibitory effects against the filamentous fungi Aspergillus niger ATCC 9763, Aspergillus flavus ATCC 9763, Nattrassia mangiferae ATCC 96293 and Fusarium verticilloides (syn. F. moniliforme) ATCC 24378. These fungi are known to be both plant and human pathogenic. The best activity was observed for the ethyl acetate extracts of the stem wood and stem bark against the growth of Fusarium verticilloides and Nattrassia mangiferae, giving diameters of inhibition zones of 19 and 20 mm, respectively, and MIC values of 250 μg/ml (Fig. 35, appendices Table 3). The Aspergillus strains were more resistant to T. brownii, and a MIC of 500 μg/ml was recorded for both the stem wood and bark ethyl acetate extracts against this fungal species. Compared to the more polar extracts, the chloroform extracts showed less activity (Fig. 35). Notably, we found that the aqueous extracts of T. brownii stem bark and wood were almost as potent as the ethyl acetate extracts against the Aspergillus strains and Fusarium verticilloides, and the aqueous extract of the stem bark was slightly more active than the ethyl acetate extracts against Nattrassia mangiferae (IZ, 19 mm) (Fig. 35). To date, there are no other reports on the growth inhibitory activity of Terminalia brownii or other species of Terminalia against Nattrassia mangiferae. This fungal species causes notable damage, such as stem and branch dieback and blossom blight on cultivated fruit trees in Sudan (Gichora et al. 2017, appendices Table 3). Thus, according to our results, water extracts of Terminalia brownii could be used to protect trees in fruit orchards from this fungal pathogen. Moreover, our results are in accordance with Opiyo et al. (2011) who also found that especially ethyl acetate extracts of the stem bark of T. brownii give good antifungal effects against Aspergillus niger and another Fusarium species, F. solani. In appendices Table 3, our results are presented in relation to the results of other authors on the antifungal effects of Terminalia spp. against filamentous fungi. As can be noted in appendices Table 3, many of the Terminalia species studied for the effects against filamentous fungi are of Asian origin, such as T. alata, T. chebula, T. bellirica and T. arjuna. In addition to T. brownii, T. spinosa and T. sericea are the only three other species with African origin that have been studied for their antifungal effects against filamentous fungi (Fabry et al. 1996; Opiyo et al. 2011). In accordance with our results for Terminalia brownii, many of the species of Terminalia studied have shown promising growth inhibitory effects against filamentous fungi (appendices Table 3), and, therefore, further studies, especially on less studied African species, are warranted. Furthermore, in order to study the phytochemical composition of the antifungal stem wood and bark ethyl acetate extracts of Terminalia brownii, we subjected these extracts to RP-18 TLC, HPLC-DAD and tandem mass spectrometry. The results of these phytochemical screenings are presented in detail in chapter 4.4.1.1. (Fig. 44). In brief, we found that the stem wood and the stem bark extracts contained a similar qualitative composition of compounds but differed in their quantities (Fig. 44 a and b). In general, the stem wood extract contained a higher quantity of compounds, and thus some them detected in this extract with our HPLC-DAD system, could perhaps not be detected in the stem bark extracts. These quantitative differences might explain the slight differences in the antifungal activity between the stem bark and stem wood extracts, the wood extracts showing slightly better antifungal activities. For example, we found two galloylated derivatives of resveratrol, trans- and cisresveratrol-3-O-β-galloylglucoside (compounds (3) and (4) in Fig. 44 a and b) in both the stem wood and bark extracts of T. brownii. However, in the stem wood they were present in much higher quantities. These resveratrol derivatives have not been found in the genus Terminalia earlier. Since resveratrol and its derivatives are known to possess good antifungal effects (Dercks and Creasy 101 1989, Houillé et al. 2014), it could be predicted that also the galloylated resveratrol derivatives we have found in T. brownii stem wood and bark could contribute to the overall good antifungal effects of these extracts, and especially for the antifungal effects of the stem wood extract, enriched in these compounds. Diameter of inhibition zone IZ Moreover, we found gallagic acid dilactone (Compound 6, Fig. 44 a and b) in the stem wood of T. brownii. This gallagic acid analogue, containing four gallic acid units, has also been found in other species of Terminalia, but has otherwise a restricted occurrence in plants. Since gallagic acid has been found to give good antifungal effects against Fusarium spp., it might also contribute to the good growth inhibitory effects of the T. brownii ethyl acetate stem wood and bark extracts against Fusarium verticilloides. Moreover, we found a number of flavonoids, including quercetin-7-β-Odiglucoside (compound 8) and quercetin-7-O-galloylglycoside (compound 10) in the stem bark and wood ethyl acetate extracts of T. brownii (Fig. 44). Of the flavonoids mentioned, quercetin-7-β-Odiglucoside was present in the highest concentration in both the stem wood and stem bark extracts, and thus is suggested to be of importance for their antifungal activities. Quercetin and its derivatives, such as quercetin-diglucoside, have accordingly been reported to give as low MIC values as 15 μg/ml against Aspergillus and Fusarium strains (Céspedes et al. 2014). 25 Aspergillus niger Aspergillus flavus 18,5 19 20 17 14 15 12 12 Nattrassia mangiferae 20 17 18 18,5 Fusarium verticillioides 19 13 18,518,5 19 17 13 18 19 18 16,5 14 12 11 10 5 0 T. brownii. W. Ch T. brownii. W. EtOAc T. brownii. W. aqu T. brownii. B. Ch T. brownii. B. EtOAc T. brownii. B. aqu Figure 35. Antifungal activity of extracts of various polarities from the stem bark and wood of Terminalia brownii against the filamentous fungi, Aspergillus niger, Aspergillus flavus, Nattrassia mangiferae and Fusarium verticillioides (Syn. F. moniliforme). Results were obtained using an agar disk diffusion method and are expressed as the diameters of the inhibition zones of triplicates. EtOAc, ethyl aceate; aqu, aquous fraction; Ch, chloroform; B, bark; W, Wood. Study II (Salih et al. 2017b). 102 4.3.3. Antimycobacterial effects of T. brownii and T. laxiflora (Study III) Mycobacterium smegmatis was chosen for the screenings since it is fast growing, safer to use and shows a high degree of genetic homology with M. tuberculosis (Tyagi and Sharma 2002, Wu et al. 1997). Globally, but especially in Africa, the HIV related cases of TB have increased during the last couple of decades, and the number of antibiotic resistant strains have became more common (Asres et al. 2001, WHO 2010). There is thus a need to explore new treatments for TB, and local plants used for the treatment of TB in traditional medicine could be potential sources for new antimycobacterial extracts and compounds. In our screenings we have chosen to investigate the antimycobacterial effects of Terminalia laxiflora and Terminalia brownii, which are both used in African traditional medicine for the treatment of symptoms related to TB. 4.3.3.1. Extracts Altogether seventy-seven extracts made from the roots, stem bark, stem wood and leaves of Terminalia laxiflora and T. brownii were screened for their growth inhibitory effects against Mycobacterium smegmatis using an agar diffusion method for the primary screening. The results of this screening are shown in Fig. 36. The MIC values of those extracts giving the best inhibitory effects in the agar diffusion screening were estimated using a microplate broth dilution method, and these results are shown in Figs. 37 A, B and C. Interestingly, when compared to the other root extracts of T. brownii, the water extracts displayed the best results in terms of the size of the diameter of the inhibition zone (IZ, 26.7 mm). However, this agar diffusion result for the decoction did not correlate well with its rather high MIC value (5000 μg/ml) (Fig. 37). Likewise, a decoction from the root of T. laxiflora showed good growth inhibition against M. smegmatis when using agar diffusion (IZ, 22 mm) but the MIC was found to be quite high, 2500 μg/ml. The agar diffusion results would justify the use of decoctions and maceration of the roots of T. brownii and T. laxiflora in Sudanese traditional medicine for the treatment of cough and other symptoms related to TB (El Ghazali et al. 2003 and 1997). To the best of our knowledge, decoctions and maceration from the roots of T. brownii and T. laxiflora have not been tested before for their in vitro growth inhibitory effects against M. smegmatis. Other extracts showing good antimycobacterial effects in our primary screening were the leaf acetone extracts of T. brownii and the leaf ethyl acetate extracts of T. laxiflora, producing inhibition zones of 27 and 23 mm, respectively (Fig. 36). The root acetone extract of T. laxiflora gave the lowest MIC value of all the extracts screened, 625 μg/ml (Fig. 37 B). Eloff (2000) has previously reported that acetone is a suitable extractant for antimicrobials in the Combretaceae, and thus our result is now in line with that finding. We discovered that the ethyl acetate extracts of both investigated species of Terminalia are enriched in ellagitannins, and this might explain the good growth inhibitory effects of the ethyl acetate extracts (MIC 1250 μg/ml) (Fig. 37). Apart from the more polar extracts mentioned, also hexane extracts from the stem bark of both species gave good growth inhibitory effects (Fig. 36 and 37). This result demonstrates that in the stem bark, there are also some antimycobacterial compounds which are of more non-polar character. 103 4.3.3.2. Sephadex LH-20 and preparative RP-18 TLC for purification of antimycobacterial fractions in Terminalia brownii and T. laxiflora Since size exclusion chromatography (Sephadex LH-20) and preparative reversed phase thin layer chromatography (RP 18-TLC) have been previously successfully used for the separation of more polar compounds, such as polyphenols (e.g. ellagitannins) in Terminalia spp. (Saleem et al. 2002), these methods were now applied in an attempt to separate fractions and, if possible, pure compounds, with improved growth inhibitory effects compared to the extracts. Sephadex LH-20 fractions of T. brownii roots For Terminalia brownii, Sephadex LH-20 separation of polyphenols from a methanolic Soxhlet root extract was performed. Two fractions were obtained, which were called the “ethanol wash” and the “acetone wash” (Fig. 38), since these fractions were eluted from the Sephadex LH-20 column with the eluents ethanol followed by acetone. When compared to the crude methanol extract of the roots (MIC 5000 μg/ml), the Sephadex fractions gave notably improved growth inhibitory effects with MIC values of 62.5 and 125 μg/ml, respectively, for the acetone and ethanol wash. The MIC of rifampicin was 3.9 μg/ml, and is thus still 16 times lower than the MIC of the acetone wash (Fig. 37 A). Moreover, the inhibition zones produced by these fractions and the crude extract (Fig. 36 A), correlated well with the MIC values, and the largest zone of 28.5 mm was shown by the acetone wash, followed by the ethanol wash, giving a zone of 25 mm, and the crude extract, giving a zone of 21 mm. When compared to the crude methanol extract of the roots of T. brownii and the ethanol wash, the acetone wash fraction was found to contain a larger proportion and higher concentrations of ellagitannins (Fig. 38 B), and especially the concentrations of methyl-(S)-flavogallonate (peak 9, tR 12.37 min) and another unknown ET at tR 15.08 min ([M-H]- at m/z 609.1088), were found to be higher than in the crude extract (Fig. 38 B). Thus, the low MIC of the Sephadex LH-20 acetone fraction could have been due to these ellagitannins and their possible synergistic effects with each other as well as with other compounds in this fraction. Methyl-(S)-flavogallonate was not available for testing as a pure compound, and thus another ellagitannin, corilagin, was tested for its growth inhibitory effects against M. smegmatis. However, we found that corilagin is only moderately active against M. smegmatis, with a MIC of 1000 μg/ml (Fig. 37 C). For polyphenols it has been found that the presence of pyrogalloyl groups (3,4,5trihydroxy-phenyl-groups) is important for their antibacterial activity (Taguri et al. 2006). Both corilagin and methyl-(S)-flavogallonate contain these pyrogalloyl groups, which could be critical for their antimycobacterial activities (Fig. 41 A and 43 A). According to our literature search, no investigations have been carried out to date on the effects of pure methyl-(S)-flavogallonate on the growth of mycobacterial species. Castalagin is a C-glycosidic ET containig a nonahydroxytriphenoyl (NHTP) group which stabilizes its chemical structure (Moilanen 2015, Quideaue et al. 2003, 2004 and 2005). Moreover, it has been found to possess antimicrobial effects against Salmonella, S. aureus and E. coli (Taguri et al. 2004), but it is not known if this activity is related to the NHTP unit of castalagin. No positive correlation between the occurrence of a flavogallonoyl unit in an ellagitannin and its antimycobacterial activity has, however, been established. An ellagitannin from strawberry leaves, HeT (1‐O‐galloyl‐2,3,4,6‐bis‐hexahydroxydiphenoyl‐β‐D‐glucopyranose), was found to interact strongly with the cell membrane of Enterococcus mundtii, without disruption of the membrane integrity (Martos et al. 2018). Moreover, mechanistic investigations revealed that HeT inhibited 104 oxygen consumption and NADH and MTT reduction in Enterococcus mundtii. Therefore, it is possible that ellagitannins such as methyl-(S)-flavogallonate which we have found in T. brownii roots, could act in a similar way on the cell membranes of Mycobacterium smegmatis. The Sephadex LH-20 ethanol wash, containing ellagic acid xyloside, ellagic acid rhamnoside and trimethyl ellagic acid along with ellagic acid as its main compounds, was four times more antimycobacterial (MIC 125 μg/ml) than the tested commercial compound, ellagic acid, which gave an MIC of 500 μg/ml against M. smegmatis (Fig. 37). This could be due to synergistic effects of the ellagic acid derivatives in the ethanol wash. The sugar part of the ellagic acid glycosides has also been thought to be important for the antimycobacterial activity of ellagic acid derivatives (Kaneko and Kaneko 2013). Accordingly, Kuete et al. (2010) reported on very promising antimycobacterial activity against M. smegmatis and M. tuberculosis MTCS2 of di-methyl ellagic acid xyloside (MIC 4.88 μg/ml) from a methanol extract of the stem bark of Terminalia superba. Moreover, also methylated ellagic acid derivatives have been found to be more antimycobacterial compared to ellagic acid (Shilpi et al. 2015). These findings strongly suggest that the good antimycobacterial effects we have found for the Sephadex LH-20 ethanol wash could be due to the growth inhibitory effects of ellagic acid glycosides and methylated ellagic acid derivatives. Preparative TLC RP-18 for separation of antimycobacterial fractions from Terminalia laxiflora roots Due to their fairly large inhibition zones (IZ) in our initial screenings (Fig. 37A and B), a methanolic Soxhlet and an ethyl acetate extract of the roots of T. laxiflora were chosen for further separation of antimycobacterial fractions using reversed phase preparative thin layer chromatography (RP-18 TLC). The fractions obtained, resulting from the preparative TLC separations and the chemical composition of these fractions, Fr5 and Fr51 isolated from the methanol and ethyl acetate extracts, respectively, is shown in Figs. 39 A and B. Since very little material was obtained for the rest of the fractions, Fr5 and Fr51 were used for the in vitro antimycobacterial tests. We found that the purifications did not improve the antimycobacterial effects of the fractions as dramatically as for the Sephadex LH-20 fractions of T. brownii, and both fractions gave an MIC value of 500 μg/ml, compared to MIC 1250 μg/ml for the crude ethyl acetate and methanol soxhlet extracts of the roots of T. laxiflora (Fig. 37 A). As is shown in Figs. 39 A and B, the HPLC-DAD chromatograms of both fractions, Fr5 and Fr51, are rich in ellagitannins and ellagic acid derivatives, and punicalagin dominates quantitatively in both of them. Punicalagin, which was isolated from the stem bark of Combretum molle, has been found to be growth inhibitory against M. tuberculosis (MIC > 600 μg/ml) (Asres et al. 2001). Therefore, we assume that the increase in the antimycobacterial activities of both TLC fractions compared to the crude extracts of Terminalia laxiflora, might be partly due to the enrichment of punicalagin in these fractions. In the TLC fraction Fr5, isolated from the methanol root extract of T. laxiflora, methyl ellagic acid xyloside and ellagic acid xyloside were found which were not present in the Fr51 fraction of the ethyl acetate root extract. These compounds did, however, not improve the antimycobacterial effects of fraction Fr5 compared to Fr51, since it’s MIC (500 μg/ml) was the same as that for the Fr5 fraction. Ellagic acid derivatives have been found to be antimycobacterial in earlier investigations. Ellagic acid from the husk of pomegranate gave an MIC value of 50 μM (0.7 μg/ml) against the growth of Mycobacterium smegmatis mc2 155 (Sridevi et al. 2017). This result is not in agreement with our MIC of 500 μg/ml for pure ellagic acid against M. smegmatis, but might be due to use of different M. smegmatis strain. Methyl groups in pteleoellagic acid have been found to be important for the good docking capacity of this compound to Mab2, an enzyme belonging to the fatty acid elongation 105 system I enzyme complex, responsible for the production of long-chain fatty acids, which are key precursors to mycolic acid in the mycobacterial cell membrane (Abrahams and Besra 2018, Shilpi et al. 2015). Thus, methyl ellagic acid xyloside in the TLC fraction Fr5 from T. laxiflora might also have an enzyme docking capacity to similar to the fatty acid elongation system I in Mycobacterium tuberculosis, but further studies regarding this are warranted. 106 IZ in mm IZ in mm 38 40 35 30 25 20 15 10 5 0 40 35 30 25 20 15 10 5 0 (B) Terminalia brownii 16 17 17 21 14 15 20 17 22 20 23 18 29 27 27 22 21 21 20 16 14 16 16 14 15 15 22 23 15 18 23 20 17 19 16 14 37 (B) Terminalia laxiflora 18 17 16 18 19 17 25 20 0 0 0 17 16 18 21 19 14 19 20 21 17 19 21 22 22 21 14 18 14 0 0 18 23 20 14 14 18 14 0 18 16 0 20 0 Figure 36. Antimycobacterial activity of extracts of various polarities from the roots, stem bark, wood and leaves of (A), Terminalia brownii and (B), T. laxiflora against Mycobacterium smegmatis ATCC 14468. Results were obtained using an agar diffusion method and are expressed as the mean of the diameters of the inhibition zones of triplicates. 107 0 6000 MIC in (μg/ml) 3000 1000 3,9 5000 62,5 5000 5000 5000 5000 2500 2500 125 5000 5000 5000 5000 1200 (C) 1000 1000 2500 1250 3,9 500 2500 2500 1250 625 Mic in μg/ml MIC in (μg/ml) 5000 2500 (B)(B) 2000 0 5000 2000 3000 1000 5000 2500 6000 4000 5000 4000 0 5000 5000 (A) 5000 800 500 500 600 400 200 0 62,5 125 500 500 250 250 250 3,9 Figure 37. Minimum inhibitory concentration of extracts, fractions and compounds against Mycobacterium smegmatis. (A) Terminalia brownii and Sephadex LH-20 fractions from its root; (B), T. laxiflora and RP-18 TLC fractions from its root (C), pure compounds identified in the antimycobacterial extracts. Results were obtained with an agar diffusion method and a turbidimetric assay. Rif, Rifampicin; brow, Terminalia brownii; Laxi, T. laxiflora; R, root; Rb, root bark; B, stem bark; W, stem wood; L, leaves; F, Fruits; MeSox, methanolic soxhlet extracts; Seph.ac, Sephadex LH20 acetone wash; Seph.EtOH, Sephadex LH-20 ethanol wash; HH2O, decoction; H2O*, maceration; acet, acetone extracts; Me*, methanol cold; EtOAc, ethyl acetate fraction; hex, hexane extracts; Dic, dichloromethane extracts. 108 (A) Crude MeOH extract, using hot Soxhlet extraction method MeSox extract Subjected to Sephadex LH-20 Fractionation (C) Ethanol wash (B) Acetone wash Figure 38. Methanolic soxhlet extract of the root of Terminalia brownii (A) and the obtained Sephadex LH-20 fractions from this extract (B and C) modified from Salih et al. 2017a (Study I). Gallic acid (1); unknown ellagitannin [M-H]- 456.9961 (2); unknown ellagitannin (3); unknown ellagitannin; (4) isomer of methyl-(S)-flavogallonate (5); gallotannin (6); ellagitannin (7); ellagitannin (8); methyl-(S)-flavogallonate (9); ellagitannin [M-H]- 609.1088 (10, ET); ellagic acid glucuronide (11, EAG); ellagic acid xyloside (12); methyl ellagic acid xyloside (13, MeX); ellagic acid (14); trimethyl ellagic acid (15); ellagitannin [M-H]- 725.4141 (16); ellagitannin [M-H]- 817.4003 (17); acetylated ellagic acid derivative (18); two ellagitannin in the same peak [MH]- 577.1369 & 577.1392 (19); ellagitannin [M-H]- 817.3999 (20). Modified from study I (Salih et al. 2017b). 109 (A) 1 R5EtoAc 1 R7 1 R6 1 R5 (C) 1 R4 Gallic acid (Rf 0.721) 1 R3 1 R2 1 R1 Corilagin (Rf 0.549) 1 (B) 4 3 2 1 R8 1 R5MeSox Ellagic acid (Rf 0.173) 1 R7 1 R6 A 1 R5 B C D 1 R4 1 R3 1 R2 1 R1 1 2 3 4 Figure 39. RP18 TLC fractionation of (A) an ethyl acetate and (B) a methanol soxhlet extract of the root of Terminalia laxiflora (modified from Salih et al 2018; Study III). TLC plates were photographed using a TLC Camaq Reprostar 3 TLC Visualizer camera and computer system. (A), thin layer chromatography finger print of ethyl acetate fractions marked as R1-R7 and TLC fingerprint of (B), the methanolic soxhlet extract. (C), standard pure compounds identified in these extracts; (1), TLC plate at daylight; (2), at 254 nm; (3), at 366 nm, (4), after sprayed with the DPPH reagent. ET, ellagitannin; MEAX, methyl ellagic acid xyloside; EAX, ellagic acid xyloside; GA, gallic acid; Rf, retardation factor. Modified from study III (Salih et al. 2018). 110 4.3.3.3. Non-polar extracts of T. laxiflora and T. brownii and their suggested antimycobacterial compounds Since we had observed in our primary screening that non-polar extracts, such as hexane extracts of the stem bark of T. laxiflora and T. brownii, give good antimycobacterial effects (Fig. 36 A and B) and MIC values of 2500 μg/ml, commercially available pure compounds, such as fatty acids and triterpenoids, some of them also found in these hexane extracts, were evaluated for their antimycobacterial effects. Of the investigated fatty acids, we found that neither stearic (18:0), nor behenic acid (22:0) were growth inhibitory against Mycobacterium smegmatis, even at a high concentration of 1000 μg/ml. This result supports the finding that saturated fatty acids are not antimycobacterial (Seidel and Taylor 2004). Conversely, it has been found that unsaturated fatty acids give good antimycobacterial effects and a high degree of unsaturation renders the fatty acids more antimycobacterial (Luo et al. 2011, Kanai and Kondo 1974, Seidel and Taylor 2004). The good antimycobacterial effects of the hexane stem bark extracts of Terminalia laxiflora and T. brownii (Fig. 37 A and B) might be partly due to the unsaturated fatty acid 1,18-octadec-9-enedioate, which is present in high quantities in the stem bark of both species (Fig. 48, peak number 1 in T. laxiflora and T. brownii GC-chromatograms). This fatty acid is suggested to interfere with the mycolic acid layer in the mycobacterial cell wall (Hill et al. 2012, Marnett 1999, Fig. 40). Moreover, interestingly, it has been found that long-chain fatty acids (C16 and C18) secreted from macrophages are bactericidal against Mycobacterium bovis, the mycobacterial strain present in the Bacillus Calmette Guerin tuberculosis vaccine (BCG) (Hemsworth and Kochan 1978). Thus, also plants could be sources of long-chained fatty acids with good anti-TB potential. Accordingly, Esquivel -Ferrino et al. (2012) have found that linoleic acid from Foeniculum vulgare gave good antimycobacterial effects. Moreover, several long-chain fatty acid compounds, including oleic and linoleic acids, have been found to inhibit the synthesis of mycolic acid in the cell wall of a number of mycobacterial strains, including Mycobacterium spp. as M. smegmatis (Morbidoni et al. 2006, Yanev et al. 2018, Kalita et al. 2018). We have, for the first time, found a number of long-chain fatty acids, such as tetracosanoic acid, hexacosanoic acid and octacosanoic acid in hexane stem bark extracts of Terminalia laxiflora and T. brownii, and these compounds are suggested to render these extracts their antimycobacterial effects (Fig. 48). In our screenings we found that β-sitosterol was only moderately growth inhibitory against M. smegmatis, showing an MIC of 500 μg/ml (Fig. 37 C). Thus, this steroidal alcohol, which we have found in both Terminalia species, might not have a very profound contribution to the growth inhibitory effects of the hexane extracts against M. smegmatis. However, other authors have reported that β-sitosterol gives a better activity against Mycobacterium tuberculosis, with a MIC of 128 μg/ml (Saludes et al. 2002). Thus β-sitosterol in non-polar extracts of Terminalia brownii and T. laxiflora could have a good growth inhibitory effect also on M. smegmatis. It could be thought that mixing plant material from these species of Terminalia into a soup or other foods containing oil or cream would extract β-sitosterol (and fatty acids) from these plants with good therapeutic effects for the treatment of TB. We detected minute quantities of betulinic acid in the non-polar extracts of the stem barks of T. laxiflora and T. brownii (Fig. 48, compound 12). Betulinic acid has been found to possess good growth inhibitory effects against Mycobacterium tuberculosis with an MIC of 32 μg/ml (Wächter et al. 1999). Thus, we suggest that betulinic acid might contribute to a significant part of the antimycobacterial effects that we have seen for the non-polar stem bark extracts of T. laxiflora and T. brownii. 111 1,18-Octadec-9-ene-dioate (Unsturated lipid) Radical lipid Lipid peroxyl radicle Lipid peroxide Figure 40. Proposed structure activity relationship (SAR) of the unsaturated fatty acid, 1,18octadec-9-ene-dioate. This fatty acid is able to bind to the mycolic acid layer in the mycobacterial cell wall and interefere with this structure. Modified from the Hill et al. 2012; Marnett 1999. We found this fatty acid in both Terminalia laxiflora and T. brownii hexane stem bark extracts (study III, Salih et al. 2018). 4.4. Results of the phytochemical studies 4.4.1. Polar and medium polar extracts 4.4.1.1. Identification of polyphenols in T. laxiflora and T. brownii using HPLC-DAD, UHPLC/QTOF-MS and LC/MSn tandem mass spectrometry (Studies I, II and III) On the basis of our results of the qualitative composition of extracts of Terminalia brownii and T. laxiflora, using HPLC-DAD and reference information from the literature (Pfundstein et al. 2010, Conrad et al. 2001), further investigations were performed on the polyphenolic composition of some antimicrobially active extracts using UHPLC/QTOF-MS. This mass spectrometric technique enables the estimation of molecular masses with four-decimal precision. Pfundstein et al. (2010) was used as key reference for the investigation on the molecular masses of ellagitannins, ellagic acid derivatives and other hydrolysable tannins in Terminalia spp. We found that the negative ion mode of mass spectrometry was favorable for the identification of polyphenols in the Terminalia spp. studied, in accordance also with Sheng et al. (2018) and Silva et al. (2000). As discussed by Pfundstein et al. (2010), the polyphenolic composition of African Terminalia species has still not been well studied. Although several species of African Terminalia have been investigated for their phytochemical constituents, we concluded that surprisingly little work has been carried out on T. 112 laxiflora and T. brownii. Consequently, these species were selected for in-depth analysis of their polyphenols. Terminalia laxiflora roots (Study I) To its polyphenolic profile, the ethyl acetate extract of the roots of T. laxiflora was found to resemble a methanol root extract of T. brownii. Altogether twenty-six compounds were identified from an ethyl acetate extract of the roots of Terminalia laxiflora, based on their HPLC-DAD retention times and UVO absorbance maxima (Fig. 42). Eighteen of these compounds were ellagitannins (18 compounds), showing the characteristic three absorbance peaks of UV light (Fig. 42). In addition, a number of ellagic acid derivatives could be detected based on their UVO absorbance maxima peaks (Fig. 42). The molecular ions could be identified for eight compounds using UHPLC/QTOF-MS. These compounds were gallic acid with a [M-H]− molecular ion at m/z 169.0136 (1), ellagic acid xyloside with a [M-H]− molecular ion at m/z 433.0407 (5) and methyl ellagic acid xyloside with a [M-H]− molecular ion at m/z 447.0568 (6), which was also found in the roots of T. brownii. Moreover, corilagin (3) (Fig. 41 A) and its isomer sanguiin H-4 (2) (at HPLC-DAD tR 12.524 min and 8,567 min, respectively and [M-H]− molecular ions of 633.0750 and 633.0743, respectively) and punicalagin (4) (at tR 15.25min, [M-H]− 1083.1541) were characterized. Although punicalagin and corilagin (Fig. 41 A and B) are known to occur in a number of Terminalia spp., and especially in Terminalia species of Asian origin (Burapadaja and Bunchoo 1995, Kinoshita et al. 2007, Tanaka et al. 1986, Chen et al. 2000), these ETs have not been identified before in T. laxiflora. Muddathir and Mitsunaga (2013) found that a methanol extract of the stem wood of T. laxiflora contained flavogallonic acid dilactone and terchebulin (appendices Table 5), but as far as we know this is the only other investigation on the polyphenolic profile of this species. Compounds identified from T. laxiflora roots Corilagin: tR HPLC-DAD 12.52 min; tR UHPLC-DAD 4.42 min; UV (MeOH) λmax: 216, 256, 380; negative ESI-Q-TOF MS m/z: 633.0750; (calc. for C27H22O18, 634.0798) (Fig. 41 A and 42 compound (3)). β-punicalagin: tR HPLC-DAD 15.25 min; tR UHPLC-DAD 5.42 min; UV (MeOH) λmax: 216, 256, 380; negative ESI-Q-TOF MS m/z: 1083.1541; (calc. for C48H28O30, 1084.0654) (Fig. 41 B and. 42, compound (4)). Sanguiin H-4 an isomer of corilagin: tR HPLC-DAD 8.57 min; tR UHPLC-DAD 3.75 min; UV (MeOH) λmax: 216, 256, 374; negative ESI-Q-TOF MS m/z: 633.0743 (Fig. 42). Ellagic acid xylopyranoside: tR HPLC-DAD 18.03 min; tR UHPLC-DAD 7.88 min; UV (MeOH) λmax: 255, 380; negative ESI-Q-TOF MS m/z: 433.0407; (calc. for C19H14O12, 434.0480) (Fig. 42, compound (5)). Also, present in T. brownii roots (Fig. 43, compound (7) and continued Fig 43 (B)). Methyl ellagic acid xyloside: tR HPLC-DAD 20.48 min; tR UHPLC-DAD 8.23; UV (MeOH) λmax: 254, 368; negative ESI-Q-TOF MS m/z: 447.0568; (calc. for C20H16O12: 448.0636) (Fig. 42, compound (6)). Also, present in T. brownii roots (Fig 43, compound (8)). 113 Ellagitannin: tR HPLC-DAD 27.88 min; tR UHPLC-DAD 12.79 min; UV (MeOH) λmax: 220, 264, 398; negative ESI-Q-TOF MS m/z: 817.4026 (Fig. 41. compound (7). Ellagitannin: tR HPLC-DAD 29.73 min, tR UHPLC-DAD 13.50 min; UV (MeOH) λmax: 220, 254, 362; negative ESI-Q-TOF MS m/z: 817.4033 (Fig. 41, compound (8)). galloyl HHDP Ellagic acid HHDP galloyl (A) HHDP (C) (B) Figure 41. Molecular structure of identified compounds of (A), corilagin; (B), α-punicalagin (R = H, R1 = OH) and β-punicalagin (R = OH and R1 = H) and (C) sanguiin H-4 found in the active extracts of T. laxiflora. (Studies I, II, III (Salih et al. 2017a, Salih et al. 2017b, Salih et al. 2018). Source of the chemical structure, CAS 2018. Figure 42. Chromatogram of the ethyl acetate extracts of Terminalia laxiflora (modified from Salih et al 2017a; Study I). (1), Gallic acid; (2) Sanguiin H-4 an isomer of corilagin; (3), Corilagin; (4) Punicalagin; (5) Ellagic acid xyloside; (6) methyl ellagic acid xyloside; (7), unknown ellagitannin at tR 27.88 min and Unknown ellagitannin at 29.73min and UVλ absorption maxima for compound (2), (3), (4) and (5). Modified from study I (Salih et al 2017b). 114 Terminalia brownii roots (Study I) An HPLC-DAD analysis of a methanolic extract of the roots of T. brownii led to the discovery of twenty-two compounds, of which the majority were ellagitannins, according to their UV λ maxima and retention time behaviour (Fig. 43). The masses of altogether 19 phenolic compounds could be determined. Nine ellagitannins, exhibiting [M-H]- ions ranging from m/z 456.9961 to m/z 817.4003 were detected. According to our literature searches, no ellagitannins with those masses we detected have been earlier reported to occur in T. brownii. Moreover, as illustrated in Fig. 43, for the first time we report the occurrence of two ellagitannins, methyl-(S)-flavogallonate (3) exhibiting an [MH]- ion at m/z 483.0745 and its isomer (2) showing an [M-H]- at m/z 483.0811 in T. brownii (tR 12.63 min, and tR 8.647 min, respectively). Methyl-(S)-flavogallonate has earlier been characterized only in one other species of Terminalia, T. myriocarpa (Marzouk et al. 2002). In addition, the masses of pure ellagic acid (9) and seven ellagic acid derivatives could be determined, among them dimethylellagic acid dixyloside (4) (tR 14.82 min, [M-H]- 595.0950), ellagic acid glucuronide (6a) (tR 17.85 min, [M-H]- 477.0651), methyl ellagic acid glucuronide (6b) (tR 17.85 min, [M-H]- 491.0829), as well as ellagic xylopyranoside (7) and methyl ellagic acid xylosides (8) at tR 18.27min [M-H]- 433.0409 and tR 19.39 min [M-H]- 447.0569, respectively (continued Fig. 43 B). Ellagic acid derivatives coupled to rhamnose have been reported before in the stem bark of T. brownii (Machumi et al. 2013, Yamauchi et al. 2016). Ellagic acid (continued Fig. 43 C), results from the lactonization of HHDP released by the hydrolysis of ellagitannins, and thus a number of ellagic acid-based derivatives are known from Terminalia spp. (Pfundstein et al. 2010). In Terminalia spp., ellagic acid derivatives occur as methylated, bound to glucuronic acid or to sugars, such as xylose of rhamnose. Moreover, a galloyl group can be bound to the ellagic acid moiety. Pfundstein et al. (2010), isolated various xylose and rhamnose coupled ellagic acid derivatives from Terminalia horrida, T. chebula and T. bellirica fruits, and some of the rhamnose coupled derivatives had not been described before. Compounds identified from T. brownii roots Isomer of Methyl-(S)-flavogallonate: tR HPLC-DAD 8.64 min, tR UHPLC-DAD 8.52 min; UV (MeOH) λmax: 216, 258, 375; negative ESI-Q-TOF MS m/z: 483.0811 (Fig. 43, compound (2)). Methyl-(S)-flavogallonate: tR HPLC-DAD 12.63 min; tR UHPLC-DAD 8.60 min; UV (MeOH) λmax: 216, 257, 381; negative ESI-Q-TOF MS m/z: 483.0795; (calc. for C22H12O13, 484.0273) (Fig. 43, compound (3)). Dimethyl ellagic acid-dixyloside: tR HPLC-DAD 14.82 min; tR UHPLC-DAD 10.39 min; UV (MeOH) λmax 254, 362; negative ESI-Q-TOF MS m/z: 595.0950 (Fig. 43, Compound (4)). Ellagitannin: tR HPLC-DAD 15.36 min; tR UHPLC-DAD 10.50 min; UV (MeOH) λmax: 216, 256, 381; negative ESI-Q-TOF MS m/z: 609.1088 (Fig. 43, Compound (5)). Ellagic acid glucuronide: tR HPLC-DAD 17.85 min; tR UHPLC-DAD 11.63 min; UV (MeOH) λmax: 254, 368; negative ESI-Q-TOF MS m/z: 477.0651 (Fig. 43, compound (6a)). Methyl ellagic acid glucuronide: tR HPLC-DAD 17.85 min; tR UHPLC-DAD 11.89 min; UV (MeOH) λmax: 254, 368; negative ESI-Q-TOF MS m/z: 491.0829 (Fig. 43, compound (6b)). 115 Ellagic acid: tR HPLC-DAD 20.36 min; tR UHPLC-DAD 12.57 min; UV (MeOH) λmax; 254, 366; negative ESI-Q-TOF MS m/z: 300.9983; (calc. for C14H6O8, 302.0060) (Fig. 43, Compound (9)). Ellagic acid derivative: tR HPLC-DAD 22.33 min; tR UHPLC-DAD 13.19 min; UV (MeOH) λmax; 248, 368; negative ESI-Q-TOF MS m/z: 343.0466 (Fig. 43, compound (10)). Ellagitannin: tR HPLC-DAD 28.31 min; tR UHPLC-DAD 14.67 min; UV (MeOH) λmax; 210, 262, 370; negative ESI-Q-TOF MS m/z: 725.4141 (Fig. 43, compound (11)). Ellagitannin: tR HPLC-DAD 28.93 min; tR UHPLC-DAD 14.98 min; UV (MeOH) λmax; 219, 254, 363; negative ESI-Q-TOF MS m/z 817.4003 (Fig. 43, compound (12)). Acetylated ellagic acid derivative: tR HPLC-DAD 33.94 min; tR UHPLC-DAD 16.10 min; UV (MeOH) λmax; 222, 348, 375; negative ESI-Q-TOF MS m/z 343.0498 (Fig. 43, compound (13)). Ellagitannin: tR HPLC-DAD 35.75 min; tR UHPLC-DAD 16.43 min; UV (MeOH) λmax; 214, 254, 372; negative ESI-Q-TOF MS m/z 577.1369 (Fig. 43, compound (14)). Ellagitannin: tR HPLC-DAD 35.75 min; tR UHPLC-DAD 16.65 min; UV (MeOH) λmax; 214, 254, 372; negative ESI-Q-TOF MS m/z 577.1320 (Fig. 43, compound (15)). Gallotannin: tR HPLC-DAD 38.98 min; tR UHPLC-DAD 17.22 min; UV (MeOH) λmax 218, 278; negative ESI-Q-TOF MS m/z: 553,1732 (Fig. 43, compound (16)). 116 Figure 43. Methanolic soxhlet extract of the root of Terminalia brownii. (1), Gallic acid; (2), Isomer of methyl-(S) flavogallonate; (3), methyl-(S)-flavogallonate; (4), Dimethyl ellagic acid di-xyloside; (5), Unknown ellagitannin; (6a), ellagic acid glucuronide; (6b), methyl ellagic acid glucorounide; (7), ellagic acid xylopyranoside; (8), methyl ellagic acid xylopyranoside; (9), ellagic acid; (10), ellagic acid derivatives; (11), (12), (14) and (15) are unknown ellagitannins; (16), gallotannin. UVλ maxima for compounds (3), (4), (7), and (8). Modified from study I (Salih et al. 2017b). Continued Figure 43. Chemical structure for the identified compound in the root extracts of Terminalia brownii (A), methyl-(S)-flavogallonate; (B), ellagic acid xylopyranoside; (C), ellagic acid and (E), Gallic acid. Besides tannins of (D), Terminalin (Gallagic acid dilactone) found in the stem bark and stem wood of T. brownii. Studies I, II, III (Salih et al. 2017a, Salih et al. 2017b, Salih et al. 2018). 117 T. brownii stem wood (Study II) The HPLC-DAD and tandem mass spectrometry (LC-MS/MS) analysis of ethyl acetate extracts of the stem bark and wood of T. brownii revealed a number of flavonoids, two stilbenes and some ellagitannins (Fig. 44A and B). The stem wood extract was found to be richer than the stem bark extract in secondary compounds, and altogether twelve compounds could be identified to their molecular masses. The same compounds were essentially present also in the stem bark, but in smaller quantities. Compounds 3 – 5, compound 7 and compounds 9 – 11, which could be characterized in the stem wood extract, could not been identified in the bark extract. We found that especially the stem wood of T. brownii contained ellagitannins (Fig. 44 B). Corilagin (9) was present at tR 18.2 min in HPLC-DAD (Fig. 44 B, Appendices, Fig 51). In the tandem mass spectrum, corilagin gave a molecular ion at m/z [M-H]- 633. In addition, MS/MS produced a fragment ion at m/z 481, corresponding to M- galloyl- gallic acid [633 (M) - 152 (galloyl) - 170 (gallic acid)]. Moreover, MS2 produced fragments of m/z 463 [M-gallic acid]. However, an ion of m/z 319 corresponding to HHDP should have been present, but could not be seen in the MSn. Instead, the ion at m/z 300, corresponding to ellagic could be seen, probably as a result of the spontaneous lactonization of HHPD [HHDP-H2O = (319-18)]-. Moreover, an ion at m/z 169 corresponding to gallic acid was present (Fig. 43, Appendices, Fig 51). In addition to the ion present from the cleavage and rearrangement of an HHDP group two fragments were present corresponding to ellagic acid fragment of m/z 275 and 301, this fragment is characteristic for the fragmentation of ellagitannins using MSn analysis. Corilagin has not been earlier characterized in Terminalia brownii, although this ET has been found in some other species of Terminalia, such as T. chebula, T. horrida and T. bellerica (Pfundstein et al. 2010), as well as in Terminalia citrina (Burapadaja and Bunchoo 1995). Based on the mass spectra obtained, we could, in addition, identify arjunglucoside I (compound 11 at tR 19.1 min in HPLC-DAD) with a molecular ion at m/z [M-H]- 725. In the MS2 mass spectrum, this compound gave a basic molecular ion at m/z [M-H]- 665 and another ion at m/z [M-H]- 503, which corresponds to the loss of a glucose unit, 162 Da [m/z 665-503]. Moreover, the same fragment of 503 showed a [M-H]- ion of 485 in the MS3 mass spectrum, which corresponds to the loss of water molecules (H2O=18), and in addition to the loss of CO2 (44) from the fragment ion [453- 409]. Further fragmentation at the MS4 level resulted in losses of carbon and oxygen atoms [M-H = 391-379 and 379-363], respectively (Appendices, Fig. 50). Arjunglucoside I has been earlier reported to occur in T. brownii (Machumi et al. 2013). Moreover, we found methyl-(S)-flavogallonate (5, C22H12O13) at tR 14.1min in the stem wood extract of T. brownii. This ellagitannin gave a molecular ion [M-H]- m/z 483, and we also identified this ET from a root extract of T. brownii (Salih et al. 2017a). Methyl-(S)-flavogallonate has not been reported earlier in T. brownii but has been found in the fruits of T. chebula, T. horrida and T. bellerica (Pfundstein et al. 2010), as well as in the leaves of T. myriocarpa (Marzouk et al. 2002). Although the T. brownii stem wood and bark are rich in ellagitannins, few investigations have been carried out this chemical class in this species of Terminalia. Yamauchi et al. (2016) reported on the occurrence of D, β-punicalagin and D, β-terchebulin in the stem bark of T. brownii, but this is the only other investigation to date on ETs in the stem of T. brownii. Deduced from our tandem mass spectrometric data, at tR 14.4 min (HPLC-DAD), we found gallagic acid dilactone (6, C28H10O16) in the ethyl acetate stem wood and bark extracts of T. brownii (Fig. 43 D and 46 A and B). Gallagic acid dilactone (terminalin) gave an [M-H]- m/z ion at 601 (continued Fig. 43 D, Appendices, Fig 52). When subjected to MS3, fragment ions of m/z 271 and 301 were observed, both of which have been described earlier for gallagic acid dilactone 118 (Pfundstein et al. 2010), the later ion corresponding to ellagic acid (continued Fig. 43 E). Gallagic acid dilactone is a phenolic acid incorporated in many ellagitannins. For example, gallagic acid dilactone is the fully lactonized form of the gallagyl unit of punicalagin and punicalin (Cerda et al. 2003, Garcia-Villalba et al. 2015). Gallagic acid dilactone has been reported to occur in fruit extracts of Terminalia chebula, T. horrida and T. bellerica (Pfundstein et al. 2010) and in T. oblongata (Oelrichs et al. 1994), but there are no previous reports on this phenolic acid in T. brownii. Our MS/MS analysis also revealed the presence of four previously known flavonoids, which have, however, not been characterized earlier in T. brownii. These flavonoids included, quercetin-7-β-Odi-glucoside (8); quercetin-7-O-galloyl-glucoside (10); naringenin-4′-methoxy-7-pyranoside (7) and 5,6-dihydroxy-3′,4′,7-tri-methoxy flavone (12), of which all were found in the stem wood. Naringenin-4′-methoxy-7-pyranoside (compound 7) was not present in the stem bark, however (Fig. 44 A). Naringenin and quercetin and their glycosidic and glucuronidic derivatives have been characterized in Terminalia spp. before, including the south east Asian species T. coriacea (Khan et al. 2017) and T. catappa (Venkatalakshmi et al. 2015). Quercetin-galloyl-glucopyranoside has earlier been isolated from the leaves of Morus alba (Moraceae) (El-Toumy et al. 2018) and from Anogeissus leiocarpus (Eltayeb et al. 2016), but to the best of our knowledge not from Terminalia spp. Two stilbenes, cis- (3) and trans-reseveratrol-3-O-β-galloylglycoside (4) were characterized for the first time in T. brownii stem wood (Fig. 44 B). In the MS spectra, both compounds showed a similar [M-H]- molecular ion at m/z 541. Fragment ions of m/z 227 and 314 were observed for both compounds in the MS3 spectra, corresponding to a resveratrol unit and a galloylhexose unit, respectively. The stilbenes mentioned have not been found in the genus Terminalia before. Some stilbenes, such as resveratrol itself (trans-3,4,5-trihydroxy-trans-stilbene) as well as the dihydroxyresveratrol-rutinoside and resveratrol-rutinoside derivatives, have been earlier reported to occur in the root bark and leaves of the African species Terminalia sericea (Joseph et al. 2007, Cock and Van Wuuren 2014) and in leaves of Terminalia prunioides (Cock and Van Wuuren 2014). Two oleanane triterpenoids (compound 1 and 2) showing [M-H]- ions at m/z 469 and 491, respectively and eluting at Rt 6.8 min were identified for the first time in the stem bark and wood extracts of T. brownii (Fig. 44 A and B). In the stem wood these triterpenoids were present in higher quantities (Fig. 44 B). Fragment ions in the MS2 chromatograms of compounds 1 and 2 indicated the loss of a carboxylic acid group for both compounds [M-H= 491- 447, corresponding to the loss of 45 molecules = COOH]. Previously, another unknown oleanane type triterpenoid at m/z [M-H]529.3651 has been characterized from the stem bark of T. brownii, along with seven other triterpenoids such as arjunic acid, sericic acid, 23-galloylarjunic acid, tomentosic acid, arjungenin, sericoside and arjunglucoside (Machumi et al. 2013). Pentacyclic triterpenoids and their glucosides are known from other species of Terminalia as well. In another African species, T. sericea, arjunic acid as well as sericic acid and its glycoside, sericoside, have been characterized (Nair et al. 2018). 119 Querder triMethfla TER Corider quergg Arjungluco I Narderi Tran Resder MeFlav Terminalin i TER Cis Resder Querder Terminalin triMethfla Figure 44. RP-HPLC-DAD chromatograms of ethyl acetate extracts of T.brownii (modified from Salih et al. 2017b; Study II). (A) stem bark and (B) stem wood extracts at 254 nm. (1) and (2) Oleanane type triterpenoids; (3) cis-resveratrol-3-O-β-galloyl-glucoside; (4) trans-resveratrol-3-O-β-galloylglucoside; (5) Methyl-(S)-flavogallonate; (6) Gallagic acid dilactone (Terminalin); (7) Naringenin-4′methoxy-7-pyranoside; (8) Quercetin-7-ß-O-diglucoside; (9) Corilagin derivative; (10) Quercetin-7-Ogalloyl-glucoside; (11) Arjunglucoside I; (12) 5,6-dihydroxy-3′,4′,7-trimethoxy flavone. Modified from study II (Salih et al. 2017b). 4.4.1.2. Polyphenols in Anogeissus leiocarpus (Study I) According to a recent review paper (Singh et al. 2016), 55 compounds have been characterized from eight species of Anogeissus, and of these, 23 have been discovered in the African species A. leiocarpus (Shuaibu et al. 2008, Mann et al. 2008, Chaabi et al. 2008, Attioua et al. 2011, Singh et al. 2016). However, most of these studies have been performed on the stem bark and leaves, and fewer investigations have been carried out on the phytochemicals of the roots (Singh et al. 2016). Our study revealed the presence of several compounds, including some stilbenes, flavonoids and gallotannins which have not been reported before in A. leiocarpus. Roots Altogether, 24 compounds were identified from a methanolic Soxhlet extract of the roots of Anogeissus leiocarpus, using the combined data of HPLC-DAD and UHPLC-DAD retention times, UVO absorption maxima (from HPLC-DAD) and negative ion mode mass spectrometry (Continued Fig. 45). Protocatechuic acid (2) (Continued Fig. 45 and 48 C), was found at tR 3.915 min, and it has been reported before in this species (Chaabi et al. 2008). In contrast to the roots of the species Terminalia studied, the roots of A. leiocarpus contained a smaller proportion of ellagitannins, and 120 only four ETs could be detected, based on their characteristic UVO maxima absorption spectra. These ellagitannins might be the same as in the stem bark extract (Fig 49, with UVλ maxima of 217, 256, 374, in compound (2); and UVλ maxima of 210, 248, 370, in compound (14). Previously, condensed tannins have been reported to occur in the root bark of A. leiocarpus (Mann et al. 2008), but no ETs are reported from the roots of this plant (Singh et al. 2016). According to our results, the roots of A. leiocarpus are rich in ellagic acid derivatives, and eight structures were recognized. Many of these ellagic acid derivatives could be derived from the ellagitannins mentioned above. Among these structures, dimethyl ellagic acid (12) ([M-H]− at m/z 329.0000) and dimethyl ellagic acid xyloside (9) ([M-H]− at m/z 461.0739) could be characterized (Continued Fig. 45). Both compounds are also present in the stem bark of this species, according to our results (Fig. 47). The ellagic acid derivatives mentioned have also been identified previously in the stem bark of A. leiocarpus (Adigun et al. 2000, Chaabi et al. 2008). Moreover, we found that the roots of A. leiocarpus are rich in flavonoids, and five flavonoids were identified which have not been reported before in this species (Continued Fig. 45). Among these previously known flavonoids are aromadendrin (syn. dihydrokaempferol (3), [M-H]- 288,0000) ampelopsin (syn. dihydromyricetin (5), [M-H]- 319,1417), taxifolin (syn. dihydroquercetin) (7) [M-H]- 303,0365) and methyltaxifolin (syn. (+)- dihydroisorhamnetin (6a), [M-H]- m/z 317,0678,) and its isomer (6b) ([M-H]- 317,0676) (Continued Fig. 45). Previously, quercetin, rutin, kaempferol and vitexin have been identified in the leaves of A. leiocarpus (Arbab 2014, Elsiddig et al. 2015). In other Anogeissus spp. some flavonoids have been identified, such as rutin, vitexin, catechin, kaempferol, quercetin and isoquercetin (Attioua et al. 2011, Singh et al. 2016). We also identified some gallotannins in the roots of A. leiocarpus, including pentagalloylglycose (9) (Continued Fig. 45 and continued 47 C) at tR 22.2 min (HPLC-DAD), giving a [M-H]− ion at m/z 939.6700 and di-galloyl-β-D-glucose (4) (Fig. 45 A and continued Fig 45) at tR 11.47 min (HPLC-DAD) and giving a [M-H]− ion at m/z 483.0808. These gallotannins have not been reported in A. leiocarpus previously. We found two stilbene compounds, pinosylvin (12) ([M-H]− 211.1339) and methylpinosylvin (15) ([M-H]− 225.0373), at tR 32.9 and 45.6 min (HPLC-DAD), respectively, in the roots of A. leiocarpus (Continued Fig. 45 and continued Fig.49 E and F). These stilbenes have not been previously reported to occur in this species. Moreover, we found that these compounds are absent from the stem bark (Fig. 47). Pinosylvin is an important phytoalexin constituent of the heartwood of Pinus spp. (Lee et al. 2005, Dinelli et al. 2006). Earlier, Rimando et al. (1994) and Eltayeb et al. (2016) have reported the occurrence of the stilbenes pterostilbene, viniferin and methylviniferin in cholorform extracts of the leaves of A. leiocarpus and in the stem of A. acuminate (continued Fig. 47 G and H). Stem bark In accordance with earlier work by Arbab (2014) and Ndjonka et al. (2014), we found that the stem bark of A. leiocarpus is rich in tannins. Moreover, we also found that compared to the roots, the stem bark contains a higher variety and quantity of tannins. We found that the majority of the larger peaks in the HPLC-DAD chromatogram of an ethyl acetate extract of the stem bark were ellagitannins and ellagic acid derivatives (Fig. 47). Six compounds were identified as ellagitannins according to their UVO absorption maxima in HPLC-DAD. Of these ETs, the masses of three compounds could be identified as follows, an 1) ET (2) at tR 8.482 min with a [M-H]− ion at m/z 121 453.1062; 2) an ET (11) at tR 26.9 min with a [M-H]− ion at m/z 833.4009; and 3) an ET (33) at tR 33.8 min with a [M-H]− ion at m/z 725.4177. Earlier, Shuaibu et al. (2008) have found that castalagin (MW 934), ellagic acid and flavogallonic acid are some of the major compounds in a methanol extract of the stem bark of A. leiocarpus (continued Fig. 43 C and 48 A and C). However, in this same paper, this author also reported that castalagin was absent from the ethyl acetate layer of the stem bark, and thus this explains why we did not find this ET in the ethyl acetate extract of the stem bark of this species. Moreover, we found the following ellagic acid derivatives in the stem bark ethyl acetate extract that we also had identified in the roots of A.leiocarpus: dimethyl ellagic acid (12) at tR 28.784 min [M-H]− 329.0000, and dimethyl ellagic acid xyloside (9) at tR 20.495 min [M-H]− 461.0766 (Fig. 47). Two other ellagic acid derivatives at tR 30 min ([M-H]− 613.0838) and at tR 35.7 min ([M-H]− 359.0436) could also be detected based on their UVO absorbtion maxima. The ellagic acid derivative which we have found in a A. leiocarpus stem bark ethyl acetate extract (12) at tR 30 min and showing a molecular ion at m/z [M-H]- 613.0838 could tentatively be assigned to 3,3`-Di-Omethyl-4-O-(n``-O-galloyl-β-D-xylopyranosyl) ellagic acid, which has been reported to occur in the fruits of Terminalia horrida (Pfundstein et al. 2010). Our results are in accordance with earlier investigations suggesting that the stem bark of A. leiocarpus is rich in ellagic acid derivatives (Jansen and Cardon 2005, Lemmens et al. 2012). Of these ellagic acid derivatives, methyl ellagic acid, dimethyl ellagic acid, dimethyl ellagic acid xyloside, and di-hydroxyl-tri-O- methylellagic acid-7-O-β-glucoside, ellagic acid-7-O-β-glucoside, ellagic acid-4'-O-β-rhamnoside, have previously been reported to occur in A. leiocarpus (Nduji and Okwute 1988, Jansen and Cardon 2005, Chaabi et al. 2008, Elsiddig et al. 2015). In addition, 3,3’,4-tri-O-methylflavellagic acid, 3,3′di-O-methylellagic acid and 3,4,3'-tri-O-methylflavellagic acid-4'-β-D-glucoside have been found in the stem bark of A. leicarpus (Nduji and Okwute 1988, Adigun et al. 2000). Our UHPLC/QTOF-MS and HPLC-DAD results revealed the presence of (epi)catechin gallate (7) at tR 16.703 min (HPLC-DAD), in the stem bark ethyl acetate extract of A. leiocarpus. This compound showed a [M-H]− molecular ion at m/z 441.0852 (Fig. 47). Earlier investigations on polyphenols in A. leiocarpus by Attioua et al. 2011, have revealed the presence of the catechin and procyanidin B5 in the leaves of this species. In addition, we found that epigallocatechin gallate (tR at UPLC 9.94, [M-H]− 457,0768) is present in the stem bark of A. leiocarpus. These two condensed tannins (CTs) have not earlier been found in A. leiocarpus. Compounds identified from A. leiocarpus roots and stem bark Aromadendrin (syn. Dihydrokaempferol) (3) (only present in roots): tR HPLC-DAD 10.47 min; tR UHPLC-DAD 3,80 min; UV (MeOH) λmax 210, 288; negative ESI-Q-TOF MS m/z: 288.0000; (calc. for C15H12O6, 288.0630) (Continued Fig. 45, compound (3)). Ampelopsin (syn. Dihydromyricetin) (in roots and stem bark): tR HPLC-DAD 12.66 min in roots and 11.700 min in stem bark; tR UHPLC-DAD 4.84 min in roots and 4.72 min in stem bark; UV (MeOH) λmax 210, 292; negative ESI-Q-TOF MS m/z: 319.1417; (calc. for C15H12O8; 320.0528) (Continued Fig. 45, compound (5)). Methyltaxifolin (syn. (+)-Dihydroisorhamnetin) (only present in roots): tR HPLC-DAD 13.44 min; tR UHPLC-DAD 5.50 min; UV (MeOH) λmax 210, 294; negative ESI-Q-TOF MS m/z: 317.0678; (calc. for C16H14O7; 318.0735) (Continued Fig. 45, compound (6a)). 122 Isomer of Methyltaxifolin (only present in roots): tR HPLC-DAD 14.82 min; tR UHPLC-DAD 5.98 min; UV (MeOH) λmax 210, 230, 288; negative ESI-Q-TOF MS m/z: 317.0676; (calc. for C16H14O7; 318.0735) (Continued Fig. 45, compound (6b)). Taxifolin (syn. Dihydroquercetin) (only present in roots): tR HPLC-DAD 17.05 min; tR UHPLCDAD 6.45 min; UV (MeOH) λmax 210, 230, 290; negative ESI-Q-TOF MS m/z: 303.0365; (calc. for C15H12O7; 304.0579) (Continued Fig. 45, compound (7)). Dimethyl ellagic acid glucoside (only present in roots): tR HPLC-DAD 21.17 min; tR UHPLCDAD 7.75 min; UV (MeOH) λmax 254, 368; negative ESI-Q-TOF MS m/z: 491.0843; (calc. for C22H20O13; 492.0897) (Continued Fig. 45, compound (8)). Pentagalloylglucose (only present in roots): tR HPLC-DAD 22.24 min; UV (MeOH) λmax 218, 284; negative ESI-Q-TOF MS m/z: 939.6700; (calc. for C41H32O26; 940.1170) (Continued Fig. 45, compound (9)). Dimethyl ellagic acid xylopyranoside (in roots and stem bark): tR HPLC-DAD 24.67 min in roots and 20.49 min in stem bark; tR UHPLC-DAD 9.65 min in roots and 13.060 min in stem bark; UV (MeOH) λmax 246, 368; negative ESI-Q-TOF MS m/z: 461.0739; (calc. for C21H18O12; 462.0792) (Continued Fig. 45, compound (10) and Fig 49, compound (10)). Dimethyl ellagic acid (in roots and in stem bark): tR HPLC-DAD 29.45 min in roots and 28.78 min in stem bark; tR UHPLC-DAD 11.25 min in roots and 14.89 min in stem bark; UV (MeOH) λmax 248, 376; negative ESI-Q-TOF MS m/z: 329.0318; (calc. for C16H10O8; 330.0372) (Continued Fig. 45, compound (11) and Fig. 47, compound (12)). Pinosylvin (only present in roots): tR HPLC-DAD 32.92 min; tR UHPLC-DAD 12.05 min; UV (MeOH) λmax 218, 308, 320; negative ESI-Q-TOF MS m/z: 211.1339; (calc. for C14H12O2; 212.0834) (Continued Fig. 45, compound (12)). 4´-Methylpinosylvin (only present in roots): tR HPLC-DAD 45.65 min; tR UHPLC-DAD 17.94 min; UV (MeOH) λmax 216, 218, 308, 318; negative ESI-Q-TOF MS m/z: 225.0373; (calc. for C15H14O2; 226.0990) (Continued Fig. 45, compound (15)) Figure 45. (A), Di-galloyl-β-D-glucose; (B), Trigalloyl glucose; (C), Pentagalloyl glucose, found in studies I and III (Salih et al. 2017a, Salih et al. 2018). 123 Continued figure 45. Methanolic soxhlet extracts of the root of Anogeissus leiocarpus (modified from Salih et al. 2017a; Study I). (1), gallic acid; (2), protocatechuic acid; (3), aromadendrin; (4), Di-galloyl-β-D-glucose; (5), ampelopsin; (6a), methyl taxifolin; (6b), isomer of methyl taxifolin; (7), taxifolin; (8), dimethyl ellagic acid glucoside; (9), pentagalloyl glucose; (10), dimethyl ellagic acid xylopyranoside; (11), dimethyl ellagic acid; (12), pinosylvin; (13), acetylated ellagic acid derivatives; (14), ellagic acid derivatives; (15), methylpinosylvin. UVλ absorption maxima for compounds (5), (7), (12), and (15). Modified from study I (Salih et al. 2017b). (B) (C) (A) Figure 46. (A), Flavogallonic acid (syn. flavogallonic acid dilactone); (B), castalagin (syn. vescalagin) and (C), protocatechuic acid (syn. dihydroxybenzoic acid) earlier characterized by Shuaibu et al. 2008, from the stem bark of Anogeissus leiocarpus (Source, CAS 2018). 124 (A) Anogeissusin A (B) Anogeissusin B (C) Anogeissinin Continued figure 46. Examples of dimeric flavano-ellagitannins, characterized in Anogeissus acuminata (Yoshida et al. 2010). (A), Anogeissusin A (C97H62O56, MW 2123.50); (B), Anogeissusin B (MW 2139.50, C97H62O57) and (C), Anogeissinin (C97H62O56, MW 2123.50). Flavan-3-ol part in the ETs in blue color. 125 Figure 47. Chromatogram of the ethyl acetate extract of the stem bark of Anogeissus leiocarpus at 270 nm (modified from Salih et al 2017; Study I). This extract contains the gallic acid (1), ampelopsin (3), epigallocatechin gallate (4), di-methyl ellagic acid xylopyranoside (9), ellagic acid derivatives (8a, 13, 15) and di-methyl ellagic acid (12) along with six unknown ellagitannins (2, 5, 6, 8b, 11 and 14) and epicatechin gallate (7). UVO absorbtion maxima are indicated for compounds 2, 9, 12 and 14. Modified from study I (Salih et al. 2017b). 126 (A) (C) (B) (E) (D) (G) (F) (H) Continued Figure 47. Flavonoid and stilbene compounds detected in Anogeissus leiocarpus roots (A, B, C, D, E and F) or stem bark (D) in this study. Taxifolin (Dihydroquercetin) (A); Methyl-taxifolin (Dihydroisorhamnetin) (B); Aromadendrin (Dihydrokaempferol) (C); Ampelopsin (Dihydromyricetin) (D); Pinosylvin (trans-Dihydroxystilbene) (E); Methyl pinosylvin (F). Identified for the first time in A. leiocarpus in our current research, study I (Salih et al. 2017a). Besides Viniferin (G) and (H) Pterostilbene have been found previously in the leaves of A. leiocarpus (Eltayeb et al. 2016) and the roots of A. acuminata (Rimando et al. 1994). Source for molecular structures: CAS, 2018. 127 4.4.2. Non-polar and medium polar extracts 4.4.2.1. Identification of terpenoids and fatty acids in T. laxiflora and T. brownii using GCMS (Study III) Lipid compounds have previously not been investigated thoroughly in T. laxiflora and T. brownii. Thus, we investigated the fatty compound composition of antimycobacterial hexane stem bark extracts of these two species of Terminalia using GC-MS analysis (Fig. 48 and continued Fig. 48). Four long-chain fatty acids and three fatty alcohols were found for the first time in the stem bark of T. laxiflora and T. brownii, namely 1) tetracosanoic acid 24:0 (C24H48O2, tR in GC/MS 9.23 min, MW. 368.6); 2) hexacosanoic acid 26:0 (C26H52O2, tR in GC/MS 10.45 min, MW 396.7); 3) octacosanol 28:0 (C28H58O, tR in GC/MS 11.18, min, MW 410.7); 4) octacosanoic acid 28:0 (C28H56O2, tR in GC/MS 11.94, MW. 424.7); 5) triacontanol (C30H62O, tR in GC/MS 12.87, MW438.81) and 6) dotriacontanol (C32H66O, tR in GC/MS 14.97, MW 438.81). In addition, the unstarurated lipid, octadec-9-ene-dioate at tR 8.30 min, MW 456 was found (Fig. 40). Several terpenoids and fatty compounds, have been reported before in African species of Terminalia and in species of the closely related genus Anogeissus. Lauric acid, heptadecanoic acid, eicosanoic acid, omega-3, omega-6, omega-9, palmitic acid, linoleic acid, oleic acid, stearic acid, arjunolic acid, sericoside, arjungenin, sericic acid, arjunetin, chebuloside, sitosterol and stigmasterol have been reported in T. sericea, , T. glaucescens and T. arjuna (Chivandi et al. 2013, Dawé et al. 2017, Bansal et al. 2017). In addition, ursolic acid, campsterol, tetradecanone, betulin, lupeol, amyrine and squalene have been found in Combretum micranthum and arjunglucoside and sericoside in Combretum molle (Bony et al. 2014, Asres et al. 2001). Hamzaoui et al. 2013 reported the occurrence of sericoside and trachelosperogenin in Anogeissus leiocarpus. 128 Abundance (1) T. laxiflora 11 6 1 4 Abun dance 2 1 5 3 7 8 9 10 * 15 14 12 13 (2) T. brownii 11 7 2 3 5 4 6 8 9 10 * 12 13 14 15 Figure 48. GC/MS chromatogram of fatty acids and triterpenes in a hexane extract of the stem bark of (A), T. laxiflora and (B), T. brownii (modified from Salih et al. 2018; Study III). TMS-1,18-octadec-9-ene dioate (1), TMS-tetracosanoic acid (2), TMShexacosanoic acid (3), TMS-octacosanol (4), TMS-octacosanoic acid (5), TMS-triacontanol (6), TMS- β-sitosterol (7), TMS- βamyrine (8), stigmast-4-en-3-one (9), TMS-dotriacontanol (10), TMS-friedelin (11), TMS-betulinic acid (12), 5α-stigmastan-3,6dione (13), TMS-oleanane-type triterpenoid (14 ) and (15). Chemical structure for the sterols found in Terminalia laxiflora and T. brownii stem bark showed in continued figure 48. Modified from study III (Salih et al. 2018). 129 (A) (B) (C) (E) (D) Continued figure 48. Chemical structure for the sterols found in Terminalia laxiflora and T. brownii stem bark using GC/MS analysis (in Figure 48), Ketosterols of (A), 5α-stigmastane-3,6-dione; (B), stigmast-4-en-3-one (sitostenone); (C), octacosanoic acid; (D), octacosanol; (E), hexacosanoic acid (cerotic acid). Source: CAS, 2018. Study III (Salih et al. 2018). 130 (A) (B) (C) (E) (D) Figure 49. Previously identified pentacyclic triterpenoid and phenolic compounds in the stem bark of Combretum hartmannianum according to Mohieldin et al., (2017) and Morgan et al., (2018). Pomolic acid (A), Trachelosperoside E-1 (B), Corosolic acid (C), Flavogallonic acid dilactone (D), and Terchebulin (E) Source: CAS, 2018 131 5. Conclusions and recommendations 5.1. Ethnopharmacological uses of species belonging to the Combretaceae family, occuring naturally in the Blue Nile and Kordofan regions, in Sudan According to our ethnobotanical and ethnopharmacological investigations, Terminalia laxiflora, T. brownii and Anogeissus leiocarpus are used for the treatment of infections and their symptoms, including cough, tuberculosis, diarrhea, wound inflammation and skin itches, among a variety of other uses. In agreement with these uses, these tree species proved to be good sources for extracts and compounds exerting growth inhibitory effects against a wide variety of human and plant pathogenic bacteria and fungi. Traditional preparations of these medicinal plants to cure infectious diseases and wounds, such as decoctions and cold water macerations, were found to give promising in vitro antimicrobial effects. 5.2. Antimicrobial effects of extracts and compounds In many cases, and in accordance with previous research on antimicrobial effects of Combretaceae species, it was observed that especially the root parts possess promising antimicrobial potential. However, also the stem wood and stem bark of the plants investigated proved to be good sources of antimicrobial compounds. A large number of extracts of varying polarities, made from the roots, stem bark, stem wood and leaves of the Terminalia, Anogeissus and Combretum species studied, were found to possess promising growth inhibitory effects against common human pathogenic bacteria as well as plant pathogenic molds, with 39 μg/ml as the lowest MIC value observed. In general, more polar extracts were found to be more antimicrobial. The antimicrobial effects of the more polar extracts, including those prepared using traditional techniques, such as decoctions and cold water macerations, are suggested to be due to ellagitannins and ellagic acid glycosides, which were found in high quantities in these extracts. Purification of a methanol extract of the roots of Terminalia brownii, using Sephadex LH-20 column chromatography, resulted in a notable increase in the activity against Mycobacterium smegmatis of an ellagitannin-enriched extract, compared to the crude methanol extract. However, for Staphylococcus aureus and Pseudomonas aeruginosa, this Sephadex LH-20 purification of a Terminalia brownii root extract did not result in an increase of the antibacterial activities of the resulting fractions. In antimicrobial n-hexane extracts of the plants studied, fatty compounds, such as fatty acids and alcohols, as well as triterpenes, were found to predominate, and thus these compounds are suggested to be responsible for the antimicrobial effects of these extracts. In summary, the in vitro antimicrobial results of this study indicate that the plants screened could be used as alcohol extracts and mixed with fat for the treatment of infectious diseases and wounds, in addition to their established uses in Sudan as water extracts and decoctions. 5.3. Phytochemical investigations HPLC-DAD results indicated that polar extracts of the species investigated, such as cold and hot methanol, aqueous (80 % methanol), ethyl acetate and acetone extracts, as well as cold water macerations and decoctions, contain a variety of polyphenolic compounds. In these extracts ellagitannins and ellagic acid derivatives predominate, but also flavonoids, stilbenes and 132 gallotannins were found to occur. A large number of unknown ellagitannins could be detected in the Terminalia species and in Anogeissus leiocarpus. In addition, the previously known ellagitannins corilagin, punicalagin and methyl-(S)-flavogallonate were found for the first time in the Terminalia species studied. Moreover, using LC-MS/MS tandem mass spectrometry and UPLC-QTOF/MS , gallagic acid dilactone (terminalin) and the stilbenes, cis- and trans- resveratrol-3-O-β-galloylglucoside, which have not been reported before in the stem wood and bark of T. brownii, where characterized. This study also led to the finding of some, previously unreported, flavonoids in Terminalia species, including quercetin-7-β-O-di-glucoside, quercetin-7-O-galloyl-glucoside, naringenin-4′-methoxy7-pyranoside and 5,6-dihydroxy-3′,4′,7-tri-methoxy flavone. Likewise, aromadendrin (syn. dihydrokaempferol), ampelopsin, taxifolin and methyl taxifolin had not been reported before in Anogeissus leiocarpus. In n-hexane extracts of T. brownii and T. laxiflora, antimycobacterial 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, and triacontanol were found for the first time. Moreover, β-sitosterol, friedelin, betulinic acid and β-amyrine, which have been previously reported in Combretaceae, were present in the hexane extracts of T. brownii and T. laxiflora. Some of the compounds mentioned were tested for their growth inhibitory effects against Mycobacterium smegmatis, and were found to be moderately active, with MIC values ranging between 250–500 μg/ml. 5.4. General recommendations 1. This study demonstrates that Terminalia laxiflora, Terminalia brownii and Anogeissus leiocarpus, used for remedy against infectious diseases and their symptoms in Sudan, might be good sources for molecular scaffolds to be used as lead molecular templates for the development of antimicrobial drugs. 2. Extensive and further research is required in order to understand the structure-activity relationship and the mechanism of actions of pure compounds and plant extracts, when used alone or in a combinations with conventional antibiotics. 3. Further studies should be performed using clinical strains of human pathogenic bacteria and yeasts as well as phytopathogenic bacteria and fungi. The research should especially focus on multidrugresistant strains. 4. Dose-response relations for in vitro and in vivo toxicity of selected extracts and compounds should be established in order to produce standardized extracts with known concentrations of antimicrobial compounds and with a minimum content of toxic compounds. 5. Scientific research on plant-derived biologically active extracts and compounds, based on traditional knowledge, should be performed according to the guidelines of the Nagoya protocol of bioprospecting and fair share of intellectual properties and bio-resources (Chiarolla et al. 2013) and the Chiang Mai and Kari-Oca declaration (Hoareau and DaSilva 1999). Special care should be taken when it comes to the eventual patenting and commercialization of the outcomes of this kind of research work, allowing the owners of the traditional knowledge (traditional healers and others) who share their knowledge, to receive a fair benefit from the eventual patenting process. 133 6. Non-wood and wood products from traditional medicinal plants growing in the natural forests in Africa are collected, harvested and exported in a large scale, within African countries and out from the continent. Therefore, increasing interdisciplinary collaboration among experts of various fields, such as economy, ecology, botany, agricultural sciences, forestry, pharmacy, medicine and others, would be needed. This kind of collaboration could help to develop a solid basis for the conservation and sustainable use of the remaining medicinal plant resources. One possible solution would be to increasingly incorporate the medicinal trees into various land-use systems for the collection of their products and as shade trees and food and nutrient providers. Useful approach might be apart from in-situ and ex-situ gene conservations, to also apply circa situm gene conservation, in which the genetic variation is preserved by using the plants in question (Appiah 2003). The results of this study suggest that it would be important to ensure the regeneration and sustainable management of important medicinal plant species belonging to the plant family Combretaceae. 7. New legislation should be adopted to control the harvesting of plant material for medicinal use, in order to avoid over-exploitation derived by the demands from pharmaceutical and cosmetic industries. 8. Traditional healers and communities should be trained to better awareness of the needs to protect and maintain their indigenous knowledge and to preserve their medicinal plants in natural stands as well as in cultivation in home gardens and agroforestry systems. 9. Association of African Medicinal Plants Standards (AAMPS) has made significant contribution with their “African Herbal Pharmacopoeia” from 2010. However, there would still be plenty of information yet to be documented regarding the ethnobotanical uses of plants in Africa. Only a fraction of the ethnomedical information about African medicinal plants has been documented and even fewer species have been studied for their biological effects and phytochemistry. 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Combretaceae Sudanese name Height Anogeissus leiocarpus*** Sahab ˃ 15m Exudate/ Smell Bark tapping produces drops of brown gum Shukheit 5m Gum exudate observed 15m A long line of gum results from the tapping 10m Exudate edible gum 12m Exudate edible gum Combretum aculeatum** Combretun ghasalense** Combretum collinum*** Combretum glutinosum ** Habil Um Ismael Habeel elgouruz Habil el-gebel Combretum hartmannianum *** Habil, Subagh 25m Combretum nigricans** Habil 12m The bark exudes gum Combretum molle** Habeel Khrisha 15m Exudate gum Terminalia brownii*** Subagh, Darot 25m Pleasent scent from the bark Sofraya, Darot 25m Pleasant scent from the bark Terminalia laxiflora* ** Exudate edible gum Darot 15m Resinous scent from the bark Darot Al-goz 12m Pleasant scent from the bark Conocarpus lancifolius* Adamas 25 m Gum excudate observed, high salt tolerance especially in dry areas Guiera senegalensis*** Gobaish 4m No gum observed Terminalia avicennioides** Terminalia macropetra** Flowering/ Fruiting September to November/ August to January March to June/ July to October November to February/ February to March January to February/ Jane to March December to March/ October to December Jane to February/ April to May February to March; April to July February to May; May to Aug April to June/ October to November February to March/ May to June/ August to November October to February/ November to April February to March/ March to August May to October/ all year September to October Twice a year all period of the year fruiting and flowering Traditional uses in Kordofan and Blue Nile regions in Sudan Root used as chewing sticks and as toothache paste, venereal infections, fever and dysentery; smoke from stem and root against rheumatism. Leaves as laxatives and for venereal disease; stem against skin infections, including leprosy; root decoction against flu Bark used for stomachache, smoke from the wood as perfume Gum used against toothache; root and leaf decoctions used against diarrhea. Root, stem and leaf decoctions reducing hypertension, as laxatives, against stomach ache , hepatitis, respiratory diseases symptoms and fever Leaves used to cure ascites, bark is mixed with gum against jaundice.; root and leaves used against TB infection For intestinal disorders, acne, jaundice, and rheumatism Leaves as incense , to induce miscarriage and relieve constipation, anthelmintic especially against hookworms Root, bark and wood used for cough and bronchitis and as fumigant Root, bark and wood used for cough and bronchitis and as fumigants Antifebrile, to cure jaundice, sores, syphilis, diarrhea and skin infections Decoction used against dysentery and diarrhea, and for skin fungal infections Gum used to cure intestinal disorder, wound healing, against dysentery Malaria, roasted fruit chewed to treat cough The most important species for curing infectious diseases in local traditional medicinal applications are labelled with three asterisks ***, such as Anogeisssus leiocarpus, Terminalia brownii and Guiera senegalensis, moderate importance with two asterisks ** and the less importance with one asterisk*. 170 Table 3. Diseases in plants, people and animals caused by filamentous fungi, common in Africa and growth inhibitory effects of extracts and/or compounds from Terminalia spp. against these fungi. Infections caused by the fungi Fungal strain a h p in animals , people , plants and others Aspergillus spp. (Ascomycota Fungi) o Pulmonary aspergillosis (aspergilloma) h (Mortensen et al. 2017); Bronchial obstruction h (Jiang et al. 2015); Pleural aspergillosis h (Bennett 1979) and allergic reactions (alveolitis) h (Edwards and Alzubaidy 1977) Potent toxins of Malformins produced by A. niger O (Blumenthal 2004) and aflatoxin by A. flavus O (Schindler et al. 1967) Growth inhibitory effects of extracts of Terminalia brownii (Salih et al. 2017b) We have found that ethyl acetate extacts of T. brownii stem wood and stem bark inhibit the growth of A. niger ATCC 9763 with an IZ of 17 mm and a MIC of 500 μg/ml. Destructive to stored plant product P (Magro et al. 2006) and caused spoilage P (Pina-Pérez et al. 2017) Caused air sac infection in poultry a (Jayanthi et al. 2015). Sinonasal infections in domestic animals (Seyedmousavi et al. 2015) References (growth inhibition of Terminalia spp. extracts) References (pure compounds isolated from Terminalia spp. with antifungal activity) T. bellirica and T. chebula against A. flavus, and A. niger (Gautam et al. 2012); T. bellirica against A. flavus and A. niger (Nilima and Deepavali 2013): T. chebula against A. niger (Zia-Ul-Haq et al. 2011); T. alata, T. arjuna, T. bellerica, T. catappa, T. chebula against A. niger and A. flavus (Shinde et al. 2011). T. avicennioides against A. niger gave MIC value of 50 μg/ml (Mann et al. 2008) T. brownii stem bark: Sitosterol, Stigmasterol, Monogynol A, Betulinic acid and Arjungenin active against A. niger (Opiyo et al. 2011); T. thorelii against A. niger (Pawar 2013); T. spinosa against A. flavus, and A. niger (Fabry et al. 1996). .); T. muelleri against A. niger (Pawar 2015). Mycotic abortion in dairy cattle a (Djønne 2007) and guttural infections in mammals a (Seyedmousavi et al. 2015). T. brownii ethyl acetate extract of the stem bark growth inhibitory against A. niger with an IZ of 15.5 mm (Opiyo et al. 2011). Fusarium spp. Produce the toxins, zearalenone, nivalenol and deoxynivalenol O (Blumenthal 2004) (Ascomycota Fungi) Contaminates agricultural plant products P,O (Chehri and Godini 2017). Causes cerebral infection in people with immunodeficiency h (Qian et al. 2011) We have found that ethyl acetate extracts of T. brownii stem wood and stem wood inhibit the growth of Fusarium verticilloides (syn. F. moniliforme) ATCC 24378 with IZ 19 and MIC 250 μg/ml Causes respiratory disease in pigs a, stomach problem in catfisha and enteritis in poultry a (Antonissen et al. 2014) 171 T. bellirica, T. sericea, T. thorelii and T. arjuna, T. chebula have been tested against F. verticilloides (Syn. F. moniliforme) (Nilima and Deepavali 2013, Samie and Mashau 2013, Pawar 2013, Tejesvi et al. 2009, Rajeswari and Ramesh 2012) T. alata root: 3,3`-di-O-methylellagic acid 4-O-β-Dglucopyranosyl-(l-4)-β-D-glucopyranosyl-(l-2)-α-Larabinopyranoside (ellagic acid glycoside). 5,7,2`-triO-methylflavanone-4`-O-α-L-rhamnopyranosyl-(1-4)β-D-glucopyranoside (arjunone, flavonone glycoside). 2α,3β,19-β,23-tetrahydroxyolean-12-en-28-oic acid3β-O-β-D-galactopyranosyl-(1-3)-β-Dglucopyranoside-28-O-β-D-glucopyranoside (triterpene saponin) active against A. niger (Srivastava et al. 2001) T. chebula stem bark: Flavonone glycoside of (5,7,2`tri-O-methyl flavanone-4`-O-β-D- galactopyranosyl (1-3)-β-D- glucopyranoside) and new saponin of 2α,19α-dihydroxy-3-oxo-12-en-28-oic-acid-O-α-Lrhamnopyranosyl-(1-4)-β-D- glucopyranoside. βamyrin, lupeol and Friedelin active against A. niger (Srivastava and Srivastava . 2004) We found that antifungal ethyl acetate extracts of T. brownii stem wood and bark contain phenolic and triterpene compounds. No literature on the activity of pure isolated or identified compounds from Terminalia species extracts against Fusarium verticilloides (Syn. F. moiliforme). Fusarium Solani causes root rot P (Toghueo et al. 2016) Nattrassia mangifera Causes Tinea unguium (Onychomycosis) h (Berger 2017), Hyperkeratosis h (SMS 2012) (Ascomycota Fungi) Causes stem dieback p (wilt diseases) and blossom blight P (FAO 2007); canker diseases P (Elliott and Robert 2008) We have found that ethyl acetate extacts of T. brownii stem wood and stem bark inhibit the growth of Nattrassia mangiferae ATCC 96293 with IZ 18.5 and MIC 250 μg/ml According to our knowledge, no literature has been recorded on the activity of extracts from Terminalia species against Nattrassia mangifera. Causes animal mycoses a (Godoy et al. 2004) have economic impact in forest products O (Blair 2015) Abbreviations: Animal a, human h, plant p and others o, IZ, diameter of inhibition zone in mm, T, Terminalia, MIC, minimum inhibition zone. 172 According to our knowledge, no literature has been recorded on the activity of pure isolated or identified compounds from Terminalia species extracts against Nattrassia mangifera Table 4. Summary of the results of this thesis work on the traditional uses of Terminalia brownii, T. laxiflora, Anogeissus leiocarpus and Combretum hartmannianum in Sudan and antimicrobial activities of various extracts, chromatographic fractions and pure compounds of the studied plants. The results are presented in relation to previous reports. Species and plant part Antibacterial effects (I) * Antifungal results against filamentous fungi (II) Antimycobacterial effects of T. laxiflora and T. brownii against M. smegmatis (III) Traditional uses in Kordofan and Blue Nile regions in Sudan ** and traditional uses according to other authors *** Reported antimicrobial activity in the literature Terminalia brownii Leaves NT NT Acetone extract of the leaves: IZ 27 mm, MIC 2500 μg/ml Decoctions and macerations of the leaves against TB, cough and respiratory tract infections** Leaves have anti-mycetoma activity (Ali et al. 2015) Stem bark P. aeruginosa (B EtOAC, IZ 21mm, MIC 39 μg/ml S. aureus (B MeSox, IZ 21mm, MIC 78 μg/ml). Aspergillus niger, A. flavus (EtOAc MIC 250 μg/ml, IZ 17 –18.5 mm) Nattrassia mangifera and Fusarium verticilloides (EtOAc MIC 500 μg/ml, IZ 19 – 20 mm); Hexane extracts of the stem bark: IZ 21mm, MIC 2500 μg/ml Stem bark decoctions against gonorrhoea, stomach pain, diarrhoea** Stem Wood S. aureus (W MeSox, IZ 20mm, MIC 312 μg/ml) M. luteus (W MeSox extracts, IZ 26mm, MIC 312 μg/ml) S. aureus (W EtOAC IZ 20mm, MIC 625 μg/ml) Aspergillus niger, A. flavus (EtOAc MIC 250 μg/ml, IZ 17 – 18.5 mm) Nattrassia mangifera and Fusarium verticilloides (EtOAc MIC 500μg/ml, IZ 18.5 – 19 mm); Stem wood decoctions, IZ 21 mm; MIC 5000 μg/ml Stem wood decoctions, macerations and fumigations for eye infections, dysentery; œdema** Stem bark extracts gave a MIC of 500 μg/ml against the Gramnegative bacterium Porphyromonas gingivalis (Mohieldin et al. 2017); stem bark extracts active against Propionibacterium acnes (Muddathir and Mitsunaga 2013); Stem wood extracts gave a MIC 500 μg/ml against Porphyromonas gingivalis (Mohieldin et al. 2017) Root P. aeruginosa (R HH2O, IZ 25mm, MIC 39 μg/ml) S. aureus (R acet, IZ 23 mm, MIC 39 μg/ml) S.epidermidis (R acet IZ 22 mm, MIC 39 μg/ml) M.luteus (R MeSox, IZ 39 mm, MIC 39 μg/ml) Root cold water macerations, IZ 26 mm, MIC 5000μg/ml) and decoctions and Soxhlet methanol extracts, IZ 22mm and MIC 5000 μg/ml for both Root for diarrhoea, venereal infections, TB and skin diseases** Root bark NT Root bark acetone extracts, IZ 23 mm, MIC 5000 μg/ml Root bark for gastric disorder; cough, gonorrhoea, TB and syphilis** Acetone Wash NT IZ 28 mm, MIC 62.5 μg/ml No reports Ethanol Wash S. aureus and P. aeruginosa (MIC 93.75 and 187.50 μg/ml, respectively) IZ 25 mm, MIC 125 μg/ml No reports Root extracts active against Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli, Bacillus anthracis, Bacillus cereus, Proteus mirabilis and of the yeasts, Candida albicans, Cryptococcus neoformans (Mbwambo et al. 2007) No reports Sephadex LH-20 fractions from the roots*: 173 Terminalia laxiflora Decoctions and macerations of the fruits against cough and respiratory tract infections** Fruits NT NT Fruit cold methanol extract, IZ 20 mm Leaves NT NT Leaves EtOAc extract, IZ 23, MIC 2500 μg/ml Against itchy skin (pruritus), fever and other symptoms related to TB such as cough** Stem bark S. aureus (B MeSox 21mm; MIC 78 μg/ml) P. aeruginosa (B EtOAc IZ 21mm MIC 312 μg/ml) S. epidermidis (B EtOAc IZ 20 mm; MIC 625 μg/ml) M. luteus (bark EtOAC IZ 27 mm, MIC 78 μg/ml) P. aeruginosa (W CHCl3 IZ 32;MIC 2500 μg/ml) S aureus Wood EtOAc IZ 23 mm, MIC 625 μg/ml NT Bark hexane extract, IZ 21 mm, MIC 2500 μg/ml Against itchy skin (pruritus), fever and other symptoms related to TB such as cough** NT Wood hexane extract, IZ 20mm; MIC 2500 μg/ml Diarrhoea, dysentery, syphilis, oedema** Root P. aeruginosa (R HH2O IZ 25mm MIC 39 μg/ml) and R EtOAc IZ 22 mm, MIC 39 μg/ml) S. aureus (R HH2O, IZ 25mm MIC 39 μg/ml) NT methanol Soxhlet extract, IZ 22; MIC 1250 μg/ml cold water extract, IZ from 21 mm; MIC 2500 μg/ml dichloromethane extract, IZ 21mm; MIC 5000 μg/ml acetone extract, IZ 20mm; MIC 625 μg/ml Venereal infection, TB, wound healing, skin infections ** RP-18 TLC fractions from the roots* NT NT TLC fractions obtained from a methanol Soxhlet and an ethyl acetate extract: MIC 500 μg/m for both Stem wood 174 Seed extract was active against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, P. aeruginosa and Proteus vulgaris (Salih 2006) Leaf extract was active against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, P. aeruginosa and Proteus vulgaris (IZ, 9 – 24 mm) (Salih 2006) No reports Stem wood extracts gave a MIC of 250 μg/ml against against Porphyromonas gingivalis (Mohieldin et al. 2017), and were active against Propionibacterium acnes (Muddathir and Mitsunaga 2013) No reports No reports Anogeissus leiocarpus Stem bark P. aeruginosa (B CHCl3 IZ 24mm, bark EtOAc IZ 22mm, MIC 625 μg/ml) S. aureus (B aqueous IZ 24 mm MIC 312 μg/ml; B MeSox IZ 24mm , MIC 39 μg/ml NT Stem bark decoction, EtOAc and aqueous extracts were moderately active, whereas the stem bark EtOAc extract was very active Stem wood P. aeruginosa (W CHCl3 IZ 28; wood MeSox IZ 26mm, MIC 1250 μg/ml) S aureus (wood MeSox IZ 20 mm, MIC 156 μg/ml) P. aeruginosa (R acet IZ 25 mm, MIC 312 μg/ml; root HH2O 24mm, MIC 78 μg/ml) S. aureus (R MeSox IZ 23mm, MIC 39 μg/ml) S. epidermidis (R MeSox IZ 28mm, MIC 39 μg/ml) NT Stem wood EtOAc, methanol Soxhlet and cold methanol extracts very active. NT Polar extracts very active, whereas the nonpolar extracts were weakly active or not active NT NT No reports NT 1000 μg/ml S. aureus (MIC 125 μg/ml); P. aeruginosa (MIC 250 μg/ml) NT 500 μg/ml Corilagin was active against C. albicans (MIC 500 μg/ml), C. glabrata (MIC 31 μg/ml), C. krusei (125 μg/ml), Cryptococcus neoformans (16 μg/ml) (Latte and Kolodziej 2000); active also against S. aureus and E. coli (Shiota et al. 2004, Hmoteh et al. 2016) No reports S. aureus (MIC 500 μg/ml) P. aeruginosa (MIC 250 μg/ml). NT active against M. smegmatis Root Decoction and maceration against cough and respiratory tract infections, diarrhoea dysentery, wound healing, skin diseases, salmonellosis, tubercolosis, cough and leprosy ** (Singh et al. 2016, Ademola and Eloff 2011, Barku et al. 2013) Venereal infections, gonorrhea, syphilis, skin diseases **. Bark exudate for dental caries and tooth ache (Singh et al. 2016) EtOAc and hexane and methanol extracts active against Bacillus subtilis and Klebsiella pneumoniae (IZ 30 and 29mm respectively) (Ali et al. 2017 and Elegami et al. 2002) Gastric ulcers, TB, fever, diarrhoea, wound infections ** Root extracts gave MIC values of 78 and 312.5 μg/ml, respectively, against Mycobacterium tuberclosis and M. bovis respectively (Mann et al. 2008a) No reports Commercial pure compounds found in the extracts and fractions: A) In polar extracts Punicalagin Tb, Tl CorilaginTb, TL, Ch Ellagic acid Tb, TL, Ch, Al Gallic acid Tb, TL, Ch, Al S. aureus (MIC 125 μg/ml) P. aeruginosa (MIC 500 μg/ml). NT 175 Gallic acid active against C. albicans (MIC 500 μg/ml) and C. glabrata (MIC 62 μg/ml), C. krusei (MIC125 μg/ml) and Cryptococcus neoformans (MIC 31 μg/ml) (Latte and kolodziej 2000) Flavonoids Tb, TL, Ch, NT NT NT Al Quercetin Apigenin NT NT Quercetin was active against S. aures and E. coli (MIC 6.25 and 100 μg/ml respectively) (Liu et al. 2010). Apigenin was also active against C. albicans and C. parapsilosis (MIC 5 μg/ml) (Lee et al. 2018) as well active against MRSA (MIC 62.5 μg/ml) and aginst Klebsiella pneumonia, Enterobacter clocea and Entercoccus faecalis (MIC 31.2 μg/ml) (Akilandeswari and Ruckmani 2016). No reports B) In hexane extracts of T. brownii and T. laxiflora stem bark Stigmasterol NT NT MIC 500 μg/ml Friedelin NT NT MIC 250 μg/ml β-sitosterol NT NT MIC 500 μg/ml Triacontanol NT NT MIC 250 μg/ml was active against Aspergillus niger (MIC 100 μg/ml) (Opiyo et al. 2011) Was active against Candidda albicans and C. parapsilosis (MIC 200 μg/ml) (Liu et al. 2009) was active against Aspergillus niger (MIC 100 μg/ml) (Opiyo et al. 2011); Mycobacterium tuberculosis, with a MIC of 128 μg/ml (Saludes et al. 2002) NO reprot Was active against Pseudomonas lachrymas (MIC 109.9 μg/ml) (Liang et al. 2015). * Results from our antimicrobial screenings; ** results from our ethnopharmacological surveys; *** result from traditional uses according to other authors; studies I, Salih et al. 2017a; II, Salih et al. 2017b; III, Salih et al. 2018. Tl, Terminalia laxiflora; Tb, T. brownii; Ch, Combretum hartmannianum; Al, Anogeissus leiocarpus. NT, not tested; No report, not reported yet in the literature; R, root; CHCl3, chloroform; Acet, acetone; EtOAc, ethyl acetate; MeSox, methanolic soxhlet; aqu, aqueous; IZ, inhibition zone diameter in mm. Sitostenone NT NT MIC 250 μg/ml 176 Table 5. Phytochemical constituents found in extracts of Combretum hartmannianum, Terminalia laxiflora, T. brownii and Anogeissus leiocarpus in this study and according to other authors. Plant species Major identified phytochemical constituents, resulting from this study* Flavonoids and Tannins and phenolic acids* stilbenes* Phytochemical constituents according to data from the literature Terpenes, fatty acids, fatty alcohols and sterols * Secondary compounds Sugars and amino acids in gum exudates Terminalia brownii Unknown ellagitannins with [M-H]molecular ions at m/z 456.9961,609.1088, 725.4141, 817.4003, 577.1369, 577.1320 and 817.3999, in antibacterial active extract of MeSox of the root part Isomer of methyl-(S)-flavogallonate [MH]- 483.08, methyl-(S)- flavogallonate [M-H]- 483.08 in antibacterial extract of MeSox of the root part Galloyl glucose derivatives, pentagalloylglucose, Gallagic acid dilactone (Terminalin) [MH]- 601, gallic acid [M-H]- 169.01, ellagic acid [M-H]- 300.99 and its derivatives (Salih et al. 2017a, Salih et al. 2018) in antibacterial, antifungal and antimycobacterial extracts of MeSox root and EtOAc stem wood parts Oleanane type triterpenoids, tetracosanoic acid 24:0-TMS, Naringenin-4′-methoxy-7pyranoside [M-H]- 433, quercetin-7-β-Odiglucoside [M-H]- 625, 5,6-dihydroxy-3′,4′,7trimethoxy-flavone [MH]- 343 (Salih et al. 2017b) Cis-resveratrol-3-O-βgalloyl-glucoside, transresveratrol -3-O-β-galloyl-glucoside [M-H]- 451 (Salih et al. 2017b) Sitosterol, stigmasterol, betulinic acid, monogynol A, arjungenin (Opiyo et al. 2011), and the chromone derivative, terminalianone (Negishi et al. 2011) 1,18-octadec-9-ene-dioate-TMS, hexacosanoic acid 26:0-TMS, octacosanoic acid 28:0-TMS, triacontanol (IOH-30:0), 30:0-TMS Octacosanol (IOH-28:0), 28:0TMS, β- sitosterol-TMS, βamyrine-TMS, stigmast-4-en-3one (Sitostenone), Dotriacontanol (IOH-32:0), 32:0TMS, friedelin (friedelanone)TMS, betulinic acid-TMS, 5αstigmastane-3,6-dione (Salih et al. 2018) 3β,24-O-ethylidenyl-2α,19α-dihydroxy-olean -12-en-28-oic acid, arjunic acid, sericic acid, 23-galloylarjunic acid, tomentosic acid, arjungenin, sericoside, arjunglucoside I and 3-O-β-D-glucopyranosyl-β-sitosterol (Machumi et al. 2013) D,β-punicalagin, a,β-terchebulin,ellagic acid-4-O- α-L-rhamnopyranoside (Yamauchi et al. 2016) 4-O-(3'',4''-di-O-galloyl-α-L-rhamnopyranosyl) ellagic acid (Schrader et al. 2016) Terminalia laxiflora 1,18-octadec-9-ene-dioate-TMS Galloyl glucose and its derivatives, gallic acid [M-H]- 169.01, ellagic acid acid [MH]- 300.99 and its derivatives, 177 Terchebulin and flavogallonic acid (Muddathir et al. 2013) Terminolic acid, trimethylellagic acid. tetramethylellagic, laxiflorin and their derivatives, sitosterol and palmitic acid (Ekong et al. 1967) Protocatechuic acid in antibacterial extract of EtOAc of the root part Octacosanoic acid 28:0-TMS, Tertracosanoic acid 24:0-TMS Hexacosanoic acid 26:0-TMS, Triacontanol (IOH-30:0), 30:0-TMS Friedelin (friedelanone)-TMS, Octacosanol (IOH-28:0), 28:0-TMS, β-sitosterol-TMS, β-amyrine-TMS, Stigmast-4-en-3-one (Sitostenone), Dotriacontanol (IOH-32:0), 32:0-TMS, Betulinic acid-TMS, 5α-stigmastane-3,6-dione (Salih et al. 2018) - Punicalagin [M-H] 1083.15, corilagin [M-H]- 633.07 and it is isomer sanguiin Unknown ellagitannins with [M-H]- ions at m/z 725,41, 817.4026 and 817.4033 (Salih et al. 2017a and b) in antibacterial and extract of EtOAc of the root Anogeissus leiocarpus Flavogallonic acid with [M-H]- 453.1062, [M-H]-, and the unknown ETs 833.4009 and [M-H]725.4177 Gallic acid, protocatechuic acid [M-H]- 153.01, galloyl glucose derivative, pentagalloyl glucose [M-H]- 639.67, Ellagic acid and its derivatives, epigallocatechin gallate [M-H]- 457.07 (Salih et al. 2017a) Taxifolin [M-H]- 303.03, isomer of methyl taxifolin [M-H]- 317.06, dihydrokaempferol [M-H]288.00, methyl- taxifolin [M-H]- 317.06, ampelopsin, [M-H]- 319.14, pinosylivin [M-H]- 211.13, (Salih et al. 2017a) Glucuronomannan of liocarpan A; water-soluble (Aspinall et al. 1969) NT Punicalagin (Watermann et al. 2015), trachelosperoside E1, trachelosperogenin E and arjungenin (Chaabi et al. 2008), castalagin and flavogallonic (Shuaibu et al. 2008) NT NT 178 Aspartic acid, arginine, alanine, cysteine, glycine, glutamic acid, histidine, hydrocyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine, valine (Anderson et al. 1987) n-hexadecanoic acid, n-octadecanoic acid, methyl hexadecanoate, methyl-7E-7octadecenoate, eicosane, methyl linoleate (Moronkola and Kunle 2014) gentisic acid (Ndjonka et al. 2014), sericoside, trachelosperogenin E, gallocatechin and (-)-epigallocatechin, 3,3'-di-O-methylellagic acid 4'O-xylopyranoside, and 3,4,3'-triO-methylflavellagic acid 4'-O-glucopyranoside (Hubert et al 2014 ), sericic acid, catechin (Hamzaoui et al. 2013) Appendix No. 1. Questionnaire form for ethnobotanical and ethnopharmacological interviews by Enass Y.A Salih This form is meant mainly for the traditional healers. However other villagers are also included, but are not requested to fill in and answer all questions. The last part of the questionnaire was meant mainly for the official responsible institute Forest National Cooperation, Sudan (FNC) to fill in. Faculty of Forestry, Department of Forest Products and Industries, University of Khartoum, Sudan P.O. Box: 13314, Khartoum-Sudan 1. Personal information of interviewed person Name:……………………………………… Residence in: Kordofan State Blue Nile State Kadogli, Al-ubied, Rashad Adamazin, Baw, Aroserous Tribe: ……………………………….. Ethnic group:…………………………. Gender: Female Male Age:……………… Education level: ………………………… Traditional knowledge of the medicinal plants: A. Inherited from generation to generation B. The owners of the traditional knowledge are only traditional healers and practitioners C. Elder women and men D. Children 2. I prefer traditional medicine 3. Wild medicinal plants mentioned during the questionnaire: modern medicine (Tablets, injection), why?.......................................... A. Sudanese Arabic name and local name if any…………………………………… B. Scientific name and Family……………………………………………………. C. At which growth stage do you collect the medicinal plants (new seedling, young, old plants)………………… D. At what time (season/day?) do you collect the medicinal plants ……………………… E. Which part of the medicinal plants do you use?……………………………for which purpose “diseases” and their symptoms (fever, cough, respiratory infections, TB, diarrhea, dysentery, stomach pain, skin rash, chest and body pains, sore throat, wound infections…………. ………..……………and how much (the dosage of the preparation)………………………………………………………………………………? F. which part of the medicinal plant are used against protozoan diseases “neglected diseases” as malaria, bilharzia (Schistosomiasis), Mycetoma, sleeping sickness (Ttrypanosomiasis) and leishmaniasis. How do you prepare the remedy?…………………………………… F. During the period of illness, how often do you use the traditional recipe (Frequency of use per a day/week or month)?............................................ G. In the case of oral application (through mouth): how does the preparation taste like? 179 Greasy Astringent Acid Salty Bitter Sweet H. In the case of external application (as ointment on the top of the skin): is there any side effects, such as changes in the skin color, irritation and inflammations? If yes explain it?.............................................................................................................. I. Does the patients (believe) like/tolerate the remedies? Yes J. Does the medicinal plant have beneficial effects as therapeutic agent and relieve the symptoms of the disease? Yes NO NO If yes, what kind of effects?................................................................... K. Have you noticed any negative effect? Yes NO If yes, describe more specifically?.................................................. L. Does this medicinal plant have an impact to increase or decrease your income? Yes NO If yes How?………………………. M. What kind of other local traditional applications does this plant species have? ……………………………………………………………………………………… N. What kind of other environmental impacts do these wild medicinal plants have? A. For domestic animals ………………………………………………………… B. For agriculture and crop cultivation………………………………………… C. For soil…………………………………………………………………………… D. For drinking water………………………………………………………………. E. For birds migration……………………………………………………………… F. For wild animals…………………………………………………………………. G. For displacement people and refugees since both of the studied area border with South Sudan and Darfur state (conflict region)…………………………………….… The below questions are meant for the official responsible Institute (FNC): 1. What is the status of the Forest? A. Natural reserve forest B. Planted reserve forest D. Private forest E. Shared forest (between local people in the study area and government (FNC) C. Community forest 2. Are there any type of agroforestry activities inside the forested area (Study areas) or near the forest? 3. A. Yes B. No 4. If yes, what type of agroforestry system? …………………………………………………… 5. How does the study area and the forest (with the focus of the studied medicinal plants) generate income to the institute (FNC) and local people? and how does this contribute to the national income of the Sudan? A. Timber B. International Carbon market D. Landscape (public gardens) C. Non- wood forest products F. International ecological market (for migrant birds, for grazing animals, for sedentary, for nomads, for wild animals and for ecological tourism). 180 6. Describe the regeneration of the studied plants? A. Natural regeneration (stump sprout, root sprout, stump coppice, root coppice or new seeds) B. Artificial regeneration (man-made, reforestation or afforestation; through seedlings or new seeds)............................................................................................... A. What are the main constraints found in the study area, especially for the forestry and other scientific researchers?……………………………………………… Author’s current address: Enass Yousif Abdelkarim Salih Viikki Tropical Resources Institute (VITRI), University of Helsinki & Faculty of Pharmacy, Division of Pharmaceutical Biosciences University of Helsinki, Finland Email: enass.salih@helsinki.fi Author’s permanent address: Enass Yousif Abdelkarim Salih Department of Forest Products and Industries Faculty of Forestry, University of Khartoum, PO BOX 13314, Shambat, Khartoum-North, Sudan Emails: enass7@yahoo.com eyabdelkareem@uofk.edu 181 Appendix 2 Compound 11, Arjunglucoside I (Study II) - hexose sugar, loss of 162 Da Figure 50. LC-MS/MS fragmentation pattern of compound 11 (in Figure 44), arjunglucoside I (syn. dotorioside I), present in an ethyl acetate extract of the stem bark of Terminalia brownii. Study II, Salih et al. 2017b, modified from Salih 2009. 186 Compound 9, ellagitannin corilagin (Study II) [M- galloyl (152)] acid - H] -18 H2O [ellagic [gallic acid] 275 [M-gallic acid (170)] Figure 51. LC-MS/MS fragmentation pattern of compound 9 (in Figure 44), corilagin present in ethyl acetate extract of the stem bark of Terminalia brownii. Study II, Salih et al. 2017b, modified from Salih 2009. 187 gallic acid part [601-300] Compound 6, terminalin (Study II) Figure 52. LC-MS/MS fragmentation pattern of compound 6 (in Figure 44), gallagic acid dilactone (terminalin), present in ethyl acetate extract of the stem bark of Terminalia brownii. Study II, Salih et al. 2017b, modified from Salih (2009). 188 UNIVERSITY OF HELSINKI Viikki Tropical Resources Institute VITRI TROPICAL FORESTRY REPORTS No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 No. 11 No. 12 No. 13 No. 14 Johansson, S. (ed.) 1989. Tutkimus ja kehitysmaiden metsät. 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Tropical dryland agroforestry on clay soils: Analysis of systems based on Acacia senegal in the Blue Nile region, Sudan. Doctoral thesis (limited distribution). Luukkanen, O., Katila, P., Elsiddig, E., Glover, E. K., Sharawi, H. and Elfadl, M. 2006. Partnership between public and private actors in forest-sector development: Options for dryland Africa based on experiences from Sudan, with case studies on Laos, Nepal, Vietnam, Kenya, Mozambique and Tanzania. Laxén, J. 2007. Is prosopis a curse or a blessing? – An ecological-economic analysis of an invasive alien tree species in Sudan. Doctoral thesis. Katila, P. 2008. Devolution of forest-related rights: Comparative analyses of six developing countries. Doctoral thesis. Reyes, T. 2008. Agroforestry systems for sustainable livelihoods and improved land management in the East Usambara Mountains, Tanzania. Doctoral thesis. Zhou, P. 2008. Landscape-scale soil erosion modelling and ecological restoration for a mountainous watershed in Sichuan, China. Doctoral thesis. Hares, M. & Luukkanen, O. 2008. Research Collaboration on Responsible Natural Resource Management, The 1st UniPID Workshop. UNIVERSITY OF HELSINKI Viikki Tropical Resources Institute VITRI TROPICAL FORESTRY REPORTS No. 37 No. 38 No. 39 No. 40 No. 41 No. 42 No. 43 No. 44 No. 45 No. 46 No. 47 No. 48 No. 49 No. 50 Husgafvel, R. 2010. Global and EU governance for sustainable forest management with special reference to capacity building in Ethiopia and Southern Sudan. Doctoral thesis. Walter, K. 2011. Prosopis, an alien among the sacred trees of South India. Doctoral thesis. Kalame, F.B. 2011. Forest governance and climate change adaptation: Case studies of four African countries. Doctoral thesis. Paavola, M. 2012. The impact of village development funds on community welfare in the Lao People’s Democratic Republic. Doctoral thesis. Omoro, Loice M.A. 2012. Impacts of indigenous and exotic tree species on ecosystem services: Case study on the mountain cloud forests of Taita Hills, Kenya. Doctoral thesis. Alam, S.A. 2013. Carbon stocks, greenhouse gas emissions and water balance of Sudanese savannah woodlands in relation to climate change. Doctoral thesis. Rantala, S. 2013. The winding road from exclusion to ownership: Governance and social outcomes in contemporary forest conservation in northeastern Tanzania. Doctoral thesis. Negash, M. 2013. The indigenous agroforestry systems of the south-eastern Rift Valley escarpment, Ethiopia: Their biodiversity, carbon stocks, and litterfall. Doctoral thesis. Kallio, M.H. 2013. Factors influencing farmers’ tree planting and management activity in four case studies in Indonesia. Doctoral thesis. Etongo, B.D. 2016. Deforestation and forest degradation in southern Burkina Faso: Understanding the drivers of change and options for revegetation. Doctoral thesis. Tekle Tegegne, Y. 2016. FLEGT and REDD+ synergies and impacts in the Congo Basin: lessons for global forest governance. Doctoral thesis. Fahmi, M.K.M. 2017. Climate, trees and agricultural practices: Implications for food security in the semi-arid zone of Sudan. Doctoral thesis (limited distribution). Abaker, W. 2018. Linkages between carbon sequestration, soil fertility and hydrology in dryland Acacia senegal plantations in Sudan: a chronosequence study. Doctoral thesis (limited distribution). Salih, E. 2019. Ethnobotany, phytochemistry and antimicrobial activity of Combretum, Terminalia and Anogeissus species (Combretaceae) growing naturally Sudan (limited distribution). ISBN 978-951-51-5420-0 (paperback) ISBN 978-951-51-5421-7 (PDF) ISSN 0786-8170 Helsinki 2019 Unigrafia