ANTIMICROBIAL EXTRACTS OF SOME EAST-AFRICAN TERMINALIA AND COMBRETUM SPECIES ARE RICH SOURCES OF

Pia Fyhrquist a,*, Enass Salih a, Into Laakso a, Heikki Vuorela a, and Riitta Julkunen-Tiitto b

a Division of Pharmaceutical Biosciences, Faculty of Pharmacy, P.O. Box 56, FIN-00014, University of Helsinki, Finland b Natural Product Research Laboratory, Department of Environmental and Biological Sciences, University of Eastern Finland, 80101 Joensuu, Finland

MAIN CONCLUSION Polar and medium-polar extracts of roots and stem bark of Terminalia kaiserana, T. sambesiaca, Combretum padoides, C. psidioides and C. zeyheri showed promising broad- spectrum antibacterial and antifungal effects. Ellagitannins were the main components in the active extracts and the good antimicrobial activities are suggested mostly to be due to ellagitannins. INTRODUCTION African Terminalia and Combretum species (Combretaceae) are a rich source of ellagitannins and many of the health beneficial effects of decoctions of these medicinal plants, among them antibacterial and antifungal effects, can be assigned to this polyphenolic group [1,2]. Ellagitannins are hexahydroxydiphenoyl esters of carbohydrates or cyclitols and in the plant family Combretaceae more specific kinds can be found, such as C-glycosidic and flavanol- based ellagitannins [3]. Ellagitannins possess promising antibacterial and antifungal effects and have been shown to be active even against antibiotic resistant bacteria such as methicillin resistant S. aureus [4]. New ellagitannins with promising biological activities could be found from the genera Combretum and Terminalia. Terminalia kaiserana, T. sambesiaca, Combretum psidioides, C. zeyheri and C. padoides, demonstrating promising antifungal and antimicrobial activities, were chosen for in depth research on their composition since studies on ellagitannins in these species are scanty. In order to validate that the antimicrobial activity of the traditional medicinal decoctions could be due to ellagitannins, we compared the polyphenolic composition of the decoctions to that of methanolic crude extracts. In ethnopharmacological approaches for finding new active principles in plants there is a danger that the traditional way of preparing extracts is omitted as the extracts to be tested usually are made due to conventional laboratory extraction techniques [5]. The compounds in these conventionally prepared extracts do not necessarily reflect the composition of compounds in traditional preparations. MATERIALS AND METHODS We identified ellagitannins and other in Terminalia and Combretum spp. based on their retention times, UV absorption maxima and masses, using high performance liquid chromatography (HPLC-UV/DAD) and ultrahigh pressure liquid chromatography based on time-of-flight mass spectrometry (UHPLC-qTOF-MS). The antibacterial and antifungal profiles of the extracts were investigated using microplate and agar diffusion methods. Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Micrococcus

* [email protected] luteus, Escherichia coli and Mycobacterium smegmatis were used as model bacteria. Candida albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis were used as model yeast species. RESULTS AND DISCUSSION Our results indicate that especially polar extracts of the roots and stem bark of the investigated Terminalia and Combretum spp., enriched in ellagitannins, possess promising broad-spectrum antibacterial and antifungal effects. The most active extracts were subjected to analysis with special emphasis on their ellagitannins. Alltogether thirteen ellagitannins, among them , , terchebulin, terflavin A, and methyl-S- flavogallonate were found for the first time in a methanolic root extract of Terminalia kaiserana. Interestingly, the decoctions of T. kaiserana roots were as rich in ellagitannins as the methanolic extracts, and a similar qualitative composition of ellagitannins could be observed. Our results justify the uses of T. kaiserana decoctions for bacterial infections and cough [6], but this species should be used with caution since it has demonstrated cytotoxicity in a brine-shrimp test [7]. A root extract of T. sambesiaca contained eight ellagitannins, among them terchebulin and . Ellagitannins have not been studied before in T. sambesiaca, but one study reports that an antifungal leaf extract of this species contains [8], although the molecular structures were not elucidated. Twenty-three ellagitannins were found in a butanol extract of the stem bark of C. padoides, with punicalagin as the main compound and an ellagitannin giving a (M-H)- molecule ion at m/z 633.0750, similar to corilagin, but with a shorter retention time, tentatively indicating the presence of a corilagin isomer in this species. The stem bark of C. psidioides contained twelve ellagitannins, among them punicalagin and corilagin. Punicalagin and three unknown ellagitannins showing (M-H)- molecular ions from m/z 613.0849 to m/z 817.4212, as well as eight other unknown ellagitannins were detected in a root extract of Combretum zeyheri. Our results show that the investigated species of Terminalia and Combretum contain a high diversity of unknown ellagitannins. The structures of these ellagitannins should be further confirmed using NMR in combination with mass spectrometry. REFERENCES [1] O. Silva and R. Serrano, FORMATEX, 2015, 236–245. [2] O. Silva et al, Fitoterapia, 2012, 83 (5), 872–876. [3] T. Okuda, Yakugaku Zasshi, 1995; 115, 81–100. [4] L. Lipińska et al. Acta Sci. Pol. Technol. Aliment., 2014, 13 (3), 289–299. [5] F. Pellati et al, J. Ethnopharmacol., 2013, 147, 277–285. [6] F. Haerdi, Acta Tropica. Supplementum, 1964, 8, 1–278. [7] M.J. Moshi et al, African Ethnomedicines network, 2006, 3 (3), 48–58. [8] P. Masoko and J.N. Eloff, Afr. J. Biotechn., 2005, 4 (12), 1425–1431. ACKNOWLEDGEMENT This study has been supported by Magnus Ehrnrooth foundation, Finska Läkaresällskapet, Societas pro Fauna et Flora Fennica, Oskar Öflund foundation, Finnish Cultural Foundation and Swedish Cultural Foundation in Finland. The first author is grateful for this support.