Antimycobacterial Activity of Ellagitannin and Ellagic Acid

South African Journal of Botany 90 (2014) 1–16

Contents lists available at ScienceDirect

South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Antimycobacterial activity of ellagitannin and ellagic acid derivate rich crude extracts and fractions of ﬁve selected species of Terminalia used for treatment of infectious diseases in African traditional medicine

P. Fyhrquist a,⁎, I. Laakso a, S. Garcia Marco b, R. Julkunen-Tiitto c, R. Hiltunen a a Faculty of Pharmacy, Division of Pharmaceutical Biology, Viikki Biocenter, P.O. Box 56, FIN-00014, University of Helsinki, Finland b Faculty of Pharmacy, Complutense University of Madrid, P.O. Box 28040, Madrid, Spain c Division of Biology, Natural Products Research Laboratory, P.O. Box 111, FIN-80101, University of Eastern Finland, Joensuu, Finland article info abstract

Article history: Ten crude extracts and their solvent partition fractions from ﬁve species of Terminalia collected in Tanzania were Received 16 May 2013 assessed for antimycobacterial effects using Mycobacterium smegmatis ATCC 14468 as a model organism. We re- Received in revised form 2 August 2013 port here, for the ﬁrst time, on antimycobacterial effects of root and stem bark extracts of Terminalia sambesiaca Accepted 21 August 2013 and Terminalia kaiserana as well as of fruit extracts of Terminalia stenostachya and leaf extracts of Terminalia Available online 9 November 2013 spinosa. T. sambesiaca gave the best effects of all the investigated species in terms of the sizes of the inhibitory Edited by V Steenkamp zones of root and stem bark extracts. A crude methanol root extract of T. sambesiaca gave lower MIC values (1250 μg/ml) than its aqueous and butanol soluble fractions (MIC 2500 μg/ml). Our preliminary HPLC–DAD Keywords: data indicates that methanol and aqueous extracts of T. sambesiaca roots are rich in ellagitannins and ellagic Antimycobacterial activity acid glycosides. Particularly, one polar ellagitannin at tR 10.3–10.9 min dominates the extracts quantitatively Terminalia and thus may be responsible for their good antimycobacterial effects. In contrast to the more polar fractions, a Tuberculosis chloroform soluble fraction of the roots of T. sambesiaca was devoid of antimycobacterial activity. Also crude Mycobacterium smegmatis methanol and aqueous extracts of the stem bark of T. sambesiaca gave promising antimycobacterial effects (MIC M. tuberculosis 1250 μg/ml). All fractions of T. kaiserana roots, except from the aqueous insoluble gave good antimycobacterial Ellagitannins effects (MIC 1250 μg/ml) and the aqueous extract showed the best effects of the fractions in terms of the size of in- Ellagic acid glycosides Gallotannins hibition zones. These results justify the uses of hot water decoctions of the roots of T. kaiserana for treatment of – Condensed tannins cough, one of the symptoms of TB. According to HPLC DAD data methanol extracts of T. kaiserana roots and their Africa aqueous fractions are rich in polar ellagitannins and ellagic acid glycosides. Quantitatively, the ellagitannins Tanzania dominate these extracts and therefore the good antimycobacterial activity of the methanol and aqueous extracts is assumed to be due to these compounds. Sephadex LH-20 CC fractions of a methanol extract of the roots of T. kaiserana inhibited the growth of M. smegmatis, giving MIC values of 1000μg/ml. Ellagic acid glycosides in these fractions must be responsible for their good antimycobacterial effects since they are present in high concentrations. Good antimycobacterial effects were also obtained with a root extract of Terminalia sericea, and especially the butanol soluble fraction was a good inhibitor of the growth of M. smegmatis (MIC 1562 μg/ml). Our preliminary HPLC–DAD results show that the roots of T. sericea are rich in ellagitannins, ellagic acid glycosides and at least one stilbene compound. Extracts of the fruits of T. stenostachya gave good antimycobacterial effects, butanol extracts being the most active. Also the leaves of T. stenostachya, and especially the butanol soluble extracts, give good antimycobacterial effects. Our HPLC–DAD data indicate that T. stenostachya leaves contain large quantities of gallic acid, ellagitannins and ellagic acid glycosides. Our results indicate that many of the investigated species of Terminalia might contain leads for development of anti-TB drugs. Standardized extracts of T. sambesiaca, T. kaiserana and T. sericea roots could be used as easily available and cheap medicines for treatment of TB in remote regions of East and South Africa. © 2013 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction

Tuberculosis (TB) is historically one of the oldest and most wide- spread diseases in history (McKinney, 2000). In recent years TB has ⁎ Corresponding author at: Faculty of Pharmacy, Division of Pharmaceutical Biology, Viikki reemerged as a major disease of global importance (Pereira et al., Biocenter, University of Helsinki, P.O. Box 56, Viikinkaari 5 E, 00014, Finland. Tel.: +358 9 191 59628. 2005). It is estimated that roughly one third of the world's population, E-mail address: pia.fyhrquist@helsinki.ﬁ (P. Fyhrquist). around 1.6–2 billion people are infected with TB and of these around

0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.08.018 2 P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16

15 million have active disease at any given time (Esimone et al., 2009). were isolated from the stem bark, roots and leaves of the West African Each year there are approximately three million deaths due to TB and species Terminalia macroptera. thus Mycobacterium tuberculosis is responsible for more human mortal- Just a small number of the thirty African species of Terminalia have ities than any other single microbial species (Corbett et al., 2003; Rogoza been investigated for growth inhibitory effects against mycobacteria. et al., 2011). In 2009 most of the cases of tuberculosis were assigned to In most cases crude extracts have been used for the screenings, and South-East Asia, Africa and Western Pacific regions, and 30% of the new just in a few cases some active principles have been isolated and charac- cases were found in Africa (World Health Organization, 2010). In Africa terized. Methylated ellagic acid derivates from the stem bark of the as well as in Eastern Europe, TB deaths are increasing after almost 40 West-African species Terminalia superba werefoundtogivegoodgrowth years of decline. The increase in new cases of TB has been considered inhibitory effects against M. tuberculosis and Mycobacterium smegmatis partly as a result of the AIDS pandemic making an increasing number (Kuete et al., 2010). Acetone extracts of the stem bark of T. sericea have of individuals susceptible to the infection, but also due to the emergence been found to effectively inhibit the growth of M. tuberculosis H37 Ra as of strains resistant to available antibiotics (Ballell et al., 2005). Further- well as the growth of a clinical strain of M. tuberculosis, but the active more, TB is a disease which affects especially women and young people compounds were not investigated (Green et al., 2010). The bark and in developing countries, partly due to poverty and therefore poor access root extracts of Terminalia glaucescens werefoundtogivesomeactivity to medicines and treatment, as well as to late presentation of the illness against Mycobacterium ulcerans (Yemoa et al., 2011). Extracts of the leading to poor prognosis (Connolly and Nunn, 1996; Mann et al., 2008). leaves, stem bark and roots of Terminalia stenostachya and the roots During the last couple of decades the HIV-related TB cases have in- and stem bark of T. glaucescens have been found to give promising creased in Africa and especially in the sub-Saharan regions (Asres growth inhibitory activity against Mycobacterium madagascariense et al., 2001) accounting for 80% of all HIV-related TB cases globally and Mycobacterium indicus (Mbawambo et al., 2011) but the active (World Health Organization, 2010). According to an estimate made by compounds in these species are still unknown. Crude extracts of the the World Health Organization the average incidence of tuberculosis leaves of T. glaucescens have shown growth inhibitory effects against in African countries more than doubled between 1990 and 2005 M. tuberculosis (H37Rv) giving MIC values of 1300 μg/ml (Nvau et al., (Chaisson and Martinson, 2008). In 2005, the WHO declared a TB emer- 2011). Hexane extracts of Terminalia avicennoides were found to give gency situation in Africa. For example in Ethiopia TB is labeled as one of very promising effects against M. tuberculosis and Mycobacterium bovis the major causes of death with 15,700 new cases of infections appearing showing MIC values of 78 μg/ml (Mann et al., 2008). Later, the in 1997 (World Health Organization, 1999). In Tanzania the number of pentacyclic triterpenoid, friedelin was isolated from a hexane fraction TB cases has steadily increased between 1983 and 2000, mainly of T. avicennoides roots and showed promising antimycobacterial effects due to HIV/AIDS (Egwaga, 2003). South Africa is among the four against M. bovis (Mann et al., 2011). These investigations demonstrate countries having the largest number of multi-drug resistant TB that the genus Terminalia includes species showing promising (MDR-TB) and extensively drug-resistant TB (XDR-TB) (World Health antimycobacterial potential and that the active compounds in these Organization, 2010). The currently available antituberculosis agents, plants should be investigated. rifampicin and isoniazid, have serious limitations and infections The aim of the present study was to investigate the antimycobacterial caused by resistant strains of M. tuberculosis are estimated to result potential of Terminalia sambesiaca, T. kaiserana, T. spinosa, T. stenostachya in up to 91% mortality among AIDS patients (Asres et al., 2001). and T. sericea. Focus was put on root and stem bark extracts since these Moreover, many of the standard antituberculosis agents cause ad- plant organs are often used as hot water decoctions for treatment of in- verse side effects in the human organism. The need for finding new fectious diseases in African traditional medicine, but in some cases leaf and more efficient low-toxic antituberculosis agents is therefore urgent and fruit extracts were investigated. For example, the dried fruits of T. (Rogoza et al., 2011). However, due to the predominance of TB in the de- sericea are used in a multi-component medication for treatment of TB veloping world there has for long been a lack of economic incentive for in Malawi (Msonthi and Mgombo, 1983), but due to the lack of fruit ma- development of new anti-TB drugs. Recently, there has been a renewed terial of T. sericea we investigated fruit extracts of the closely related T. interest in finding new agents for treatment of TB (Global Alliance for stenostachya for their antimycobacterial potential. The species of TB Drug development, 2001; Pauli et al., 2005; Rogoza et al., 2011). Terminalia were selected on the basis of their traditional uses for treat- Antimycobacterial active compounds have been found among many dif- ment of bacterial infections/TB (Kokwaro, 1976; Msonthi and Mgombo, ferent chemical structures in higher plants (Mitscher and Baker, 1998). 1983; Chhabra et al., 1989; Fyhrquist et al., 2002) and due to criteria of Especially tropical plants are richinusefulsecondarycompoundsand not or only to a limited extent being investigated before for new antitubercular agents which might act on other sites than the con- antimycobacterial activity. The species of Terminalia used in this study ventional antibiotics. TB drugs might be found from medicinal plants were collected in Tanzania in spring 1999 and are typical for East and used in traditional medicine for treatment of tuberculosis and other South Africa. The occurrence of T. kaiserana is restricted to East Africa, bacterial infections. The influence of herbal (traditional) medicine and and this might be the reason for why this species of Terminalia has not natural products upon drug discovery has been profound, with a number been studied before for its antimycobacterial effects. Crude methanol of clinically active drugs derived from natural products or having a extracts giving good antimycobacterial activity were fractionated by natural product pharmacophore (Koehn and Carter, 2005). solvent partition in order to find fractions giving high antimycobacterial In Africa the genus Terminalia (Combretaceae) comprises about thirty activity. Our study also focused on HPLC–UV–DAD profiling of the species (Wickens, 1973) of small, medium or large trees, occurring in antimycobacterially most potent extracts of Terminalia.Thespecies Miombo and dry woodlands as well as rainforests. Studies on the chosen for this study have given good to excellent antibacterial and ethnopharmacological uses as well as on biological activities on extracts antifungal effects in our previous studies (Fyhrquist et al., 2002, of some African Terminalia species indicate that they could have 2004) and therefore we assumed to find species of Terminalia giving promising medical applications for treatment of infectious diseases, good antimycobacterial activity as well. both as standardized crude extracts (phytomedicines) and as sources Due to the slow growth rate and the virulence of M. tuberculosis we of (new) active compounds. Some compounds with antibacterial used the more rapidly growing saprophytic species M. smegmatis ATCC activities have been isolated from African species of Terminalia.Anti- 14468 as a surrogate screen for testing the antimycobacterial effects of bacterial resveratrol-3-β-rutinoside (Joseph et al., 2007), anolignan extracts, solvent partition and column chromatography fractions of B, termilignan B and arjunic acid (Eldeen et al., 2005, 2008)have Terminalia spp. The drug sensitivity of M. smegmatis is very close to been isolated from the roots and stem bark of Terminalia sericea. that of M. tuberculosis and thus screenings against this mycobacterial Ellagitannins (Conrad et al., 2001; Silva et al., 2000, 2002)and species give predictive results for indicating anti-tuberculosis activity pentacyclic triterpenoids (Conrad et al., 1998) with antibacterial effects (Newton et al., 2002). P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16 3

2. Material and methods 2.4.2. TLC method for ellagitannins and gallic/ellagic acid in Terminalia species

2.1. Plant material Aluminium-backed RP-18 F254s plates (Merck, Darmstadt, Germany) were used for investigating more polar compounds, such as ellagitannins The plant species were collected from Mbeya and Iringa districts and ellagic acid glycosides, in extracts of stem bark and roots of in Tanzania in February–March 1999. A voucher specimen is depos- T. sambesiaca, T. kaiserana and T. sericea. Sephadex LH-20 fractions ited in the Botanical Museum (H) of the Finnish Museum of Natural of the roots of T. kaiserana were also investigated by this method. History in Helsinki, Finland and at the Herbarium of the University of 1 μl of extracts and solvent partition fractions (50 mg/ml) or 5 μl Dar-es-Salaam, Tanzania. Details of the species of Terminalia used in Sephadex LH-20 fractions (20 mg/ml) were applied 1 cm from the this investigation and information on their uses in African traditional base of the plates with a microcapillary pipette. 1 μl of ellagic acid, medicine as well as results on their antimycobacterial and antimicrobial gallic acid and (+)-catechin (Merck, 1 mg/ml) were applied as stan- activities and isolated compounds are summarized in Table 1. dards. The plates were developed in distilled water + methanol + orthophosphorous acid, 5 + 5 + 0.1 (v/v) and developed to a distance of 8cm. The plates were documented using a Camaq Video documenta- 2.2. Extraction tion system and operated with Video store software. The plates were photographed at 254 and 366 nm. Some of the plates were sprayed 2.2.1. Soxhlet extraction of the plant material with a solution of vanillin:sulfuric acid, developed in an oven at 20 g shade dried and finely milled plant material (roots, stem bark, + 120 °C for 5 min and observed for colored spots. The Rf values of the leaves and fruits) was extracted with 500ml MeOH in a Soxhlet appara- compounds in the extracts were compared to those of the standards. tus for 4 h. The resulting extract was reduced to dryness under vacuum at +40 °C using a rotary evaporator. The extract was then freeze-dried in a lyophilisator for 1–2 days. The resulting residue was redissolved in 2.4.3. Sephadex LH-20 column chromatography MeOH (50 mg/ml) for antimycobacterial testing. AMeOHrootextractofT. kaiserana was fractionated using Sephadex LH-20 column chromatography. Sephadex LH-20 (Pharmacia, Uppsala) was swollen in MeOH overnight before use. A glass column (40 cm in 2.2.2. Solvent fractionation length, 1.8 cm in diameter) was filled with a slurry of Sephadex LH-20. 1500 mg lyophilized crude Soxhlet MeOH extracts of different plant 650 mg of the dry methanol extract of the roots of T. kaiserana was ap- organs of the Terminalia species were dissolved in 50ml water for 30min, plied to the column. Gradient elution was performed starting with 100% half of which in an ultrasonic bath. The extracts were centrifuged and the MeOH and continuing with MeOH:EtOH combinations increasing the fi aqueous part was used for further fractionation; rstwith3×25mlCHCl3: percentage of EtOH to 96% in the end of the fractionation. Nine fractions EtOH (2:1) and then with 3 × 25 ml n-BuOH, resulting in CHCl3,n-BuOH, were collected (20 ml of each) and pooled to four fractions following aqueous and aqueous-insoluble fractions. The fractions were evaporated analytical RP-18 thin layer chromatography: F1 (152 mg), F2 (111 mg), to dryness and re-dissolved in MeOH to stock solutions of 50 mg/ml for F3 (25 mg), and F4 (63 mg). antimycobacterial testing.

2.4.4. HPLC–UV/DAD method I 2.3. Acid-butanol assay for determining proanthocyanidins in root extracts HPLC analyses using the method of Dastmalchi et al. (2007) were of T. kaiserana and T. sambesiaca performed for qualitative analysis of extracts of Terminalia species. The liquid chromatographic system (Waters 600 E) consisted of a pump and Acid hydrolysis for indication of condensed tannins (proxantho- a controller coupled to a 991 PDA detector and a 717 plus autosampler cyanidins) to yield red anthocyanidines was performed according to under control of Millennium 2000 software (Waters Corp., Milford, Hagerman (2002). 20 mg of dried methanolic extracts of the roots USA). Samples of 20 μl(20mg/mlinMeOH)wereinjected.Separations of T. kaiserana and T. sambesiaca were dissolved in 1 ml MeOH. The ex- were performed on a reversed phase Hypersil BDS-C-18 analytical tracts were diluted 1:9 so that the final concentration was 2mg/ml. 1ml column (250 mm × 4.6 mm ID 5 μm). Elution was performed as gradient of these extracts were hydrolyzed using 6ml acid butanol reagent (5% v/v elution using A) 0.1% formic acid in water and B) 100% acetonitrile. The conc. HCl in n-butanol) and 250 μl iron reagent (2% w/v ferric am- gradient started with 85% A and ended with 100% B and the length of monium sulfate in 2 M HCl). Hydrolysis was performed in boiling therunswere50–60min. The flow rate was 1ml/min. UV chromatograms water bath for 45 min. Tubes were cooled and absorbance was measured were constructed at 254, 280 and 340 nm. UVλ absorption maxima spec- at 550 nm. tra of the compounds of interest were recorded between 200 and 400nm using Millennium 2000 software and compared to reference com- 2.4. Chromatography and mass spectrometry pounds such as ellagic acid, gallic acid and epigallocatechin gallate (Sigma-Aldrich). 2.4.1. TLC method for catechins and oligomeric proanthocyanidins in T. sericea roots 2.4.5. HPLC–UV/DAD method II A TLC method according to Vovk et al. (2002) was used. Glass-backed Qualitative HPLC analyses were performed using a second HPLC- cellulose plates (Merck, Art. 5716) pre-developed with water were used. method according to Julkunen-Tiitto et al. (1996), developed especially 10μl of the aqueous, water-insoluble and BuOH fractions of T. sericea roots for detection of polyphenols. HPLC-system consisted of a Waters 600 E (10 mg/ml) were applied as a thin line at the base of the plate using pump and a controller coupled to a 991 PDA detector and an autosampler Linomat.Theextractswereapplied2×sothatthefinal volume was 20 under control of Agilent Chemstation software (Waters Corp., Milford, μl. 10 μl of gallocatechins, catechins and epicatechins (Sigma-Aldrich, 1 USA). Samples of 10μl (2mg/ml in 50% MeOH) were injected. Separations mg/ml) were used as standards. The plates were dried with a hair dryer were performed on a reversed phase Hypersil Rp C-18 analytical column and were put in a horizontal TLC chamber. The plates were developed (length: 10 mm; ID: 2 mm). Gradient elution was performed by using in 1-butanol+water+acetic acid, 4+2+1 (v/v). Development distance solvent systems as follows: A) Aqueous 1.5% tetrahydrofuran + 0.25% was 5cm. The plate was then dipped in vanillin: H3PO4 solution and dried orthophosphoric acid and B) 100% MeOH. The flow rate was 2 ml/min. carefully. The compounds of interest became visible as red spots. The Rf UV chromatograms were constructed at 220, 270, 280, 320 and 360 nm. values of compounds in the fractions and extracts of T. sericea were UVλ absorption maxima spectra of the compounds of interest were compared to those of the standard compounds. recorded between 200 and 400 nm using Agilent Chemstation software. 4 .Fhqite l ot fia ora fBtn 0(04 1 (2014) 90 Botany of Journal African South / al. et Fyhrquist P.

Table 1 Terminalia species collected in Tanzania and included in this study. Summary of traditional uses as well as antimicrobial/antimycobacterial effects and identiﬁed compounds of the species. Results obtained by our research group marked with an asterisk *.

Terminalia species (collector voucher specimen) Uses of plant species in TM Way of preparation in TM Antibacterial, Identiﬁed compounds antifungal and antimycobacterial effects

T. sericea Burch ex. DC. Tuberculosis: In Malawi the Dried fruit for tuberculosis, Leaf extracts antibacterial (Eloff, 1999; Triterpenes sericoside and arjunglucoside in roots and (P.Fyhrquist&L.Mwasumbi, dried fruit is used in a but the way of preparation Fyhrquist et al., 2002*). stem bark (Bombardelli et al., 1974, 1974, 1986; voucher 1697045) multi-component recipe not published (Msonthi and Very promising antibacterial and Maeda and Fukuda, 1996). (Msonthi and Mgombo, 1983; Mgombo, 1983). antifungal proﬁle of especially aqueous Resveratrol-3-O-β-D-rutinoside in roots Moshi and Mbwambo, 2005). Hot water decoctions of roots andMeOHextractsofrootsandstem and stem bark (Bombardelli et al., 1975). Diarrhea, colic, pneumonia, for diarrhea and colic as well bark (Fyhrquist, 2002, 2004*; Innocent, Antibacterial resveratrol-3-O-β-D-rutinoside, arjungenin, gonorrhea, cough,skin as for venereal diseases 2002; Steenkamp et al., 2004; Moshi and stigmasterol and β-sitosterol in root diseases (Drummond and (Neuwinger, 1996, 2000). Mbwambo, 2005; Tshikalange et al., 2005; bark (Joseph et al., 2007). Coates-Palgrave, 1973; Outer layer of roots boiled in Eldeen et al., 2005). Antibacterial anolignan B in ethylacetate extracts Hedberg et al., 1982). hot water and decoction Resveratrol-3-O-β- of roots (Eldeen et al., 2005). In Tanzania: Diarrhea, fever, drunk for pneumonia rutinoside exhibited a weak antibacterial Antibacterial Termilignan B and arjunic acid in roots –

hypertension, bacterial infections (Drummond and activity against E. coli (Joseph et al., 2007). (Eldeen et al., 2008). 16 (Fyhrquist et al., 2002).* Coates-Palgrave, 1973). Ethanol, ethyl acetate and dichloromethane Antimicrobial compounds in leaves unknown. In Tanzania roots, stem root and bark extracts antimycobacterial Ellagic acid derivates, ellagitannins, gallic acid, bark and leaves made giving MICs of 1.56–3.12 mg/ml catechins and a stilbene in BuOH extracts of roots.* into hot water decoctions (Eldeen and Van Staaden, 2007). or mixed in maize porridge Acetone extracts of stem bark to treat diarrhea, fever antimycobacterial (MIC 25 μg/ml) and hypertension (Green et al., 2010). (Fyhrquist et al., 2002). * The crude MeOH extract of the roots and especially the BuOH fraction of this extract inhibit effectively the growth of M. smegmatis (Activity index compared to rifampicin is 0.68; MIC 1.56 mg/ml). * T. sambesiaca Engl. & Diels. Bloody diarrhea, appendicitis, Hot water decoctions Promising antibacterial (Chhabra et al., 1989; May contain saponin-like compounds in the leaves (P.Fyhrquist&L.Mwasumbi, stomach ulcers, cancer of stem bark and leaves Fyhrquist et al., 2002*) and antifungal proﬁle (Masoko et al., 2005; Masoko and Eloff, 2005). voucher 1697061) (Chhabra et al., 1989). to treat cancer, stomach (Fyhrquist et al., 2004*; Masoko et al., 2005; Ellagic acid glycosides, ulcers and appendicitis, Shai et al., 2008)ofMeOHextractsoftheroots ellagitannins and gallic acid in methanol powdered root bark and stem bark as well as acetone, extracts of roots and stem bark. * for bloody diarrhea hexane and dichloromethane extracts of (Chhabra et al., 1989). the leaves. Terminalia species (collector voucher specimen)

Uses of plant species in TM Way of preparation in TM Antibacterial, antifungal and antimycobacterial effects Identiﬁed compounds

Gave the most promising antimycobacterial results of the ﬁve species of Terminalia we used for our investigation. Root and stem bark crude (MIC 1.25 mg/ml) and aqueous extracts (MIC 2.5 and 1.25 mg/ml) gave activity indexes of 0.70–0.71 compared to rifampicin *. T. kaiserana F. Hoffm. Gonorrhea, bilharzia, Hot water decoctions Promising antibacterial and antifungal proﬁle Ellagitannins, ellagic acid glycosides, (P.Fyhrquist&L.Mwasumbi, scabies, headache, cough, of the roots are used (Fyhrquist et al., 2002, 2004). To the best of our gallotannins and gallic acid as well as voucher 1697066) cardiac problems (Haerdi, 1964; for headache, cough, knowledge there are no other reports on the condensed tannins in roots. * Kokwaro, 1976; Chhabra et al., 1989). backache, gonorrhea, antimicrobial effects of extracts/ In Tanzania: diarrhea, gonorrhea bilharzias and as diuretics compounds of this and vomiting (Fyhrquist et al., 2002).* (Haerdi, 1964; Kokwaro, species despite of its frequent use in traditional 1976; Chhabra et al., 1989). medicine in East Africa. * In Tanzania dried leaves, Cytotoxic in brine shrimp test (Moshi etal., 2006). roots or stem bark are made into hot water decoctions and Especially the aqueous part as well as the crude tea or mixed with Ugali for MeOH extract of roots, both enriched in polar diarrhea, ellagitannins, are active against M. smegmatis

gonorrhea and vomiting (MIC 1.25 mg/ml). Ellagic acid glycoside rich 1 (2014) 90 Botany of Journal African South / al. et Fyhrquist P. (Fyhrquist et al., 2002).* Sephadex LH-20 fractions gave promising antimycobacterial effects (MIC 1 mg/ml). * T. stenostachya Engl. & Diels. For treatment of epilepsy and poisoning Root decoctions for Extracts of stem bark and leaves have Leaf extracts contain gallic acid in high (P.Fyhrquist&L.Mwasumbi, (Gelﬂand et al., 1985; Neuwinger, 2000). epilepsy/poisoning given good antibacterial (Fyhrquist et al., 2002) concentrations, ellagitannins, gallotannins voucher 1697028) (Gelﬂand et al., 1985; and antifungal (Fyhrquist et al., 2004)results*. and ellagic acid glycosides. * Neuwinger, 2000) Stem bark, root and leaf extracts of this species effectively inhibited the growth of Mycobacterium madagascariense and M. indicus,aswellasthe growth of some gram-negative and gram- positive bacteria (Mbawambo et al., 2011). BuOH fractions of extracts of leaves and fruits active against M. smegmatis.* T. spinosa Engl. For treatment of malaria, Fresh leaves are pounded Good antibacterial activity of young branches on Tannins, coumarins (Okemo, 1996; Omulokoli, 1997) (P.Fyhrquist&L.Mwasumbi, fever and wounds and the juice drank for S. aureus and Enterococci, but no inhibition of Gallic acid, gallotannins, ellagitannins voucher 1697047) (Kokwaro, 1976; malaria and stem bark is Mycobacterium tuberculosum and ellagic acid glycosides in the leaves. * Heine and Brenzinger, 1988; chewed or added to tea growth (Fabry et al., 1998). Chhabra et al., 1989) to reduce fever A BuOH fraction of a MeOH extract of the (Heine and Brenzinger, 1988). leaves inhibited the growth of M. smegmatis.*

Plant uses for tuberculosis or its symptoms, such as cough marked with bold text. TM, traditional medicine. – 16 5 6 P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16

2.5. Assays for testing antimycobacterial activity Table 2 Antimycobacterial effects of extracts and their solvent partition fractions of species of Terminalia collected in Tanzania in spring 1999. Results obtained with an agar diffusion 2.5.1. Agar disk diffusion methodA. * Activity index in relation to rifampicin. Amodified agar disk diffusion method based on the methods of Barry and Thornsberry (1991) and Rauha et al. (2000) was used. M. smegmatis Bacterial species M. smegmatis ATCC 14468 A.I.* ATCC 14468 was grown for five days at +37 °C on Löwenstein–Jensen Terminalia species, agar slants (Becton–Dickinson & Company, USA). 200 μl bacterial culture crude extracts and their fractions 8 containing 1.0 × 10 CFU/ml was inoculated on Petri dishes (∅ =14cm, T. sambesiaca RCr 37.0 ±0.5 0.70 Bibby Sterilin, UK) containing 30 ml Middlebrook 7H10 agar (Difco) T. sambesiaca WS 34.0 ±0.5 0.65 BuOH supplemented with oleic acid, albumin, dextrose and catalase (OADC T. sambesiaca 25.0 ±1.3 0.48 T. sambesiaca WI 29.5 ±1.0 0.56 supplement, Difco) as top layer and 30 ml Base agar (Difco) as base T. sambesiaca CHCL3 NA NA ∅ SCr layer. Filter paper disks ( = 12.7 mm, Schleicher & Schuell) loaded T. sambesiaca 26.0 ± 0.0 0.50 with 200μl extracts/fractions (50mg/ml and 20mg/ml), 200μlrifampicin T. sambesiaca WS 37.5 ±0.3 0.71 (10 mg/ml, Sigma-Aldrich) and 200 μl MeOH (as negative control) were T. sambesiaca BuOH 34.5 ±0.8 0.66 WI allowed drying completely before placing them equidistantly from each T. sambesiaca 22.0 ±2.0 0.42 T. sambesiaca CHCL3 17.0 ± 0.0 0.32 other on the Petri dishes. Prior to incubation the dishes were kept in T. stenostachya SCr 14.0 ± 0.0 0.27 +4 °C to facilitate diffusion of the extracts into the agar. The dishes T. stenostachya WI NA NA were then incubated at +37 °C for five days. Each extract/fraction was T. stenostachya Ws NA NA BuOH tested in triplicate. The diameter of zones of inhibition (IZ) was measured T. stenostachya 17.5 ± 0.3 0.33 T. stenostachya CHCL3 17.0 ± 1.5 0.32 with a caliper and the mean of 3 diameters ±SEM was calculated. For T. stenostachya FCr 31.5 ±0.5 0.60 estimating minimum inhibitory concentration (MIC) two-fold dilutions T. stenostachya WS 27.0 ±1.5 0.51 of the extracts and fractions from 195 μg/ml to 25 mg/ml and from 78 T. stenostachya WI 14.0 ± 0.0 0.27 μg/ml to 20mg/ml, respectively, were performed. For rifampicin dilutions T. stenostachya BuOH 35.5 ±0.3 0.68 LCr ranging from 2.44 μg/ml to 5 mg/ml were used. The MIC was considered T. stenostachya 20.5 ±1.2 0.39 T. stenostachya WI NA NA as the lowest concentration of extract, fraction or antibiotic giving a T. stenostachya Ws 24.0 ±0.0 0.46 small (1 mm) clear zone of growth inhibition around the disk. Activity T. stenostachya BuOH 25.0 ±0.3 0.48 index of the plant extracts and fractions was measured in relation to T. stenostachya CHCL3 14.0 ± 1.5 0.27 RCr rifampicin according to Singariya et al. (2012). T. sericea 31.5 ±0.3 0.60 T. sericea WS 27.0 ±1.0 0.51 WI Inhibition zone of the plant extract T. sericea 14.0 ± 0.0 0.27 AIðÞ¼ Activity index : T. sericea BuOH 35.5 ±0.8 0.68 Inhibition zone of Rifampicin T. sericea CHCL3 ** T. sericea LCr 16.0 ± 0.5 0.30 T. sericea WS NA NA 2.5.2. Microplate dilution method for MIC estimations WI fi T. sericea NA NA Amodi ed microplate assay based on Collins and Franzblau (1997) T. sericea BuOH 17.0 ± 0.0 0.32 was used for measuring minimum inhibitory concentrations of the T. sericea CHCL3 16.5 ± 0.3 0.31 extracts and fractions of the most promising species of Terminalia. T. kaiserana RCr 27.5 ±3.5 0.52 WI M. smegmatis ATCC 14468 was grown for five days at +37 °C on T. kaiserana 14.0 ± 0.0 0.27 T. kaiserana Ws 25.0 ±1.6 0.48 Löwenstein–Jensen agar slants. After completion of growth a small T. kaiserana BuOH 19.5 ± 2.0 0.37 amount of culture was transferred into Dubos broth and the absorbance T. kaiserana CHCL3 20.0 ± 0.0 0.38 of the samples was measured at 625 nm. The absorbance at 625 nm T. kaiserana LCr 18.0 ± 0.0 0.34 was adjusted to 0.1 (approx. 1.0×108CFU/ml) using Dubos broth supple- T. kaiserana WI NA NA T. kaiserana BuOH 21.5 ±0.5 0.41 mented with Dubos broth albumin (Difco). This suspension was diluted CHCL3 fi 5 T. kaiserana NA NA further so that the nal number of cells in inoculum was 5.0 × 10 T. spinosa LCr 21.5 ±0,3 0.41 CFU/ml. A portion of 100 μl of inoculum was added to the wells of the T. spinosa WS 23.0 ±1,0 0.44 microplate (Nunc, Nunclone, Denmark). Dilutions of 100 μlofeachof T. spinosa WI NA NA BuOH the plant extracts (9.76μg/ml–5 mg/ml in Dubos broth) and of rifampicin T. spinosa 22.5 ±0.3 0.43 CHCL3 (0.9 μg/ml–1 mg/ml in Dubos broth) were added to the wells of the T. spinosa 17.0 ± 0.0 0.32 Rifampicin 52.5 ±0.5 100.00 microplate, so that the final volume/well was 200 μl. Thus, the final number of bacterial cells/well at starting point of experiment was *Crude extracts in MeOH; Cr, crude extract; R, roots; S, stem bark; F, fruits; L, leaves; WS, 5 aqueous fraction; WI, water insoluble fraction; BuOH, butanol fraction; CHCL3, chloroform 2.5 × 10 CFU/ml. Wells containing all tested concentrations of plant ex- fraction; NA, not active. A Two hundred μl extracts/fractions (50 mg/ml) and rifampi- tracts, fractions or rifampicin in broth without bacterial inoculum were cin (10 mg/ml) were applied on filter paper disks. Diameter of inhibition zones in mm used as controls in order to measure background absorbance produced as mean of triplicates (n = 3) ± SEM of three experiments. Most promising results (IZ ≥ by the extracts, fractions and the antibiotic. The solvent control; MeOH 20 mm) indicated by bold text. Activity index results obtained using disk diffusion data. at 5% or less did not affect the growth of M. smegmatis. The plates sealed with parafilm were incubated for four days at +37 °C, whereafter turbid- ity of the wells at 620 nm was measured using a Victor (Wallac, Finland) treatment of tuberculosis or its symptoms in African traditional medicine spectrophotometer. The results were measured as mean percentage (Table 1), were screened for their growth inhibitory effects against inhibition of three replicate samples ±SEM. The minimum inhibitory M. smegmatis ATCC 14468. M. smegmatis is an important test model concentration (MIC) was considered as the smallest concentration of organism for the initial, primary screening for antimycobacterial the extracts/fractions as well as rifampicin inhibiting 90% or more of activity in the search for drugs with potential anti-tuberculosis the growth of M. smegmatis. effects. Inhibitory effects against this bacterial strain indicate that the extracts/fractions also to a certain extent inhibit the growth of the closely 3. Results and discussion related M. tuberculosis. M. smegmatis grows faster than Mycobacterium tuberculosum and does not present the pathogenic properties of the Altogether 48 extracts and solvent partition fractions of different latter (Pauli et al., 2005; Carneiro et al., 2008). M. smegmatis has polarities from five species of Terminalia, some of them used for been found to be rather resistant to rifampicin and it has been suggested P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16 7

Table 3 Minimum inhibitory concentrations of antimycobacterially promising crude methanol extracts of Terminalia species and their solvent partition fractions, as well as Sephadex-LH 20 fractions of a root extract of Terminalia kaiserana against Mycobacterium smegmatis ATCC 14468. MIC values were obtained both using microdilution and agar diffusion methods.

Crude extracts and their solvent partition fractions as well as fractions obtained with Sephadex LH-20 MIC microdilution (μg/ml) MIC agar diffusion (μg/ml)

T. sambesiaca roots:

Crude methanol extract 1250 (IC99)1562

Aqueous fraction 2500 (IC96)3125

Aqueous-insoluble fraction 2500 (IC95)3125 T. sambesiaca stem bark:

Crude methanol extract 1250 (IC98)1562

Aqueous fraction 1250 (IC95)1562 T. kaiserana roots:

Crude methanol extract 1250 (IC98)3125

Aqueous fraction 1250 (IC99)1562

Butanol soluble fraction 1250 (IC99)1562

Chloroform soluble fraction 1250 (IC100)1562 Sephadex LH-20 fractions of T. kaiserana rootsa:

F1 4000 (IC90)5000

F2 1000 (IC94)1250

F3 1000 (IC92)1250

F4 1000 (IC91)1250 T. sericea roots: Crude *3125 Butanol soluble fraction *1562

Rifampicin 3.90 (IC98)78 * Not tested due to lack of material. a Fractions F1–F4 obtained with Sephadex LH-20 column chromatography from a methanol root extract of Terminalia kaiserana. In brackets the percentage of the bacterial cell growth inhibited by indicated concentration. IC = inhibitory concentration. that this natural resistance is due to presence of efﬂux pumps (Piddock drug resistant strains of M. tuberculosis. Tables 2 and 3 present the re- et al., 2000). Thus, good inhibitory results of plant extracts against this sults on antimycobacterial activity of extracts and fractions of the inves- species of Mycobacterium might indicate promising results against tigated species of Terminalia.

Fig. 1. HPLC profile at 280 nm of a crude methanol extract of the roots of Terminalia sambesiaca obtained using the HPLC method II. The extract is rich in ellagitannins. Ellagitannins at tR 10.978, 12.415 and 15.357 min show UV absorption spectra with three absorption maxima peaks, typical for ellagitannins (the two first mentioned demonstrated in figure). Ellagic acid glycosides occur at tR 18.3, 19.7, 20.6 and 21.5 min. 8 P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16

Fig. 2. HPLC proﬁle at 220 nm of an aqueous fraction of a MeOH extract of the roots of Terminalia sambesiaca. Obtained using the HPLC method II. This extract is enriched in polar ellagitannins (tR = 4.4, 5.0, 6.3, 7.2, 8.8 and 10.3 min) and thus these compounds could be responsible for the good antimycobacterial effects of this extract. UVλ absorption maxima of the dominating ellagitannin in this extract, at tR 10.325 min is illustrated in ﬁgure.

3.1. Terminalia sambesiaca of T. sambesiaca might inhibit the growth of Mycobacterium with some other mechanisms, not necessarily entering the cell. Although T. sambesiaca is not used for the treatment of tuberculosis Very little is known about the chemical composition of T. sambesiaca in African traditional medicine, this species gave the most promising apart from one investigation (Masoko et al., 2005) in which saponin-like antimycobacterial effects of the ﬁve species of Terminalia we investigated, compounds were found in the leaves, using TLC as method. To our knowl- in terms of sizes of inhibition zones. Both extracts of the roots and stem edge the chemical composition of the roots of T. sambesiaca has not been bark effectively inhibited the growth of M. smegmatis giving clear inhibi- investigated before. Our results obtained using the acid butanol assay tion zones of 37.0 and 26.0 mm for crude methanolic root and stem bark show that the roots of T. sambesiaca contain moderate amounts of − extracts, respectively (Table 2). This is the ﬁrst time T. sambesiaca is condensed tannins; 9.2 mg ± 0.008 mg g 1, mean ± SD. According reported to give promising antimycobacterial effects. The roots contain to our results obtained by HPLC–UV–DAD, root extracts of T. sambesiaca many antimycobacterial compounds of different polarities since both contain gallic acid, ellagic acid glycosides and ellagitannins (Fig. 1). the aqueous and the aqueous-insoluble fractions gave growth inhibition. MIC was 2500 μg/ml for the aqueous and aqueous-insoluble fractions (Table 3) and diameters of inhibition zones were 34.0 mm and 29.5 mm, respectively (Table 2). The crude methanol extracts of the rootswereevenmoreeffectivethanthefractionsgivingMICvaluesof 1250 μg/ml (Table 3). Some of the compounds in the crude extract might act synergistically, enhancing the antimycobacterial effects of each other. The antimycobacterial effects of the stem bark of T. sambesiaca were mainly concentrated to the more polar fractions, such as the aqueous and butanol soluble fractions giving inhibition zones of 37.5 and 34.5 mm, respectively (Table 2). Also the aqueous insoluble part of the stem bark gave some antimycobacterial effects. Chloroform-soluble fractions of the roots gave no antimycobacterial activity, whereas the same fraction of the stem bark gave slight antimycobacterial activity. Fig. 3. Catechins and procyanidins in solvent partition fractions of a MeOH root extract of Terminalia sericea separated by cellulose-thin layer chromatography (TLC). Standards and The lipid-rich hydrophobic cell wall of mycobacteria makes them fractions from the left to right: procyanidin B 1, procyanidin B 2, (+)-catechin, epicatechin, generally more susceptible to less polar compounds. Thus, the more gallocatechin, epigallocatechin, epicatechin-gallate, a mixture of catechins, aqueous frac- polar compounds in the aqueous fraction and crude methanol extracts tion of a root extract of T. sericea, aqueous-insoluble fraction, and a BuOH fraction. P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16 9

Fig. 4. HPLC proﬁle obtained with the HPLC method II and recorded at 280 nm of a butanol fraction of the crude root extract of Terminalia sericea. This extract is rich in ellagitannins.

Figure shows UVλ absorption maxima spectra of an ellagitannin at tR 15.249 min and a stilbene at tR 17.049 min. Compound at tR 20.286 min is an ellagic acid derivate.

Quantitatively, one ellagitannin at tR 10.978 min is dominating the crude antibiotics (Elegami et al., 2002). Punicalagin showed potent growth methanol extract of the roots, and is also present in high quantities in inhibitory effects against both Gram-negative and Gram-positive bacte- the aqueous extract. This compound shows three UVλ absorption ria (among them MRSA) as well as against fungi (Reddy et al., 2007). maxima peaks, which is typical for ellagitannins, the absorption Furthermore, ellagitannins have been found to act synergistically with maxima occurring at 217, 260 and 380 nm (Fig. 1). Compounds at ampicillin against S. aureus and to inhibit growth of this bacterium by tR 9.840, 12.415 and 15.357 min are also ellagitannins. Compound at tR causing lysis of the cell walls (Din et al., 2012). There exists only one 12.415 min seems to be closely related to compound at tR 10.978 min, publication on the growth inhibitory effects of ellagitannins against and shows UVλ absorption maxima at 216, 257 and 382 nm (Fig. 1). Mycobacteria, in which punicalagin was shown to be responsible for Ellagitannins occur in high quantities also in an aqueous fraction of the the good antimycobacterial effects of a stem bark extract of Combretum crude root extract, which is enriched in polar ellagitannins (Fig. 2). This molle (Asres et al., 2001). means that they must be readily extractable also in hot water decoctions, T. sambesiaca has several uses in African traditional medicine, among the way this plant is used in African traditional medicine. The ellagitannin them for treatment of bloody diarrhea and stomach ulcers (Table 1; at tR 10.3–10.9 min, present in both the aqueous fraction and the crude Chhabra et al., 1989), but it has not been reported to be used for the extract in high concentrations might partly be responsible for the treatment of tuberculosis. Thus, our results indicate that hot water promising antimycobacterial effects of the aqueous and crude extracts. decoctions of the roots and stem bark of this species could be used as Ellagitannins, such as punicalagin, punicalin, chebulanin, chebulagic readily available and affordable treatment of tuberculosis in East and acid, chebulinic acid, corilagin, terﬂavin, terchebulin and tellimagrandin, South African rural regions. These decoctions contain ellagitannins and as well as some condensed tannins have been characterized from ellagic acid derivates which may be the causative agents of the good Terminalia species (Burapadaja and Bunchoo, 1995; Silva et al., 2000; antimycobacterial effects we can demonstrate. T. sambesiaca roots and Pfundstein et al., 2010). The co-occurrence of punicalagin, punicalin, stem bark are potential sources of new antimycobacterial ellagitannins chebulinic and chebulagic acid seems to be a chemotaxonomic feature and ellagic acid glycosides, and research should be conducted on their of the genus Terminalia (Yoshida et al., 2010). Several authors have re- growth inhibitory effects on different strains of M. tuberculosis. ported that ellagitannins possess powerful antimicrobial effects: Corilagin and tellimagrandin have been found to reduce the minimum inhibitory 3.2. Terminalia sericea concentration of β-lactams in methicillin-resistant Staphylococcus aureus (Shiota et al., 2004) and to dose-dependently affect membrane per- The crude methanol extract of the roots of T. sericea and especially the meability of Candida albicans and Escherichia coli, but not of S. aureus n-butanol fraction of this extract gave good growth inhibitory effects (Li et al., 2012). Tannin rich extracts of Terminalia species have proved to against M. smegmatis showing inhibition zones of 31.5 and 35.5 mm, be effective inhibitors of the growth of the Gram-negative bacterium respectively and an MIC of 1562 μg/ml for the butanol fraction Pseudomonas aeruginosa, showing resistance against many conventional (Tables 2 and 3). An aqueous-insoluble partoftherootextractgave 10 P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16

Fig. 5. HPLC–UV–DAD chromatograms of a crude methanol extract of Terminalia kaiserana and its fractions obtained using Sephadex LH-20 column chromatography. HPLC-chromatograms were recorded at 280 nm using the HPLC method I. The crude extract contains several ellagitannins (tR 8.3–22.7 min) absent from the Sephadex fractions. The dominating compounds in fractions 2–4 are ellagic acid and methyl-ellagic acid glycosides (tR 25 and 28 min, respectively). slight antimycobacterial effects, the inhibition zone being 14.0 mm. Our acid, isolated from ethyl acetate extracts of the roots of T. sericea have results are in agreement with York et al. (2012) who demonstrated MIC given promising antibacterial effects against Gram-positive and values of 670 and 2670 μg/ml for an organic solvent and aqueous extract, Gram-negative bacteria, but only slight activity against the growth respectively, of mature stem bark of T. sericea against M. smegmatis ATCC of Mycobacterium aurum A+ (Eldeen et al., 2008). Therefore, the 14468. antimycobacterially active compounds in the roots of T. sericea In contrast to the root extracts, we found that the leaf extracts gave might belong to some other chemical categories than lignans and just slight growth inhibitory effects against M. smegmatis.Theinhibitory pentacyclic triterpenes, or then these compounds possibly act in synergy effects of the leaves were concentrated to the butanol-soluble and in the extracts losing their effects when puriﬁed. chloroform fractions, whereas the aqueous and aqueous-insoluble Our preliminary results on the chemical composition of T. sericea roots extracts did not give any effects (Table 2). obtained using thin layer chromatography on cellulose indicate that the In our earlier investigations T. sericea root and leaf methanolic ex- aqueous and the aqueous-insoluble fractions of a crude methanol extract tracts gave good effects against several Gram-positive bacteria and might contain (+)-catechin. The butanol fraction of the crude root Enterobacter aerogenes (Fyhrquistetal.,2002). Many other authors extract might contain epicatechin, epigallocatechin and gallocatechin have also reported good antibacterial effects of T. sericea leaf, stem (Fig. 3). T. sericea has not been reported to contain catechins before, bark and root extracts (Eloff, 1999; Innocent, 2002; Steenkamp et al., although there are some reports on other species of Terminalia containing 2004; Moshi and Mbwambo, 2005). Acetone extracts of the stem bark this class of compounds. Catechin and epicatechin have been isolated of T. sericea have been reported to demonstrate good activities against from the stem bark of a Terminalia mollis (Liu et al., 2009)and

M. tuberculosis H37 Ra and the multi drug resistant clinical strain Mtb 2 (−)-catechin from the trunk bark of Terminalia glabrescens (Garces with MIC values of 25 μg/ml (Green et al., 2010), but the active com- et al., 2003). Catechins have been found to possess interesting antimicro- pound (s) in these extracts were not investigated. Eldeen and Van bial effects, and for example bactericidal epigallocatechin-gallate was Staaden (2007) reported good growth inhibitory effects of ethyl acetate, shown to cause damage to bacterial membranes (Ikigai et al., 1993). dichloromethane and ethanol extracts of the roots and stem bark of Catechins are also known to inactivate bacterial enzymes (Friedman T. sericea against Mycobacterium fortuitum (MICs 1560–3120 μg/ml). et al., 2006). Catechin has been found to give inhibitory effects against

There are scanty publications on the chemical structures of antibac- the growth of M. tuberculosis H37Rv showing MIC values of 200 μg/ml, terial compounds of this plant. Up to date four compounds with antibac- whereas epigallocatechin was found to be inactive (Martins et al., 2011). terial effects have been isolated from T. sericea roots and stem bark. The Our HPLC–UV–DAD results show that a butanol fraction of the crude promising antimicrobial compound, anolignan B (Eldeen et al., 2005) extract of the roots contains ellagic acid glycosides, ellagitannins, gallic and resveratrol-3-O-β-rutinoside showing some antibacterial activity acid and at least one stilbene (Fig. 4). There are no reports on ellagitannins (Bombardelli et al., 1975; Joseph et al., 2007) have been characterized or ellagic acid derivates characterized from T. sericea. Ellagitannins might from the stem bark and roots of T. sericea. Termilignan B and arjunic be responsible for the good antimycobacterial effects of methanol and P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16 11

Fig. 6. HPLC chromatogram of a methanolic root extract of Terminalia kaiserana recorded at 280 nm using the HPLC method II. According to HPLC-retention times as well as UVλ maxima data the extract contains gallic acid (tR 1.699 min, UVλ absorption maxima spectrum in figure), ellagitannins (tR 3.090, 7.504, 7.952, 8.491, 9.819, 10.930, 11.696, 12.491, 15.281 and 15.597 min) and ellagic acid glycosides (tR 20.340, 20.510 and 24.207 min). butanol extracts of the roots of T. sericea, since according to Eldeen Tothebestofourknowledgetherearenoreportsonsecondary et al. (2008), lignans and pentacyclic triterpenoids in this plant have compounds in T. kaiserana up to date. We have initiated work on charac- not shown very promising antimycobacterial activity. The good terization and isolation of antimicrobial compounds from the root, which antimycobacterial activity found by Green et al. (2010) for acetone is the most commonly used part of this plant for traditional medicine. extracts of the stem bark of T. sericea may be due to ellagitannins and Also preliminary identification of compounds in the stem bark has ellagic acid glycosides, since this kind of compounds are also extracted been performed. According to our results obtained using an acid butanol with acetone. test, the roots and stem bark of T. kaiserana contain a high concentration T. sericea is reported to be used for the treatment of tuberculosis in of condensed tannins, 35.9±0.042 and 48.1±0.272mgg−1,mean±SD, African traditional medicine (Table 1). In Malawi the fruits are made respectively. For comparison, the total extractable tannin content in into a multi-component treatment for this disease (Msonthi and Terminalia brownii leaves was found to be 125 mg g−1 dry weight Mgombo, 1983). Roots of T. sericea are not reported to be used for the (Osuga et al., 2006). It is possible that condensed tannins partly give treatment of TB, but hot water decoctions of the outer layer (bark) of the good antimycobacterial effects of both stem bark and root extracts the roots are used for curation of pneumonia and cough (Drummond of T. kaiserana. Some condensed tannins from Terminalia spp. are and Coates-Palgrave, 1973). Our results indicate that in addition to the known to be very toxic, such as terminalin A, which was isolated from fruits also the roots of T. sericea could be used for the treatment of Terminalia oblongata (Oelrichs et al., 1994). Thus, Terminalia species mycobacterial infections. have to be used with caution as medicinal plants. Standardized extracts in which the tannin concentration is controlled, should be developed 3.3. Terminalia kaiserana for medicinal uses. Condensed tannins are known to give promising antimicrobial effects against Campylobacter jejuni (Anderson et al., We report for the first time good antimycobacterial activities of both 2012), Salmonella typhimurium (Costabile et al., 2011) and methicillin- root and leaf extracts of T. kaiserana. Especially the crude methanol ex- resistant S. aureus (Hatano et al., 2005), among others, and thus can tract of the roots and its aqueous fraction, enriched in polar ellagitannins, form a vital component of the herbal medicine. Condensed tannins gave promising results, giving inhibition zones of 27.0 and 25.0 mm, re- have, to the best of our knowledge, not been screened for activity against spectively and MIC values of 1250 μg/ml for both (Tables 2 and 3). In Mycobacterium species. addition a BuOH fraction of the leaves gave inhibition zones of 21.0 mm We also found that the methanolic root extract of T. kaiserana against M. smegmatis (Table 2). Interestingly, also a chloroform-soluble contains several ellagitannins, ellagic acid glycosides and methylated fraction of the roots, enriched in ellagic acid derivates, gave good ellagic acid glycosides according to HPLC retention times and UVλ antimycobacterial activities (MIC 1250 μg/ml), thus indicating that absorbance maxima data (Figs. 5, 6 and 7). The quantitatively most this plant contains antimycobacterially active compounds of a range of prominent ellagitannin occurs at tR 15.280 min and has three UV ab- different polarities (Tables 2 and 3). sorption maxima at 216, 256 and 380nm (Figs. 6 and 7). Another tannin 12 P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16

Fig. 7. UVλ absorption maxima spectra of ellagitannins at a) tR = 12.5 min and b) tR = 15.2 min and c) ellagic acid glycoside at tR = 20.2 min in root extracts of Terminalia kaiserana.

at tR 12.491min also occurs in relatively high quantities, and showed UV against P. aeruginosa (Sarabhai et al., 2013). Ellagic acid has been absorption maxima at 217, 258 and 382 nm (Fig. 7). An ellagic acid found to inhibit plasma coagulation caused by S. aureus as this bacteria glycoside and its isomer occur in high quantities at tR 20.339 and produce fibrin-rich biofilms to prolong infection (Akiyama et al., 2001). 20.513 min, respectively (Figs. 6 and 7). In an attempt to separate Ellagic acid-like compounds bind to proteins thus inactivating microbial antimycobacterially active polyphenols and ellagic acid derivates, metha- adhesions, enzymes, cell membrane transport proteins, etc. (Scalbert, nol extracts of the roots of this plant were subjected to Sephadex-LH 20 1991; Haslam, 1996). This warrants further studies on antibacterial column chromatography. Nine fractions were pooled together to four and antimycobacterial ellagic acid derivates and ellagitannins in yet un- fractions according to RP-18 TLC qualitative data. Especially fractions studied species of Terminalia. A number of ellagic acid glycosides have F2, F3 and F4 gave good antimycobacterial effects showing MIC values been isolated from natural sources, but their synthesis is very difficult of 1000 μg/ml, whereas fraction F1 was active only at 4000 μg/ml due to the O-glycosylation step (Asami et al., 2003). Therefore, higher (Table 3). According to HPLC–DAD data, fraction F1 contains predomi- plants are the cheapest source of these compounds. nantly gallotannins and condensed tannins, whereas fractions F2–F4 are Our results on the good antimycobacterial activity of root and leaf enriched in ellagic acid glycosides, showing two dominating compounds extracts of T. kaiserana are in accordance with the use of hot water at tR 25 and 28 min, as well as one compound at 26.5 min in fraction F4, decoctions of the roots and the leaf sap of this species in African tradi- using the HPLC method I (Fig. 5). The retention times of peaks at 25 and tional medicine for treatment of cough, possibly related to tuberculosis

28 min correspond to those at tR 19.1 min (UVλmax = 254 and 364 nm, (Table 1; Haerdi, 1964). Moshi et al. (2006) have found that this species ellagic acid glycoside) and 22.4 min (UVλmax = 254 and 364 nm, showed cytotoxicity in a brine shrimp test and thus extracts of this plant tentatively identiﬁed as methyl ellagic acid glycoside), when we used should be used with caution in traditional medicine. It would therefore theHPLCmethodII.ThecompoundattR 26.5min in fraction F4 is possibly be important to investigate which component/-s in the extract is/are an isomer of compound at tR 25 min. Since the ellagic acid glycosides in toxic and to develop standardized extracts for safer medicinal use. fractions F2–F4 are dominating quantitatively, they must be responsible for the good antimycobacterial effects of these fractions. This data is in 3.4. Terminalia stenostachya accordance with an investigation of Kuete et al. (2010) who reported that a methyl derivate of ellagic acid, 3,4′-di-O-methylellagic 3′-O-β-D- Crude methanol extracts of fruits and leaves of T. stenostachya xylopyranoside, isolated from the stem bark of the West African species as well as their butanol soluble fractions inhibited the growth of T. superba, gives excellent growth inhibitory effects against several M. smegmatis. The butanol fractions of the fruits gave the best effects strains of M. tuberculosis and M. smegmatis. of the investigated organs showing clear inhibition zones of 35.0 mm Ellagic acid as well as its glycosidic derivates is known to possess (Table 2). Also the butanol soluble fractions of the leaves gave clear antibacterial effects mainly against Gram-positive bacteria (Akiyama inhibition zones of 25.0 mm (Table 2). To the best of our knowledge et al., 2001; Soh et al., 2009; Panichayupakaranant et al., 2010)butalso fruit extracts of T. stenostachya have not been screened before for P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16 13

Fig. 8. HPLC chromatogram at 280 nm of a methanol extract of the leaves of Terminalia stenostachya.HPLCmethodIIwasused.Gallicacid(tR 1.701 min, UVλ max is shown in ﬁgure) concentration is high in the leaves of this species. Ellagitannins (for example compound at tR 10.9 min) and gallotannins (compound at tR 13.6 min) occur in this extract. Compound at tR 20.608 min is an ellagic acid glycoside which shows two distinct UVλ absorption peaks (shown in ﬁgure).

antimycobacterial effects. There exists only one report on the antimyco- (Fabry et al., 1998) and thus it seems that the leaves contain some bacterial effects of T. stenostachya to date; Mbawambo et al. (2011) compounds with antimycobacterial activity which are present only found that stem bark, root and leaf extracts of this species effectively in small concentrations or are absent in the twigs. inhibited the growth of M. madagascariense and M. indicus. In contrast We have tentatively identified gallic acid, gallotannins, ellagitannins to the findings of Mbawambo et al. (2011) we found that a stem bark and an ellagic acid glycoside in the leaves of this plant and found that extract of this species did not possess significant antimycobacterial the leaves are especially rich in different gallotannins (Fig. 9). Tannins activity. This could be due to the use of different test organisms, and coumarins have been reported from this plant (Okemo, 1996; M. smegmatis differing in sensitivity to M. madagascariense and M. indicus. Omulokoli et al., 1997). Our preliminary results on the chemical composition of T. spinosa is frequently used in African traditional medicine for treat- T. stenostachya, obtained using HPLC–UV–DAD, show that methanolic ment of malaria, fever, wounds and jaundice (Table 1; Kokwaro, 1976; leaf extracts contain gallic acid in high concentrations, ellagitannins Heine and Brenzinger, 1988; Chhabra et al., 1989; Beentje, 1994). The and ellagic acid glycosides (Fig. 8). To the best of our knowledge the ways of preparation of T. spinosa for traditional medicine vary. Leaf chemical composition of T. stenostachya leaves has not been investigated juice from pounded leaves is reported to be used as well as teas (Heine before. and Brenzinger, 1988) and cold water infusions of the leaves (Beentje, Root decoctions of T. stenostachya have been reported to be used for 1994). The cold water infusions and teas extract the gallotannins we treatment of epilepsy and poisoning in African traditional medicine have identified in the leaves of this plant (Fig. 9). Gallotannins might (Table 1; Gelfland et al., 1985; Neuwinger, 2000)andnowourfinding give leaf extracts of T. spinosa their good antimycobacterial potential. indicates a new potential use of the leaves and fruits of this plant for Gallotannins, and especially penta- to heptagalloylated gallotannins, the treatment of mycobacterial infections in East Africa. have been shown to potently inhibit the growth of Gram-negative and Gram-positive bacteria (Tian et al., 2009). Gallotannins have been 3.5. Terminalia spinosa found to induce changes in bacterial membrane permeability when binding to proteins embedded in these membranes (Engels et al., We found that a crude methanol extract of the leaves of T. spinosa as 2011), and they are able to penetrate bacterial membranes and coagulate well as its butanol soluble fraction gave growth inhibitory effects the cell contents (Li, 2006). Gallotannins bind to enzymes non- against M. smegmatis showing clear inhibition zones with diameters specifically and thus render them inactive (Konishi et al., 1993). The 21.5 and 23.0 mm, respectively (Table 2). Extracts of the young twigs inhibition of bacterial growth by gallotannins might also be due to their have been found to be inactive against the growth of M. tuberculosis ability to complex with metal ions (Scalbert et al., 1999). 14 P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16

Fig. 9. HPLC chromatogram at 280 nm of a butanol extract of the leaves of Terminalia spinosa. The HPLC method II was used. This extract contains gallic acid (tR 1.695 min), ellagitannins

(compound at tR 10.8 min), an ellagic acid derivate (tR 20.627 min) and gallotannins (tR 15.0, 17.9, 19,6 and 22.4min). The ﬁgure shows an UV absorption spectrum of the main compound, agallotanninattR 22.412 min.

4. Conclusions Acknowledgments

Due to the increasing incidence of antibiotic resistant tuberculosis, The first author is grateful for the financial support for this research both globally and in Africa south of Sahara, it would be important to project from the Finnish Cultural Foundation, Swedish Cultural Founda- find new and effective medicines against this disease. These antimyco- tion in Finland, Oskar Öflund Foundation, Societas pro Fauna et Flora bacterially active compounds can be found from higher plants, and Fennica and the Finnish Medical Association (Finska Läkaresällskapet). especially from plants which have uses in traditional medicine for treat- Thanks are due to Professor Raimo Hiltunen and Professor Riitta ment of tuberculosis and bacterial infections in general. Julkunen-Tiitto for providing excellent laboratory facilities. Sincere grati- African species of Terminalia are potential sources of antimyco- tude goes to Professor Riitta Julkunen-Tiitto for sharing her expertise in bacterial and antibacterial ellagitannins, ellagic acid derivates, gallotan- HPLC–UV/DAD and mass spectrometry. nins and condensed tannins. Species belonging to these genera are often used as hot water decoctions or water extracts for traditional medicine, References and thus it can be predicted that large amounts of tannins and ellagic acid glycosides are extracted using these methods of preparation. Anderson, R.C., Vodovnik, M., Min, B.R., Pinchak, W.E., Krueger, N.A., Harvey, R.B., Nisbet, Ellagitannins are a group of hydrolyzable tannins which have not been D.J., 2012. Bactericidal effect of hydrolysable and condensed tannin extracts on Campylobacter jejuni in vitro. Folia Microbiologica 57, 253–258. subjected to thorough investigation on their antimycobacterial potential. Akiyama, H., Kazuyasu, F., Yamasaki, O., Oono, T., Iwatsuki, K., 2001. Antibacterial action of Up to date there exists only one publication on the antimycobacterial several tannins against Staphylococcus aureus. Journal of Antimicrobial Chemotherapy ellagitannin punicalagin, extracted from the stem bark of C. molle 48, 487–491. Asami, Y., Ogura, T., Otake, N., Mishimura, T., Xinsheng, Y., Sakurai, T., Nagasawa, H., (Asres et al., 2001). Since this group of tannins show a high diversity in Sakuda, S., Tatsuta, K., 2003. Isolation and synthesis of a new bioactive ellagic acid molecular structure it is expected that new antitubercular agents could derivate from Combretum yunnanensis. Journal of Natural Products 66, 729–731. be found from this group of secondary metabolites. Von Martius et al. Asres, K., Bucar, F., Edelsbrunner, S., Kartnig, T., Höger, G., Thiel, W., 2001. Investigations (2012) found a positive correlation between the total phenolic/tannin on antimycobacterial activity of some Ethiopian medicinal plants. Phytotherapy Research 15, 323–326. content and antimicrobial activity of Eucalyptus extracts. This can be Ballell, L.R., Field, A., Duncan, K., Young, R.J., 2005. New small molecule synthetic true for Terminalia species as well. The relative amounts of hydrolyzable antimycobacterials. Antimicrobial Agents and Chemotherapy 49, 2153–2163. and condensed tannins did not determine the antimicrobial effects of Barry, A.L., Thornsberry, C., 1991. Susceptibility Tests: Diffusion Test Procedures. In: Balows, A. (Ed.), Manual of Clinical Microbiology. American Society of Microbiology, Eucalyptus species (Von Martius et al., 2012), but this remains to be Washington DC. investigated in species of Terminalia. More research would be needed Beentje, H.J., 1994. Kenya Trees, Shrubs and Lianas. National Museums of Kenya, Nairobi. to determine the individual antimycobacterial and antimicrobial effects Bombardelli, E., Martinelli, E.M., Mustich, G., 1974. Plants of Mozambique. VII. Phytochemistry 13, 2559–2562. of different kinds of tannins in extracts of Terminalia spp. used in African Bombardelli, E., Martinelli, E.M., Mustich, G., 1975. A new hydroxystilbene glycoside from traditional medicine. Terminalia sericea. Fitoterapia 46, 199–201. P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16 15

Bombardelli, E., Crippa, F., Pfifferi, G., 1986. Sericoside, a New Glucoside in Functional Haslam, E., 1996. Natural polyphenols (vegetable tannins) as drugs: possible modes of Cosmetics. Preprints of the 14th F.S.C.C. congress, Barcelona (1986), pp. 975–986. action. Journal of Natural Products 59 (2), 205–215. Burapadaja, S., Bunchoo, A., 1995. Antimicrobial activity of tannins from Terminalia citrina. Hatano, T., Kusuda, M., Inada, K., Ogawa, T.O., Shiota, S., Tsuchiya, T., Yoshida, T., 2005. Effects Planta Medica 61, 365–366. of tannins and related polyphenols on methicillin-resistant Staphylococcus aureus. Carneiro, A.L.B., Teixeira, M.F.S., Aruijo de Oliveira, V.M., Cristo Fernandez, O.M., Socorro Phytochemistry 66, 2047–2055. de Barros Cauper, G., Pohlit, A.M., 2008. Screening of Amazonian plants from the Hedberg, I., Hedberg, O., Madati, P., Mshigeni, K.E., Mshiu, E.N., 1982. Inventory of plants used Adolpho Ducke forest reserve, Manaus, state of Amazonas, Brazil, for antimicrobial in traditional medicine in Tanzania. I. Plants of the families Acanthaceae-Cucurbitaceae. activity. Memorias do Instituto Oswaldo Cruz, Rio de Janeiro 103 (1), 31–38. Journal of Ethnopharmacology 6, 29–60. Chaisson, R.E., Martinson, N.A., 2008. Tuberculosis in Africa – combating and HIV – driven Heine, B., Brenzinger, M., 1988. Plants of the Borana. Kölner Beiträge zur crisis. The New England Journal of Medicine 358, 1089–1092. Entwichlingsgeshichtforschung, vol. 8 (part 4. Germany). Chhabra, S.C., Mahunnah, R.L.A., Mshiu, E.N., 1989. Plants used in traditional medicine Ikigai, H., Nakae, T., Hara, Y., Shimamura, T., 1993. Bactericidal catechins damage the lipid in Eastern Tanzania. II. Angiosperms (Capparidaceae-Ebenaceae). Journal of bilayer. Biochimica et Biophysica Acta 1147, 132–136. Ethnopharmacology 25, 339–359. Innocent, E., 2002. Antimicrobial Flavonoids and Stilbene Glycosides from Uvaria scheffleri Collins, L.A., Franzblau, S.G., 1997. Microplate alamar blue assay versus BACTEC 460 system and Terminalia sericea. M.Sc. Dissertation University of Dar es Salaam, p. 143. for high-troughput screening of compounds against Mycobacterium tuberculosis Joseph,C.C.,Moshi,M.J.,Innocent,E.,Nkunya,M.H.H.,2007.Isolation of a stilbene and Mycobacterium avium. Antimycobacterial Agents and Chemotherapy 41 (5), glycoside and other constituents of Terminalia sericea.AfricanJournalofTraditional, 1004–1009. Complementary, and Alternative Medicines 4 (4), 383–386. Connolly, M., Nunn, P., 1996. Women and tuberculosis. World Health Statistics Quarterly Julkunen-Tiitto, R., Rousi, M., Bryant, J., Sorsa, S., Keinänen, M., Sikanen, H., 1996. Chemical 49 (2), 115–119. diversity of several Betulaceae species: comparison of phenolics and terpenoids in Conrad, J., Vogler, B., Klaiber, I., Roos, G., Walter, U., Kraus, W., 1998. Two triterpene esters northern birch stems. Trees 11, 16–22. from Terminalia macroptera bark. Phytochemistry 48 (4), 647–650. Koehn, F.E., Carter, G.T., 2005. The evolving role of natural products in drug discovery. Conrad, J., Vogler, B., Reeb, S., Klaiber, I., Roos, G., Vasquiez, E., Setzer, M.C., Kraus, W., Nature Reviews. Drug Discovery 4, 206–220. 2001. Isoterchebulin and 4,6-O-isoterchebuloyl-D-glucose, novel hydrolysable tannins Kokwaro, O., 1976. Medicinal Plants of East Africa. East African Literature, Nairobi. from Terminalia macroptera. Journal of Natural Products 64, 294–299. Konishi, K., Adachi, H., Ishigaki, N., Kanamura, Y., Adachi, I., Tanaka, T., Nishioka, I., Nonaka, Corbett, E.L., Watt, C.J., Walker, N., Mather, D., Williams, B.G., Ravigliione, M.C., Dye, C., G.-I., Horikoshi, I., 1993. Inhibitory effects of tannins on NADH dehydrogenases of 2003. The growing burden of tuberculosis: global trends and interactions with the various organisms. Biological and Pharmeutical Bulletin 16, 716–718. HIV epidemic. Archives of Internal Medicine 163, 1009–1021. Kuete, V., Tabopda, T.K., Ngameni, B., Nana, F., Tshikalange, T.E., Ngadjui, B.T., 2010. Costabile, A., Sanghi, S., Martin-Pelaez, S., Mueller-Harvey, I., Gibson, G.R., Rastall, R.A., Antimycobacterial, antibacterial and antifungal activities of Terminalia superba Klinder, A., 2011. Inhibition of Salmonella Typhimurium by tannins in vitro.Journal (Combretaceae). South African Journal of Botany 76, 125–131. of Food, Agriculture & Environment 9 (1), 119–124. Li, C.Y., 2006. Extraction and Purification of Proanthocyanidin from Grape Seed, its Dastmalchi, K., Dorman, D., Laakso, I., Hiltunen, R., 2007. Chemical composition and Structure and Functionality. Ph. D. Dissertation Jiangnan University, China. antioxidative activity of Moldavian balm (Dracocephalum moldavica L.) extracts. Li, N., Luo, M., Fu, Y., Zu, Y., Wang, W., Zhang, L., Yao, L., Zhao, C., Sun, Y., 2012. Effect of Food Science and Technology 40 (9), 1655–1663. corilagin on membrane permeability of Escherichia coli, Staphylococcus aureus and Din, W.M., Jin, K.T., Ramli, R., Khaitir, T.M.N., Wiart, C., 2012. Antibacterial effects of Candida albicans. Phytotherapy Research 1–7(November2012). ellagitannins from Acalypha wilkensiana var macafeana hort.: surface morphology Liu, M., Katerere, D.I., Gray, A.I., Seidel, V., 2009. Phytochemical and antifungal studies on analysis with environmental scanning electron microscopy and synergy with antibiotics. Terminalia mollis and Terminalia brachystemma. Fitoterapia 80, 369–373. Phytotherapy Research, October 2012, 1–8. Maeda N., Fukuda M., 1996. Skin lightening preparations containing sericoside. Patent-Japan Drummond, R.B., Coates-Palgrave, K., 1973. Common Trees of the Highweld. ISBN, Rhodesia. Kokai Tokkyo Koho-08133 (6), 951. Egwaga, S.M., 2003. The impact of HIV on transmission of tuberculosis in Tanzania. Mann, A., Amupitan, J.O., Oyewale, A.O., Okogun, J.I., Ibrahim, K., Oladosu, P., Lawson, L., Tuberculosis 83, 66–67. Olajide, I., Nnamdi, A., 2008. Evaluation of in vitro antimycobacterial activity of Eldeen, I.M.S., Elgorashi, E.E., Mulholland, D.A., Van Staden, J., 2005. Anolignan B: a bioactive Nigerian plants used for treatment of respiratory diseases. African Journal of compound from the roots of Terminalia sericea. Journal of Ethnopharmacology 103 (1), Biotechnology 7 (11), 1630–1636. 135–138. Mann, A., Ibrahim, K., Oyewale, A.O., Amupitan, J.O., Fatope, M.O., Okogun, J.I., 2011. Eldeen, I.M.S., Van Staaden, J., 2007. Antimycobacterial activity of some trees used in Antimycobacterial Friedelin-Terpenoid from the root bark of Terminalia avicennioides. South African traditional medicine. South African Journal of Botany 73, 248–251. American Journal of Chemistry 1 (2), 52–55. Eldeen, I.M.S., Van Heerden, F.R., Van Staaden, J., 2008. Isolation and biological Martins, A., Vasas, A., Viveiros, M., Molnàr, J., Hohmann, J., Amaral, L., 2011. Antibacterial activities of termilignan B and arjunic acid from Terminalia sericea roots. Planta Medica properties of compounds isolated from Carpobrotus edulis. International Journal of 74, 411–413. Antimicrobial Agents 37, 438–444. Elegami, A.A., El-Nima, E.I., El-Tohami, Muddathir, A.K., 2002. Antimicrobial activity of Masoko, P., Eloff, J.N., 2005. The diversity of antifungal compounds of six South African some species of the family Combretaceae. Phytotherapy Research 16, 555–561. Terminalia species (Combretaceae) determined by bioautography. African Journal of Eloff, J.N., 1999. The antibacterial activity of 27 southern African members of Biotechnology 4 (12), 1425–1431. Combretaceae. South African Journal of Science 95, 148–152. Masoko, P., Picard, J., Eloff, J.N., 2005. Antifungal activities of six South African Terminalia Engels, C., Schieber, A., Gänzle, M.G., 2011. Inhibitory spectra and modes of antimicrobial species (Combretaceae). Journal of Ethnopharmacology 99, 301–308. action of gallotannins from Mango Kernels (Mangifera indica L.). Applied Environmental Mbawambo, Z.H., Erasto, P., Nondo, R.O., Innocent, E., Kidukuli, A.W., 2011. Antibacterial Microbiology 77, 2215–2223. and cytotoxic activities of Terminalia stenostachya and Terminalia spinosa. Tanzania Esimone, C.O., Nworu, C.S., Onuigbo, E.B., Omeje, J.U., Nsirim, K.L., Ogbu, J.C., Ngwu, M.I., Journal of Health Research 13 (2), 1–8. Chah, K.E., 2009. Anti-mycobacterial activity of root and leaf extracts of Anthocleista McKinney, J.D., 2000. In vivo veritas: the search for TB drug targets goes live. Nature djalonensis (Loganiaceae) and Diospryros mespiliformis (Ebenaceae). International Medicine (New York) 6, 1330–1333. Journal of Green Pharmacy 3, 201–205. Mitscher, L.A., Baker, W.R., 1998. A search for novel chemotherapy against tuberculosis Fabry, W., Okemo, P., Ansorg, R., 1998. Antibacterial activity of East African medicinal amongst natural products. Pure Applied Chemistry 70 (2), 365–371. plants. Journal of Ethnopharmacology 60, 79–84. Moshi, M.J., Mbwambo, Z.H., 2005. Some pharmacological properties of extracts of Friedman, M., Henika, P.R., Levin, C.E., Mandrell, R.E., Kozukue, N., 2006. Antimicrobial Terminalia sericea roots. Journal of Ethnopharmacology 97, 43–47. activities of tea catechins and theaflavins and tea extracts against Bacillus cereus.Journal Moshi, M.J., Mbwambo, Z.H., Nondo, R.S.O., Masimba, P.J., Kamuhabwa, A., Kapingu, M.C., of Food Protection 69, 354–361 (2006). Thomas, P., Rickard, M., 2006. Evaluation of ethnomedical claims and brine Fyhrquist, P., Mwasumbi, L., Hæggström, C.-A., Vuorela, H., Hiltunen, R., Vuorela, P., 2002. shrimp toxicity of some plants used in Tanzania as traditional medicines. Ethnobotanical and antimicrobial investigation of some species of Terminalia and African Ethnomedicines network 3 (3), 48–58. Combretum (Combretaceae) growing in Tanzania. Journal of Ethnopharmacology Msonthi, J.D., Mgombo, D., 1983. Medicinal herbs in Malawi and their uses. Hamdard 26 79, 169–177. (2), 94–100. Fyhrquist, P., Mwasumbi, L., Haeggström, C.-A., Vuorela, H., Hiltunen, R., Vuorela, P., Neuwinger, H.D., 1996. African Ethnobotany Poisons and Drugs Chemistry (Germany). 2004. Antifungal activity of selected species of Terminalia, Pteleopsis and Chapman & Hall GmbH, D-69469 Winheim (Bundes Republik Deutschland). Combretum (Combretaceae) collected in Tanzania. Pharmaceutical Biology 42 Neuwinger, H.D., 2000. African Traditional Medicine. A Dictionary of Plant Use and (4–5), 308–317. Applications.Medpharm Scientific Publishers, Stuttgart, Germany 589. Garces, F.R., Garces, W.S., Miguel, D.L.S., Serea, A.A.T., Prado, F.C., 2003. Chemical Newton,S.M.,Lau,C.,Gurcha,S.S.,Besra,G.S.,Wright,C.W.,2002.The evaluation constituents from Terminalia glabrescens. Journal of the Brazilian Chemical Society 14 of forty three plant species for in vitro antimycobacterial activities; isolation of (3), 461–465. active constituents from Psoralea corylifolia and Sanguinaria canadensis.Journal Gelfland, M., Mavi, S., Drummond, R.B., Ndemera, B., 1985. The traditional medicinal of Ethnopharmacology 79, 57–67. practitioner in Zimbabwe. Mambo Press, Gweru, Zimbabwe. Nvau, J.B., Oladosu, P.O., Orishadipe, A.T., 2011. Antimycobacterial evaluation of some Global Alliance for TB Drug Development, 2001. Tuberculosis. Scientificblueprintfor medicinal plants used in the plateau State of Nigeria for treatment of tuberculosis. tuberculosis drug development. Tuberculosis (Edinburgh, Scotland) 81 (Suppl. 1), 1–51. Agriculture and Biology Journal of North America 2 (9), 1270–1272. Green, E., Samie, A., Obi, C.L., Bessong, P.O., Ndip, N.R., 2010. Inhibitory properties of Oelrichs, P.B., Pearce, C.M., Zhu, J., Filippich, L.J., 1994. Isolation and structure determination selected South African Medicinal plants against Mycobacterium tuberculosis.Journal of terminalin A, a toxic condensed tannin from Terminalia oblongata. Natural Toxins 2 of Ethnopharmacology 130, 151–157. (3), 144–150. Haerdi, F., 1964. Die Eingeborenen-Heilpflanzen des Ulanga-Distriktes Tanganjikas Okemo, P.O., 1996. Antimicrobial Efficacy of Selected Medicinal Plants used by Kenyan (Ostafrika). Acta Tropica. Supplementum 8, 1–278. Herbal doctors. Ph. D. thesis Kenyatta University of Nairobi. Hagerman, A.E., 2002. Tannin Handbook. Miami University, Oxford OH 45056. www.users. Omulokoli, E., Khan, B.,Chhabra,S.C.,1997.Antiplasmodial activity of four Kenyan medicinal muohio.edu/hagermae/. plants. Journal of Ethnopharmacology 56, 133–137. 16 P. Fyhrquist et al. / South African Journal of Botany 90 (2014) 1–16

Osuga,I.M.,Abdulrhazak,S.A.,Nishino, N., Ichinohe, T., Fujihara, T., 2006. Potential nutritive Silva,O.,Gomes,E.T.,Wolfender,J.L.,Marston,A.,Hostettman,K.,2000.Applica- value of selected browse species from Kenya using in vitro gas production technique tion of high performance liquid chromatography coupled with ultraviolet and polyethylene glycol. Livestock Research for Rural Development 18 (12). spectroscopy and electrospray mass spectrometry to the characterization of Panichayupakaranant, P., Tewtrakul, S., Yuenyongsawad, S., 2010. Antibacterial, anti- ellagitannins from Terminalia macroptera roots. Pharmaceutical Research 17 inflammatory and anti-allergic activities of standardised pomegranate rind extract. (11), 1396–1401. Food Chemistry 123, 400–403. Silva, O., Ferreira, E., Vaz Pato, M., Canica, M., Gomes, E.T., 2002. In vitro anti-Neisseria Pauli, G.F., Case, R.J., Inui, T., Wang, Y., Cho, S., Fisher, N.H., Franzblau, S.G., 2005. New gonorrheae activity of Terminalia macroptera leaves. FEMS Microbiology letters 211 perspectives on natural products in TB drug research. Life Science 78, 485–494. (2), 271–274. Pereira, M., Tripathy, S., Indamdar, V., Ramesh, K., Bhavsar, M., Date, A., Iyyer, R., Singariya, P., Kumar, P., Mourya, K.K., 2012. An activity guided isolation and evaluation of Acchammachary, A., Mehendale, S., Risbud, A., 2005. Drug resistance pattern of various solvent extracts of the leaves of Anjan grass. Hygeia: Journal for Drugs and Mycobacterium tuberculosis in seropositive and seronegative HIV-TB patients in Medicines 4 (2), 49–56. Pune, India. Indian Journal for Medicinal Research 121, 235–239. Soh, R.F., Bankeu, J.K., Lenta, B.N., Mba`ning, B.M., Ngouela, S., Tsamo, E., Sewald, N., 2009. Pfundstein, B., Desouky, S.K.L., Hull, W.E., Haubner, R., Erben, G., Owen, R.W., 2010. Poly- Antibacterial ellagic acid derivatives and other constituents from Pancovia phenolic compounds in the fruits of Egyptian medicinal plants (Terminalia bellerica, pedicellaris. Zeitschrift für Naturforschung 64b, 1070–1076. Terminalia chebula and Terminalia horrida): characterization, quantitation and deter- Steenkamp, V., Matihva, E., Gouws, M.C., Van Rensberg, C.E.J., 2004. Studies on antibacterial, mination of antioxidant capacities. Phytochemistry 71, 1132–1148. antioxidant and fibroblast growth stimulation of wound healing remedies from South Piddock, L.J.V., Williams, K.J., Ricci, V., 2000. Accumulation of rifampicin by Mycobacterium Africa. Journal of Ethnopharmacology 95, 353–357. aurum, Mycobacterium smegmatis and Mycobacterium tuberculosis. Journal of Antimi- Tian, F., Li, B., Ji, B., Zhang, G., Luo, Y., 2009. Identification and structure–activity relationship crobial Chemotherapy 45, 159–165. of gallotannins separated from Galla chinensis.LWT— Food Science and Technology 42, Rauha, J.-P., Remes, S., Heinonen, M., Hopia, A., Kähkönen, M., Kujala, T., Pihlaja, I.K., Vuorela, 1289–1295. H., Vuorela, P., 2000. Antimicrobial effect of Finnish plant extracts containing the flavo- Tshikalange, T.E., Meyer, J.J.M., Hussein, A.A., 2005. Antimicrobial activity, toxicity, and the noids and other phenolic compounds. International Journal of Food Microbiology 56, isolation of a bioactive compound from the plants used to treat sexually transmitted 3–12. diseases. Journal of Ethnopharmacology 96, 515–519. Reddy, M.K., Gupta, S.K., Jacob, M.R., Khan, S.I., Ferreira, D., 2007. Antioxidant, antimalarial Von Martius, S., Hammer, K.A., Locher, C., 2012. Chemical characteristics and and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids antimicrobial effects of some Eucalyptus kinos. Journal of Ethnopharmacology from Punica granatum L. Planta Medica 73, 461–467. 144, 293–299. Rogoza, L.N., Salakahutdinov, N.F., Tolstikov, G.A., 2011. Anti-tubercular Activity of Natural Vovk, I., Simonovska, B., Vuorela, P., Vuorela, H., 2002. Optimization of separation of (+)- Products: Recent Developments. Opportunity, Challenge and Scope of Natural products catechin and (–)-epicatechin on cellulose TLC plates. Journal of Planar Chromatogra- in Medicinal Chemistry, pp. 103–120. phy 15 (6), 433–436. Sarabhai, S., Sharma, P., Capalash, N., 2013. Ellagic acid derivates from Terminalia chebula Wickens, G.E., 1973. Flora of Tropical East Africa. Combretaceae.East African Community Retz. downregulate the expression of quorum sensing genes to attenuate Pseudomonas (99 pp.). aeruginosa PAO 1 virulence. PLoS One 8 (1), 1–11. World Health Organization, 1999. Global Tuberculosis Control. WHO Report 1999.Geneva, Scalbert, A., 1991. Antimicrobial properties of tannins. Phytochemistry 30, 3875–3883. WHO. Scalbert, A., Mila, I., Expert, D., Marmolle, F., Albrecht, A.-M., Hurrell, R., Huneau, J.-F., World Health Organization, 2010. Global Tuberculosis Control Report. WHO report Tome´, D., 1999. Polyphenols, Metal Ion Complexation and Biological Consequences, 2010.WHO, Geneva 218. In: Gross, G.G., Hemingway, R.W., Yoshida, T. (Eds.), 1st ed. Plant Polyphenols 2: Yemoa, A., Gbenou, J., Affolabi, D., Moudachirou, M., Bigot, A., Anagonou, S., Portaels, F., Chemistry, Biology, Pharmacology, Ecology, vol. 66. Kluwer Academic/Plenum Pub- Quetin-Leclercq, J., Anandi, M., 2011. Buruli ulcer: a review of in vitro tests to screen lishers, New York, p. 545 (1999). natural products for activity against Mycobacterium ulcerans. Planta Medica 77, Shai, L.J., McGaw, L.J., Masoko, P., Eloff, J.N., 2008. Antifungal and antibacterial activity of 641–646. seven traditionally used South African plant species active against Candida albicans. York, T., Van Vuuren, S.F., de Wet, H., 2012. An antimicrobial evaluation of plants for treat- South African Journal of Botany 74, 677–684. ment of respiratory infections in rural Maputaland, Kwazulu-Natal, South Africa. Jour- Shiota, S., Shimizu, M., Sugiyama, J., Morita, Y., Mizushima, T., Tsushiya, T., 2004. Mecha- nal of Ethnopharmacology 144, 118–127. nisms of action of Corilagin and Tellimagrandin I that remarkably potentiate the ac- Yoshida, T., Amakura, Y., Yoshimura, M., 2010. Structural features and biological proper- tivity of β-lactams against methicillin-resistant Staphyloccus aureus. Microbiology ties of ellagitannins in some plant families of the order myrtales. International Journal and Immunology 48 (1), 67–73. of Molecular Sciences 11, 79–106.